Veterinary Parasitology 202 (2014) 319–325
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Use of ponazuril paste to treat coccidiosis in shelter-housed cats and dogs A.L. Litster a,∗ , J. Nichols a , K. Hall a , J. Camp b , A.S. Mohamed b,1 a Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA b Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 725 Harrison Street, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 18 February 2014 Accepted 1 March 2014
Keywords: Cat Coccidia Dog Ponazuril Shelter Cystoisospora spp.
a b s t r a c t Cystoisospora (synonym Isospora) spp. infections are common in dogs and cats worldwide, especially in crowded or unsanitary environments. Ponazuril (toltrazuril sulfone) is a widely used oral treatment, but protocols that will produce oocyst excretion below the detection limit in shelter-housed animals have not been determined. The aim of this study was to determine the efficacy of ponazuril paste at each of three dosages (dosage 1, 50 mg/kg q24 h for 3 days, dogs n = 14, cats n = 16; dosage 2, 50 mg/kg as a single dose, dogs n = 13, cats n = 25; or dosage 3, 20 mg/kg as a single dose, dogs n = 16, cats n = 23) in shelter-housed dogs (n = 43) and cats (n = 64) with confirmed coccidiosis. Fecal oocyst counts and identification and fecal consistency scoring was performed pre-treatment (Day 1) and again at Day 3–4 and Day 8. There were higher proportions of animals with oocyst excretion below the detection limit at both Day 3–4 and Day 8 in the dosage 1 group (dogs 92.9%, cats 87.5%) than in the other two groups (dosage 2, dogs 76.9%, cats 80.0%; dosage 3, dogs 68.8%, cats 47.8%). Animals with high fecal oocyst counts at Day 1 were significantly more likely to be infected at Day 3–4 (dogs, P < 0.001; cats, P = 0.013). Fecal consistency score at Day 3–4 was not significantly related to infection status (dogs, P = 0.898; cats, P = 0.136). Further studies are warranted to investigate a ponazuril protocol that can safely reduce fecal oocyst burdens in infected dogs and cats to levels below the detection limit. Environmental decontamination is also important to reduce the likelihood of re-infection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Coccidia are obligate intracellular protozoan parasites commonly found in the gastrointestinal tract of dogs and cats (Lappin, 2005; Dubey et al., 2009; Gates and Nolan, 2009; Lucio-Forster and Bowman, 2011). The most
∗ Corresponding author. Tel.: +1 765 4183186; fax: +1 765 4961108. E-mail address: [email protected] (A.L. Litster). 1 Current address: Department of Public Health, Maricopa County, Phoenix, AZ 85012, USA. http://dx.doi.org/10.1016/j.vetpar.2014.03.003 0304-4017/© 2014 Elsevier B.V. All rights reserved.
commonly diagnosed infections in dogs are Cystoisospora (synonym Isospora) canis and Cystoisospora ohioensiscomplex (C. ohioensis + Cystoisospora burrowsi), while Cystoisospora felis and Cystoisospora rivolta predominate in the cat (Gates and Nolan, 2009). Transmission occurs via the fecal–oral route or by ingestion of the infected tissues of a transport host (e.g. mice, rats, hamsters and other vertebrates; Companion Animal Parasite Council, 2014), and infection rates are higher in crowded and/or unsanitary environments (Lappin, 2005). Coccidiosis is typically a disease of puppies and kittens 1 year of
320
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
age (Gates and Nolan, 2009). In most cases, diarrhea, if present, is self-limiting or rapidly responsive to treatment for coccidiosis (Dubey et al., 2009), but the presence of enteric protozoans in diarrheic stool does not prove a causal association, and reinfection with Cystoisospora spp. is common; therefore an apparent lack of response to therapy should be a prompt for further evaluation (Lappin, 2005). Large- or mixed-bowel diarrhea and abdominal discomfort are sometimes the result of intestinal cell destruction by Cystoisospora spp. (Lappin, 2005) and in young and immunocompromised animals, severe clinical signs, such as watery or hemorrhagic diarrhea, can occur, leading to anemia and severe dehydration if left untreated (Altreuther et al., 2011; Petry et al., 2011). Sulfadimethoxine is the only drug that has been approved for the treatment of coccidiosis in dogs and cats (Companion Animal Parasite Council, 2014), but since sulfonamides are coccidiostatic, a low level of persistent infection is possible after treatment (Dubey et al., 2009). Trimethoprim sulfonamide, furazolidone and amprolium are also commonly used drugs (Lappin, 2005). Ponazuril (toltrazuril sulfone) is currently the treatment used by many clinicians for Cystoisospora spp. infections in small animals and is available in the USA in paste form (Marquis® paste, Bayer Animal Health) as a treatment for Sarcocystis neurona infection in horses (Dubey et al., 2009). Its mechanism of action remains unknown (Wise et al., 2012), but it has been shown to interfere with normal parasite division (Mitchell et al., 2003). Toltrazuril is known to act on the respiratory chain and pyrimidine pathways (Harder and Haberkorn, 1989). Various protocols for toltrazuril have been tested under experimental and field conditions for canine and feline Cystoisospora spp. infections (Daugschies et al., 2000; Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007; Dubey et al., 2009; Altreuther et al., 2011; Petry et al., 2011). The drug appears to be well tolerated even in very young animals (Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007), but depending on the protocol used, infection and oocyst shedding might not be completely eliminated and repeated re-infection can occur (Dubey et al., 2009). Sporulated Cystoisospora spp. oocysts are extremely resistant to routine disinfection protocols, so additional measures, such as control of transport hosts, thorough environmental cleaning with either 10% ammonia left in contact with contaminated surfaces for at least 10 min or with steam, treatment of all animals, and prophylactic treatment of incoming animals, might be required in shelters, kennels or catteries, or where there are recurrent problems (Dubey et al., 2009). The aim of this study was to determine the efficacy of treatment with ponazuril paste at each of three dosages in shelter-housed dogs and cats with confirmed coccidiosis. 2. Materials and methods Dogs and cats were recruited for the study from PAWS Chicago, a large adoption-guarantee animal shelter in inner-city Chicago. On the day of arrival, all animals underwent a full physical examination by a shelter veterinarian. Vaccination was performed with core vaccines (Richards et al., 2006; American Animal Hospital Association (AAHA)
Canine Vaccination Task Force, 2011) and the animals were prophylactically dewormed using pyrantel pamoate (Strongid-T® , Zoetis) and praziquantel (compounded) at standard dosages (Plumb, 2011). Selamectin (Revolution® , Zoetis) was administered topically as a prophylactic measure at shelter intake. Feces were collected on Day 1 (pre-treatment) and consistency scored using an illustrated scoring chart (Nestlé-Purina fecal scoring system, 2007). Specimens were then centrifuged with zinc sulfate and examined microscopically for evidence of intestinal parasites. All animals with fecal specimens containing coccidia oocysts were provisionally enrolled and feces from the same specimen were sent to the Purdue University College of Veterinary Medicine Diagnostic Parasitology Laboratory (PVMDPL), where standard qualitative flotation, zinc sulfate flotation and quantitative flotation were performed by technicians masked to the provisional infection status of the specimens. A centrifugal fecal flotation procedure with zinc sulfate solution was used for the qualitative determination of oocyst presence and the Modified McMaster’s test was used for the quantitative procedure (Zajac and Conboy, 2012). The sensitivity of the Modified McMaster’s was 100 oocysts/g of feces. Enrollment was confirmed once coccidiosis was identified by the PVMDPL personnel. Animals with fecal samples containing coccidia oocysts at provisional enrollment were bathed once on Day 1 using shampoo and water to help reduce the risk of reinfection. On Day 1, after provisional enrollment, animals were allocated to one of three treatment groups using oral ponazuril in paste form (Marquis® paste, Bayer Animal Health) – 50 mg/kg q24 h for 3 days (dosage 1), 50 mg/kg as a single dose (dosage 2), or 20 mg/kg as a single dose (dosage 3). Treatment allocation was performed at provisional enrollment from a list of predetermined numbers and was unrelated to fecal parameters or any attributes of the animals enrolled. Because we were particularly interested in investigating the efficacy of dosage 2 and dosage 3 for the treatment of infected cats so that shelter costs could be minimized, the list of predetermined numbers allowed for more cats to be allocated to these two groups. After provisional enrollment, including ponazuril treatment and bathing, animals were either individually housed (dogs – n = 21; cats – n = 52) or were co-housed (dogs – n = 21 co-housed; cats – n = 13) at PAWS Chicago, or were placed individually with foster families for the duration of the study (dogs – n = 1; cats – n = 2). If animals were cohoused, individual fecal specimens were collected on Day 1, before co-housing commenced. At Day 3–4 and Day 8, the identification of individual fecal specimens was made possible by the oral administration of colored non-toxic plastic glitter in gelatin capsules 24–48 h before fecal collection (Griffin, 2002). Follow up fecal specimens were collected at Day 3–4 and at Day 8. These were scored and sent to the PVMDPL for analysis as per Day 1. Technicians performing fecal analysis were unaware of the provisional or previous infection status of specimens and treatment allocation. Animals that had coccidia oocysts in their feces at Day 3–4 were divided into two groups – those that received follow-up treatment with dosage 1 commencing on Day 3–4 and those that received no further treatment. Allocation to these two
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
321
Table 1 Ages and body weights of dogs and cats with coccidiosis and fecal oocyst count (oocysts/g) and fecal scores at enrolment (Day 1–pre-treatment). Dogs (n = 43)
Median age in months (range) Median bodyweight in kg (range)a Fecal scoreb (1–7/7; range)c
Cats (n = 64)
Dosage 1 (n = 14)
Dosage 2 (n = 13)
Dosage 3 (n = 16)
Dosage 1 (n = 16)
Dosage 2 (n = 25)
Dosage 3 (n = 23)
2.0 (2.0–36.0)
4.0 (2.0–48.0)
3.0 (2.0–36.0)
12.0 (2–121)
4.0 (1.5–60.0)
3.5 (2.0–24.0)
2.0 (1.5–15.0)
6.2 (3.6–23.0)
6.7 (1.2–25.0)
2.9 (0.5–6.3)
1.6 (0.6–6.2)
1.5 (0.4–3.8)
5.0 (3–6)
4.0 (3–6)
4.5 (3–6)
3.5 (2–7)
4.0 (2–6)
4.0 (3–6)
Dosage 1 – 50 mg/kg q24 h for 3 days; dosage 2 – 50 mg/kg as a single dose; dosage 3 – 20 mg/kg as a single dose. a Median bodyweight at Day 1 was lower in dogs from the dosage 1 group than in dogs from the dosage 2 or 3 groups (P = 0.003 and P = 0.015, respectively). b Nestlé-Purina Fecal Scoring System, where 1/7 denotes very dry hard feces and 7/7 denotes watery feces. c Fecal score at Day 1 was higher in dogs from the dosage 1 group than in dogs from the dosage 2 or 3 groups (P = 0.018 and P = 0.014, respectively).
groups depended on the availability of shelter resources. If housing was available, animals that were oocyst-positive at Day 3–4 received no further treatment to determine if the Day 8 fecal specimens would eventually have oocyst counts below the limit of detection. However, if the shelter housing resources were limited, animals that were oocystpositive at Day 3–4 were treated with dosage 1 so that the chances of parasite clearance were maximized and the animal could be moved into a foster home or adoptive home. Statistical comparisons among groups at Day 1 on age, body weight, fecal oocyst count and fecal consistency scores were made using Mann–Whitney tests. Mann–Whitney tests were also used to compare Day 1 fecal oocyst counts and Day 3–4 fecal consistency scores among dogs and cats that were either infected or noninfected at Day 3–4. 2 test or Fisher’s exact tests, where appropriate, were used to compare the proportions of individually housed or co-housed dogs and cats that were either infected or non-infected at Day 3–4. A commercially available software program was used for statistical calculations (GraphPad Prism 5.0). 3. Results Table 1 shows ages (months) and body weights (kg), fecal oocyst counts (oocysts/g) and fecal consistency scores (1–7/7) at Day 1. At Day 1, dogs from the dosage 1 group weighed less and had a higher fecal consistency score than those in the other two groups (bodyweight P = 0.003 and P = 0.015; fecal consistency score P = 0.018 and P = 0.014; comparisons with dosage 2 and 3 groups, respectively). Dogs from the dosage 1 group also had lower fecal oocyst counts than those from dosage 3 at enrollment (P = 0.009). There were no statistically significant differences among cat groups on any of the variables reported at Day 1. No adverse events were observed that were thought to be related to treatment with ponazuril. Fecal flotation results for each of the groups throughout the study period are summarized in Table 2. Dogs and cats that were coccidia oocyst-positive at Day 3–4 had significantly higher Day 1 fecal oocyst counts than those that were coccidia oocyst-negative at Day 3–4 (dogs, P < 0.001; cats, P = 0.013; Table 3). There were no significant differences in Day 1 age (dogs, P = 0.974; cats, P = 0.845), or bodyweight (dogs, P = 0.935; cats, P = 0.629), or Day 3–4 fecal consistency scores (dogs, P = 0.898; cats, P = 0.136), when animals
that were coccidia oocyst-positive at Day 3–4 were compared with those that were coccidia oocyst-negative at Day 3–4. Fecal consistency score at Day 1 was not significantly different in dogs that were oocyst-positive at Day 3–4 compared with those that were oocyst-negative at Day 3–4 (P = 0.848), but was statistically lower in cats that were oocyst-positive at Day 3–4 (median 3, range 2–6) than in those that were oocyst-negative at Day 3–4 (median 4, range 2–7; P = 0.002). Dogs and cats that were co-housed or foster-housed rather than individually housed during the study period were not more likely to have oocysts in their feces at Day 3–4 (dogs P = 0.41; cats P = 0.49). Information on species identified at each fecal test in animals that were oocyst-positive at Day 3–4 and/or Day 8 is shown in Table 4. 4. Discussion This study demonstrated that in shelter dogs and cats naturally infected with coccidiosis, ponazuril administered at dosages up to 50 mg/kg q24 h for 3 consecutive days (dosage 1) was not always enough to reduce fecal oocyst counts to levels below the detection limit by 3–4 days after the initiation of treatment. This was a surprising finding, since it is a higher dosage than that used in previous studies (Daugschies et al., 2000; Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007; Altreuther et al., 2011; Petry et al., 2011), but some of those studies reported experimental infections (Daugschies et al., 2000; Reinemeyer et al., 2007; Altreuther et al., 2011; Petry et al., 2011) and in some cases the outcome measure was a significant reduction in fecal oocyst count rather than oocyst excretion below the detection limit. In our study, for both cats and dogs, the proportion of animals that did not have oocysts on fecal flotation at both Day 3–4 and Day 8 increased as the dosage of ponazuril increased (Table 2). In five dogs and seven cats that were still infected at Day 3–4, follow up treatment with dosage 1 cleared the infection, but this was not the case for three of the cats. Treatment efficacy was also related to fecal oocyst count at Day 1, and animals with a high initial oocyst count were statistically more likely to remain infected on Day 3–4. It is therefore surprising that the efficacy of dosage 1 compared to the other two treatment groups appeared similar for both dogs and cats (Table 2), since Day 1 fecal oocyst
322
Table 2 Fecal flotation results from dogs and cats with coccidiosis and treated with ponazuril at one of three different dosage regimens. Dogs (n = 43) Dosage 1 (n = 14)
Dosage 2 (n = 13)
Dosage 3 (n = 16)
Dosage 1 (n = 16)
Dosage 2 (n = 25)
Dosage 3 (n = 23)
200 (100–3700)
400 (100–25400)
1100 (100–115000)
850 (100–62800)
200 (100–11600)
400 (100–43700)
13 (92.9)
10 (76.9)
11 (68.8)
14 (87.5)
20 (80.0)
11 (47.8)
1 (7.1) – Neg at Day 8
1 (7.1) – Neg at Day 8
3 (18.8) – All neg at Day 8
2 (12.5) – Both neg at Day 8
2 (8.0) – Both neg at Day 8
6 (26.1) – 3 neg and 3 pos at Day 8
0 (0.0)
0 (0.0)
2 (12.5) – Both pos at Day 8
0 (0.0)
1 (4.0) – Neg at Day 8
4, (17.4) – 2 neg and 2 pos at Day 8
0 (0.0)
2 (14.3)
0 (0.0)
0 (0.0)
2 (8.0)
2 (8.7)
C. ohioensis-complex (3), C. canis (8), C. ohioensis-complex and C. canis (3)
C. ohioensis-complex (5), C. canis (4), C. ohioensis-complex and C. canis (4)
C. ohioensis-complex (9), C. canis (4), C. ohioensis-complex and C. canis (3)
C. rivolta (5), C. felis (8), C. rivolta and C. felis (3)
C. rivolta (6), C. felis (11), C. rivolta and C. felis (8)
C. rivolta (6), C. felis (12), C. rivolta and C. felis (5)
neg, negative; pos, positive; FFl, fecal flotation. Dosage 1 – 50 mg/kg q24 h for 3 days; dosage 2 – 50 mg/kg as a single dose; dosage 3 – 20 mg/kg as a single dose. a Fecal oocyst burden at Day 1 was lower in dogs from the dosage 1 group than in dogs from the dosage 3 group (P = 0.009).
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
Fecal oocyst count in oocysts/g at Day 1 (range)a FFl oocyst negative at Day 3–4 and at Day 8; n (%) FFl oocyst positive at Day 3–4; repeat treatment with Dosage 1 from Day 3–4; n (%) FFl oocyst positive at Day 3–4; no further treatment; n (%) FFl oocyst negative at Day 3–4; FFl positive at Day 8; n (%) Coccidia species identified at Day 1 (n)
Cats (n = 64)
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
323
Table 3 Comparison of median fecal oocyst counts at Day 1 in animals that were coccidia oocyst-positive at Day 3–4 and coccidia oocyst-negative at Day 3–4.
Dogs – oocyst-negative at Day 3–4 Dogs – oocyst-positive at Day 3–4 Cats – oocyst-negative at Day 3–4 Cats – oocyst-positive at Day 3–4
Median fecal oocyst count at Day 1 (oocysts/g)
n (%)
300 4100 200 1500
36/43 (83.7) 7/43 (16.3) 49/64 (76.6) 15/64 (23.4)
counts were statistically lower in dogs from the dosage 1 group (Table 1). Future studies should aim to determine the minimum dosage of ponazuril necessary to reduce oocyst excretion to below the detection limit, either by increasing the dose rate or by continuing treatment for a longer period. The balance between the cost of treatment and the length of the course of treatment is an important consideration in busy shelters with limited resources. Another interesting aspect of the results is detailed in Table 4, which shows that six animals (Cats G and H; Dogs A–D) had different species identified at Day 1 and Day 3–4. It is possible that the species identified at Day 3–4 were from newly acquired infections from environmental contamination at the shelter, although perhaps the species identified at Day 3–4 were also present at Day 1, but oocyst excretion was below the detection limit. Cat H was infected with C. rivolta at Day 1, C. felis at Day 3–4 and both species at Day 8, but in the remaining five animals, fecal testing was oocyst-negative at Day 8. It is possible that these Day 8 results were the result of spontaneous cessation of oocyst excretion (Lappin, 2005) rather than a treatment-related effect. Additionally, two cats (P and Q) tested oocyst-negative at Day 3–4, but were oocystpositive at Day 8 (Table 4). It is possible that this was the result of a temporary coccidiostatic effect of ponazuril at Day 3–4. Cystoisospora spp. oocysts remain infective for up to 1 year in moist, protected environments unless exposed to freezing or very high temperatures (Companion Animal
P
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Use of ponazuril paste to treat coccidiosis in shelter-housed cats and dogs A.L. Litster a,∗ , J. Nichols a , K. Hall a , J. Camp b , A.S. Mohamed b,1 a Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA b Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 725 Harrison Street, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 18 February 2014 Accepted 1 March 2014
Keywords: Cat Coccidia Dog Ponazuril Shelter Cystoisospora spp.
a b s t r a c t Cystoisospora (synonym Isospora) spp. infections are common in dogs and cats worldwide, especially in crowded or unsanitary environments. Ponazuril (toltrazuril sulfone) is a widely used oral treatment, but protocols that will produce oocyst excretion below the detection limit in shelter-housed animals have not been determined. The aim of this study was to determine the efficacy of ponazuril paste at each of three dosages (dosage 1, 50 mg/kg q24 h for 3 days, dogs n = 14, cats n = 16; dosage 2, 50 mg/kg as a single dose, dogs n = 13, cats n = 25; or dosage 3, 20 mg/kg as a single dose, dogs n = 16, cats n = 23) in shelter-housed dogs (n = 43) and cats (n = 64) with confirmed coccidiosis. Fecal oocyst counts and identification and fecal consistency scoring was performed pre-treatment (Day 1) and again at Day 3–4 and Day 8. There were higher proportions of animals with oocyst excretion below the detection limit at both Day 3–4 and Day 8 in the dosage 1 group (dogs 92.9%, cats 87.5%) than in the other two groups (dosage 2, dogs 76.9%, cats 80.0%; dosage 3, dogs 68.8%, cats 47.8%). Animals with high fecal oocyst counts at Day 1 were significantly more likely to be infected at Day 3–4 (dogs, P < 0.001; cats, P = 0.013). Fecal consistency score at Day 3–4 was not significantly related to infection status (dogs, P = 0.898; cats, P = 0.136). Further studies are warranted to investigate a ponazuril protocol that can safely reduce fecal oocyst burdens in infected dogs and cats to levels below the detection limit. Environmental decontamination is also important to reduce the likelihood of re-infection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Coccidia are obligate intracellular protozoan parasites commonly found in the gastrointestinal tract of dogs and cats (Lappin, 2005; Dubey et al., 2009; Gates and Nolan, 2009; Lucio-Forster and Bowman, 2011). The most
∗ Corresponding author. Tel.: +1 765 4183186; fax: +1 765 4961108. E-mail address: [email protected] (A.L. Litster). 1 Current address: Department of Public Health, Maricopa County, Phoenix, AZ 85012, USA. http://dx.doi.org/10.1016/j.vetpar.2014.03.003 0304-4017/© 2014 Elsevier B.V. All rights reserved.
commonly diagnosed infections in dogs are Cystoisospora (synonym Isospora) canis and Cystoisospora ohioensiscomplex (C. ohioensis + Cystoisospora burrowsi), while Cystoisospora felis and Cystoisospora rivolta predominate in the cat (Gates and Nolan, 2009). Transmission occurs via the fecal–oral route or by ingestion of the infected tissues of a transport host (e.g. mice, rats, hamsters and other vertebrates; Companion Animal Parasite Council, 2014), and infection rates are higher in crowded and/or unsanitary environments (Lappin, 2005). Coccidiosis is typically a disease of puppies and kittens 1 year of
320
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
age (Gates and Nolan, 2009). In most cases, diarrhea, if present, is self-limiting or rapidly responsive to treatment for coccidiosis (Dubey et al., 2009), but the presence of enteric protozoans in diarrheic stool does not prove a causal association, and reinfection with Cystoisospora spp. is common; therefore an apparent lack of response to therapy should be a prompt for further evaluation (Lappin, 2005). Large- or mixed-bowel diarrhea and abdominal discomfort are sometimes the result of intestinal cell destruction by Cystoisospora spp. (Lappin, 2005) and in young and immunocompromised animals, severe clinical signs, such as watery or hemorrhagic diarrhea, can occur, leading to anemia and severe dehydration if left untreated (Altreuther et al., 2011; Petry et al., 2011). Sulfadimethoxine is the only drug that has been approved for the treatment of coccidiosis in dogs and cats (Companion Animal Parasite Council, 2014), but since sulfonamides are coccidiostatic, a low level of persistent infection is possible after treatment (Dubey et al., 2009). Trimethoprim sulfonamide, furazolidone and amprolium are also commonly used drugs (Lappin, 2005). Ponazuril (toltrazuril sulfone) is currently the treatment used by many clinicians for Cystoisospora spp. infections in small animals and is available in the USA in paste form (Marquis® paste, Bayer Animal Health) as a treatment for Sarcocystis neurona infection in horses (Dubey et al., 2009). Its mechanism of action remains unknown (Wise et al., 2012), but it has been shown to interfere with normal parasite division (Mitchell et al., 2003). Toltrazuril is known to act on the respiratory chain and pyrimidine pathways (Harder and Haberkorn, 1989). Various protocols for toltrazuril have been tested under experimental and field conditions for canine and feline Cystoisospora spp. infections (Daugschies et al., 2000; Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007; Dubey et al., 2009; Altreuther et al., 2011; Petry et al., 2011). The drug appears to be well tolerated even in very young animals (Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007), but depending on the protocol used, infection and oocyst shedding might not be completely eliminated and repeated re-infection can occur (Dubey et al., 2009). Sporulated Cystoisospora spp. oocysts are extremely resistant to routine disinfection protocols, so additional measures, such as control of transport hosts, thorough environmental cleaning with either 10% ammonia left in contact with contaminated surfaces for at least 10 min or with steam, treatment of all animals, and prophylactic treatment of incoming animals, might be required in shelters, kennels or catteries, or where there are recurrent problems (Dubey et al., 2009). The aim of this study was to determine the efficacy of treatment with ponazuril paste at each of three dosages in shelter-housed dogs and cats with confirmed coccidiosis. 2. Materials and methods Dogs and cats were recruited for the study from PAWS Chicago, a large adoption-guarantee animal shelter in inner-city Chicago. On the day of arrival, all animals underwent a full physical examination by a shelter veterinarian. Vaccination was performed with core vaccines (Richards et al., 2006; American Animal Hospital Association (AAHA)
Canine Vaccination Task Force, 2011) and the animals were prophylactically dewormed using pyrantel pamoate (Strongid-T® , Zoetis) and praziquantel (compounded) at standard dosages (Plumb, 2011). Selamectin (Revolution® , Zoetis) was administered topically as a prophylactic measure at shelter intake. Feces were collected on Day 1 (pre-treatment) and consistency scored using an illustrated scoring chart (Nestlé-Purina fecal scoring system, 2007). Specimens were then centrifuged with zinc sulfate and examined microscopically for evidence of intestinal parasites. All animals with fecal specimens containing coccidia oocysts were provisionally enrolled and feces from the same specimen were sent to the Purdue University College of Veterinary Medicine Diagnostic Parasitology Laboratory (PVMDPL), where standard qualitative flotation, zinc sulfate flotation and quantitative flotation were performed by technicians masked to the provisional infection status of the specimens. A centrifugal fecal flotation procedure with zinc sulfate solution was used for the qualitative determination of oocyst presence and the Modified McMaster’s test was used for the quantitative procedure (Zajac and Conboy, 2012). The sensitivity of the Modified McMaster’s was 100 oocysts/g of feces. Enrollment was confirmed once coccidiosis was identified by the PVMDPL personnel. Animals with fecal samples containing coccidia oocysts at provisional enrollment were bathed once on Day 1 using shampoo and water to help reduce the risk of reinfection. On Day 1, after provisional enrollment, animals were allocated to one of three treatment groups using oral ponazuril in paste form (Marquis® paste, Bayer Animal Health) – 50 mg/kg q24 h for 3 days (dosage 1), 50 mg/kg as a single dose (dosage 2), or 20 mg/kg as a single dose (dosage 3). Treatment allocation was performed at provisional enrollment from a list of predetermined numbers and was unrelated to fecal parameters or any attributes of the animals enrolled. Because we were particularly interested in investigating the efficacy of dosage 2 and dosage 3 for the treatment of infected cats so that shelter costs could be minimized, the list of predetermined numbers allowed for more cats to be allocated to these two groups. After provisional enrollment, including ponazuril treatment and bathing, animals were either individually housed (dogs – n = 21; cats – n = 52) or were co-housed (dogs – n = 21 co-housed; cats – n = 13) at PAWS Chicago, or were placed individually with foster families for the duration of the study (dogs – n = 1; cats – n = 2). If animals were cohoused, individual fecal specimens were collected on Day 1, before co-housing commenced. At Day 3–4 and Day 8, the identification of individual fecal specimens was made possible by the oral administration of colored non-toxic plastic glitter in gelatin capsules 24–48 h before fecal collection (Griffin, 2002). Follow up fecal specimens were collected at Day 3–4 and at Day 8. These were scored and sent to the PVMDPL for analysis as per Day 1. Technicians performing fecal analysis were unaware of the provisional or previous infection status of specimens and treatment allocation. Animals that had coccidia oocysts in their feces at Day 3–4 were divided into two groups – those that received follow-up treatment with dosage 1 commencing on Day 3–4 and those that received no further treatment. Allocation to these two
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
321
Table 1 Ages and body weights of dogs and cats with coccidiosis and fecal oocyst count (oocysts/g) and fecal scores at enrolment (Day 1–pre-treatment). Dogs (n = 43)
Median age in months (range) Median bodyweight in kg (range)a Fecal scoreb (1–7/7; range)c
Cats (n = 64)
Dosage 1 (n = 14)
Dosage 2 (n = 13)
Dosage 3 (n = 16)
Dosage 1 (n = 16)
Dosage 2 (n = 25)
Dosage 3 (n = 23)
2.0 (2.0–36.0)
4.0 (2.0–48.0)
3.0 (2.0–36.0)
12.0 (2–121)
4.0 (1.5–60.0)
3.5 (2.0–24.0)
2.0 (1.5–15.0)
6.2 (3.6–23.0)
6.7 (1.2–25.0)
2.9 (0.5–6.3)
1.6 (0.6–6.2)
1.5 (0.4–3.8)
5.0 (3–6)
4.0 (3–6)
4.5 (3–6)
3.5 (2–7)
4.0 (2–6)
4.0 (3–6)
Dosage 1 – 50 mg/kg q24 h for 3 days; dosage 2 – 50 mg/kg as a single dose; dosage 3 – 20 mg/kg as a single dose. a Median bodyweight at Day 1 was lower in dogs from the dosage 1 group than in dogs from the dosage 2 or 3 groups (P = 0.003 and P = 0.015, respectively). b Nestlé-Purina Fecal Scoring System, where 1/7 denotes very dry hard feces and 7/7 denotes watery feces. c Fecal score at Day 1 was higher in dogs from the dosage 1 group than in dogs from the dosage 2 or 3 groups (P = 0.018 and P = 0.014, respectively).
groups depended on the availability of shelter resources. If housing was available, animals that were oocyst-positive at Day 3–4 received no further treatment to determine if the Day 8 fecal specimens would eventually have oocyst counts below the limit of detection. However, if the shelter housing resources were limited, animals that were oocystpositive at Day 3–4 were treated with dosage 1 so that the chances of parasite clearance were maximized and the animal could be moved into a foster home or adoptive home. Statistical comparisons among groups at Day 1 on age, body weight, fecal oocyst count and fecal consistency scores were made using Mann–Whitney tests. Mann–Whitney tests were also used to compare Day 1 fecal oocyst counts and Day 3–4 fecal consistency scores among dogs and cats that were either infected or noninfected at Day 3–4. 2 test or Fisher’s exact tests, where appropriate, were used to compare the proportions of individually housed or co-housed dogs and cats that were either infected or non-infected at Day 3–4. A commercially available software program was used for statistical calculations (GraphPad Prism 5.0). 3. Results Table 1 shows ages (months) and body weights (kg), fecal oocyst counts (oocysts/g) and fecal consistency scores (1–7/7) at Day 1. At Day 1, dogs from the dosage 1 group weighed less and had a higher fecal consistency score than those in the other two groups (bodyweight P = 0.003 and P = 0.015; fecal consistency score P = 0.018 and P = 0.014; comparisons with dosage 2 and 3 groups, respectively). Dogs from the dosage 1 group also had lower fecal oocyst counts than those from dosage 3 at enrollment (P = 0.009). There were no statistically significant differences among cat groups on any of the variables reported at Day 1. No adverse events were observed that were thought to be related to treatment with ponazuril. Fecal flotation results for each of the groups throughout the study period are summarized in Table 2. Dogs and cats that were coccidia oocyst-positive at Day 3–4 had significantly higher Day 1 fecal oocyst counts than those that were coccidia oocyst-negative at Day 3–4 (dogs, P < 0.001; cats, P = 0.013; Table 3). There were no significant differences in Day 1 age (dogs, P = 0.974; cats, P = 0.845), or bodyweight (dogs, P = 0.935; cats, P = 0.629), or Day 3–4 fecal consistency scores (dogs, P = 0.898; cats, P = 0.136), when animals
that were coccidia oocyst-positive at Day 3–4 were compared with those that were coccidia oocyst-negative at Day 3–4. Fecal consistency score at Day 1 was not significantly different in dogs that were oocyst-positive at Day 3–4 compared with those that were oocyst-negative at Day 3–4 (P = 0.848), but was statistically lower in cats that were oocyst-positive at Day 3–4 (median 3, range 2–6) than in those that were oocyst-negative at Day 3–4 (median 4, range 2–7; P = 0.002). Dogs and cats that were co-housed or foster-housed rather than individually housed during the study period were not more likely to have oocysts in their feces at Day 3–4 (dogs P = 0.41; cats P = 0.49). Information on species identified at each fecal test in animals that were oocyst-positive at Day 3–4 and/or Day 8 is shown in Table 4. 4. Discussion This study demonstrated that in shelter dogs and cats naturally infected with coccidiosis, ponazuril administered at dosages up to 50 mg/kg q24 h for 3 consecutive days (dosage 1) was not always enough to reduce fecal oocyst counts to levels below the detection limit by 3–4 days after the initiation of treatment. This was a surprising finding, since it is a higher dosage than that used in previous studies (Daugschies et al., 2000; Lloyd and Smith, 2001; Charles et al., 2007; Reinemeyer et al., 2007; Altreuther et al., 2011; Petry et al., 2011), but some of those studies reported experimental infections (Daugschies et al., 2000; Reinemeyer et al., 2007; Altreuther et al., 2011; Petry et al., 2011) and in some cases the outcome measure was a significant reduction in fecal oocyst count rather than oocyst excretion below the detection limit. In our study, for both cats and dogs, the proportion of animals that did not have oocysts on fecal flotation at both Day 3–4 and Day 8 increased as the dosage of ponazuril increased (Table 2). In five dogs and seven cats that were still infected at Day 3–4, follow up treatment with dosage 1 cleared the infection, but this was not the case for three of the cats. Treatment efficacy was also related to fecal oocyst count at Day 1, and animals with a high initial oocyst count were statistically more likely to remain infected on Day 3–4. It is therefore surprising that the efficacy of dosage 1 compared to the other two treatment groups appeared similar for both dogs and cats (Table 2), since Day 1 fecal oocyst
322
Table 2 Fecal flotation results from dogs and cats with coccidiosis and treated with ponazuril at one of three different dosage regimens. Dogs (n = 43) Dosage 1 (n = 14)
Dosage 2 (n = 13)
Dosage 3 (n = 16)
Dosage 1 (n = 16)
Dosage 2 (n = 25)
Dosage 3 (n = 23)
200 (100–3700)
400 (100–25400)
1100 (100–115000)
850 (100–62800)
200 (100–11600)
400 (100–43700)
13 (92.9)
10 (76.9)
11 (68.8)
14 (87.5)
20 (80.0)
11 (47.8)
1 (7.1) – Neg at Day 8
1 (7.1) – Neg at Day 8
3 (18.8) – All neg at Day 8
2 (12.5) – Both neg at Day 8
2 (8.0) – Both neg at Day 8
6 (26.1) – 3 neg and 3 pos at Day 8
0 (0.0)
0 (0.0)
2 (12.5) – Both pos at Day 8
0 (0.0)
1 (4.0) – Neg at Day 8
4, (17.4) – 2 neg and 2 pos at Day 8
0 (0.0)
2 (14.3)
0 (0.0)
0 (0.0)
2 (8.0)
2 (8.7)
C. ohioensis-complex (3), C. canis (8), C. ohioensis-complex and C. canis (3)
C. ohioensis-complex (5), C. canis (4), C. ohioensis-complex and C. canis (4)
C. ohioensis-complex (9), C. canis (4), C. ohioensis-complex and C. canis (3)
C. rivolta (5), C. felis (8), C. rivolta and C. felis (3)
C. rivolta (6), C. felis (11), C. rivolta and C. felis (8)
C. rivolta (6), C. felis (12), C. rivolta and C. felis (5)
neg, negative; pos, positive; FFl, fecal flotation. Dosage 1 – 50 mg/kg q24 h for 3 days; dosage 2 – 50 mg/kg as a single dose; dosage 3 – 20 mg/kg as a single dose. a Fecal oocyst burden at Day 1 was lower in dogs from the dosage 1 group than in dogs from the dosage 3 group (P = 0.009).
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
Fecal oocyst count in oocysts/g at Day 1 (range)a FFl oocyst negative at Day 3–4 and at Day 8; n (%) FFl oocyst positive at Day 3–4; repeat treatment with Dosage 1 from Day 3–4; n (%) FFl oocyst positive at Day 3–4; no further treatment; n (%) FFl oocyst negative at Day 3–4; FFl positive at Day 8; n (%) Coccidia species identified at Day 1 (n)
Cats (n = 64)
A.L. Litster et al. / Veterinary Parasitology 202 (2014) 319–325
323
Table 3 Comparison of median fecal oocyst counts at Day 1 in animals that were coccidia oocyst-positive at Day 3–4 and coccidia oocyst-negative at Day 3–4.
Dogs – oocyst-negative at Day 3–4 Dogs – oocyst-positive at Day 3–4 Cats – oocyst-negative at Day 3–4 Cats – oocyst-positive at Day 3–4
Median fecal oocyst count at Day 1 (oocysts/g)
n (%)
300 4100 200 1500
36/43 (83.7) 7/43 (16.3) 49/64 (76.6) 15/64 (23.4)
counts were statistically lower in dogs from the dosage 1 group (Table 1). Future studies should aim to determine the minimum dosage of ponazuril necessary to reduce oocyst excretion to below the detection limit, either by increasing the dose rate or by continuing treatment for a longer period. The balance between the cost of treatment and the length of the course of treatment is an important consideration in busy shelters with limited resources. Another interesting aspect of the results is detailed in Table 4, which shows that six animals (Cats G and H; Dogs A–D) had different species identified at Day 1 and Day 3–4. It is possible that the species identified at Day 3–4 were from newly acquired infections from environmental contamination at the shelter, although perhaps the species identified at Day 3–4 were also present at Day 1, but oocyst excretion was below the detection limit. Cat H was infected with C. rivolta at Day 1, C. felis at Day 3–4 and both species at Day 8, but in the remaining five animals, fecal testing was oocyst-negative at Day 8. It is possible that these Day 8 results were the result of spontaneous cessation of oocyst excretion (Lappin, 2005) rather than a treatment-related effect. Additionally, two cats (P and Q) tested oocyst-negative at Day 3–4, but were oocystpositive at Day 8 (Table 4). It is possible that this was the result of a temporary coccidiostatic effect of ponazuril at Day 3–4. Cystoisospora spp. oocysts remain infective for up to 1 year in moist, protected environments unless exposed to freezing or very high temperatures (Companion Animal
P
