1. health and fitness
2. disease

DC Academy Notes Dwight D. Bowman, MS, PhD Department of Microbiology & Immunology, College of Veterinary Medicine, Cornell University Giardia, Cryptosporidium, and Coccidia Some intestinal parasitic infections of the dog and cat will just not cure. It is fairly easy these days to clear these pets of their infections with nematodes, trematodes, and cestodes, because the pharmaceutical industry has provided excellent means of combating these parasites with wonder drugs, such as, fenbendazole, ivermectin, and praziquantel. Low doses kill worms and the remarkable levels of safety due to selection for products that are highly lethal to the worms while remaining well below toxic levels in the host are fantastic compared to what used to be. However, certain parasites are either hard to treat, infections refuse to clear, or they just refuse to be controllable in certain environments. Some of this is due to the lack of efficacy of all drugs as occur with Cryptosporidium, others like Giardia just do not seem to respond to repeated treatments, and finally there is Cystoisospora that has stages in the mucosa that perhaps reactivate for some reason later in the life on the animal. Cryptosporidium Dogs are host to their own species of this genus of parasite, Cryptosporidium canis Fayer, Trout, Xiao, Morgan, Lal, and Dubey, 2001. Cats have their own species as well, Cryptosporidium felis Isaki, 1979. These two parasites only have minimal zoonotic potential, having been found in people, in spite of their living in close contact with their pets, only on very few occasions. However, due to common knowledge amongst both veterinarians and the public as to the potential threat of treating people with Cryptosporidium, the treatment of these infections. Thus, attempts have been made to clear these hosts of their infection with a number of products. However, as with people, treatment has been less than satisfactory. Animals seem to remain infected, in spite of repeated treatment. Interestingly, and probably because of the drug treatment failures, there have been basically no reports on treating dogs and cats for cryptosporidiosis; with a single report on trying to treat cats with paromomycin. There are two possible reasons why treatment is so difficult. It may be because of the nature of the relationship of the parasite to the host cell. It may also be that Cryptosporidium is not the same as other coccidia, and thus, the drugs that work against Eimeria and other coccidial parasites just do not work the same against this species. The organisms causing cryptosporidiosis are barely within the cell that they infect. These parasites live just inside the host-­‐cell membrane and develop a thickened boundary between themselves and the host cell cytoplasm. Thus, it is considered by some that they do not respond to typical drug treatments because the drugs just cannot get there through the regular host cell machinery. Another thought is that it is because the genus Cryptosporidium has been removed from the coccidia proper and placed within the gregarines (Barta and Thompson, 2006; Carreno et al, 1997). This group of parasites is known to be related to the coccidia and other Apicomplexan parasites like the hemoparasitic Apicomplexa, such as, Plasmodium and Babesia. Most gregarines are found within the intestinal tracts of marine, freshwater, and terrestrial vertebrates. They tend to be very large cells, and they do not enter host cells like coccidia, instead they attach by their front ends to the surface of the intestinal hold cell and appear to feed from the attachment site, a process known as myzocytosis. Thus, they remain for the most part extracellular to the host cell. Cryptosporidium seems to be a miniature form of 1 gregarine that infects vertebrates and in its biology goes one step further in that it actually enters the host cell, but barely. It is possible that one reason the common anticoccidials drugs are not successful in treating cryptosporidiosis is that they were all developed for related but markedly different Apicomplexan parasites, the coccidia and allies. There has been some evidence that form Colorado that dog park attending dogs may have a greater prevalence of Cryptosporidium and Giardia in their feces than dogs not attending such parks, but the numbers of animals in this study was quite small (Wang et al, 2012). The one isolate of Cryptosporidium recovered from these dogs was C. canis. Prevalence of infection has been examined on several occasions recently, but remains problematic in that many animals only shed for a short period of time. An examination of 1,322 fecal samples from shelter cats in New York State revealed that 3.8% of the cats were shedding at the time they were examined (Lucio-­‐Forster and Bowman, 2011). In a survey of cats with and without diarrheic feces in northern California, 3.2% of 190 liquid samples were positive for Cryptosporidium while none of 54 normal fecal samples were positive; however the number of samples was such that the association was not statistically significant (Queen et al., 2012). Of course many cats are likely to have been positive at some point in their lives, and the infection has gone unnoticed. There is not any truly successful treatment for cryptosporidiosis in people or animals. The Companion animal parasite Council lists some of treatments that have been utilized (CAPC website). These include: Paromomycin: 150 mg/kg SID for 5 days (dogs and cats); Tylosin: 10 to 15 mg/kg TID for 14 to 21 days (cats); azithromycin: 5 to 10 mg/kg BID for 5 to 7 days (dogs); 7 to 15 mg/kg for 5 to 7 days (cats); and nitazoxanide (Alinia™): 100 mg BID for 5 days in animals 24 to 47 months old and 200 mg BID for 5 days in animals 4 to 11 years old. The efficacy of nitazoxanide in dogs and cats is not known. Giardia The species of Giardia infecting mammals have undergone a bit of nomenclatural confusion because old species were named, and these get in the way of naming new species. They are now divided for the most part between hosts by an assignment of a letter code that has been determined by molecular identification. Dogs are most commonly infected with what has been identified and named as Assemblages C and D; cats most commonly by Assemblage F; and people get Assemblage B and also a form of A. There does appear to be some minimal cross-­‐over of the assemblages between the different hosts, but most of the time, the assemblages seem to stay in their host. Again, although there are different opinions, it appears that for the most part the zoonotic potential of the Giardia assemblages infecting pets is minimal. Recent work from the United States and Canada seems to support that dogs and cats are infected most commonly with Assemblages C&D and F, respectively. It was shown in Canada that of samples submitted to a university hospital for diagnosis, 68% of 75 canine samples were D, 31% D, and a single sample contained B (the human associated assemblage) (McDowall et al., 2011). Of 13 cats sampled, 100% were Assemblage F. Similarly, of 5 samples from dogs in Colorado, all 5 samples were C or D (Wang et al., 2012). The one exception has been from 128 samples examined from dogs in the western United States where of 128 samples, 15% were B, 16% D, 28% A, and 41% B, with 83% of the dogs having mixed infection with different Assemblages (Covacin et al., 2011); this appears to be the only report with such a wide away of genotypes being observed. Cats have been examined as to Assemblage 2 less than dogs recently in the USA, in 2006, the examination of Giardia cysts being shed by cats in a colony, revealed that 8 of the 18 cats were all shedding Assemblage F (Fayer et al., 2006). Unlike for Cryptosporidium, a number of products have been used to treat infected dogs and cats. These include fenbendazole and febantel, albendazole, tinidazole, oxfendazole, mebendazole, furazolidone, quinacrine, and azithromycin. Also, unlike for Cryptosporidium, these products have been used in a number of published laboratory trials and have been reported on as to their success in a number of publications. REPORTS ON TREATMENT: DOGS Metronidazole and other nitroimidazoles: Work has appeared on these compounds for treating giardiasis in dogs for the last 30 years. In none of these studies does metronidazole appear to be 100% curative in all dogs. Cure rates (Percentage of dogs that stop shedding organisms in their feces) have been reported in a number of studies: for tinidazole, metronidazole, and ornidazole cure rates were reported as less than 50% (Nesvadba et al., 1980); metronidazole curing 67% and tinidazole curing 68% (Zimmer and Burrington, 1986); 100% cure of 6 dogs after 18 days from the first treatment with metronidazole using cyst recovery, but only 5 of 6 dogs, 83%, were cured using antigen detection (Decock et al., 2003); again with metronidazole, 72% were cured 1 week after treatment, 75% at 10 days after treatment, and 100% by 2 weeks after treatment with cure being assessed by cyst recovery with the control dogs remaining positive (Chon and Kim 2005); in another study by the same authors, after 7 days of treatment with metronidazole, on days 8, 9, and 10 only 5 of the 7 treated dogs were clear of cysts (Kim and Chon, 2005). Benzimidazoles: Using benzimidazoles to treat canine giardiasis began about 10 years after the first reports on treatment with nitroimidazoles. Initial work with albendazole showed promise in using this formulation in treating giardiasis (Barr et al., 1983), but this work has not been pursued further due to the development of bone-­‐marrow toxicosis in some treated dogs (Meyer, 1998). Fenbendazole: Fenbendazole is now approved in most European Union countries for the treatment of giardiasis in dogs. The work that is published with fenbendazole shows fairly good efficacy. Treatment of 6 dogs daily for 3 days or 3 times a day for 3 days cleared 100% of these 12 dogs when 3 fecal samples were examined for cysts after the last day of treatment with 1 day between samples; cure rates of 100% (Barr et al, 1994). Another study with 10 dogs treated for 3days found that with 12 fecal samples being examined for cysts over the next 25 days that 9 dogs remained negative, but one dog had a single positive sample the third week after treatment; cure rate of 90% (Zajac et al, 1998). In another study (the same one that above found a 100% cysts cure in 6 dogs treated with metronidazole) found only 50% of dogs treated with fenbendazole cured relative to cysts by day 18 after treatment, and 0% cured relative to antigen clearance (Decock et al., 2003). All 7 dogs treated for seven days were found to be cyst free when examined on days 8, 9, and 10 after the first treatment – a cure rate of 100% (Kim and Chon, 2005). In a clinical trial in which fenbendazole was given in a combination product along with praziquantel for 3 days, of 34 dogs house in groups, the cure rate was 65%; for dogs house individually, the cure rate was 85% (Beelitz et al., 2006). Of 26 clinical canine cases in Japan that were treated (11 with fenbendazole for 3 days; 8 with febantel combination for 3 days; 7 with albendazole BID for 2 days), clearance was reported as 100% in all cases after one or two treatment regimens (Itoh et al., 2002). Febantel: There has been an equivalent or slightly greater number of publications on febantel in combination praziquantel and pyrantel than there have been on fenbendazole. Febantel appears in different concentrations in different formulations, thus dosages must be compared more carefully for this compound than the others. For, five naturally infected, lab-­‐reared beagles treated for three days in a row or 5 lab-­‐beagles treated with the same dose only once; based on fecal examination for cysts daily 3 for 10 days, the cure rate for the thrice treated dogs was 100%, while the cure rate for the other treated group was only 40% (Barr et al., 1998). Using a copro-­‐antigen test to verify clearance, in dogs treated for two days in a row at the anthelmintic dosage, 100% of 14 dogs less than 1 year were leared, 89% of 1-­‐2 year-­‐old dogs were cleared, and 86% of dogs over 2 years of age were cleared (Bartuzki et al., 2002). Using naturally infected, lab-­‐reared beagles, 4 were treated for 3 days with a febantel containing combination product and 5 were treated for 5 days; 19 days after the last treatment, 75% of the 4 3-­‐day treated dogs were cured as determined by antigen and cyst detection, and 80% of the dogs treated for 5-­‐days were cleared (Payne et al, 2002). In another study, 67% of 5 dogs treated daily for 3 days were cleared of cysts and 17% were cleared with respect to antigen (Decock et al., 2003). Of 26 clinical giardiasis cases in Japan that were treated (8 with febantel combination for 3 days; 11 with fenbendazole for 3 days; 7 with albendazole BID for 2 days), clearance was reported as 100% in all cases after one or two treatment regimens (Itoh et al., 2002). Oxfendazole: Using dogs from kennels, oxfendazole was given to dogs in four kennels known to be shedding cysts in their feces. The dogs were treated for 3 days orally with oxfendazole liquid at a 1X and a 2X dose; there were 2 kennels with less than adequate sanitation and 2 kennels with excellent sanitation and the dogs were kept in their runs with the cages being washed with ammonia or bleach after the administration of the first of the three treatments. Using cysts recovery as the measure of detection, with both doses, dogs in the 2 kennels with inadequate sanitation (Kennel 1 with 6 dogs treated at the 1X dose; Kennel 2 with 11 dogs (incl.: 6 not cleared at Kennel 1 and 5 more dogs) treated at the 2X doses) had cysts reappear in the feces by Day 12 or sooner. Thus clearance at Day 12 was 0%. In the other two kennels, the 10 dogs in Kennel 3 and the 9 dogs in Kennel 4 all remained negative 9 days after the first treatment indicting 100% clearance (Villeneuve et al., 2000). In the one other trial with oxfendazole, there were 5 dogs treated at the 1X dose kept in cages that were cleaned weekly with quaternary ammonia (Decock et al., 2003). There were two dogs negative for cysts and antigen 5 days after the first treatment, but all dogs were positive from Day 10 by one or both assays. By day 18 after treatment, all dogs were positive by antigen detection (0% clearance) and 4 of the 5 dogs by cyst detection (20% cleared). Acridine derivatives: In people, historically about 90% of patients are cleared of their infections with a 5-­‐day course of quinacrine treatment (Atabrine) (Beaver et al., 1984). There have been few studies with this drug in dogs with giardial infections, but the studies are successful enough that they probably warrant further attention. In one study, 18 dogs that were shedding cysts were treated with quinacrine BID for 5 days. None of the treated dogs shed cysts for 3 weeks after infection (the pens housing the dogs and the exercise yards had a quaternary ammonium-­‐based detergent-­‐disinfectant applied daily as a layer of foam to the walls, floors, and fences where it was allowed to sit for at least 15 minutes before rinsing) (Zimmer and Burrington, 1986). The study also looked at intestinal trichomonads that were not affected by the quinine. In a second group of dogs that was treated with quinine and metronidazole BID for 5 days, all dogs were cleared of both parasites with stools being examined for cysts for 3 weeks. There was some very early work using quinacrine in clinically ill dogs in Canada where the drug was given TID on the first day and BID for 5 more days with excellent results; in this case the readout was the presence of trophozoites in the stool (Croquette, 1950). Macrolide antibiotics: Azithromycin has been used to treat a single dog in Poland for diarrhea that was infected with Giardia (10 mg/kg per os, SID for 5 days) (Zygner et al., 2008). This diarrhea ceased in this dog and examination of the stool by microscopic and PCR examination could no longer detect the parasite after treatment was completed. REPORTS ON TREATMENT: CATS Metronidazole and other nitroimidazoles: Again there have been a number of reports with these compounds with varying cure rates, but it appears that metronidazole may seem to be more effective in 4 cats than in dogs: for tinidazole, metronidazole, and ornidazole cure rates were reported as less than 50% (Nesvadba et al., 1980); it was reported that treatment with metronidazole at 22 mg/kg, BID, for 5 days was 100% curative in 24 cats by cyst examination with 17 days after treatment (Zimmer, 1987); treatment of 26 cats with 25 mg/kg, BID for 5 days was 100% curative in 26 treated cats as determined by fecal examination with IFA (Scorza and Lappin, 2004). Benzimidazoles: Fenbendazole: Treating 8 cats with fenbendazole (50 mg/kg, PO, SID, for 5 days) resulted in only a 50% cure rate as determined by finding cysts in the feces after treatment using IFA (Keith et al., 2003. Febantel: Using febantel at 37.8 mg/kg PO, SID for 5 days or at 12.5 mg/kg PO, SID for 5 days, cured only 3% of 10 cats of their Giardia infections, while treatment of 6 cts with 56.5 mg/kg, PO, SID for 5 days cured 66% of 6 cats of their infection (Scorza et al., 2006) RESISTANCE: For whatever reason, giardiasis sometimes will just not clear in an infected dog and cat. Repeated treatments with different products fail to clear animals with formed stools of their infections, may simply remove signs from animals with loose stools, or may simply not seemingly have any effect on the outcome of a persistent infection. There is some indication from human medicine that Giardia in some instances is perhaps truly resistant to metronidazole and benzimidazoles (Abboud et al., 2001 for metronidazole and albendazole; Jimenes-­‐Cardose et al., 2004 for albendazole; Leitsch et al., 2011 for nitroimidazoles generally). This is of concern in veterinary medicine considering how often these products are used to treat giardiasis in dogs and cats, but it may go a long way to explain the failures seen in the field. Unfortunately, there is no good marker that is currently in place to detect whether the Giardia with which one is dealing, whatever the given Assemblage, is resistant or not. It is possible that in the next few years, molecular markers may be developed by various reference laboratories that will be able to report whether or not a strain appears to be of a resistant variety or not. Cystoisospora Cystoisospora canis and C. felis along with several other species within this genus are what everyone used to consider as being within the genus Isospora. The reason for the name change is that these parasites have a cyst-­‐like stage, a cystozoite, which occurs in the tissues of a host that ingests an infective oocyst containing sporozoites (Barta et al., 2005). Thus, the parasite can persist in the tissue of paratenic hosts like sparrows, rabbits, and mice that ingest the oocysts. They can then pass the infection along, when dogs eat the oocysts. This is totally unlike the coccidial infections of herbivores and chickens where there is no such stage. When dogs and cats persistently shed these forms in their feces, recurrently over time, it is hard to know if it is reinfection or if it is reactivation of the latent cystozoites in the tissues. At this time, there is no good evidence of cystozoite reactivation, but it does appear to be a possibility. However, when coccidial infections in dogs and cats will not clear after treatment, it must also be remembered that the endogenous stages within the intestine of these animals, tho oocysts that are produce may be deep in the lamina propria, and may take awhile to erode their way to the surface for excretion in feces. The Companion Animal Parasite Council sites a number of treatments for coccidiosis in dogs and cats that have been described and used in the past (CAPC website). The only drug approved in terms of coccidiosis for dogs and cats is sulfadimethoxine that is actually labeled for treatment of the enteritis associated with coccidiosis. In Europe, Bayer Animal Health has received a label against the replication 5 and shedding of coccidial oocysts in dogs and puppies using emodepside plus toltrazuril suspension (Procox® suspension for dogs) at 0.45 mg emodepside and 0.9 mg toltrazuril per kg body weight. This product also treats adult, immature adult and larval stages of Toxocara canis and adult stages of Uncinaria stenocephala and Ancylostoma caninum (Altreuther et al., 2011). Finally, it should be remembered that we know that coccidial parasites of other hosts are highly likely to develop resistance in the face of continued drug pressure, so, concern should be given to the routine treatment of shelter animals for coccidial infections that are not causing clinical signs. Conclusions It would seem that there will continue to be problems treating cryptosporidiosis in dogs and cats. Giardia is can be treated, but the cure rate is often not 100%. For coccidia in puppies, ponazuril/toltrazuril is recognized by many veterinarians to be the choice for treating infections with this group of parasites. IN the case of Giardia and Cryptosporidium, due to concerns about potential resistance developing, concern should be given to the need to treat all animals that are without signs and the goal of having every dog and cat being treated within a shelter or when it is in the process of being adopted. REFERENCES Cryptosporidium Barta JR, Thompson RCA. 2006. What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends Parasitol 22:463-­‐468. Carreno RA, et al., 1994. Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-­‐subunit ribosomal RNA gene sequences. Parasitol Res 85:899-­‐904. Companion Animal Parasite Council http://www.capcvet.org/capc-­‐recommendations/cryptosporidia/ Lucio-­‐Forster A, Bowman DD. 2011. Prevalence of fecal-­‐borne parasites detected by centrifugal flotation in feline samples from two shelters in upstate New York. J Fel Med Surg 13: 300-­‐303. Queen EV, et al 2012 Prevalence of selected bacterial and parasitic agents in feces from diarrheic and healthy control cats from northern California J Vet Intern Med 26: 54-­‐60. Wang A, et al 2012 Prevalence of Giardia and Cryptosporidium species in dog park attending dogs compared to non-­‐dog park attending dogs in one region of Colorado Vet Parastiol 184: 335-­‐340. Giardia Abboud P, et al., 2001. Successful treatment of metronidazole-­‐ and albendazole-­‐resistant giardiasis with nitazoxanide in a patient with acquired immunodeficiency syndrome. Clin Inf Dis 32: 1792-­‐1794. Barr SC, et al. 1993. Efficacy of albendazole against giardiasis in dogs. Am J Vet Res 54: 926-­‐928. Barr SC, et al. 1994. Efficacy of fenbendazole against giardiasis in dogs. Am J Vet Res 55: 988-­‐990. Barr SC, et al. 1998. Efficacy of a drug combination of praziquantel, pyrantel pamoate, and febantel against giardiasis in dogs. Am J Vet Res 59: 1134-­‐1136. Barutzki D. 2002. Efficacy of pyrantel embonate, febantel and praziquantel against Giardia spp. in naturally infected dogs. In Giardia: the cosmopolitan parasite, Olson BE et al eds, Pages: 177-­‐180 Beaver PC et al. 1984. Clinical Parasitology, Lea & Febiger, Philadelphia, PA 484 pages Beelitz P, et al. 2006. Giardia-­‐Infektionen bei Hunden in Deutschland: Bewertung von Behandlungsregimen in unterschiedlichen Haltungsformen und Pravalenz. Prakt Tierartz 87: 597-­‐
603. Chon SK, Kim NS. 2005. Evaluation of silymarin in the treatment on asymptomatic Giardia infections in dogs. Parasitol Res 97:445-­‐451. Covacin C, et al., 2011. Genotypic characterisation of Giardia from domestic dogs in the USA. Vet Parasitol 177:28-­‐32. 6 Croquette LPE. 1950. Canine giardiasis and its treatment with atebrin. Can J Comp Med 14:230-­‐235. Decock C, et al. 2003. Evaluation de quatre traitements de la giardiose canine. Rev Med Vet ue de Medecine Veterinaire 154:763-­‐766. Fayer R, et al. 2006. Detection of Cryptosporidium felis and Giardia duodenalis Assemblage F in a cat colony. Vet Parasitol 140: 44-­‐53. Itoh N, et al. 2002. Treatment of canine giardiasis with benzimidazoles. J Jap Vet Med Assoc 55:739-­‐
743. Jimenez-­‐Cardose E, et al. 2004. In vitro activity of two phenyl-­‐carbamate derivatives, singly and in combination with albendazole against albendazole-­‐resistant Giardia intestinalis. Acta Tropica 92: 237-­‐244 Keith CL, et al 2003. Evaluation of fenbendazole for treatment of Giardia infection in cats concurrently infected with Cryptosporidium parvum. Am J Vet Res 64: 1027-­‐1029. Kim NS, Chon SK. 2005. The efficacy of albendazole, fenbendazole and metronidazole for treatment of canine Giardia. J Vet Clin 22:239-­‐243. Leitsch D, et al. 2011. Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-­‐
nitroimidazole activation while flavin metabolism is linked to 5-­‐nitroimidazole resistance in Giardia lamblia. J Antimicrob Chemother 66: 1756-­‐1765. McDowall RM, et al. 2011. Evaluation of the zoonotic potential of Giardia duodenalis in fecal samples from dogs and cats in Ontario. Can Vet J 52: 1329-­‐1333. Meyer EK. 1998. Adverse events associated with albendazole and other products used for treatment of giardiasis in dogs. J Am Vet Med Assoc 213: 44-­‐46. Nesvadba J et al., 1980. Beitrag zur klinischen Manifestation und Therapie der Giardiasis. Deutsche Veterinarmedizinische Gesellschaft Fachgruppe Kleintierkrankheiten in Verbindung mit der deutschsprachigen Gruppe der WSAVA, 13-­‐15 November 1980 in Karlsruhe. pp 223-­‐232. Payne PA, et al. 2002. Efficacy of a combination febantel-­‐praziquantel-­‐pyrantel product, with or without vaccination with a commercial Giardia vaccine, for treatment of dogs with naturally occurring giardiasis. J AM Vet Med Assoc 220: 330-­‐333. Scorza AV, et al. 2006. Efficacy of a combination of febantel, pyrantel, and praziquantel for the treatment of kittens experimentally infected with Giardia species. J Fel Med Surg 8: 7-­‐13. Scorza AV, Lappin MR. 2004. Metronidazole for the treatment of feline giardiasis. J Fel Med Surg 6: 157-­‐160. Villeneuve V, et al. 2000. Efficacy of oxfendazole for the treatment of giardiosis in dogs. Experiments in dog breeding kennels. Parasite 7: 221-­‐226. Wang A, et al 2012 Prevalence of Giardia and Cryptosporidium species in dog park attending dogs compared to non-­‐dog park attending dogs in one region of Colorado Vet Parasitol 184: 335-­‐340. Zajac AM, et al., 1998. Efficacy of fenbendazole in the treatment of experimental Giardia infection in dogs. Am J. Vet Res 59: 61-­‐63. Zimmer JF, Burrington DB. 1986. Comparison of four protocols for the treatment of canine giardiasis. J AM Anim Hosp Assoc 22: 168-­‐172. Zygner W, et al. 2008. Azithromycin in the treatment of a dog infected with Giardia intestinalis. Polish J Vet Sci 11: 231-­‐234. Cystoisospora Altreuther G, et al. 2011. Efficacy of emodepside plus toltrazuril suspension (Procox oral suspension for dogs) against prepatent and patent infection with Isospora canis and Isospora ohioensis-­‐complex in dogs. Parastiol Res 109: S9-­‐S20. Barta JR, et al. 2005. The genus Atoxoplasma (Garnham 1950) as a junior objective synonym of the genus Isospora (Schneider 1881) species infecting birds and resurrection of Cystoisospora (Frenkel 1977) as the correct genus for Isospora species infecting mammals. J Parasitol 91: 726-­‐727.
7 Toxoplasmosis Toxoplasma gondii is a widespread zoonotic protozoan that infects most, if not all, species of birds and mammals. As the definitive hosts for this organism, felines are the only animals that pass oocysts in their feces, although intermediate hosts harbor infective tissue cysts. Most feline infections occur post-­‐
natally through ingestion of infected tissue cysts or rarely oocysts, although congenital infections can occur (Dubey and Jones, 2008). Feline infections are typically subclinical; congenitally infected kittens are the most likely to have clinical signs of infection, but previously clinically healthy adult cats may can also be affected Dubey and Jones, 2008; Vollaire et al, 2003). Common symptoms of T. gondii infection in cats can include fever, ocular inflammation, anorexia, lethargy, abdominal discomfort and neurologic abnormalities. Cats are more likely to shed oocysts following ingestion of tissue cysts rather than tachyzoites or oocysts (Dubey 2009). The ingestion of one bradyzoite will lead to feline infection, whereas a feline must ingest 1000 oocysts to develop an infection. Following the ingestion of tissue cysts containing bradyzoites, some bradyzoites convert to tachyzoites and some to T. gondii schizonts, which replicate asexually in the intestinal tissue before beginning sexual reproduction. Although most cats only shed oocysts once in their lives following infection, experimental infection and immunosuppression resulted in repeated shedding by kittens 20–21 days following the immunosuppressive event (Malmasi, 2009). In addition, oocyst shedding was induced in a non-­‐
immunosuppressed kitten following a second inoculation with infected mouse brain homogenate (Malmasi, 2009). It is thought that infection with one genotype of T. gondii confers immunity to all genotypes, but this phenomenon has not been explored in cats. Toxoplasma gondii infection can cause severe neurologic or ocular disease in the fetus during human pregnancy. Humans acquire their infections from ingestion of oocyst-­‐contaminated soil and water, from tissue cysts in undercooked meat, by transplantation, blood transfusion, laboratory accidents, or congenitally (Dubey and Jones, 2008). Most people infected after birth are asymptomatic; however, some may develop fever, malaise, and lymphadenopathy. Congenital toxoplasmosis often results in debilitating ocular disease, causing (among other manifestations) retinochoroiditis and anterior uveitis (Commodoro et al., 2009). Historically, women demonstrating exposure to T. gondii prior to pregnancy through serology were considered safe from future infection and risk to the fetus. However, apparent recrudescent infections during pregnancy can occur in immunocompetent mothers, although few cases have been reported (Elbez-­‐Rubenstein 2009). A case of maternal T. gondii infection and subsequent fetal infection was reported in a 31-­‐year old French woman with serological evidence of previous T. gondii exposure. The strain isolated from this pregnancy is likely to be the same strain as in the original infection (Elbez-­‐Rubenstein 2009), suggesting that exposure prior to pregnancy does not automatically confer protection from future infections that occur following recrudescence of T. gondii (Elbez-­‐Rubenstein 2009). Post-­‐natally infected humans, especially those with immunosuppression, can develop ocular complications such as retinochoroiditis. Toxoplasmic encephalitis, pulmonitis, or other systemic disease can be seen in patients with immunosuppressive disorders and those undergoing immunosuppressive therapy for circumstances such as organ transplantation. The current prevalence of T. gondii in humans within the United States based upon serology performed on 17,692 people examined in the National Health and Nutrition Examination Survey (NHANES IV) from 1999-­‐2004 revealed a prevalence of 10.8% which was down from 14% in the NHANES III study with sera collected from in the prior decade, 1988-­‐1994 (Jones et al., 2007). It has also been reported that it is now possible to differentiate infections obtained from oocysts versus infections obtained from meet using a stage specific antibody test (Boyer et al., 2011). Using this test, it was found that 59 of 76 mothers of congenitally infected infants (77.6%) had obtained their infections from 8 oocysts. This argues that more infection occur from oocyst than from meat which supports the very low levels of infection that have been reported in commercial beef, poultry, and chicken from grocery stores in the United States (zero of 2,094 beef samples, zero of 2,094 chicken samples, an 8 of 2,094 pork samples) (Dubey et al., 2005). Oocysts are also known to be a major source of infection in marine mammals, such as sea otters, that most likely become infected by the ingestions of marine snails and possibly abalone that have acquired oocysts washed into the ocean in rainwater that have become trapped in the tissue of their gills (Johnson et al., 2009). Dubey and Carpenter (1993a) examined 100 cases of histologically confirmed toxoplasmosis in domestic cats and provide the definitive report on clinical toxoplasmosis in cats. Eleven of 100 cats were purebred, cats ranging in age from 2 weeks to 16 years, and 65 were male, 34 were female and the sex of 1 was not determined. Of the 100 cats 36 had generalized, 26 had pneumonic, 16 had abdominal, 7 had neurologic, 9 had neonatal, 2 had hepatic, 2 had cutaneous, 1 had pancreatic, and 1 had cardiac toxoplasmosis. Fever (40.0 to 41.7 C) is present in many cats with toxoplasmosis. Clinical signs of dyspnea, polypnea, icterus, and signs of abdominal discomfort are frequent findings. Gross and microscopic lesions are found in many organs but are most common in the lungs. Gross lesions in the lungs consisted of diffuse edema and congestion, failure to collapse, and multifocal areas of firm, white to yellow, discoloration. Pericardial and abdominal effusions may be present. The liver is the most frequently affected abdominal organ and diffuse necrotizing hepatitis may be visible grossly. Gross lesions associated with necrosis can also be observed in the mesenteric lymph nodes and pancreas. Ocular lesions are also common in cats but the actual prevalence is not known. Most lesions are in the anterior segment (Lappin et al. 1989c). Cats with ocular lesions have a higher seroprevalence than cats with normal eyes. Ocular findings are varied: they include aqueous flares, hyphema, velvety iris, mydriasis, anisocoria, retinal hemorrhages, retinal atrophy, retinochoroiditis and slow pupillary reflex. Central nervous system toxoplasmosis is not common in cats. However, neurological signs including hypothermia, partial or total blindness, stupor, incoordination, circling, torticollis, anisocoria, head bobbing, ear twitch, atypical crying, and increased affectionate behavior have been reported (Dubey and Carpenter, 1993a). The severe central nervous system involvement observed in congenitally infected infants and AIDS patients and the tendency of tissue cysts to develop in the brains of humans and mice have led to the erroneous assumption by many that toxoplasmosis in all animals is a central nervous system disease. Central nervous system infections do occur in cats but neurologic signs are not the most common clinical sign of infection in cats (Dubey and Carpenter, 1993a). Congenital toxoplasmosis occurs in cats, but the frequency is not known. Disease in congenitally infected kittens can be severe and fatal (Dubey and Carpenter, 1993b). The most common clinical signs are anorexia, lethargy, hypothermia and sudden death (Dubey et al., 1995b). The diagnosis of clinical feline toxoplasmosis requires that 3 criteria be fulfilled (Lappin, 1990). The cat must have clinical signs consistent with toxoplasmosis, serological evidence of recent or active infection, and the cat must respond to anti-­‐T. gondii treatment or have T. gondii demonstrated in its tissues or body fluids. Toxoplasmosis should be suspected in cats with anterior uveitis, retinochoroiditis, fever, dyspnea, polypnea, abdominal discomfort, icterus, anorexia, seizures, ataxia and weight loss. Fecal examination only rarely detects oocysts in cats and most cats with clinical toxoplasmosis will not be excreting oocysts at the time of presentation. Thoracic radiographs may be helpful. Diffusely disseminated and poorly demarcated foci of increased radiodensity caused by interstitial and alveolar pneumonia are suggestive of but not definitive for T. gondii in febrile cats. Several serological tests are available for the diagnosis of active toxoplasmosis in cats. Titers obtained in one type of test may not correlate with titers obtained in other tests (Patton et al., 1991, Dubey and Thulliez, 1989, Lappin and Powell, 1991). Most tests rely on the detection of IgG antibodies 9 which do not develop until about 2 weeks postinfection and may remain at high levels for several years to the life of the cat (Dubey et al., 1995a). Therefore, diagnosis of active toxoplasmosis in cats using an IgG based test requires that a rising titer be demonstrated (Lindsay et al., 1997a). Diagnostic tests based on detection of IgM antibodies (Lappin et al., 1989a; 1989c, Lin and Bowman, 1991), circulating parasite antigens (AG) (Lappin et al., 1989b) or acetone-­‐fixed (AF) tachyzoite antigens (Dubey et al., 1995a) can detect early infections at 1 to 2 weeks post exposure. The T. gondii-­‐
specific IgM levels in cats peak at 3 to 6 weeks and drop to negative by 12 weeks post exposure in the IgM-­‐ELISA teat. However, some cats will have sporadic low IgM-­‐ELISA levels for up to 1 year post exposure. Peak detection of circulating T. gondii antigens occurs about 21 days post exposure but some cats will have circulating T. gondii antigens for at least 1 year in the AG-­‐ELISA; overall, the test is not very useful in diagnosis (Lappin et al., 1989b). Reactivity to AF-­‐tachyzoites in the modified direct agglutination test (MAT, normally formalin-­‐fixed [FF] tachyzoites are used) remains present for up to 70 months (Dubey et al., 1995a). The IgA-­‐ELISA produces variable results in detecting serum antibodies in cats and is not used to detect early infections (Burney et al., 1995). The use of an early detection test coupled with an IgG detection test can provide valuable information on the kinetics of the T. gondii infection. For example, a high IgM-­‐ELISA titer and a negative or low IgG-­‐ELISA titer would indicate active infection. The reverse would be true for a chronic infection. Serology can often be difficult to interpret and should never be the sole bases for diagnosis. Detection of T. gondii antibodies in aqueous humor has been used as an aid in the diagnosis of ocular toxoplasmosis in cats (Patton et al., 1991, Lappin et al., 1992c, 1995, Lin et al., 1992b). Calculating the Goldman-­‐Witmer coefficient (C-­‐value) helps correct for antibodies that may have leaked across a damage vasculature and not been produced directly in the eye (Lappin et al., 1992c) Experimentally infected cats begin to have detectable IgA and IgG levels in aqueous humor at 4 weeks post exposure while IgM is either not present or at levels too low to detect (Lappin et al., 1995); however, all 3 antibody isotypes have been found in the aqueous humor of naturally infected cats. Cats with C-­‐values <1 are considered to have antibodies that have leaked across a damaged vasculature while C-­‐values of 1 to 8 are highly suggestive of clinical ocular toxoplasmosis (Lappin et al., 1992c, 1995). Cats with C-­‐values >8 are considered to have conclusive evidence of ocular antibody production due to T. gondii infection (Chavkin et al., 1994). Most cats with C-­‐values >1 will respond to specific antitoxoplasmal treatment (Lappin et al., 1992c). Although not conclusive, a trend toward association of T. gondii-­‐specific IgA in the serum of cats with ocular disease has been reported (Burney et al., 1995). Toxoplasma gondii antibodies have been demonstrated in the cerebrospinal fluid (CSF) of cats with experimental infections but no clinical signs of encephalitis using the FF-­‐MAT (Patton et al., 1991) a modified ELISA (Lin et al., 1992b) and IgG-­‐ ELISA (Muñana et al., 1995). No IgM was detected in the CSF of experimentally infected cats using the IgM-­‐ELISA. Little else is available on the diagnosis toxoplasmic encephalitis in cats using CSF. Because T. gondii-­‐specific IgG has been observed in the CSF of clinically normal cats, it has been suggested that the diagnosis of central nervous system toxoplasmosis in cats not be based solely on detection of intrathecally synthesized T. gondii-­‐ specific IgG (Muñana et al., 1995). Neonatal toxoplasmosis is difficult to diagnosis antemortem because the clinical signs are vague and kittens will have nursed prior to examination. Serological indications can be inferred in some cases by comparing titers in queens with their kittens (Dubey et al., 1995b) Transplacental transfer of T. gondii antibodies does not occur in cats (Dubey et al., 1995b). If the queen is seronegative then it is unlikely that the kittens have toxoplasmosis because transplacental transmission is unlikely if the queen has acquired the infection with less than 2 weeks left in pregnancy which is the time it takes or a detectable antibody response. If the queen has a positive IgM titer or the queen and kittens have rising IgG titers then transplacental or lactogenic transmission is possible. Western blot analysis of serum from the queen and kitten can be helpful in diagnosing neonatal toxoplasmosis in kittens (Cannizzo et al., 1996). 10 Antigen recognition patterns are different for congenitally infected kittens when compared to queens or kittens that have maternally acquired antibody. Serum for congenitally infected kittens usually will usually recognize an antigen with a molecular mass between 27 to 29 kD (Cannizzo et al., 1996). Direct demonstration of T. gondii stages can be used to make a method of antemortem diagnosis. Examination of bronchiolar lavage material or material collected by abdominocentesis can be used to detect suspected cases of disseminated toxoplasmosis in cats or neonatal toxoplasmosis in kittens. Examination of CSF may also demonstrate organisms in cases of encephalitis. The polymerase chain reaction (PCR) has been widely used in human medicine to detect T. gondii in secretions and fluids and methods are now is routinely used in cats (Stiles et al., 1996; Lappin et al., 1996b; Burney et al., 1998) The primers have been developed that amplify portions of the parasites B1 gene and used to detect tachyzoites in serum, blood, aqueous humor, and CSF. The PCR test can detect DNA from as few as 10 tachyzoites in serum, CSF, and aqueous humor (Stiles et al., 1996, Lappin et al., 1996a&b) and DNA from as few as 100 tachyzoites in blood (Stiles et al., 1996).The use of PCR combined with traditional antibody testing maybe useful in the antemortem diagnosis of toxoplasmosis in cats. Results of PCR testing alone should never be used as the sole method of diagnosis of toxoplasmosis. Postmortem diagnosis can be made by demonstration of the parasite in tissue sections using routine methods or by supplementing histopathologic examinations with immunohistochemical staining for specific for T. gondii. Other methods, such as, bioassays in cats or mice can be used but are not practical. Treatment of cats at this time for toxoplasmosis would most likely be most successful with the administration of ponazuril at 50 mg/kg daily for 3 days. This anticoccidial product that is marketed in the United States for treating equine protozoal myeloencephalitis in horses, has proven excellent in the killing of tachyzoites and bradyzoites in experimentally infected animals (Mitchell et al, 2004). REFERENCES Boyer KM, et al. 2011. Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America. Clin Infect Dis 53: 1081-­‐1089. Burney DP, et al. 1998. Polymerase chain reaction for the detection of Toxoplasma gondii within aqueous humor of experimentally-­‐inoculated cats. Vet Parasitol 79:181-­‐186. Burney DP, et al. 1995. Detection of Toxoplasma gondii-­‐specific IgA in the serum of cats. Am J Vet Res 56:769-­‐773. Cannizzo KL, et al. 1996. Toxoplasma gondii antigen recognition by serum immunoglobulins M, G, and A of queens and their neonatally infected kittens. Am J Vet Res 57:1327-­‐1330 Chavkin MJ, et al. 1994. Toxoplasma gondii-­‐specific antibodies in the aqueous humor of cats with toxoplasmosis. Am J Vet Res 55:1244-­‐1249 Chavkin MJ, , et al. 1992. Seroepidemiology and clinical observations of 93 cases of uveitis in cats. Prog Vet Comp Ophthalmol 2:29-­‐36. Commodaro AG, et al. 2009. Ocular toxoplasmosis—an update and review of the literature. Mem Inst Oswaldo Cruz 104: 345–350 Dubey JP 2009. History of the discovery of the life cycle of Toxoplasma gondii. Int J Parasitol 39: 877–
882 Dubey JP, Carpenter JL. 1993a. Neonatal toxoplasmosis in littermate cats. J Am Vet Med Assoc 203:1546-­‐1549. Dubey JP, Carpenter JL. 1993b. Histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-­‐
1990). J Am Vet Med Assoc 203:1556-­‐1566. Dubey JP, Jones JL. 2008. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 38: 1257–1278 Dubey JP, et al. 1995b. Diagnosis of induced toxoplasmosis in neonatal cats. JAVMA 207:179-­‐185. 11 Dubey JP, et al. 1995a. Long-­‐term antibody responses of cats fed Toxoplasma gondii tissue cysts. J Parasitol 81:887-­‐893. Dubey JP, et al. 2005. Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers. J Parasitol 91: 1082-­‐1093. Dubey JP, Thulliez P. 1989. Serologic diagnosis of toxoplasmosis in cats fed Toxoplasma gondii tissue cysts. JAVMA 194:1297-­‐1299. Elbez-­‐Rubenstein A. 2009 .Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J Infect Dis 199: 280–285 Johnson CK, et al. 2009. Prey choice and habitat use drive sea otter pathogen exposure in a resource-­‐
limited coastal system. Proc Natl Acad Sci USA 106: 2242-­‐2247. Jones JL et al. 2007. Toxoplasma gondii infection in the United States, 1999-­‐2004, decline from the prior decade. Am J Trop Med Hyg 77: 405-­‐410. Lappin MR, et al. 1996a. Polymerase chain reaction for the detection of Toxoplasma gondii in aqueous humor of cats. Am J Vet Res 57:1589-­‐1593. Lappin MR, et al. 1995. Detection of Toxoplasma gondii-­‐specific IgA in the aqueous humor of cats. Am J Vet Res 56:774-­‐778. Lappin MR, et al. 1996b. Primary and secondary Toxoplasma gondii infections in normal and feline immunodeficiency virus infected cats. J Parasitol 82: 733-­‐742. Lappin MR, et al. 1989a. Diagnosis of recent Toxoplasma gondii infection in cats by use of an enzyme-­‐
linked immunosorbent assay for immunoglobulin M. Am. J. Vet. Res. 50:1580-­‐1585. Lappin MR, et al. 1989b. Enzyme-­‐linked immunosorbent assay for the detection of circulating antigens of Toxoplasma gondii in the serum of cats. Am. J. Vet. Res. 50:1586-­‐1590. Lappin MR, et al. 1989c. Clinical feline toxoplasmosis: Serological diagnosis and theraputec management of 15 cases. J. Vet. Int. Med. 3:139-­‐143. Lappin MR, Powell CC. 1991. Comparison of latex agglutination, indirect hemagglutination, and ELISA techniques for the detection of Toxoplasma gondii-­‐specific antibodies in the serum of cats. J Vet Intern Med 5:299-­‐301. Lappin MR, et al. 1992c. Enzyme-­‐linked immunosorbent assays for the detection of Toxoplasma gondii-­‐
specific antibodies and antigens in the aqueous humor of cats. JAVMA 201:1010-­‐ 1016. Lappin MR. 1990. Challenging cases in internal medicine: What’s your diagnosis? Vet Med 84:448-­‐455. Lin DS, Bowman DD. 1991. Cellular responses of cats with primary toxoplasmosis. J. Parasitol. 77:272-­‐
279. Lin DS, et al. 1992b. Antibody responses to Toxoplasma gondii antigens in aqueous and cerebrospinal fluids in cats infected with T. gondii and FIV. Comp Immuno Microbiol Infect Dis 15:293-­‐299. Lindsay DS, et al. 1997a. Feline toxoplasmosis and the importance of the Toxoplasma gondii oocyst. Comp. Contin. Ed. Pract. Vet. 19:448-­‐461. Malmasi A, et al. 2009. Prevention of shedding and re-­‐shedding of Toxoplasma gondii oocysts in experimentally infected cats treated with oral clindamycin: a preliminary study. Zoonoses and Public Health. 56, 102–104 Mitchell SM, et al. 2004. Efficacy of ponazuril in vitro and in preventing and treating Toxoplasma gondii infections in mice. J Parasitol 90:639-­‐642. Muñana KR, et al.1995. Sequential measurement of Toxoplasma gondii-­‐ specific antibodies in the cerebrospinal fluid of cats with experimentally induced toxoplasmosis. Prog Vet Neuorl 6:27-­‐31. Patton S, et a. 1991. Concurrent infection with Toxoplasma gondii and feline leukemia virus. J Vet Intern Med 5:199-­‐201. Stiles J, et al. 1996. Detection of Toxoplasma gondii in feline and canine biological samples by use of the polymerase chain reaction. Am J Vet Res 57:264-­‐267. 12 Vollaire MR et al. 2005. Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the United States. Am J Vet Res 66:874–877
13 Cuterebriasis; Fleas, Ticks, and Associated Disease Cuterebriasis Cuterebra, a genus of dipteran obligate parasites of rodents and lagomorphs in North America, undergoes an obligatory deep migration through the tissues of its host before the third-­‐instar larva appears in a subcutaneous boil where it undergoes rapid growth before dropping to the soil to pupate (Sabrosky, 1986). Surprisingly in the rodent and lagomorph hosts, there is actually very little loss of fitness of the host from infections with these parasites that can appear strikingly large in comparison to the host (Slanksy, 2007). There are 34 species of Cuterebra throughout North America (Sabrosky, 1986), and dogs and cats Cuterebra larva removed from the retro-­‐
are known to sometimes have the larvae develop into mature larvae within the lesions in their skin, orbital region of a cat in upstate New York
nasal cavity, or eyelid (Scott et al., 2001). In the northeastern United States there is the seasonal appearance of neurologic disease in cats that has been associated with the migration of Cuterebra larvae through the spinal cord or brain (Glass et al., 1998). The larvae cannot be identified to species, and while third-­‐stage larvae can be identified to subgenus, first and second instars cannot be identified morphologically beyond the generic level (Sabrosky, 1986). Thus, it is unknown if the disease in cats seen in the northeastern USA is due to one or more than one of the six species of Cuterebra that occur in this area: three species in the subgenus Trypoderma that use lagomorphs as their typical hosts, C. T. abdominalis, C. T. buccatta, and C. T. cuniculi, and three in the subgenus Cuterebra that used rodents as the typical hosts, C. C. emasculator, C. C. fontinella, and C. C. americana. The flies all have univoltine life cycles with the females laying eggs on blades of grass in spring or early summer near the entrance to the host's burrow, and the larva then leaves the egg to get onto the host when it is passing by and enters the host through one of the body's orifices. The migratory patten in mice experimentally infected with C. C. fontinella revealed that whether the larvae first entered the host via the nares or anus, they migrated first to the trachea and thoracic region before migrating through the abdomen to the site of development in the subcutaneous tissues of the postero-­‐ventral abdominal region (Gingrich, 1981). A mature larva in a rodent or rabbit requires some 3 to 8 weeks including the migratory phase of the life cycle (Bowman et al., 2002). In cats, the disease usually presents between late June and the first killing frost in October. In most cats, and dogs, it seems that the larvae ultimately reach the subcutaneous tissues and mature (although they seem incapable of producing viable adults after pupation), and in these cases the diagnosis of infection is simply the finding of the larva in the subcutaneous boil. Unfortunately, in some cats, the infection produces respiratory signs followed by neurologic disease that is often fatal. In late summer to early fall, cats can develop an acute onset of neurologic disease that may be preceded by upper respiratory signs one to two weeks previously. These cats can present with depression, blindness, and behavioral changes. Lesions may be in the cerebrum or cerebellum in association with feline ischemic encephalopathy, but in some cases the larvae are found within the spinal canal. Diagnosis is typically based on signs, response to treatment with high doses of ivermectin and corticosteroids, imaging with computerized axial tomography or magnetic resonance, or by necropsy. Treatment remains high-­‐dose ivermectin, often with corticosteroids, or surgical removal of the offending bot. Work is underway to develop a means of assisting the diagnosis with the utilization of an 14 ELISA for Cuterebra-­‐specific IgG and IgM in cats with known and suspected disease. It is hoped that this will ultimately prove useful in ruling out the infection in cases where cats present with signs other than a bot within a subcutaneous lesion. References Birkenheuer, A. J., Le, J. A., Valenzisi, A. M., Tucker, M. D., Levy, M. G., Breitschwerdt, E. B. 2006. Cytauxzoon felis infection in cats in the mid-­‐Atlantic states: 34 cases (1998-­‐2004). J Am Vet Med Assoc 228, 568-­‐571 Birkenheuer, A. J., Marr, H. S., Warren, C., Acton, A. E., Mucker, E. M., Humphreys, J. G., Tucker, M. D. 2008. Cytauxzoon felis infections are present in bobcats (Lynx rufus) in a region where cytauxzoonosis is not recognized in domestic cats. Vet Parasitol 153, 126-­‐130 Blouin, E. F., Kocan, A. A., Glenn, B. L., Kocan, K. M., Hair, J. A., Doyle, R. T. 1984. Transmission of a Cytauxzoon-­‐like parasite by Dermacentor variabilis from a naturally infected bobcat to domestic cats. [Abstract]. 58th Ann Meeting Am Soc Parasitol, San Antonio, TX, pp. 50 Bowman D. D, Hendrix, C. M, Lindsay D. S., Barr, S .C. 2002. Cuterebridae. In: Feline Clinical Parasitology. Iowa State University Press, Ames, IA, 430-­‐442 pp. Brown, H. M.; Lockhart, J. M.; Latimer, K. S.; Peterson, D. S. 2010. Identification and genetic characterization of Cytauxzoon felis in asymptomatic domestic cats and bobcats. Vet Parasitol 172, 311-­‐316 Cohn, L. A., Birkenheuer, A. J., Brunker, J. D., Ratcliff, E. R., Craig, A. W. 2011. Efficacy of atovaquone and azithromycin or imidocarb dipropionate in cats with acute cytauxzoonosis. J Vet Intern Med 25, 55-­‐60 Gingrich, R. E. 1981. Migratory kinetics of Cuterebra fontinella (Diptera: Cuterebridae) in the white-­‐
footed mouse, Peromyscus leucopus. J Parasitol 67, 398-­‐402. Glass, E. N., Cornetta, A. M., de Lahunta, A., Center, S. A., Kent, M. 1998. Clinical and clinicopathologic features in 11 cats with Cuterebra larvae myiasis of the central nervous system. J Vet Int Med 12, 365-­‐368. Harrison, R. L. 2006. A comparison of survey methods for detecting bobcats. Wildl Soc Bul 34, 548-­‐552 Jackson, C. B., Fisher, T. 2006. Fatal cytauxzoonosis in a Kentucky cat (Felis domesticus). Vet Parasitol 139, 192-­‐195 Meier, H. T., Moore, L. E. 2000. Feline cytauxzoonosis: a case report and literature review. J Am An Hosp Assoc 36, 493-­‐496 Reichard, M. V., Meinkoth, J. H., Edwards, A. C., Snider, T. A., Kocan, K. M., Blouin, E. F., Little, S. E. 2009. Transmission of Cytauxzoon felis to a domestic cat by Amblyomma americanum. Vet Parasitol 161, 110-­‐115 Sabrosky, C. W. 1986. North American species of Cuterebra, the rabbit and rodent bot flies (Diptera: Cuterebridae). Thomas Say Foundation Monograph, Entomological Society of America, College Park, MD, 240 pp. Scott, D. W., Miller, W. H., Griffin, C .E. 2001. Parasitic Skin Diseases, In: Muller & Kirk's Small Animal Dermatology, 6th ed., W.B. Saunders, Philadelphia, PA, pages 423-­‐516. Slansky, F. 2007. Insect/mammal associations: effects of cuterebrid bot fly parasites on their hosts. Ann Rev Entomol 52, 17-­‐36. Snider, T. A., Confer, A. W., Payton, M. E. 2010. Pulmonary histopathology of Cytauxzoon felis infections in the cat. Vet Pathol 47, 698-­‐702 Wagner, J. E. 1976. A fatal cytauxzoonosis-­‐like disease in cats. J Am Vet Med Assoc 168, 585-­‐588 Williams, K. J., Summers, B. A., de Lahunta, A. 1998. Cerebrospinal cuterebriasis in cats and its association with feline ischemic encephalopathy. Vet Pathol 35, 330-­‐343 15 FLEAS Younger veterinarians today have no idea what flea control was like just 20 years ago. It was about then that veterinarians received the capability of making a major difference in flea control through products that were placed in or on pets that significantly and very successfully reduced flea populations on pets. This has made a huge difference in how pets are protected from these parasite infestations. The fleas that typically bother the cat and dog, Ctenocephalides felis (and the less common Ctenocephalides canis) have similar life cycles. Fleas have 6 life stages: eggs, three larval stages that look like caterpillars, a pupal stage, and the adult stage. Within the pupal stage the caterpillar undergoes a metamorphosis and develops into an adult flea, the stage that is familiar to most pet owners. In this life cycle, the eggs produced by the female (up to 50 eggs a day at the maximum reproductive potential of the female flea) fall off the pelage of the host and drop to the ground in the area where the pet rests. If conditions are near perfect, the larvae can hatch from the egg and develop to the adult stage in just over a week. It is in the pupal stage that the flea waits for its next host (this is the stage most resistant to desiccation), and the flea will eclose from its cocoon when it senses the heat of a passing host. The adult flea will then jump onto the passing host where it will feed and continue the cycle. The large numbers of eggs produced by a female are the reason why numbers can become massive in a short time. Ten female fleas can produce 3,500 eggs in a week. The current products work either through interfering with the development of fleas in the egg or the larval stage or by directly affecting the nervous system of the fleas as adults. Some of the products act upon fleas by direct contact, and others require that fleas first bite the host to obtain the active ingredient in the insecticide. The concern remains that fleas may develop resistance to these products. A flea larval bioassay was developed by an international team of scientists to monitor the susceptibility of fleas (Ctenocephalides felis) to imidacloprid (Advantage, Bayer Animal Health). The assay was validated using laboratory and field isolates of C. felis. Flea eggs representing different field isolates of C. felis have been collected by veterinarians in the United States, United Kingdom, and Germany. Of the 972 flea isolates obtained during the 5-­‐year study reported in 2006, 768 contained sufficient numbers of eggs to conduct the larval bioassay. Greater than 5% survival occurred for only six of the field isolates evaluated. Further evaluation and analysis of these isolates demonstrated that they did not differ significantly in their susceptibility to imidacloprid from the reference strains used to develop the assay. Collections of field flea isolates are continuing in an attempt to detect and document any change in the susceptibility of field flea populations to imidacloprid. It might be necessary at some time in the future to combine flea treatments where fleas are attacked by both adulticides and larvicides in order to maintain the same great level of protection that is currently provided. However, it would appear that we are still holding our own in the battle against this pest, and hopefully, we will not have to return to the days when fleas are the curse that they once were. We can only hope that with the help of the various corporations developing new products and application systems that we can stay ahead in the race. Fleas as Vectors Controlling flea infestation in companion animals facilitates more than just the removal of annoying pests; the flea species that infest our pets play active roles spreading multiple interspecies and zoonotic disease agents. These agents include Mycoplasma spp., Dipylidium caninum and potentially Feline Leukemia Virus within a species and cat-­‐scratch disease (Bartonella henselae), flea-­‐borne rickettsioses (Rickettsia typhi, Rickettsia felis) and plague (Yersinia pestis) to humans. Flea associated diseases can 16 occur anywhere throughout the United States, although certain diseases do show some geographic specificity. Young children and immunocompromised individuals represent two demographics that are most severely affected by flea-­‐associated diseases however these diseases are infective to all populations. Understanding the zoonotic potential and risk factors involved in these diseases can help veterinarians make appropriate recommendations to owners concerned about their vulnerability to these infections. Cat-­‐Scratch Disease Bartonella henselae (Gram-­‐negative) is the infectious bacterial agent responsible for cat-­‐scratch disease (CSD). This bacterium is spread between cats and to humans by the cat flea, Ctenocephalides felis. C. felis first becomes infective after ingesting a bloodmeal from a bacteremic cat. The bacteria is then shed through the feces of the flea, which is often stored in the nail bed of the cat, and is inoculated into another cat or person through contamination of an open wound with the contaminated flea feces. An alternative route of transmission is through direct inoculation of a wound with a bacteremic cat’s saliva, such as through a bite. B. henselae then colonizes in erythrocytes and endothelial cells of the infected individual. Cat-­‐scratch disease is one of the most common causes of regional lymphadenopathy in children and young adults. The majority of cases in the nation occur between September and January; well after the time that most people believe that flea control is not necessary. However, no flea feces; NO transmission. So, if pets are protected through Fall, there should be a marked reduction in cases. In addition to regional lymphadenopathy, other clinical signs reported included erythema, fever and mild headaches and in 5-­‐15% of patients include encephalitis, retinitis and endocarditis. More severe clinical signs are seen in immunocompromised individuals and may include tumor-­‐ like growths of blood vessels on the external skin surface and hepatitis, bacillary angiomatosis. Control over flea infestation is fundamental towards controlling cat-­‐scratch disease because serologic tests do not properly identify bacteremic cats as potential reservoirs. Most cats have been exposed to B. henselae and will possess residual antibodies against the organism regardless of its bacteremic status so a positive serologic test does not necessarily translate to a bacteremic cat. Furthermore, because the primary route of transmission to humans is through flea feces, eliminating fleas will eliminate the primary source of infection. PCR and blood culture tests can be done to diagnose the presence of Bartonella spp. in a cat but false negatives are not uncommon and positive results do not always correlate with clinical illness. Treatment for cats with Bartonella spp. is generally not recommended as cats do not show clinical signs but if a cat tests positive on a culture test and treatment is indicated (often due to an immunocompromised individual that is in close proximity to the cat), then treatment with doxycycline or amoxicillin-­‐clavulanate in conjunction with flea control for 2 weeks is the recommended therapy. Rickettsioses Several rickettsial (Gram-­‐ negative) agents, such as Rickettsia typhi and Rickettsia felis, are transmitted to humans from animals usually through an intermediate flea vector and by a method similar to the transmission of B. henselae. Rickettsia typhi is the agent known to cause murine typhus in humans, a disease characterized by high fever, headaches, chills and malaise with concurrent gastrointestinal, respiratory and neurologic symptoms and a maculopapullary rash that is reported to present in 50% of infected patients. R. felis causes a disease that presents with clinically similar symptoms to R. typhi and PCR is needed to distinguish between the agents as serologic evidence alone cannot. It is important to distinguish between the agents causing the “typhus symptoms” because of the information if provides on the transmission of the disease, particularly the involvement domestic cats 17 play in transmission. Rickettsia typhi is maintained primarily in rat reservoirs and spread by the rat flea, Xenopsylla cheopis but an alternative cycle involving opossums, cats and cat fleas, C. felis, has been proposed. Rickettsia felis, however, is thought to circulate between opossums and cats and has been isolated from cat fleas. Both of these cycles suggest that the domestic cat plays a role in maintaining these bacterial agents however the exact role cats play in transmission of the disease is unclear-­‐ identifying the agent causing the symptoms may help clarify their role. Serological evidence shows that cats can be exposed to these bacteria without showing overt clinical signs but studies using PCR to isolate these agents from serologically positive cats have been unsuccessful. This suggests that it is unlikely that cats directly transmit the agents. What is likely, however, is that cats serve as a feeding and mechanical source for both parasites (X. cheopis and C. felis) acting as a bridge between fleas and people. Prophylactic flea control in cats could help protect them from these parasites and therefore decrease the propagation and spread of these agents. Plague Plague is a life-­‐threatening disease caused by the Gram-­‐negative bacteria, Yersinia pestis. This disease is endemic in the western United States with a southwest focus in northern Arizona, northern New Mexico and southern California and a West Coast focus in California, southern Oregon and western Nevada. Yersinia pestis is maintained mainly in wild rodent reservoirs including rats, rock squirrels, ground squirrels, prairie dogs and chipmunks and is transmitted by two rodent fleas, Xenopsylla cheopis and Oropsylla montana, as well as through direct exposure to the tissues, secretions and respiratory droplets of infected animals. In the United States, approximately 20 cases of plague are reported each year of which 7.7% are associated with transmission from cats. Plague can present in one of three forms, bubonic, pneumonic or septicemic. Roughly 80-­‐90% of cases reported worldwide are of the bubonic form but in 2006, of the 17 cases reported in the US, 35% were classified as primary septicemic plague. Humans with cutaneous exposure usually develop the bubonic form where as humans exposed through inhalation develop the pneumonic form. If left untreated the bubonic form can spread to the lungs, develop into the pneumonic form allowing the infected individual to spread the disease to another person. If this form of plague left untreated for 3-­‐4 days it can be fatal. Domestic cats have been implicated in the transmission of plague to people in two ways. First, people who are exposed to infected cats can directly contract the disease from their tissues and secretions. Importantly, although most cats are very susceptible to plague and show signs of clinical illness, one study that experimentally infected 16 cats with Y. pestis, showed that 19% of the animals did not show clinical signs. This suggests that it is possible that a cat is spreading the bacteria without evidence of infection, making it hard for a person to recognize it as a threat. An alternative way cats propagate the transmission to humans is through their hunting behaviors. Cats that hunt and carry dead rodents into the vicinity of humans expose them to infected rodents and the fleas that they carry. We also now know that people get plague from sleeping in the same bed with an unprotected dog that has been roaming about in areas with plague. The infection is due to the hitchhiker rodent fleas. People who go west for vacation and sleep with their dog in the tent or camper need to think about flea protection for their pet. For treatment doses in animals as well as treatment for human symptoms please refer to the article, “Flea-­‐associated diseases of cats in the USA: bartonellosis, flea-­‐borne rickettsioses and plague” by Kristina M. McElroy et al (2010). Veterinarians should be particularly vigilant with their hospital flea control regimens as to mitigate personal exposure as well as transmission of these diseases to other animals in the hospital. 18 TICKS Overview: The population of ticks in the Unites States is probably at levels higher than when your parents went to school. The reasons are manifest, but most stem from environmental stewardship. But often, the best of intentions does not have all of the desired consequences. • Ticks appear more populous than they were fifty years ago. • Tick pressure and associated tick diseases are increasing in people and dogs; this is not simply increasing awareness, but it is also due to increasing contact between pets, owners, and ticks. • We have excellent means of diagnosing many tick infections • We have tick prevention products that are amazingly good, but the pressure is very high these days. There are more ticks today? It would seem so. Not certain that we have real data as to tick numbers, but there sure seem to be more ticks now than ever before. My youth was spent in the woodlands of the Midwest, never had a tick. As a graduate student in Louisiana, many hours spent in the forests and swamps collecting reptilian specimens and flies, never had a tick. Three years living on an old farmhouse just south of Madison, Wisconsin, and I never had a tick. Ithaca NY for the last two decades, and I am surrounded by people, including one of my own family members, who get Lyme disease from a tick in the backyard. One of the fellows working in the lab next door, has complained repeatedly to me about the ticks bothering him (and his dog) all winter long in his own backyard. It sure seems like there are many more ticks now than there were not very long ago. There are more deer and other wildlife: This is a fact. So, we do not have great data on ticks, but there is good data on other wildlife species. The numbers of deer are up dramatically. As a child in Ohio, people had to go to the "wilds" of Pennsylvania or around and south of Chillicothe, OH, to see a deer. Now, like in NY, to see a deer all you have to do is put up your electric garage door. Deer were once extinct in Kansas; the people of the state imported deer from other states to repopulate the state. Coyotes were once found west of the Mississippi, now they are throughout the lower 48 United States. Raccoons in the northeastern US have recently had their numbers reduced by raccoon rabies, but they are still numerous and are even more numerous now in the Midwest and western states. Similarly bobcat numbers are up, red fox numbers are up, even brown bear numbers are up. It is believed that the re-­‐establishment of large wild turkey populations has assisted in the increase in the numbers of ticks in some areas. The number of people who hunt is down. The wildlife has bounced back extremely well since the 1950s. Much of the wildlife that has returned is that which does well in suburban and peri-­‐
urban environs. Places where people and pets like to live and play. Wildlife and Ticks: These two worlds come together to provide more hosts upon which ticks can feed. Ticks other than Rhipicephalus sanguineus are not that fastidious in their host choice. The other ticks, most of which are three-­‐host ticks as well, often prefer small things like rodents as larvae, while the nymphs feed on larger things like turkeys, rabbits, and fox sized creatures, and the adults feed often on the largest adult mammals like deer. It is because ticks feed on multiple hosts that they are great vectors. Many of the diseases transmitted to dogs and people are picked up from rodents by the larval or nymphal ticks. Thus, the increased number of hosts has probably served to increase the number of ticks. If nothing else, it has changed the numbers of different types of ticks such that populations that 19 feed on deer may be out-­‐competing those that do not feed on deer before the cervine populations exploded. Work has also shown in some island communities that deer eradication will reduce tick numbers; this is a method that would be socially unacceptable to many. People, pets, and ticks: People have chosen to live in areas that are great sources of ticks. To many in the nation, the days of happily living in a sterile suburban neighborhood of manicured lawns where each house looks similar to it neighbor is considered an anathema. Similarly, the days when the lawns of these houses were treated with various insecticides have long passed. Thus, back in the old days, these non-­‐glorious areas of suburban residence were also considered an anathema to ticks. Ticks need a semi-­‐
varied environment for the different stages, different hosts for different stages, and plenty of hosts on which to feed; instead, they once had sterility coated with various pesticides. Now, the vast majority of people (and pets probably like it better as well) want to live in a residence that is conducive to the presence of ticks. Ticks have never had it better, and we now design our residence units so that they reside directly in just about the best tick environment possible, artificial forest margins with a diversity of plants, habitats, and wildlife. Tick Pressure: This can be amazing, Dr. Dryden of KSU shows areas in Kansas where if he takes a Beagle on an 80 minute walk, the average Beagle gets 40 ticks. That means that ticks are jumping onto hosts at the rate of one every two minutes, and some veterinarians from Arkansas and Texas think this is not even real pressure. Dogs with ticks come into practices requiring transfusions to keep them alive until the ticks can be removed. I saw a picture that I thought initially was a pinecone until a closer look revealed it to be the ear of a golden retriever; there was no visible ear. People in deer blinds around Ithaca, NY, can find themselves passing the time, but not paying attention to the job at hand, as they regularly pick Ixodes ticks off their pant legs. TICK ASSOCIATED DISEASES: Dogs and people can share disease agents from tick bites that will cause infection and disease; shared agents are: Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. Dogs and people are not the major reservoirs for these infections (with the exception of E. canis and perhaps E. ewingii that might be found mainly in dogs), instead the infection is maintained in wildlife, often rodents. People, dogs, and cats typically become infected by the bite of a tick that fed upon one of these hosts in an earlier developmental stage, e.g., as a larva or a nymph. In the case of Hepatozoon americanum, the dog eats either a tick or a paratenic host to become infected. Lyme Disease Infection with Borrelia burgdorferi continues to be one of the main driving forces in tick control around the nation. People remain highly afraid of the infection and the potential surreptitious sequelae that can vary from mild arthritis like symptoms to sever neurologic disease. Thus, although disease can develop in pets, the typical driving force is that people are more concerned that they personally might be infected by the bite of the Ixodes tick. They want their pet protected, but they also want themselves protected. Fortunately, it appears to remain fairly well isolated to certain geographic areas in the northeastern, north central, and Pacific coastal areas of the USA. Ehrlichiosis Ehrlichia canis in dogs can cause fever, myalgia, depression, leucopenia, and thrombocytopenia which may lead to bleeding diatheses, particularly epistaxis. Infections of humans 20 with E. canis have occurred. E. chaffeensis usually causes human monocytotropic ehrlichiosis with symptoms including a febrile, flu-­‐like illness that can be fatal, and dogs seem to be rarely infected with a subclinical infection with this same species. Ehrlichia ewingii typically causes a mild febrile disease in both dogs and people, and in dogs, this might be associated with a polyarthritis. Many dogs infected with E. ewingii are asymptomatic, and this subclinical infection may put pet owners at risk. The vector of E. canis is the brown dog tick, Rhipicephalus sanguineus. Amblyomma americanum, the lone star tick, is the major vector for both E. chaffeensis and E. ewingii. E. chaffeensis appears to mainly be an ehrlichiosis of deer that is transmitted occasionally to the unfortunate dog and human. A. americanum is also in the major vector of E. ewingii, although the rickettsial organism has also been detected in Dermacentor variabilis and R. sanguineus. It appears that the reservoir hosts of E. ewingii are both dogs and deer. People are most commonly infected with E. chaffeensis and E. ewingii in the southern US, where the lone star tick is more common. Doxycycline remains the treatment of choice for ehrlichiosis in both dogs and people. However, data is emerging showing that dogs infected with E. canis may transmit the infection to ticks even after antibiotic treatment. Thus, there is concern that these infections may not clear appropriately with current treatment regimes. Babesiosis Dogs in Florida may be infected with either (or both) of two species of Babesia, Babesia canis vogeli and Babesia gibsoni. Both are transmitted by the Brown Dog Tick, Rhipicephalus sanguineus. B. canis is the larger form, with pear-­‐shaped trophozoites being found in pairs in the erythrocytes. B. gibsoni is smaller and usually round to oval in shape. A survey of greyhounds in Florida has shown that large numbers (46% of 383 greyhounds) have antibodies to B. canis, but most do not have clinical signs. Clinical signs when present include depression, anorexia, anemia, and splenomegaly. The strain of Babesia canis vogeli in Florida appears mainly to cause disease in puppies, for which the major diagnostic feature is anemia. In the US, babesiosis in dogs typically manifests with anemia, anorexia, and lethargy. In a recent survey of 673 canine blood samples tested for Babesia DNA by PCR, the 144 positive samples came from 29 states and one Canadian province. Of these samples, 91% (131) were recognized as the small form Babesia, B. gibsoni, and 10 were recognized as the larger Babesia form, B. canis vogeli (three samples did not match current recognized species). Almost all the samples representing B. gibsoni (122 of 131) were from American pit bull terriers. Six of the 10 B. canis vogeli cases were in greyhounds. Dogs with babesiosis due to Babesia canis vogeli are most commonly treated with either a single intramuscular injection of 3.5 mg/kg diminazene (Berenil) or subcutaneous injections of 15 mg/kg phenamidine (Ganaseg) per kilogram; neither of which are commonly available in the US. B. gibsoni infections are not as readily curable with these drugs as is B. canis, and recently, a combination of atovaquone and azithromycin has been recently recommended as a treatment for dogs with B. gibsoni. Hepatozoonosis This is a horrible destructive disease of dogs caused by the Apicomplexan Hepatozoon americanum that is acquired by the dog eating an Amblyomma maculatum tick containing infective oocysts in its body cavity. Recent work has shown that dogs can also get infected by eating paratenic hosts, rabbits or rodents, that have eaten an infectious tick. This disease is found in Tennessee, Alabama, Georgia, Mississippi, Florida, Louisiana, Oklahoma, and Texas The disease, characterized by periodic or persistent fever, weakness, muscle atrophy, generalized pain or hyperesthesia, reluctance to move, mucopurulent ocular discharge, and gradual deterioration of body condition, is often fatal. Laboratory findings include neutrophilic leukocytosis (leukocyte counts can exceed 200,000/µL), mild to moderate nonregenerative anemia, mild elevation in serum alkaline phosphatase, and occasional hyperglobulinemia. Radiographs may demonstrate periosteal proliferation 21 of various bones, including the ilium, humerus, radius, ulna, femur, tibia, fibula, and vertebrae. These lesions occur most often in younger dogs. There is not treatment effective in eliminating H. americanum from infected dogs. Treatment can increase survival time, improve the quality of life, and decrease the number and severity of clinical relapses. Supportive care and nonsteroidal anti-­‐inflammatory drugs can ensure hydration and assist with pain control. Either of two acute parasiticidal treatments may be administered: Ponazuril may be administered at a dose of 10 mg/kg PO q12h for 14 day. A triple-­‐combination therapy consists of trimethoprimsulfadiazine (15 mg/kg PO q12h for 14 days), pyrimethamine (0.25 mg/kg PO q24h for 14 days), and clindamycin (10 mg/kg PO q8h for 14 days). Still required will be a prolonged, at least 2-­‐year course of therapy with a quinolone. Decoquinate (Deccox®, Alpharma Inc., Fort Lee, NJ) at a dose of 10 to 20 mg/kg mixed in the food twice daily. This is equivalent to 0.5 to 1.0 teaspoon/10kg (22 lb) of formulated decoquinate administered twice daily. Cytauxzoon felis This parasite first appeared in domestic cats in Missouri in 1973, and the concern at that time was that it was an introduced species of importance to agriculture, perhaps related to Theileria parva (Wagner, 1976). It was also at this time described as a new species: Cytauxzoon felis Kier, 1979. Cytauxzoon felis used to be thought to be transmitted to cats from bobcats, Lynx rufus, by the bite of the American Dog Tick, Dermacentor variabilis. We have more recently learned that the vector is the lone star tick, Amblyomma americanum. This matches what is known about where the disease is located, it occurs in the overlapping ranges of the bobcat and the lone star tick, not in the overlapping areas of the Dermacentor and the bobcat. The sexual stages occur in the tick which inoculates sporozoites into the host when it bites. So now, the reservoir host is the bobcat, and the vector is the lone star tick. But, just to keep things confusing, seems that there may now be some strains transmitted by D. variabilis in the field. In the cat, there are two important life cycle stages, schizonts and merozoites. Schizonts are found in histiocytes and macrophages of the bone marrow, veins, and venules of various organs, including the lungs, liver, spleen, lymph nodes, brain, and kidneys. Merozoites occur in circulating red blood cells later in the infection, and therefore, often will not be found in cats dying of acute disease. Disease occurs in cats that are bitten by the vector. In the cat, schizonts develop within monocytes, which become markedly enlarged. Cats with acute disease typically develop anemia, depression, fever, dehydration, and icterus. The majority of cats die within nine to 15 days of infection. The cause of death is occlusion of veins and venules with schizont-­‐laden macrophages. Hematologic changes may be severe, and result from displacement of hematopoietic tissue within the bone marrow. If the cat survives for more than six days, erythrocytes become infected and the merozoite stage develops, typically with no more than 1% to 4% of red blood cells infected. In the case of the bobcat, the schizogonous stage is shortened, and they become prolonged carriers of the erythrocytic stage. In a survey of cases from the mid-­‐Atlantic states of the US, of 34 cats infected with C. felis, 32 succumbed to the infection. The most common signs are pancytopenia and icterus. During the acute disease, organisms can be identified in smears of bone marrow or in biopsy specimens. Chronic disease is diagnosed by finding the merozoites in red blood cells. No treatments are consistently efficacious during the acute stage of the disease. 22 Natural cases of cytauxzoonosis in cats have typically been described from the southeastern and south central United States, cases being reported from Kansas, Oklahoma, Missouri, Arkansas, Texas, Louisiana, Mississippi, Georgia, and Florida. More recently, C. felis has been diagnosed in Kentucky, Indiana, Tennessee (70 cases), coastal North Carolina and South Carolina. Thus, it is becoming obvious that cytauxzoonosis is spreading beyond its typical confined range in the south-­‐central United Stated up into the lower Midwest. In the series of 80 described cases, the most common historical complaints were lethargy (n = 78), and anorexia (n = 60). Vomiting (usually once or twice) was reported in 6 cats. Other complaints that were reported were unsteady gait (3), abnormal behavior (1), abortion (1), and hematuria (1). The most common abnormalities on physical examination were hyperthermia (temperature >39.2 C; n = 72), icterus (31), elevated nictitans (31), dehydration (22), the presence of ticks (22), tachypnea (respiratory rate >40 breaths per minute; 20), tachycardia (heart rate >200 beats per minute; 13), pallor (9), murmur (8), vocalization (5), discomfort on abdominal palpation (5), lymphadenomegaly (5), and splenomegaly (5). Abortion, stupor, gallop rhythm, muscle wasting, ear mites, abscess, and disorientation were each found in a single cat. Temperature range in the 78 cats with legible recorded temperature was 38.3–
41.7 C (101.0–107.01F). Diagnosis has been improved through the development of PCR assays (Brown et al, 2010), and this has led to the ability to detect infections in asymptomatic cats. It may be that there are different strains of C. felis circulating in the wild and domestic populations with some being deadly and some being relatively benign. However, the risk remains. This is actually a very good argument for cats being kept indoors in areas where the disease is known to occur. Treatment now appears to be best through the use of atovaquone (15 mg/kg PO q8h) and azithromycin (10 mg/kg PO q24h, with heparin, fluids, and supportive care. Previously treatment had been with imidocarb (3.5 mg/kg IM). In an open-­‐label, randomized prospective study, of 53 cats treated with the atovaquone and azithromycin, 60% survived to discharge. Unfortunately for the 27 cats treated with imidocarb, only 26% of the cats survived to discharge. The mean temperature of cats randomized to receive atovaquone and azithromycin (104.5 + 1.1 F; 40.3 C) was identical to that of cats randomized to receive imidocarb (104.5 + 1.1 F; 40.3 C). Of 80 cats included in data analysis, 39 survived and 41 died. Of the 41 that died, 5 were euthanized because of severe clinical deterioration and moribund condition. Twenty-­‐four of 41 cats that died did so the day of or the day after presentation for care; only 3 cats died or were euthanized more than 3 days after presentation. Overall, survival was greater in cats treated with atovaquone and azithromycin than in cats treated with imidocarb (P = 0.0036; odds ratio 7.2; 95% CI 2.2, 24.0). It would seem that in areas where this disease is present that the best prevention for cats is an indoor existence. The disease often has a highly fatal outcome, and we as of yet do not have a perfect tick preventive for cats. If cats do go outside in these areas, they should have some form of tick prevention applied, but the lone star tick is one that is hard to dissuade from attacking and biting a host if it so chooses. Tick Transmitted Viruses – More and more, I think we are going to see the importance of tick-­‐
transmitted viruses in the case of people and pets in the USA. In Europe, people have long been concerned about various tick-­‐transmitted viruses: Russian Spring and Summer Encephalitis Virus, Crimean Congo Hemorrhagic Fever Virus, etc. We have had associated viral infections, and in 2009, a fatality in Maine due to Powassan virus – a virus in this same group. In Europe, they have recently begin to examine dogs with CNS disease and have realized that they may like people develop encephalitis from these deer-­‐tick viruses. We know nothing at this point about what occurs in cats. CONTROL 23 Excellent Tick Products: Dogs and cats should be prevented from acquiring tick-­‐transmitted infections, and if possible should be treated with products that will minimize their risk of carrying potentially infectious ticks back into the home. Cats in areas with cytauxzoonosis may be most wisely relegated to a life of confinement to an indoor existence. With the safe and efficacious products available, it is prudent to prevent tick infestations the entire year. The tick products available today are safer and better than anything before. However, the adversary just keeps coming after the pet. The products are excellent, but they have never been tested against the application of 60 to 100 ticks an hour being trickled onto a dog over a 4 or 5 hour period. Thus, the pressure is more than almost any product can bear. In areas of high tick numbers, veterinarians will recommend more than one product. There is almost no other way to win. In these circumstances it is hard for any product to successfully hold off the attack for a month. Thus, some people are utilizing applications with two products every other week or the use of tick collars along with the application of a monthly topical. These are not simple scenarios, because the products are tested as stand-­‐alone products, and the concern remains as to how safe are multiple applications of different products to a pet. Tick preventive products are EPA approved products, so they are considered to be applied externally and to remain external to the dog (or cat) to which they are applied. However, there is always concern about their potential internalization and associated reactions. We will review the different products and the studies that have been performed with each and the comparisons that have made between products. However, the most important thing to remember is that in some areas it is going to be almost impossible to apply a product that will stop every single tick from getting onto a host, and very likely that it will be impossible to stop every single tick from attaching and attempting to feed. 24 Heartworms, macrocyclic lactones, and the specter of resistance to prevention in the United States Text from article published in the Open Access Parasites & Vectors 2012, 5:138 (9 July 2012) Review Emerging knowledge has brought to the forefront the real possibility that resistance to macrocyclic lactones in the canine heartworm Dirofilaria immitis is being observed and could become a threat in the not too distant future. This information appears to contradict what has been observed (or more specifically, not observed) in human medicine, where more than two decades of use of these compounds against similar human parasites have not, thus far, resulted in significant problems of resistance. As study progresses on the apparent loss of efficacy of macrocyclic lactones in canine heartworm prevention, an understanding of the background (e.g., details of the preventive products and their use, issues of compliance, genetics, and other considerations) is necessary to ascertain the presence of true resistance in the heartworm population. Heartworm prevention in dogs with macrocyclic lactones and the choice of target doses for preventive treatment The goal of heartworm preventive therapy in dogs has been to stop infection by Dirofilaria immitis by killing the stage that is deposited by the mosquito and first enters the dog, the third-­‐stage larva (L3), as well as the young and maturing fourth-­‐stage larva (L4). The selection of product doses to achieve this goal has often been focused on a minimum effective dose, determined by dose-­‐titration studies using experimentally infected dogs; the dose-­‐limiting target organism, however, has not always been heartworms. Two types of studies have been established as the routine method for testing monthly heartworm preventives: dose-­‐titration studies to determine the minimum effective dose against the helminth in question, D. immitis, and dose-­‐confirmation studies to verify that this minimum dose is effective against two different isolates of the helminth. The testing of the compounds is similar in all cases as per requirements of the Center for Veterinary Medicine (CVM) of the United States Food and Drug Administration (FDA). Thus, the basic study design is the same in almost all cases (with one exception that will be discussed below). Efficacy is determined by giving dogs 30 to 100 L3 of D. immitis, and then 30 days later (since the drug is labeled to be given monthly and most products provide very little in the way of residual compound within the dog after just a few days), the dogs in the treatment groups are given the test compound whereas control dogs either remain untreated or are placebo-­‐treated. Approximately five months after infection, all dogs in both groups are euthanatized and necropsied, and the number of worms present in each of the dogs is counted. This is the basic design for both the dose-­‐
titration and dose-­‐confirmation trials. In the dose-­‐titration trials, now being called dose-­‐determination trials by the CVM/FDA, there are typically four groups of dogs: one that is treated at the expected target dose, one that is lower than the expected target dose, one that is higher than the expected target dose, and an untreated control group. In the dose-­‐confirmation trials, there are only two groups of dogs – dogs treated with the target dose and untreated controls. Dose-­‐confirmation trials are usually done as replicate studies in two different laboratories, each using a different isolate of the parasite. Due to the perfect efficacy originally afforded by ivermectin, the efficacy for approval of heartworm preventive drugs was set at 100%. Thus, a single worm in a dose-­‐confirmation trial would preclude a drug from receiving approval. The single exception to this basic experimental design for the testing of all the products on the market in the United States is ProHeart® 6, a slow-­‐release injectable product designed to prevent infection for six months. Thus, for the ProHeart® 6 studies, dogs in the treatment group are administered the product, and then six months later both the treated dogs and the untreated control 25 dogs are inoculated with 30 to 100 L3; five months after infection, these dogs are euthanatized and necropsied to determine worm burdens. The difference in design is because this drug is expected to be present in the dogs for six months, and thus, at six months it must still provide 100% protection against newly acquired infections. To reiterate, with most preventive products the drug is cleared fairly rapidly, and the dogs are dosed monthly to kill any larvae that have been acquired during the last 30 days. In contrast, the level of ProHeart® 6remaining within the dogs six months after treatment is intended to be sufficient to provide 100% protection against incoming larvae. These are the two basic formulas for all the trials presented below For Heartgard 30® (As marketed: tablets formulated to supply a minimum effective dose band of 6 mcg ivermectin/kg and a maximum dose of 12 mcg/kg), the target was heartworm prevention. In the original NADA 138–412 [1], "Of the 83 dogs treated at monthly intervals in natural infection trials, or treated 30 days after induced infection, with doses of ivermectin at 3.0 mcg/kg or greater, only 2 dogs developed infections [Author’s note: the 2 dogs that were positive were dogs that had each received 3.0 mcg/kg on day 30 after having been infected with approximately 50 L3]. Even when the treatment interval was extended to 45 or 60 days following infection, only 2 of 88 dogs given ivermectin at 6.0 mcg/kg or more developed infections." Dogs receiving 6 mcg/kg at 45 and 60 days were negative at necropsy. Thus, the minimal effective dose of 6.0 mcg/kg monthly was chosen. In the case of Interceptor® (As marketed: formulated in a tablet to provide a dose band of 0.5-­‐1.0 mg milbemycin oxime/kg) (NADA 140–915), 0.05 mg/kg was not always 100% protective against experimental heartworm infections when administered 30 days post inoculation with L3 [2]. However, a single treatment with 0.1 mg/kg or more at 30 days post infection appeared 100% effective [2]. The higher dose of 0.5 mg/kg was eventually chosen for this product in order to achieve >90% efficacy against hookworms. In the case of Revolution™ (As marketed: formulated as a topically applied product to deliver a dose band of 6–13 mg selamectin/kg) (NADA 141–152) [3], the dose was driven by flea control where "6 mg/kg of selamectin was selected as a minimum dose for effectiveness against fleas on dogs 30 days following a single topical administration." For heartworms, "Selamectin applied topically as a single dose of 3 or 6 mg/kg was 100% effective in preventing the maturation of heartworms in dogs following inoculation with infective D. immitis larvae 30 or 45 days prior to treatment, and 6 mg/kg was 100% effective in preventing maturation of heartworms following inoculation of infective larvae 60 days prior to treatment." In the case of ProHeart™ Tablets (As marketed: tablets formulated to provide a dose band of 3–6 mcg moxidectin/kg) (NADA 141–051) [4], "Moxidectin was 100% effective in preventing the development of a one month-­‐old heartworm infection of 50 L3 larvae of D. immitis in dogs when administered as a single oral treatment at 1.5, 3.0, and 6.0 mcg/kg." In this NADA there were no dose-­‐
confirmation studies with dogs receiving the 3.0 mcg/kg target dose a month after experimental infection with 30 to 100 heartworm larvae. However, there was a second dose-­‐determination trial where “Moxidectin was 100% effective in preventing the development of a two month-­‐old heartworm infection of 50 L3 larvae of D. immitis in dogs when administered as a single oral treatment at 0.5 mcg/kg.” The target dose chosen for ProHeart™ Tablets was 3 mcg/kg. For ProHeart® 6 injectable (As marketed: formulated as an injectable slow-­‐release product providing an initial dose of 0.17 mg moxidectin/kg; there is no dose band due to the formula allowing precise dosing) (NADA 141–189) [5], dosage titrations were performed with initial doses of 0.06, 0.17, and 0.50 mg/kg. In one study no heartworms were recovered from any dogs receiving any of the moxidectin-­‐
containing products, while in the other study one dog in the lowest dose group was infected. Thus, the minimum dose of 0.17 mg/kg was chosen. This dose was also found to have excellent efficacy against hookworm infections. 26 For Advantage Multi® for dogs (As marketed: formulated in a topical application to provide a combination product with a dose band of 2.5-­‐6 mg moxidectin/kg for heartworms and 10–25 mg imidacloprid/kg for flea control) (NADA 141–251) [6], the dosage was chosen based on intestinal nematodes -­‐ "Dosage Characterization for the Prevention of Heartworm Disease: Refer to section e., Dosage Characterization for the Treatment and Control of Intestinal Nematodes, which establishes a minimum effective dose for moxidectin." There was not a dose titration performed since the target parasite was not heartworm. In three trials, however, this minimum dose of 2.5 mg moxidectin/kg protected all 44 dogs that were treated 33 to 34 days after inoculation and 12 dogs treated 45 days after inoculation. . Control of filariid infections in humans In human medicine, the target dose for the control of the human filariid nematodes Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi has not been chosen for the purpose of preventing infection by killing L3 and L4. Rather, the goal has been to suppress the levels of microfilariae in the blood and skin of infected individuals so that, although the adult worms survive, there is no transmission between people. Also, the suppression of the skin-­‐dwelling microfilariae of O. volvulus prevents the damaging effects of the microfilariae on the cornea -­‐ the cause of river blindness. The dose chosen for microfilarial suppression has been 200 mg/kg administered orally every 6–12 months [7]. This increased dosage is necessary because microfilariae are typically less susceptible to macrocyclic lactones than are immature (L3, L4) filariid larvae. Although the campaign against human filariasis has now been active for more than twenty years with millions of doses being given to people in the developing world, resistance has not emerged as a significant problem; however, there are some very recent indications that the duration of microfilarial suppression may not be as long as it once was after treatment [8]. As yet, these effects do not appear to be hampering the progress of control in the field. Factors that may influence the development of resistance in heartworms Two factors in the prevention of heartworm infection are both likely to play major roles in how the different drugs might lead to resistance or how they might differ in their ability to affect resistant worms. First, as discussed above, canine heartworm preventives were designed to prevent infection by killing L3 and young L4. They were not designed to suppress microfilariae as in the control of human filarial infections. If drugs at preventive doses are given to dogs with existing infections, microfilariae will not be suppressed and many will survive in spite of the fact that they have encountered a macrocyclic lactone; these drug-­‐selected microfilariae can then be transmitted between dogs by mosquitoes. Thus, use of preventives in microfilaremic dogs is a less than prudent course since it might select for resistance through the exposure of microfilariae to sublethal concentrations of macrocyclic lactones. Secondly, some of the heartworm preventives on the market had other targets, such as fleas and intestinal nematodes, that required higher doses for killing than did heartworm, resulting in an increased minimum dosage for the combination product. Very typically in the case of resistance, increased doses of drug will continue to be efficacious against resistant strains until selection is so marked that the treatments become toxic to the host before the worms are killed. In either case, with a resistant phenotype, if dogs are infected by L3 that develop to patency in spite of exposure to a preventive, the ever-­‐hardier microfilariae will be capable of transmission by mosquitoes to other dogs. The concern is whether or not resistant phenotypes of heartworms have already appeared in the field. What is resistance? Resistance is defined by there being "a greater frequency of individuals within a population able to tolerate doses of a compound than in a normal population of the same species and is heritable……” [9]. Resistance to macrocyclic lactones is a well known phenomenon amongst intestinal nematodes. It has appeared in Haemonchus contortus populations [10]; in populations of Parascaris equorum [11]; and recent concerns have arisen relative to the small strongyles of horses [12]. In these cases, the target for control has been the adult worms using formulations with 90% to 98% efficacy. Resistance is selected by 27 the repeated treatment of all animals, placing significant selection pressure on the populations within the intestine of the host. The worms that survive treatment exposure are the only worms producing eggs for a period of time, and this provides them with a competitive edge. In the case of Haemonchus contortus, resistant isolates have been maintained in sheep, and the heritable resistance trait is known to pass from generation to generation [13]. Potential arguments against the development of resistance within heartworm populations might be the long generation time, i.e., the 7-­‐month-­‐long life cycle, large numbers of untreated refugia (stray dogs and, in the USA, coyotes), and the perfect efficacy of the preventive products against the highly susceptible L3. However, the long life cycle and refugia are unlikely to play a role if, for the former, resistant worms do not make a significant trade-­‐off in return for fitness, and for the latter, because full reversion (i.e., loss of resistance) -­‐ as sometimes occurs in bacteria when the drug pressure is removed -­‐ has not been observed in nematodes [14]. Also, the killing of a highly susceptible L3 stage by low doses of product may not be of actual significance in delaying resistance if, in the field, another more numerous and more resistant stage, such as the microfilaria, becomes the target of misdirected off-­‐label treatment. That resistance persists through worm developmental stages is indicated by the success of the larval migration assay for detecting resistance of adult H. contortus to macrocyclic lactones [15]. Since drug resistance in nematodes does not seem to impair fitness, the detection of this trait in any nematode population is of significant concern. Treatment failures in the field may be due to resistance, but often the true cause is hard to pinpoint due to potential problems with recording, lack of compliance, underdosing, reinfection (or preinfection in the case of heartworm preventives), etc. For some compounds, it is possible that resistance is not inevitable for a plethora of factors that may be genetic or related to management. As resistance of nematodes to various compounds, including macrocyclic lactones, has been reported in a number of important nematode parasites, it would seem that macrocyclic lactone resistance amongst filariid nematodes is a real threat. The current debate is whether or not the recent reports of lack of efficacies (LOEs) with heartworm preventives are due to the appearance of resistant forms or a confluence of confounding factors such as poor compliance The Mississippi delta and lack of efficacies (loes) In the area extending along the Mississippi River from Tennessee through Louisiana, there are many practitioners who contend that the heartworm preventives they have been using are no longer protecting dogs from infection. On the other hand, it has been reported that a very high percentage of these product failures are occurring in pets where compliance has not been as good as initially perceived by the claimant [16]. This speaks to the continuing problem of trying to identify resistance in the field, where treatment failures may or may not accurately reflect an underlying issue of resistance. However, other knowledge has come to light in the past 18 months that suggests that the veterinary community should be concerned about the threat of heartworm resistance. Microfilarial studies related to resistance phenotypes Microfilariae have been purified from the blood of dogs in the Mississippi Delta that were purportedly infected with heartworms while on preventive therapy. These microfilariae were examined for their ability to survive in different concentrations of several macrocyclic lactones [17]. Some of the isolates showing reduced susceptibility to macrocyclic lactones were grown to the L3 stage in mosquitoes and used to infect dogs. When the microfilariae from these latter dogs were examined in the same assay, they were found to similarly lack susceptibility to macrocyclic lactones. Thus, this observed lack of microfilarial susceptibility to macrocyclic lactones is a form of genetically inherited resistance; however, this trait may or may not be linked to the ability of later stages (i.e., L3 and young L4) to grow to adulthood in dogs on preventive therapy. With the isolation of these strains, experiments can now be designed to test whether these isolates can successfully infect dogs on preventive therapy. Persistent microfilaremia in a Katrina rescue dog taken to Canada and treated for its heartworm infection 28 Anecdotal reports by practitioners have circulated about the inability to clear dogs of their microfilariae after the adults are removed with Immiticide® (melarsomine dihydrochloride). Recently, a detailed case description was published concerning one such case [18]. This was a dog from the southern United States that was rescued following hurricane Katrina and relocated to Canada where it was treated with Immiticide® for its heartworm infection on two separate occasions, 5 months apart [18]. The dog remained microfilaremic after the first treatment in spite of being placed on a macrocyclic lactone preventive product. Eight months after the first adulticidal treatment, the dog had become antigen negative, but it remained positive for microfilariae in spite of having received a second round of Immiticide® and multiple treatments with macrocyclic lactones including two doses of ivermectin at 200 mcg/kg. The dog was then treated every other week with milbemycin oxime, ultimately near the top of the preventive dose band at 1.1 mg/kg, and finally daily for 7 days at 2.0 mg/kg followed again a month later by the daily administration of 2.0 mg/kg for 8 days. The dog remained antigen negative and microfilariae positive until just recently when the microfilariae finally disappeared, more than two years after the second adulticidal treatment (Personal communication with one of the authors). This appears to be independent verification that microfilariae that are refractory to macrocyclic lactones exist [17]. However, microfilariae from this infection were not grown to L3 and used to infect another dog, so it is not known whether this resistance trait was inheritable, and again, there is no proof that the phenotype of microfilarial resistance translates into resistance of L3 when they are inoculated into dogs receiving macrocyclic lactones. Susceptibility of third-­‐stage larvae from mosquitoes to macrocyclic lactones Infective-­‐stage heartworm larvae have been examined for resistance to macrocyclic lactones using an assay where L3 grown in mosquitoes are allowed to migrate through fine mesh in the presence of different macrocyclic lactone concentrations (a method originally developed to measure the prevalence of resistant phenotypes in horse and sheep strongylids). L3 grown from the apparently resistant microfilarial isolates from the Mississippi Delta were found to be as susceptible to macrocyclic lactones as L3 grown from non-­‐resistant microfilariae [19]. However, as with microfilarial susceptibility, it remains unclear whether the phenotype of decreased L3 susceptibility to macrocyclic lactones is related to the ability to infect a dog on heartworm preventive therapy. Lab-­‐based studies with heartworms in dogs relative to preventive product failures In 2004, Pfizer Animal Health sent a letter to all veterinary practitioners in the United States that informed them that "An additional experimental study evaluating the performance of Revolution® involved mixed breed dogs challenged with a rigorous infection of 50 heartworm larvae and treated 30 days later with a single dose of Revolution™. …. The results indicated some treated dogs harbored 1 or 2 adult heartworms five months following this laboratory challenge, all untreated control dogs exhibited substantial worm burdens (14–43 adult worms per dog)." This was the first report of laboratory studies in which heartworms developed in dogs given a single dose of a marketed preventive at the prescribed dosage 30 days after inoculation with infective larvae of a laboratory strain. The development of Trifexis® In 2011, two papers and the Freedom of Information summary for the New Animal Drug Application (NADA) appeared on the development of a new milbemycin oxime-­‐containing heartworm (HW) preventive product, Trifexis® (As marketed: tablets formulated to provide a dose band of 0.5-­‐1.0 mg milbemycin oxime/kg ion for heartworm with 30–60 mg spinosad/kg for flea control) [20-­‐22]. "To achieve a FDA-­‐CVM-­‐approved label claim for HW prevention as determined in dose confirmation (DC) testing protocols for these pioneer ML [macrocyclic lactones], dogs were generally inoculated with 50 infective D. immitis third stage larvae (L3) obtained from experimentally infected mosquitoes, and then 30 days later administered a single dose of the preventive being tested. A corresponding nontreated control group was used to confirm adequacy of infection of the HW isolate. Five to 6 months later, after surviving worms have had a chance to mature, necropsy examinations determine the effectiveness of 29 the product. The presence of a single HW in any of the treated dogs would have prevented approval because 100% prevention was necessary to obtain a label claim based on this dosing protocol. ….. As part of the development program of an ML for HW prevention, 2 separate recently isolated HW isolates were tested according to current FDA-­‐CVM requirements. While one isolate was fully susceptible to this ML [macrocyclic lactone]providing 100% prevention after a single dose administered 30 days after inoculation with HW L3, efficacy of a single treatment against the second isolate was <100% [20]." In this study, 3 of the 10 infected dogs treated with Trifexis® had 3 worms at necropsy, 1 had 1 worm and 2 had 2 worms. This work was done with a strain identified as MP3 -­‐ a heartworm isolate named after the naturally infected dog from Georgia, USA from which it was isolated, “Miss Piggy.” The manufacturer of Trifexis® pressed forward with its development and reported "A study was undertaken with this second field isolate to assess the effectiveness of currently marketed ML, in this case administering a single dose of IVM [ivermectin] or MBO [milbemycin oxime], in dogs challenged with HW L3 1 month before treatment [21]." The products examined for preventive efficacy were Heartgard Plus Chewables for Dogs® (As marketed: dose band of 6–12 mcg ivermectin/kg), and Interceptor Flavor Tabs for Dogs & Cats® (As marketed: dose band of 0.5-­‐1.0 mg milbemycin oxime/kg). This work was done in dogs with the MP3 strain using a similar challenge model as already described [20]. There was one worm found in one dog in each of the Heartgard Plus®-­‐ and Interceptor®-­‐treated groups of dogs (comprising 14 dogs each). Thus, neither product was 100% efficacious in preventing infection by the MP3 strain in this historical method of preventive testing. In the development of Trifexis®, additional studies were conducted with this strain examining the effects of multiple treatments at 30 and 60 days after infection and at 30, 60, and 90 days after infection. In the 10 dogs treated twice with Trifexis®, there was a single worm present at necropsy at 150 days postinfection (1 worm in 1 dog), whereas no worms were observed in the dogs treated three times. Thus, the Trifexis® label has the statement "For heartworm prevention, give once monthly for at least 3 months after exposure to mosquitoes “[22]. Continuing treatment for three months following the end of the mosquito season should protect dogs from infection by killing any worms acquired during the last month of transmission. MP3 and Advantage Multi® versus Heartgard Plus®, Interceptor Flavor Tabs®, and Revolution™ In the introduction to a study that compared the efficacy of these four products in dogs with a single treatment 30 days after inoculation with 100 L3 of the MP3 heartworm isolate, it is disclosed that the sponsor of the research, Bayer Animal Health, LLD, as part of product development, was working on a new macrocyclic lactone-­‐containing preventive (two formulations of ivermectin-­‐containing products with target minimum doses of 6 mcg/kg and 9 mcg/kg; personal communication as to dose bands used) [23]. In this work using the same dose of ivermectin as an already marketed product or one that was at a dose 1.5 times higher, "a strain of D. immitis (MP3 laboratory strain; TRS Laboratories Inc., Athens, GA) was used to evaluate a potential new anthelmintic product." It was found "In that study, the MP3 laboratory strain was less susceptible to traditionally effective doses of an ivermectin-­‐based preventive in a limited number of dogs." Thus, Bayer decided to examine the efficacy of four commercial monthly products against this problematic strain. The study design in this subsequent trial used 100 L3, treatment with the labeled monthly dose 30 days later, and necropsies 150 days after treatment. There were 8 dogs in each group. The results were such that 7 dogs developed infections with adult worms in each of the groups treated with Heartgard Plus® (0–7 worms), Interceptor® (0 to 6 worms), and Revolution™ (0–9 worms), whereas no worms were recovered from any 8 of the Advantage Multi®-­‐treated dogs. Thus, again, with the MP3 isolate, not all dogs were afforded 100% protection by all products. In these single treatment studies, only Advantage Multi® was 100% efficacious in preventing development of the MP3 larvae to adulthood. Summary of the above studies utilizing MP3 30 Among the studies discussed above, where the MP3 strain was used, there have been eight reported instances in which dogs have become infected in spite of being given a single heartworm preventive treatment 30 days after infection: 1 study with Trifexis®, 2 studies with Heartgard Plus®, 2 studies with Interceptor®, 1 study with Revolution™, and 2 studies with ivermectin in a product undergoing development. There was also one study where one dog had one adult heartworm at necropsy after having received two Trifexis® treatments 30 and 60 days after infection. Although F1 generations of MP3 have been established (personal communications to the author), there have been no reports as of yet on whether the resistance trait seen with MP3 is heritable. One might surmise, however, that worms that survive a single treatment are perhaps more likely to produce offspring with a greater chance of surviving repeated macrocyclic lactone preventive therapy than those killed by a single treatment. Of course, it depends on whether the trait is present in the genome of the worm and how it is inherited by offspring when the worms mate. Additional studies with lack of efficacy There has recently been another report of dogs not being protected with milbemycin oxime at the same dosage as provided in Interceptor®, Sentinel®, and Trifexis®. In this case, Sentinel® Spectrum® Tasty Chews (a combination product of 0.5-­‐1.0 mg milbemycin oxime/kg, lufenuron, and praziquantel) was being brought to market in the USA (NADA 141–333) [24]. The report in the NADA approved in December of 2011 includes a trial wherein the dogs were treated monthly for 6 months beginning 30 days after infection with 50 D. immitis L3. In this study, all treated dogs were protected. However, the NADA states: "A 6-­‐consecutive monthly dosing regime was selected for effectiveness studies against D. immitis infections. Neither one dose nor two consecutive doses of SENTINEL SPECTRUM provided 100% effectiveness against induced heartworm (D. immitis) infections in dogs". The label for this product states: "For heartworm prevention, give once monthly beginning within 1 month of the dog’s first seasonal exposure to mosquitoes and continuing until at least 6 months after the dog’s last seasonal exposure." At this point, it is unclear if this study was done using the MP3 strain, but the work was performed in the laboratory where the strain was first isolated and maintained, so it is a possibility. If the strain was MP3, it would mean that there is an additional study where milbemycin oxime was not effective in preventing the development of heartworms after one or two treatments. Molecular biological examination of heartworms as related to resistance Recently the molecular phenotypes of heartworms have been examined in an effort to see if various molecular markers may be associated with treatment failures [25,26]. The amount of polymorphism has been examined in different genes associated with macrocyclic lactone resistance in other nematodes, e.g., the ABC transporter gene -­‐ best known to veterinarians as the PGP (P-­‐
glycoprotein-­‐like) gene or the MDR1 (Multi-­‐Drug Resistance 1) gene -­‐ that is defective in dogs of the collie breed, leading to avermectin sensitivity. It was first shown that in heartworms collected from around the world, there were small differences in several of these genes that appeared to be randomly distributed throughout the population, i.e., the worms were polymorphic within the gene examined. [Author’s note: A gene exhibits no polymorphism if any change in the gene is fatal to the worm, and if there is no polymorphism in a gene, then it is of no value in determining whether or not drug selection has an effect on the frequency of polymorphism. Selection will produce reduced polymorphism in a gene within a population; this is the classic selection for pea color that led Mendel to his understanding of genetic heritability.] In the case of D. immitis, these authors found that the microfilariae produced by isolates from the Mississippi Delta were markedly reduced in their polymorphism for the genes examined. The authors concluded that this indicated that selection, likely via macrocyclic lactones, had driven worms toward genetic similarity in those genes. Any selection in nematodes is believed to be housed solely in the genome, so any reduced polymorphism would also appear in all life stages. These same investigators found a reduced polymorphism in the microfilariae from the Katrina rescue dog in Canada whose microfilariae did not clear in the presence of high doses of milbemycin oxime [18]. 31 However, they have also examined these genes in MP3 and have found that, based on the polymorphisms examined, the genes from the MP3 strain appear susceptible but with some degree of ML selection in its history but that they have not undergone a high degree of loss of polymorphism when compared to that of the Mississippi Delta isolates and the Katrina rescue dog [27]. So – do we have resistance? Resistance can come about in several ways. It can be due to a spontaneous mutation. It can be induced by mutagens, e.g., irradiation or chemical exposure. It can be due to continued selection pressure -­‐ such as by repeated treatment of worms with the same drug – causing a particular phenotype to rise in frequency within a population. In the case of heartworms, we can probably rule out a mutagen-­‐induced introduction of resistance genes; thus, it is most likely that the resistance is due to either a spontaneous mutation or via selection of a rare or uncommon phenotype. Mp3 as a spontaneous mutation If with MP3 the resistance is heritable, i.e., microfilariae produced by dogs infected with L3 derived from Miss Piggy’s microfilariae are capable of infecting dogs receiving macrocyclic lactone preventives, then this is a strain that is resistant to macrocyclic lactones. (It can be argued that it is not fully resistant because multiple preventive doses are protective, but it can also be readily argued that it is a phenotype that undoubtedly has a reduced susceptibility to macrocyclic lactones compared to other strains that have been historically examined.) It does not matter whether Miss Piggy’s population of adult worms and their microfilariae ever saw macrocyclic lactones before or not. If these microfilariae – which have been shown to be less susceptible to heartworm preventives – grow to adulthood and produce offspring that can routinely infect dogs on preventives just like their parents, they are resistant worms. This could be due to a one-­‐time chance event occurring from the pairing of one male and one female worm within Miss Piggy producing microfilariae with a resistant phenotype. It may be that MP3 was simply a rare capture of a spontaneous mutation. MP3 chosen by Selective Drug Pressure It is also possible that the genes for resistance have been in the genetic make-­‐up of a small population of D. immitis somewhere in the field for a very long time, and via the widespread use of macrocyclic lactones, worms in dogs were selected that have a resistant phenotype. These already resistant worms then found their way via a mosquito into Miss Piggy. Since both the ancestors of filarial nematodes and the Actinobacteria producing the macrocyclic lactones occur within soil ecosystems, genes supporting resistance to these bacterial products have likely provided a survival advantage to nematodes for eons. The genes only became important relative to drug resistance in nematodes like Parascaris equorum and D. immitis when we started using purified products from these Actinobacteria to treat or prevent nematode infections. Then, these resistant phenotypes were promoted by selection. In this scenario, the genes for resistance were already present in the population and just never had a chance to show their survival potential until challenged, such as occurred when the MP3 isolate was captured and tested in the studies described above. When parasitologists think and talk about resistance to anthelmintics they are usually considering the drug-­‐induced selection of resistant forms. The selection of MP3 and possibly other heartworm strains in the United States with resistance to macrocyclic lactones could occur in at least two different ways. In one case, where selection pressure may have been applied through regular preventive therapy, worms like MP3 -­‐ representing a very small portion of the population -­‐ might sneak through and survive in a few dogs on preventive. These infections might remain undetected if dogs with developed adults and microfilariae did not receive annual status checks, and the patent infections could then be spread by mosquito transmission. In another case, the adulticidal treatment of dogs with macrocyclic lactones (Slow-­‐Kill or Soft-­‐Kill) rather than Immiticide® might, by subjecting the off-­‐target microfilariae to prolonged drug exposure, select for populations of resistant circulating microfilariae that are spread to 32 new dogs by mosquitoes. In the first case, selection is at the level of L3 and L4; in the second case, selection would be occurring at the level of the microfilariae. Conclusions At this point, it is not known if the F1 generation of worms produced from microfilariae of LOE dogs in the Mississippi Delta can develop to adults in dogs on preventive therapy. We do not even know at this time if they can survive following a single preventive treatment, as has MP3 in clinical drug trials. Based on the data to date, it appears that the microfilarial assay used to evaluate these isolates may measure a lack of susceptibility in microfilariae, but this may not correlate with increased survival of L3 (grown from those same microfilariae) in the presence of these anthelmintics. Also, the isolates from the Mississippi Delta and the Katrina dog that have reduced sensitivity to macrocyclic lactones were different than MP3 in that MP3 microfilariae appeared to be more similar to the microfilariae of fully susceptible forms based on certain gene polymorphisms. It is possible that the phenotype being examined in the microfilarial susceptibility assay might not be the same as that required for resistance in larval and adult heartworms. If the offspring of MP3 are found to behave the same way as the original isolate in similar drug efficacy trials, the trait is heritable, and resistance could be assumed. It does not matter if the trait was due to spontaneous mutation or selection. Again, because heritability of this trait has yet to be shown for MP3, it cannot be stated whether the resistant phenotype will persist into the next generation. A spontaneous inheritable mutation event would seem likely to occur with less frequency than cases selected for by large-­‐scale drug use in a population where some worms already carry resistance genes. If the problem with selamectin as reported by Pfizer’s 2004 letter was due to a difference in the isolate used in those trials, rather than due to some defect in product absorption or other product-­‐associated problem, then this could be considered evidence of either spontaneous mutations occurring on more than one occasion or the existence of a survival-­‐promoting genetic trait in a number of heartworms within the general heartworm population. Again, from the original Heartgard® 30 NADA 138–412, there were two dogs that developed heartworm infections out of 83 dogs treated at 3.3. mcg/kg administered 30 days after infection, and 2 dogs given 12 mcg/kg ivermectin developed adult heartworm infections with 1 and 5 worms each, when product was administered at 45 days and 60 days after infection (dogs getting 6 mcg/kg at 45 and 60 days were negative at necropsy). These results from sometime before 1987 suggest that there is polymorphism in the worms relative to a potentially resistant phenotype. This would lead to the conclusion that there are resistance-­‐associated phenotypes occurring at some low level in the D. immitis population. Recently, there has been another study of the genetics of heartworms in the United States. Using 11 polymorphic microsatellite loci and 192 individual heartworms from 9 different geographic locations in the United States and Mexico, the genetics and population structure of heartworms were examined and 4 major genetic clusters were identified [28]. The clusters were associated with the eastern United States, central USA, western USA, and Mexico. There was a low level of heterozygosity observed in general, and high levels of reciprocal gene flow between the populations in the eastern US and the central US. It appears that geographic barriers impede significant gene flow between these areas and California and Mexico. Germane to the conclusion of this review is a conclusion reached by these authors on the relationship between gene flow and resistance: “This pattern of gene flow could certainly influence the spread of alleles beneficial to canine heartworm. In an area where there is a significant amount of gene flow, such as the Gulf Coast, the dispersal of drug resistance alleles would occur rapidly. Those resistance alleles would not necessarily need to arise in that geographic region, but could arrive there via dispersal from some other area.” At this point, careful study has begun into the issue of resistance in heartworms, and the potential of resistance existing has been taken seriously in many different laboratories. The life cycle of 33 heartworms is long, and it takes at least 6 months to develop adult worms and produce circulating microfilariae in a dog, and then another 6 months to determine if these microfilariae, when grown to L3 in mosquitoes and used to infect additional dogs, produce resistant worms. It also must be remembered that worms, unlike bacteria, have to undergo genetic recombination through mating between males and females to produce offspring. What if there is only one adult male in the MP3 population containing the resistance trait? There is a good chance that a similar male may not develop in the group of 30–50 worms in the next infected dog. At this time, we do not even know that the worms from an MP3 infection are capable of producing microfilariae if the infections develop and persist in the face of preventive therapy; the assumption is that they would, but they might not. Overall, a prudent approach remains similar to what has been recommended in the past: Vigilance through testing dogs before initiating heartworm prevention; vigilance through testing dogs annually for heartworms to avoid letting any MP3 (or other potentially resistant strain) slip by, live, and produce microfilariae while the dog is on prevention; and vigilance through treating dogs with adult worms with Immiticide® rather than a macrocyclic lactone, to reduce the chance of producing or selecting microfilariae with a resistant phenotype. Furthermore, it would be prudent to keep all dogs on year-­‐round prevention since this should reduce selection of worms that have seen only one dose of drug due to a miscalculation of the end of the transmission season, or because it would appear that some strains, such as MP3, may require three consecutive doses of preventive to be fully eliminated. It is critical that dogs on preventive be tested annually and, if found infected, that the infection be cleared using Immiticide®. There is some indication that doxycycline administration to dogs with adult worm infections might suppress the infectivity to the next canine host of L3 that develop from microfilariae produced by these doxycycline-­‐
treated worms [29], but the sample size from this work is still very small, so it may not be wise to count on this quite yet. Overall, at this time, there is reason for concern, a lot of research that still needs to be performed, a critical need for veterinarians to impress upon their clients that they need to pay attention to keeping current on their prevention, and a need for veterinary practitioners to practice careful record keeping while continuing to report all LOEs to the FDA. Competing interests The author has, in the past, received financial remuneration from the manufacturers of all products mentioned in this review. Acknowledgements The author would like to thank Drs. Alice Lee and Araceli Lucio-­‐Forster (College of Veterinary Medicine, Cornell University) and Dr. Michael Labare (United States Military Academy) for their careful reading of the manuscript. References 1. NADA: 138–412 Heartgard-­‐30®.: ; http://www.fda.gov/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummarie
s/ucm111205.htm?utm_campaign=Google2&utm_source=fdaSearch&utm_medium=website&utm_
term=nada%20138-­‐412&utm_content=1. 2. NADA: 140–915 Interceptor®.: ; http://www.fda.gov/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummarie
s/ucm054857.htm?utm_campaign=Google2&utm_source=fdaSearch&utm_medium=website&utm_
term=nada%20140-­‐915&utm_content=5. 3. NADA: 141–152 Revolution™.: ; http://www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADr
ugSummaries/ucm117201.pdf. 4. NADA: 141–051 ProHeart™ for Dogs.: ; http://www.fda.gov/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummarie
s/ucm116537.htm. 34 5. NADA: 141–189 ProHeart®6.: ; http://www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADr
ugSummaries/ucm117588.pdf. 6. NADA: 141–252 Advantage Multi® for Dogs.: ; http://www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADr
ugSummaries/UCM051438.pdf. 7. Cupp EW, Bernardo MJ, Kiszewski AE, Collins RC, Taylor HR, Aziz MA, Greene BM: The effects of ivermectin on transmission of Onchocerca volvulus. Science 1986, 231:740–742. 8. Churcher TS, Pion SD, Osei-­‐Atweneboana MY, Prichard RK, Awadzi K, Boussinesq M, Collins RC, Whitworth JA, Basanez MG: Identifying sub-­‐optimal responses to ivermectin in the treatment of River Blindness. Proc Natl Acad Sci USA 2009, 106:16716–16721. 9. Prichard RK, Hall CA, Kelly JD, Martin ICA, Donald AD: The problem of anthelmintic resistance in nematodes. Aust Vet J 1980, 56:239–250. 10. Williamson SM, Storey B, Howell S, Harper KM, Kaplan RM, Wolstenholme AJ: Candidate anthelmintic resistance-­‐associated gene expression and sequence polymorphisms in a triple-­‐
resistant field isolate of Haemonchus contortus. Mol Biochem Parasitol 2011, 180:99–105. 11. Reinemeyer CR, Prado JC, Nichols EC, Marchiondo AA: Efficacy of pyrantel pamoate against a macrocyclic lactone-­‐resistant isolate of Parascaris equorum in horses. Vet Parasitol 2010, 171:111–
115. 12. Traversa D, Samson-­‐Himmelstjerna Gvon, Demeler J, Milillo P, Schurmann S, Barnes H, Otranto D, Perrucci S, Regalbono AFdi, Beraldo P, Boeckh A, Cobb R: Anthelmintic resistance in cyathostomin populations from horse yards in Italy, United Kingdom and Germany. Parasit Vectors 2009, 2(Suppl 2):S2. doi:10.1186/1756-­‐3305-­‐2-­‐S2-­‐S2. 13. Kaminsky R, Bapst B, Stein PA, Strehlau GA, Allan BA, Hosking BC, Rolfe PF, Sager H: Differences in efficacy of monepantel, derquantel and abamectin against multi-­‐resistant nematodes of sheep. Parasitol Res 2011, 109:19–23. 14. Wolstenholme AJ, Fairweather I, Prichard R, Samson-­‐Himmelstjerna Gvon, Sangster NC: Drug resistance in veterinary helminths. Trends Parasitol 2004, 20:469–476. 15. Kotze AC, le Jambre LF, O’Grady J: A modified larval migration assay for detection of resistance to macrocyclic lactones in Haemonchus contortus, and drug screening with Trichostrongylidae parasites. Vet Parasitol 2006, 137:294–305. 16. Atkins C: Current controversies and dilemmas in heartworm disease: defining the issues by close examination.: ; [https://www.aahanet.org/Education/WebConference.aspx?key=428166ce-­‐8c29-­‐
462d-­‐ad51-­‐f7b9bd3b1350]. 17. Blagburn BL, Vaughan JL, Butler JM, Mount JD, Spencer JA, Carmichael J, Schenker R: Evaluation of susceptibility of heartworm (Dirofilaria immitis) biotypes to macrocyclic lactones using microfilariae-­‐based single dose and dose-­‐mortality regression assays [abstract]. In Proceedings of the AAVP 56th Annual Meeting. MO: St. Louis; 2011:110. 16–19 July 2011. 18. Bourguinat C, Keller K, Bhan A, Peregrine A, Geary T, Prichard R: Macrocyclic lactone resistance in Dirofilaria immitis. Vet Parastiol 2011, 181:388–392. 19. Moorhead AD, Evans CC, Wolstenholm AJ, Storey BE, Blagburn BL, Vaughan JL, Schenker R, Carmichael J, Kaplan RM: In vitro bioassay for measuring anthelmintic susceptibility in Dirofilaria immitis [abstract]. In Proceedings of the AAVP 56th Annual Meeting. MO: St. Louis; 2011:109. 16–19 July 2011. 20. Snyder DE, Wiseman S, Cruthers LR, Slone RL: Ivermectin and milbemycin oxime in experimental adult heartworm (Dirofilaria immitis) infection of dogs. J Vet Intern Med 2011, 25:61–64. 35 21. Snyder DE, Wiseman S, Bowman DD, McCall JW, Reinemeyer CR: Assessment of the effectiveness of a combination product of spinosad and milbemycin oxime on the prophylaxis of canine heartworm infection. Vet Parasitol 2011, 180:262–266. 22. NADA: 141–321 Trifexis.: ; http://www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADr
ugSummaries/UCM252248.pdf?utm_campaign=Google2&utm_source=fdaSearch&utm_medium=w
ebsite&utm_term=nada trifexis&utm_content=1. 23. Blagburn BL, Dillon AR, Arther RG, Butler JM, Newton JC: Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis. Vet Parasitol 2011, 176:189–194. 24. NADA: 141–333 Sentinel® Spectrum®.: ; http://www.fda.gov/downloads/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADr
ugSummaries/UCM292003.pdf. 25. Bourguinat C, Keller K, Prichard RK, Geary TG: Genetic polymorphism in Dirofilaria immitis. Vet Parasitol 2011, 176:368–373. 26. Bourguinat C, Keller K, Blagburn B, Schenker R, Geary TG, Prichard RK: Correlation between loss of efficacy of macrocyclic lactone heartworm anthelmintics and P-­‐glycoprotein genotype. Vet Parasitol 2011, 176:374–381. 27. Bourguinat C, Keller K, Bhan A, Peregrine AS, Blagburn BL, Geary TG, Prichard RK: Macrocyclic lactone resistance in Dirofilaria immitis [abstract]. In Proceedings of the AAVP 56th Annual Meeting. MO: St. Louis; 2011:108. 16–19 July 2011. 28. Belanger DH, Perkins SL, Rockwell RF: Inference of population structure and patterns of gene flow in canine heartworm (Dirofilaria immitis). J Parasitol 2011, 97:602–609. 29. McCall JW, Genchi C, Kramer L, Guerrero J, Dzimianski MT, Supakorndej P, Mansour AM, McCall SD, Supakorndej N, Grandi G, Carson B: Heartworm and Wolbachia: therapeutic implications. Vet Parasitol 2008, 158:204–14. 36 Internal Helminth Control in Dogs and Cats DOGS Year-­‐round prevention also helps with internal parasite control. Parasites have evolved to with life cycles that are designed to get into carnivores. The most important internal parasites of the dog and cat are roundworms (Toxocara canis and Toxocara cati), hookworms (Ancylostoma caninum, Ancylostoma tubaeforme, and Ancylostoma braziliense), and whipworms (Trichuris vulpis). The days have passed when it is socially acceptable for pets to have these infections. People now basically want their pets to be worm free just like their children or grand children. Hookworms and roundworms live in carnivores and have life cycles adapted to this type of host. They both utilize invertebrate and vertebrate paratenic hosts to get to the carnivorous host. Transmission routinely occurs in the winter – yes routinely. Rodents, birds, and rabbits that are ingested are perfect sources of infective larvae for these hosts. The whipworms do not utilize paratenic hosts, but the eggs survive very well in the environment, and if an egg is infective when the cold comes, it can be infective when ingested in midwinter if the host has access to soil. If you think about where dogs and cats live over winter, the ground upon which they sleep is often far from frozen solid. Thus, all three of these nematodes are designed for transmission in the frozen north. Toxocara spp. Toxocara canis is probably the most common and well known parasite of the dog around the world. The worms are passed in the feces sporadically or after treatment, and the eggs are easily detected using standard fecal flotation methods. The adult worms live in the small intestine, the eggs pass in the feces of the host into the environment. In the soil the eggs become infective in about 9 days at 26-­‐30°C. Dogs become infected by eating embryonated eggs or paratenic hosts (e.g., mice, chickens, and lambs). Invertebrate paratenic hosts, like earthworms, probably help the worms make their way to the vertebrate paratenic hosts. The most significant mode of transmission in the dog is transplacental transmission of larvae. Dogs infected prenatally can develop pulmonary lesions. Light infections cause mild petechial hemorrhaging, while heavy infections may cause pneumonia. Mature in heavy infections can be found in the stomach, bile duct, and peritoneal cavity. Heavily infected puppies can develop mucoid enteritis and intestinal impaction. Often the migrating larvae are arrested in the tissues forming granulomas. Gut penetration by larvae can cause severe anemia as a prelude to death. Adult dogs (and probably cats) can easily be infected experimentally by feeding them only a few eggs, the misunderstanding that adult animals cannot be so infected was based on early work where infections with massive numbers of eggs failed to produce patent infections. The infection of adult dogs has since been verified and re-­‐verified on several occasions. Clinical signs occur only in young dogs following the ingestion of eggs: coughing and nasal discharge that usually subsides after about 3 weeks. Heavy infections can cause vomiting, anorexia, abdominal 37 distension, mucoid diarrhea, debilitation, reduced growth rate, allergic pruritus, and a characteristic foul oral odor. Adult dogs typically are without clinical signs. Toxocara canis causes visceral larva migrans in humans. Eggs in the soil require about two weeks to embryonate, but can then remain infective for several years. Larval toxocariasis manifests in a spectrum of syndromes that includes visceral, neural, ocular, covert, and asymptomatic toxocariasis. Asymptomatic toxocariasis is the most common form, but clinical cases do still occur. The most well-­‐
recognized source of infection is ingestion of contaminated soil by toddlers. Anti-­‐Toxocara antibodies among the U.S. population was recently measured to be 13.9% using sera from the Third National Health and Nutrition Examination Survey. Having a positive titer was associated with a low-­‐level of education, being born in a foreign country, living in overcrowded conditions and in conditions of poverty. Pet ownership was not associated with increased seroprevalence. Toxocara cati is found wherever there are cats. Adult worms live in the small intestine of the cat and pass characteristic single-­‐celled, thick-­‐shelled eggs into the environment in the feces of the host. The egg passed in feces develops to the infectious stage in the environment; the infectious egg contains a third-­‐stage (L3) larva within. The eggs require several days to weeks in aerated and moist soil to become infectious and develop more slowly to the infective stage than the eggs of Toxascaris leonina. The cat may become infected in one of two ways. First, after ingestion of infective eggs, the larvae hatch in the stomach and migrate into the lining of the stomach, as well as through the gut wall, to muscles, the liver and the lungs. As larvae that reach the lungs develop, they grow and breakout into the alveolar spaces, they are then coughed up by the host and swallowed, only to reach the small intestine once more. Here, they develop through the 4th stage into adults, which live in the small intestinal lumen. The prepatent period for this route of transmission is 40-­‐56 days, although the adult worms are present in the small intestine by day 28. The second route of infection is via a paratenic host. A small rodent (typically a mouse) that has eaten an infective egg will have larvae hatch in the stomach and migrate to tissues (such as the liver) where they arrest development. Upon ingestion by the cat, the larvae within the tissue of the mouse are freed and develop in the gut of the cat into adults. In this case, there is no somatic migration taking place. Transmammary transmission is now thought to only occur in the cat when the mother receives a very large number of eggs just prior to or after she begins nursing. The mode of infection determines the rate of worm development in the cat. The fourth-­‐stage larvae will appear after about 10 days in cats fed infected mice and in about 14 days in cats fed infective eggs. These stages are found in the stomach contents, intestinal wall, and intestinal contents. It is believed that immature adult worms first appear around 28 days after infection with infective eggs. Accordingly, the prepatent periods are shortened depending on the route of infection. The longest prepatent period will be after the ingestion of eggs, and infection via paratenic hosts will result in prepatent periods somewhere in between. For a long time it was believed that the typical cause of visceral larval migrans was only Toxocara cati, but more recently the potential for T. cati to also be associated with this infection has increased based upon what is known of the life cycle and serology done on humans in parts of the world other than the United States. Ancylostoma spp. In the United States there are four hookworms of dogs and cats. Ancylostoma caninum is the common hookworm of the dog. Ancylostoma tubaeforme is the common hookworm of the cat. Ancylostoma braziliense is a hookworm of the dog and cat shared by cats in the southeastern United States. The worm Uncinaria stenocephala is a hookworm of dogs in the northern United States; this hookworm occurs in dogs and cats in Europe and has been reported from cats in the United States, but it seems to be quite rare in cats in the United States. These nematodes utilize cockroach paratenic hosts and various vertebrates as the paratenic host that carries the infection to the carnivore. Ancylostoma tubaeforme larvae, it seems, are poor skin 38 penetrators, so cats are probably most commonly infected by predation. Similarly, although the dog hookworm, Ancylostoma caninum can penetrate via the skin (as can Ancylostoma braziliense) both these nematodes also routinely utilize vertebrate paratenic hosts. Finally, the dog hookworm is very successful at infecting puppies by the reawakening of the larvae during lactation. Hookworms cause blood loss by the host. The order of blood loss per worm from most to least is Ancylostoma caninum, Ancylostoma tubaeforme, Ancylostoma braziliense, and Uncinaria stenocephala. A mature female A. caninum will cause about 40 µl of blood to be lost by a dog each day; so, 25 worms means the loss of a ml of blood each day. Thus, 100 worms leads to the loss of 4 ml each day. A. braziliense causes only about 1 µl of blood loss per day or only about one tenth of an ml per 100 worms. U. stenocephala causes only about 0.3 µl of blood lost per worm a day; so just about one, one hundredth the blood lost from A. caninum. In cats, A. tubaeforme is the most dangerous of the worms. There have not been studies measuring the blood lost due to a single A. tubaeforme in cats, but it has been shown that around 200 hookworms will cause acute disease in a cat infected with this species. Trichuris vulpis Whipworms live in the cecum and colon. The worms live with the anterior end threaded through a cellular syncytium within the mucosa, while the posterior end is free within the lumen. The eggs leave the female from the vulva located at about the level where the body thickens behind the esophagus. The males have a coiled tail and a single spicule. The eggs will develop in the soil to contain infective first-­‐stage larvae in 9-­‐10 days at 33º-­‐38ºC and 25-­‐26 days at 19º-­‐25ºC. There are three potential life-­‐cycle scenarios after egg ingestion that have been described, and each has some experimental support: (1) larvae enter the mucosa of the small intestine for 8 to 10 days before reentering the lumen and moving to the cecum where they complete development; (2) larvae enter the mucosa of the small intestine and migrate within the mucosa to the cecum; or (3) larvae enter the mucosa of the cecum and remain there to maturity. The prepatent period is 70 to 100 days, and adult worms live months to years. In light infections, worms are usually found only in the cecum, and a few worms typically cause no noticeable signs. As the number of worms increases towards one or several hundred, the worms also are found in the mucosa of the colon. Disease, bloody diarrhea, dehydration, and anemia, occurs mainly in heavier infections Trichuris vulpis is on occasion stated to be a zoonotic infection, but the data is not convincing. Human whipworms (Trichuris trichiura) sometimes produce large eggs that are similar in size to Trichuris vulpis, and these abnormal eggs, when alongside the regular-­‐sized and smaller Trichuris trichiura eggs in a human fecal, can resemble the eggs of Trichuris vulpis. Adult worms have been recovered and examined from these patients and have been determined to be Trichuris trichiura. In dogs also, some Trichuris vulpis eggs are enlarged compared to regular eggs. Whipworms are common nationally in dogs from shelters of all ages. For dogs <6 months of age, the prevalence was 7.84%; 6-­‐12 months, 16.83%; 1-­‐3 years, 15.38%; 3-­‐7 years, 15.48%; and >7 years 11.74%. Adult dogs are about as susceptible to infection as younger dogs. Also, foxes and coyotes are both regular hosts for this parasite and act as reservoirs of canine infections. CONTROL We have excellent products that can now provide guaranteed protection to pets if they are administered on a regular monthly basis. Also, some products are now capable of also killing the stages developing within the animal rather than just the adults, thus, we may now be able to completely suppress egg production between the monthly product applications. Work currently underway by Dr. Blagburn at Auburn has shown that the levels currently being diagnosed in shelter animals is still not much different than it was in the first half of the 1990's. Thus, pets are still at risk, and these infections remain common in the unprotected pet population. It seems, however, that with the regular care of as 39 many pets as possible that we may be able to bring these parasites under control, at least, among those pets that routinely see their veterinarian. CATS Introduction This report will cover feline parasites, it will look at the more common feline hookworms and roundworms, but it will also touch upon tapeworms and lungworms. Also, it will examine the presence of heartworms in cats, which are considered typically of minimal importance in the western US. Finally touched upon will be fleas and also Cuterebra, which is potentially a greater problem for cats in the western US than in the eastern states. The Common Feline Roundworm, Toxocara cati Data from the maps on the www.capcvet.org website (using data from the laboratories of IDEXX, Antech, and Banfield Pet Hospitals) has data on roundworms. Nationally, the prevalence of T. cati eggs in the feces of cared-­‐for cats was 6.1% based on 780,000 samples (Table 1). For the 4 million dog samples, there were 2.9% containing the eggs of T. canis. Nationally, there were only 4 states, Alaska, Arizona, Nevada, and California where there was a lower percentage of cats infected than dogs. The southwest, highlighted on Table 1, was the lowest area for the infection of cats with roundworms, but it must be remembered that these results are for cats seeing veterinarians. Table 1. Toxocara cati – Fecal results from the CAPC website for 2010 Results of Fecal Examinations on 667,913 Cats around the United States Number Number. Percent Number Number Percent STATE samples positive Positive STATE Samples Positive Positive AK 1,368 25 1.8% MT 820 49 6.0% AL 2,919 163 5.6% NC 20,096 1,101 5.5% AR 555 42 7.6% ND 96 67 69.8% AZ 9,697 53 0.5% NE 1,996 129 6.5% CA 80,737 1,860 2.3% NH 14,519 967 6.7% CO 13,690 164 1.2% NJ 44,625 4,512 10.1% CT 30,593 2,755 9.0% NM 1,595 17 1.1% DC 1,224 45 3.7% NV 6,099 33 0.5% DE 4,617 312 6.8% NY 63,865 6,327 9.9% FL 51,389 890 1.7% OH 28,098 1,941 6.9% GA 17,559 926 5.3% OK 4,264 327 7.7% HI 2,024 70 3.5% OR 15,309 612 4.0% IA 2,263 192 8.5% PA 51,431 4,760 9.3% ID 510 30 5.9% RI 6,917 502 7.3% IL 23,527 1,998 8.5% SC 7,420 385 5.2% IN 17,952 1,098 6.1% SD 310 60 19.4% KS 3,506 203 5.8% TN 6,275 397 6.3% KY 5,406 263 4.9% TX 38,211 1,245 3.3% LA 2,669 88 3.3% UT 2,858 91 3.2% MA 47,538 3,152 6.6% VA 33,304 1,546 4.6% MD 34,842 2,452 7.0% VT 1,600 222 13.9% ME 2,328 252 10.8% WA 28,829 1,129 3.9% MI 14,511 1,630 11.2% WI 6,909 861 12.5% MN 10,045 683 6.8% WV 444 73 16.4% 40 MO MS 12,169 540 633 41 5.2% 7.6% WY Total 35 780,103 3 47,376 8.6% 6.1% What is often not realized is the effect that Toxocara cati has on the lungs of the infected cat. The majority of work done in this regard was published only in the form of a thesis submitted in 1969.1 In this work, it was presented in great detail that the experimental infection of cats with T. cati induced severe lesions, medial hypertrophy of the pulmonary vessels, in the lungs. It was concluded that these studies showed "that the lesion is produced earlier and is much more extensive than in feline lungworm. Aelurostrongylus abstrusus, infection. The earliest change noted in the arteries was mild intimal proliferation 2 weeks post infection, and medial hyperplasia was observed as early as 3 weeks and was severe by 6 weeks postinfection." It was felt that the observed lesions seen in random-­‐sourced cats was probably most commonly caused by T. cati. Also, the lesions did not clear in cats that had been infected, and it was considered that the induced lesions were irreversible. Also noted in these cats, like with A. abstrusus infections, was the occurrence of peribronchial mucus gland hyperplasia. A single egg inoculum caused the lesion, and repeated reinfection simply caused them to worsen and to persist for longer periods. Swerczek also reported on the disease induced in cats after their experimental infection with the eggs of the canine roundworm, Toxocara canis. About this infection, he stated that "the focal pneumonia associated with the migration of T. canis larvae was much more severe than that seen in T. cati infection." and "Muscular hyperplasia of the bronchioles and terminal alveolar ducts was also seen and was more severe in cats with evidence of sever ascarid infection." A cat was found naturally infected with larvae identified as those of T. canis with very large granulomas in the kidneys that also had significant lesions is the lungs similar to those described by Swerczek in his thesis.2 The lesions in the cat were similarly reproduced in cats that were experimentally infected with Toxocara canis, and these lesions also included severe lung disease with medial hypertrophy of the pulmonary vessels.3 Hookworms – Ancylostoma tubaeforme Hookworms are fairly rare in cats in the states west of Texas. It is difficult to understand why it remains so low in the coastal western states, because hookworms do very well along the eastern seaboard and around the Gulf of Mexico. California and other western coastal states should be concerned about the potential introduction of Ancylostoma braziliense. It seems to be restricted to the southeastern coastal areas of the United States, but its introduction into California could have significant impacts on beaches and to the tourist community. Aelurostrongylus abstrusus – the feline lungworm The feline lungworm of cats, Aelurostrongylus abstrusus, is a parasite that is often considered 41 Fecal results of 1322 cats from two shelters in upstate NY
rather rare and unusual. It would appear that this may not actually be the case. In a survey of two shelters from Cortland and Tompkins County New York, of the 1,322 samples that were examined from individual cats, 6.2% were found to contain larvae of A. abstrusus.4 In this work, it is expected that since the manner of diagnosis was centrifugal flotation with zinc sulfate flotation that the number of cases detected was actually lower than the true prevalence. The biology and disease caused by A. abstrusus has been fairly well described. Cats get infected with this lungworm by eating molluscan intermediate hosts common in most gardens (slugs or land-­‐
snail, e.g., Deroceras, Arion, Helicella) or various paratenic vertebrate hosts, such as amphibians, reptiles, birds, and small mammals. Larvae are shed in the feces of infected cats in slightly more than a month after the infection is initiated. The larvae are only infective to another host if they pass through a snail. The adult worms live in the terminal respiratory bronchioles and alveolar ducts. The disease is due to the presence of the worms and, probably even more so, to the eggs that are laid in the surrounding tissue in which the larvae develop and hatch. Cats with mild infections often have only minimal clinical signs, but heavily infected cats can present with severe bronchopneumonia and open-­‐
mouthed abdominal breathing. This worm seems restricted to felids, and does not pose any known risk to people. We know that we can use certain products off-­‐label to treat cats infected with A. abstrusus. Cats can be successfully treated with either a single treatment with moxidectin at the routine dose as in the monthly preventive for cats and by a three-­‐day course of fenbendazole.5 Thus, it would appear that Advantage Multi for cats may act as a preventive for this infection. We do not know if the dose in the heartworm products containing ivermectin, selamectin, or milbemycin oxime would be effective in treating and preventing feline infections with this lungworm. Diagnosis of lungworm infection in cats is not always easy. It is by finding the larvae in the feces. Methods used include direct smears, zinc-­‐sulfate or sugar flotation, and the use of a Baermann funnel method used to harvest the motile larvae from larger volumes of fresh feces. The direct smear and funnel method have the advantage that they provide the observer with actively motile larvae that aid in identification. The larvae obtained in zinc-­‐sulfate flotations are characterized by the dorsal spine on the tail. In sugar, the larvae may be hard to visualize, because they are crenated by the osmotic pressure of the sugar solution, but they can often still be recognized if the tail can be found. A. abstrusus is only one example of a good reason why it is necessary for pets that are on good all-­‐
year-­‐round health parasite prevention to have regular fecal exams. An indoor cat may be more likely to eat an infected mouse in the winter than the summer, so seasonality may not play a major role in when infections with this parasite appear. A cat that gets to go outdoors is always at risk of getting infection if it hunts. Feline Heartworm – Dirofilaria immitis in Cats is More Common than Expected Recent data compiled into maps on the CAPC website (www.capcvet.org) reveals that feline heartworm is much more common in cats around the nation than previously recognized (Table 2). The fact that 2.7% of the cats tested around the nation are antibody positive for heartworm is an indication that a fairly large percentage of cats entering veterinary clinics around the United States have been exposed to heartworms. That cats are infected with heartworms fairly regularly is supported by surveys examining shelter cats at necropsy for the presence of heartworms. (Ryan and Newcomb, 1995). If you look at the numbers from Arizona California, Colorado, New Mexico, Nevada, and Texas, you can see that these states are not the lowest prevalence regions – with the exception of Arizona, they are all at or about the national average of 2.7% (CO, is 2.6% , but close enough to 2.7%). 42 Table 2. Results of Heartworm Antibody Tests on 249,597 Cats around the United States Number Number. Percent Number Number Percent STATE samples positive Positive STATE Samples Positive Positive AK 108 3 2.8% MT 0 0 0.0% AL 3,638 178 4.9% NC 13,279 364 2.7% AR 58 1 1.7% ND 0 0 0.0% AZ 4540 84 1.9% NE 713 8 1.1% CA 27,943 823 2.9% NH 574 9 1.6% CO 6,657 170 2.6% NJ 8,299 221 2.7% CT 4,519 102 2.3% NM 1,445 50 3.5% DC 757 15 2.0% NV 1,916 53 2.8% DE 1,678 44 2.6% NY 11,398 299 2.6% FL 43,147 1,390 3.2% OH 4,514 59 1.3% GA 11,639 407 3.5% OK 2,311 59 2.6% HI 634 33 5.2% OR 3,389 47 1.4% IA 251 5 2.0% PA 10,214 180 1.8% ID 0 0 0.0% RI 747 19 2.5% IL 6,515 116 1.8% SC 5,304 161 3.0% IN 3,129 56 1.8% SD 0 0 0.0% KS 679 10 1.5% TN 4,745 221 4.7% KY 1,263 24 1.9% TX 17,567 535 3.0% LA 1,761 78 4.4% UT 793 11 1.4% MA 8,856 202 2.3% VA 9,729 210 2.2% MD 7,114 128 1.8% VT 373 21 5.6% ME 0 0 0.0% WA 3,628 45 1.2% MI 5,605 113 2.0% WI 510 6 1.2% MN 4,647 54 1.2% WV 0 0 0.0% MO 2,662 74 2.8% WY 0 0 0.0% MS 349 9 2.6% Total 249,597 6,697 2.7% We now also are aware that cats suffer from disease due to the presence of young adult heartworms in the heart even if they do not mature; the disease has come to be called HARD for Heartworm Associated Respiratory Disease.7 Results indicate that lesions and signs of are associated with death of pulmonary stages of D. immitis resemble those of other diseases such as feline asthma or other cause of tracheitis/bronchitis and interstitial lung disease. Thus, we now know that heartworm infections in cats even if the worms are not their long enough to stimulate an antigenemia in infected cats are still likely to induce disease in these cats. It should not be forgotten when considering feline heartworm disease that the worms that enter the heart of the cat after 70 to 100 days of development are 2 to 3 cm long when they arrive at the pulmonary arteries. Again, cats tend to over react to the presence of nematodes in their lungs with the indication of sever lung changes which in HARD are also associated with medial hypertrophy of the pulmonary vessels. Both these findings, the high number of cats seeing infective larvae and the present of HARD in cats, are further verification of the need to provide cats with heartworm prevention. This lung disease is preventable if the cats are on heartworm preventives. These preventives are also likely to protect cats from infections from pulmonary disease due to lungworms and roundworms. 43 Feline Tapeworms – Dipylidium caninum and Taenia taeniaeformis There are only two tapeworms in cats. In areas where the cat flea, Ctenocephalides felis, is rare, it can be expected that these parasite is also rare. It has been seen in cats in Utah,8 but no one has looked recently. Taenia taeniaeformis can persist wherever there are cats and rodents, and they seem to go together quite naturally. Surveys of for larvae in rodents have revealed the presence of T. taeniaeformis in Utah, northeastern California, and eastern New Mexico.8-­‐10 Tapeworms do cause disease in cats and have been the cause of string foreign body-­‐like disease.11 Treatment for tapeworms in cats is relative easy now with formulations of products containing praziquantel designed for oral, injectable, and topical delivery. Feline References 1. Swerczek, Thomas W. (1969) Medial hyperplasia of the pulmonary arteries of cats. PhD Dissertation, University of Connecticut, 199 pages 2. Parsons, J.C., et al. (1988) Disseminated granulomatous disease in a cat caused by larvae of Toxocara canis, J Comp Pathol 99: 343-­‐346 3. Parsons, J.C., et al. (1989) Pathological and haematological responses of cats experimentally infected with Toxocara canis larvae, Int J Parasitol 19: 479-­‐488 4. Lucio-­‐Forster, A., and D.D. Bowman (2011) Prevalence of fecal-­‐borne parasites detected by centrifugal flotation in feline samples from two shelters in upstate New York, J. Fel Surg Med 13: 300-­‐303 5. Traversa, D., et al. (2009) Efficacy and safety of imidacloprid 10%/moxidectin 1% spot-­‐on formulation in the treatment of feline aelurostrongylosis, Parasitol Res 105: 55-­‐62 6. Ryan, W. G., and K.M. Newcomb (1995) Prevalence of feline heartworm disease -­‐ a global review. Proceedings of the heartworm symposium '95, Auburn, Alabama, USA, 31 March-­‐2nd April, 1995, Pages: 79-­‐86 7. Blagburn, B. L. (2009) Canine and feline heartworm disease: what you need to know. Proceedings of the North American Veterinary Conference, Orlando, Florida, USA, 17-­‐21 January, 2009, Pages: 1164-­‐1167 8. Sawyer, T.W., et al. (1977) Helminth parasites of cats and dogs from central Utah, Great Basin Natur 36: 471-­‐474 9. Pfaffenberger, G.S., and D. de Bruin (1988) Parasites of the hispid cotton rat, Sigmodon hispidus (Cricetidae), and population biology of the cotton rat louse, Hoplopleura hirsuta (Hoplopleuridae: Anoplura), in eastern New Mexico, including an annotated host-­‐parasite bibliography, Tex J Sci 40: 369-­‐399. 10. Theis, J.H., and R.G. Schwab (1992) Seasonal prevalence of Taenia taeniaeformis: relationship to age, sex, reproduction and abundance of an intermediate host (Peromyscus maniculatus), J Wildl Dis 28: 42-­‐50 11. Wilcox, R.S., et al (2009) Intestinal obstruction caused by Taenia taeniaeformis infection in a cat, J Am An Hosp Assoc 45: 93-­‐96 44