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Prevalence of Cryptosporidium parvum infection in southwestern Ontario and its association with diarrhea in neonatal dairy calves Lise A. Trotz-Williams, Brenna D. Jarvie, S. Wayne Martin, Kenneth E. Leslie, Andrew S. Peregrine Abstract — Cryptosporidium parvum infection was detected in 203 (40.6%) of 500 Ontario dairy calves aged 7 to 21 d, on a convenience sample of 51 farms with a history of calf diarrhea. Withinfarm prevalence ranged from 0% to 70%, and both shedding and intensity of shedding were significantly associated with diarrhea. This parasite appears to be common in Ontario dairy calves and important as a cause of dairy calf scours in the province. Résumé — Prévalence de l’infection à Cryptosporidium parvum dans le sud-ouest de l’Ontario et son association avec la diarrhée chez les veaux laitiers nouveau-nés. Une infection à Cryptosporidium parvum a été détectée chez 203 (40,6 %) veaux laitiers ontariens sur 500, âgés de 7 à 21 jours, dans 51 fermes choisies selon un critère de commodité et présentant une histoire de diarrhée. La prévalence dans chaque ferme variait de 0 à 70 % et l’élimination du parasite et l’intensité de cette élimination étaient toutes deux significativement associées à la diarrhée. Ce parasite semble fréquent chez les veaux laitiers de l’Ontario et constitue une cause importante de diarrhée des veaux laitiers dans cette province. (Traduit par Docteur André Blouin) Can Vet J 2005;46:349–351
ryptosporidium parvum, a coccidian intestinal C parasite, is commonly diagnosed as a cause of diarrhea in neonatal dairy calves in many parts of the world. Cryptosporidiosis (scouring due to C. parvum infection) most often affects calves under 1 mo of age, and affected calves may shed large numbers of infective oocysts in the feces (1). While infection is generally self-limiting, fatalities associated with cryptosporidiosis have been reported. Cryptosporidium parvum is also a recognized zoonotic agent, and persons in contact with infected animals are at risk of contracting C. parvum infection. Cryptosporidium parvum is a ubiquitous pathogen and has been reported from many geographical regions of the world. A study of 7369 calves from 1103 dairy farms in the United States detected infection in 48% of the calves between 7 and 21 d of age, with at least 1 calf testing positive on 59% of the farms (2). Similarly, 59% of Department of Population Medicine (Trotz-Williams, Jarvie, Martin, Leslie); Department of Pathobiology (Peregrine), Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1. Funding was received from the Ontario Ministry of Agriculture and Food (OMAF), and National Sciences and Engineering Research Council of Canada (NSERC). Address all correspondence to Lise Trotz-Williams; e-mail:
[email protected] Reprints will not be available from the authors. Can Vet J Volume 46, April 2005
386 dairy calves up to 24 wk of age on 20 farms in British Columbia were found to be shedding C. parvum oocysts (3); infected calves were found on 80% of the farms. In Québec, infection was detected in calves on 88% of 505 dairy farms (4). When present on a farm, the prevalence of C. parvum infection may be very high. Furthermore, a Cryptosporidium sp. is often the only pathogen detected in diarrheic calves (5). Although C. parvum is known to be common in many areas of North America, no prevalence studies have been reported for dairy calves in Ontario. In addition, the importance of this parasite as a cause of calf scours in the province has yet to be established. However, C. parvum has often been detected in fecal samples from scouring calves sent to the Animal Health Laboratory at the University of Guelph (6). To determine the prevalence of C. parvum infection among dairy calves in southwestern Ontario, and to investigate the importance of this parasite as a cause of calf scours in this region, fecal samples were obtained from 500 calves on a convenience sample of 51 dairy farms from May to August 2002. Herds were selected with the assistance of 5 veterinary practices in the vicinity of Guelph. Many of the farms had a current history of scouring in preweaned calves, as farms were selected based on a current or recent history of neonatal calf diarrhea. Farms were visited every other week throughout the course of the study and a single fecal sample was collected from each calf aged 7 to 21 d. No calf in the 349
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Herd identification number Figure 1. Percentage of Cryptosporidium parvum-infected calves by herd, ordered by increasing prevalence. The number of calves sampled per farm ranged from 2 to 102, with fewer than 20 calves sampled on most farms.
study was sampled more than once. Fecal consistency was scored on a 4-point scale as described by Larson et al (7). For this study, a score of 3 or 4 indicated the presence, and a score of 1 or 2 the absence, of diarrhea. Fecal samples were stored at 4°C and were examined at the Ontario Veterinary College for C. parvum oocysts within 48 h of collection by a standardized sucrose wet mount method. In brief, 1.0 g of feces was mixed with 3 mL of sucrose solution (specific gravity 1.32) to form a homogeneous mixture. One drop of the mixture was then placed on a microscope slide using a disposable transfer pipette (Fisher Scientific, Pittsburgh, Pennsylvania, USA) and covered with a 22-mm 22-mm coverslip (Fisher Scientific), with the drop being allowed to fill the entire area under the coverslip. The area under the coverslip was then examined at 400 magnification using a light microscope (Leica; Opti Tech Scientific, Scarborough, Ontario). Cryptosporidium parvum oocysts were identified as spherical, pink-tinged refractile cysts approximately 4 m 5 m in size. Oocysts were quantified by counting the number of oocysts in 10 fields and multiplying the average number per field by 2304 (the number of fields in the coverslip area). Results of this test have shown substantial agreement with the results of nested polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) analysis at the Cryptosporidium oocyst wall protein (COWP) gene locus (kappa = 0.77 for 110 samples; authors’ unpublished data), with respect to classification of samples as negative or positive. Analysis by PCR-RFLP of the COWP gene, followed by gel electrophoresis, also 350
enabled the identification of all amplified DNA as that of C. parvum (8). Statistical analysis was performed using statistical software (Stata, version 8.0; Stata Corporation, College Station, Texas, USA) to determine the prevalence of C. parvum infection and diarrhea, and to investigate the relationships between infection, diarrhea, and intensity of oocyst shedding by infected calves. The number of calves sampled per farm varied from 2 to 102. Of the 500 calves sampled, 402 were heifers and 98 were bulls. There was no statistically significant difference in the risk of infection between male and female calves (P 0.05). Two hundred and three calves (40.6%) were shedding C. parvum oocysts at the time of sampling. Infected calves were found on 76% of the farms. Within-herd prevalence of infection ranged from 0% to 72% (Figure 1), and the intraclass correlation coefficient was 0.21. Three hundred and nineteen (63.8%) of the calves had fecal scores indicative of diarrhea. Of these diarrheic calves, 161 (50.5%) were shedding C. parvum oocysts. In contrast, only 23.5% of the 181 nondiarrheic calves were shedding oocysts. There was no statistically significant difference between the mean ages of calves in the diarrheic and nondiarrheic groups. Analysis of the relationship between diarrhea and C. parvum infection revealed an odds ratio of 3.07 (95% confidence limits 1.93, 4.88) with an associated P-value of 0.0001, when controlling for herd in a mixed effects logistic regression model. This indicated a strong and highly significant association between infection with C. parvum and the occurrence of diarrhea in the calves, Can Vet J Volume 46, April 2005
with calves shedding oocysts having 3 times the risk of being diarrheic as uninfected calves. Regressing diarrhea status on a log transformation of the oocyst count, while controlling for herd in a mixed effects logistic model, gave an odds ratio of 1.18 (95% confidence limits 1.11, 1.24), with an associated P-value of 0.0001. This suggested a highly significant association between the intensity of shedding and the presence of diarrhea. The requisite assumption of linearity for the logistic model was evaluated by generating a scatterplot of the log odds of the outcome against the categorized predictor; this plot showed no evidence of nonlinearity. The findings of this study show that C. parvum is commonly found among 7- to 21-day-old dairy calves from farms in southwestern Ontario with a history of calf diarrhea. The within-farm prevalence of oocyst shedding was also highly variable, with some farms having a much higher prevalence of shedding than others. This fact is illustrated by both the range of within-farm prevalence figures and the moderate intraclass correlation coefficient, which indicated that the variation in risk of C. parvum infection between herds was relatively large compared with that between calves. The results reported here also illustrate that scouring among 7- to 21-day-old calves in Ontario is significantly associated with C. parvum infection. From work conducted elsewhere, it is well known that this parasite is one of the pathogens most commonly found in scouring calves and that it may be detected either alone or with other enteropathogens (9–11). Furthermore, if the level of oocyst shedding is regarded as indicative of the intensity of infection, our findings show a significant positive association between the intensity of infection in calves and the presence of diarrhea, further emphasizing the importance of this parasite as a cause of scouring in young dairy calves. Since C. parvum appears to be a common infection on dairy farms in southwestern Ontario, and is associated with scouring in young calves, attention needs to be paid to reducing the frequency and intensity of this infection in dairy calves in Ontario. Further research at the Ontario Veterinary College will therefore be directed towards investigating the risk factors for C. parvum infection and disease in dairy calves in Ontario. Furthermore, the study reported here provided information on the prevalence of C. parvum in the summer months only; any seasonal variation in the prevalence of C. parvum will be explored in our future study. In addition, the prevalence evaluation
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described in this report did not include an investigation of the influence of other enteropathogens on the relationship between C. parvum infection and diarrhea; future work will address this question. The potential importance of oocyst shedding by infected livestock as a source of infection among humans in Ontario will also be assessed. Given the findings of this study, it is important that practitioners in Ontario consider C. parvum as a differential diagnosis when investigating the etiology of scouring in calves under 1 mo of age.
Acknowledgments The authors thank Grazyna Adamska-Jarecka for laboraCVJ tory diagnostic support.
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