key: cord-0026070-jft610pa
authors: LEE, Hsu-Hsun; LEE, Jui-Yu; LIU, Shyh-Shyan; CHEN, Chen-Chih; HSU, Huan-Yu
title: Cryptosporidium parvum infection and management-based risk factors of dairy calves in Taiwan
date: 2021-10-14
journal: J Vet Med Sci
DOI: 10.1292/jvms.21-0366
sha: fd0e1715a399cc527f3cdde35803b6352ff3bcf1
doc_id: 26070
cord_uid: jft610pa

Cryptosporidiosis is one of the major causes of diarrhea in calves. Cryptosporidium parvum is considered the most important calf diarrhea pathogen in the Cryptosporidium species. Not only could infected calves spread C. parvum, but infected adult cattle could also shed oocysts. The objectives of this study were (1) to investigate the prevalence of C. parvum in dairy herds in Taiwan, including calves, the dams in delivery enclosure, the floor, and the drinking water; (2) to clarify the relationship of diarrhea, management, and C. parvum infection. Twenty dairy herds in Taiwan were selected by random sampling, including 226 calves and 198 dams, and other environmental samples were collected. A questionnaire was filled out by the farm owners to collect information regarding the management of calves and the delivery enclosure. Hierarchical logistic regression was used to analyze the risk factors for C. parvum infection. The prevalence of C. parvum infection in calves was 26.5% (60/226), while in dams, it was 19.7% (39/198). The C. parvum infection in calves increased with environmental contamination of C. parvum and clinical signs of diarrhea, while it decreased with a yard provided in the delivery enclosure. In conclusion, the management of the delivery enclosure appears to be more important for preventing cryptosporidiosis in calves in Taiwan.

comprised a fecal survey via the modified Ziehl-Neelsen method and an immunofluorescence assay in cattle and goats, showing a prevalence of 37.6% (173/460) and 35.8% (44/123), respectively [25] . The prevalence in the abovementioned research included all species of Cryptosporidium, and thus the real status of pathogenic Cryptosporidium, which is C. parvum in Taiwan, could have been overestimated. Therefore, the prevalence of C. parvum in dairy cattle in Taiwan is still unknown. The objectives of this study were (1) to investigate the prevalence of C. parvum in dairy herds in Taiwan, including calves and the dams in the delivery enclosure; (2) to clarify the relationship of diarrhea, management, and C. parvum infection.

From April 2017 to February 2018, the feces of 226 calves from 20 dairy herds in North, Middle, South, and East Taiwan. Farms were selected by random sampling, the average cow number was 366 ± 201 with 6 to 40 under 1.5 months old calves from each farm ( Supplementary Table) . All of the calves under 1.5 months old in the selected farms were sampled. The calves' feces samples were defined as being either normal or diarrhea according to Uga's research [10] . Next, 179 and 206 swab samples were taken from the calves' drinking water and rearing enclosure, respectively. From dams in the delivery enclosure, 198 feces samples were taken, while 32 and 30 samples were taken from the drinking water and floor in the delivery enclosure, respectively. All owners agreed samples collection in this study. All sampling procedures were executed by veterinarians under the Animal Protection Act from the Council of Agriculture in Taiwan. No extra animals were intentionally used or purchased in this study.

Feces were collected from calves via the hand (with latex gloves) to stimulate the rectum to induce defecation. The drinking water of the calves was collected from the tank used to provide water to the calves. The swab samples of the rearing enclosure were collected using plastic spoons. If the calves were fed in independent cages, samples were collected from the upper, middle, and lower sites of the front and rear fences. If the calves were fed in groups, the sampling sites were taken in duplicate from the front, middle, and rear of all the feeding area. Samples from different sites of the same cages or feeding areas were mixed as the rearing enclosure swab sample for all of those calves that stay in that cage or feeding area. Feces from dams were collected via rectal palpation using a long plastic glove. The collected samples were stored at 0°C in an ice box and transferred to a −20°C refrigerator within 24 hr.

All solid samples were filtered by gauze to remove most of the fiber. Then, the oocysts were concentrated by salt flotation after filtration. Depending on the samples having been collected from calves or dams, a 10 or 20 g sample was mixed with 30 or 50 ml of reverse osmosis water, and then filtered by gauze to remove most of the fiber. Thirty milliliters of the mixed sample were moved to a centrifuge tube and centrifuged at 3,500 g for 10 min. The supernate was removed and then reverse osmosis water was added to reach 30 ml. Centrifugation was repeated three times; for the third centrifugation, saturated salt solution was added to reach 30 ml, then mixed and rested for 15 min. Following this, 3 ml of the supernate was collected and then reverse osmosis water was added to reach 30 ml in a new centrifuge tube. The tube was centrifuged at 3,500 g for 10 min, and then the supernate was removed; this process was repeated three times to remove the salt. After the third centrifugation, 5 ml of ultrapure water was added to the tube with precipitate. The tube was shaken to mix it completely, ready for DNA extraction. The FastDNA ® SPIN Kit for Soil (MP Biomedicals, Carlsbad, CA, USA) was used to extract DNA from the samples. For the water samples without visible fiber, salt flotation after filtration was used to concentrate the oocytes without filtration, and the same DNA extraction step as for the solid samples was used.

The extracted genomic DNA sample was used as a template to amplify the 18S rRNA of C. parvum by nested PCR [21] . The first-step primers for nested PCR were the forward primer EF:5′-TTCTAGAGCTAATACATGCG-3′ and the reverse primer ER:5′-CCCATTTCTTCG AAACAGGA-3′. The second-step primers were the forward primer CphF:5′-AGAGTGCTTAAAGCAGGCATA-3′ and the reverse primer IR:5′-AAGGAGTAAGGAACAACCTCCA-3′. The first step of nested PCR was performed using 3 µl of extracted DNA, 10 µl of 2X Taq PCR MasterMix (Genomics Bioscience and Technology Co., Ltd., New Taipei City, Taiwan), 0.5 µl (10 µM) of each forward (EF) and reverse (ER) primer, and 6 µl of pure water in a 0.2 ml Eppendorf. The PCR conditions were as follows: Denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 45 sec, 49°C for 45 sec, 72°C for 1 min, and extension at 72°C for 7 min. The second step of nested PCR was performed using 3 µl of the PCR product from the first reaction rather than the extracted DNA and the same cycling conditions as the first step. The final PCR products were analyzed by gel electrophoresis on a 2% agarose gel and visualized under blue light. The final target was a 305 bp fragment of 18S rRNA of C. parvum, as shown in Fig. 1 . 

A questionnaire was filled out by the farm owners to collect information regarding the management of calves and the delivery enclosure (Fig. 2) . The diarrhea episodes of the calves were recorded at the time of sample collection. Whether a yard for prepartum cows were provided or not and the bedding materials in their delivery enclosure were also recorded.

We first estimated the prevalence of C. parvum infection and its 95% confidence interval (CI) in both calves and cows. Furthermore, we adopted hierarchical logistic regression to analyze the relationship between C. parvum infection in calves and other environmental explanatory variables (Table 1) . We transformed all of the categorical explanatory variables into dummy variables, and centered and standardized the numerical variables. In addition, the farms were designed as a random effect for the model to evaluate the variance of intercept between the different farms. The hierarchical logistic regression was fit by maximum likelihood with Laplace approximation [16] . The computing environment R with the package lme4 [3, 19] was used for model construction.

For model construction, we first fitted the model with each univariable analysis and retained the variables with P-values of less than 0.1 for further multivariable model construction.

Multicollinearity between explanatory variables was evaluated using the variance inflation factor (VIF) [13] . The VIF threshold was set as 10 to avoid problematic effects of multicollinearity on the parameter estimations [1] . Variables were discarded from model construction if their VIF value was larger than 10.

The explanatory variables selected in the models were based on the Wald test with a P-value threshold set at 0.05 [4, 8] . The backward stepwise method was adopted for variable selection. Values of the Akaike information criterion (AIC) and the AIC weights were used to assess the models' fit and to select the best-fitted model [4, 8] . Based on the fitted model, the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) was calculated to evaluate the discriminate ability of the model.

The prevalence of C. parvum infection in calves was 26.5% (60/226; 95% CI: 20.7-32.3%) and 19.7% (39/198; 95% CI: 14.2-25.2%) in dams. The percentage of C. parvum-positive herds was 90.0% (18/20; 95% CI: 76.9-100%). The prevalence of C. parvum in North, Middle, South, and East Taiwan is shown in Table 2 .

The proportion of positives in the rearing enclosure swab samples was 18% (38/206; 95% CI: 12.8-23.2%), while it was 2.2% (4/178; 95% CI: 0-4.1%) in the drinking water of the calves. The samples from the floor and drinking water in the delivery enclosure had a positive proportion of 16.7% (5/30; 95% CI: 3.6-30%) and 6.3% (2/32; 95% CI: 0-14.7%), respectively. The C. parvum-positive proportion of calves based on different environmental explanatory variables is shown in Table 3 . 

The explanatory variables chosen for model construction with a P-value below 0.1 in the univariable analysis were Diarrheal=Yes, Rearing enclosure=Positive, BirthF-Clean=2 (once every two-three days), and Yard=Yes ( Table 4) .

The final fitted model included the explanatory variables of Diarrheal=Yes, Rearing enclosure=Positive, and yard provided had the lowest AIC value and AIC weight of 0.59 (Table 5) . Furthermore, the AIC value indicated that the null model of hierarchical logistic regression fit better than that of logistic regression (Table 5) .

Based on the final fitted model, the probability of C. parvum infection in calves increased with proportion of positives in the rearing enclosure of C. parvum and clinical signs of diarrhea, while it decreased with a yard provided in the delivery enclosure ( Table 4 ). The estimated odds ratios of each univariable analysis and the final fitted variables in the hierarchical logistic regression model are shown in Table 4 . We created a ROC curve (Fig. 3) and estimated the AUC based on the final fitted model. The computed value of AUC was 77.62% (95%CI: 70.45-84.88%), which indicated that the discriminate ability of fitted model was considered acceptable to excellent [17, 18] .

This research comprised a survey of C. parvum infection in calves younger than 1.5 months and prepartum cows in dairy herds in Taiwan, which were analyzed using nested PCR. This is the first research of the prevalence of C. parvum in dairy cattle in Taiwan. The results show that the prevalence of C. parvum infection was 26.5% and 19.7% in calves and dams, respectively, and positivity rate in herds was 90.0%. The prevalence of C. parvum in pre-weaned claves was reported from 3.4 to 96.6% in different areas [23] . The differences mainly based on their different experiment designs and the detection methods of these studies, which resulted in diverse prevalence of C. parvum. Longitudinal study and sampling diarrheic calves only presented higher prevalence in the past studies. This study present point-prevalence and collect both diarrheic and non-diarrheic calves under 1.5 months old. The prevalence of this study was 26.5%, which showed the infection of C. parvum is severe in calves in Taiwan. In addition, hierarchical logistic regression showed that diarrhea in calves was positively related to C. parvum, indicating that the diarrhea in calves caused by C. parvum infection in Taiwan dairy herds is an important issue. C. parvum has been confirmed as not only a harmful pathogen in calves, but also being able to possibly infect humans-especially children [6] . Therefore, understanding the risk factors of C. parvum infection is very important for human and herd health. The rearing enclosure, which was investigated for C. parvum, was positively related to C. parvum infection in calves, showing that infected calves shed oocytes into environment. Even cleaning the cage every day or using disinfectant could not decrease the C. parvum infection rate in calves. This suggests that prevention of infection in dams and decontamination of delivery enclosures are more important for preventing cryptosporidiosis in calves in Taiwan. This study also indicated that 19.7% of dams had been infected, with the floor and drinking water in the delivery enclosure having a C. parvum positive proportion of 16.7% and 6.3%, respectively. Even concrete flooring, which is commonly used in delivery enclosures in Taiwan and is easy to clean, the delivery enclosures were contaminated with C. parvum. A previous study indicated that the floor and drinking water in the delivery enclosure having a Cryptosporidium oocyte proportion of 80% and 64% in a high contaminated farm [9] . These results support the insight that C. parvum can infected neonatal calve soon after delivery [26] , suggesting that famers should consider more effective cleaning methods in delivery enclosures to decrease C. parvum infection in neonatal calves in Taiwan. However, the frequency of cleaning and the use of disinfectant were not related to C. parvum infection, perhaps because most disinfectants have limited efficacy against C parvum, including those recorded in this study [2] . Cleaning the environment and feeding tools by hot water or heating was more efficient for killing oocyte of C. parvum [14] , but this kind of cleaning procedure was rarely performed in Taiwan dairy herds. That might explained no relationship between the frequency of cleaning (in delivery and calves' enclosure) and C. parvum infection in this study. Although there were several studies indicated that large size farm has higher risk of C. parvum infection, the farm size did not relate to C. parvum infection in this study [20, 24] . It might because of few large farms (>500 adult cows) in Taiwan and only one farm had more than 500 adult cows in this study. So the farm size was not a significant factor of C. parvum infection in Taiwan.

On the contrary, provided yard for prepartum cows showed a lower infection rate for C. parvum in calves and tended to be negatively related to C. parvum infection. Even there is no study describing the relationship between providing yard for prepartum cows and C. parvum infection directly, minimizing the exposure of calves to C. parvum was indicated as an effective strategy for preventing infection of C. parvum [14] . A yard for prepartum cows could provide more space in calving area and decrease the opportunity to contact oocyte of C. parvum as the calves born. There was another report indicated the industrial farming system, compared to gracing farming system has higher risk for C. parvum infection [15] . That result also suggested large space in calving area may decrease the risk for C. parvum infection.

In conclusion, the relationship between diarrhea and C. parvum infection in calves is very high in Taiwanese dairy herds, and the management of the delivery enclosure such as providing a yard appears to be more important for preventing cryptosporidiosis in calves in Taiwan. CONFLICTS OF INTEREST. The authors declare that there is no conflict of interest.

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ACKNOWLEDGMENTS. We thank all farmers for their cooperation in providing information and samples for use in this study. This study was also financially supported by the Research Center for Animal Biologics, from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education and the Ministry of Science and Technology (MOST 109-2634-F-020-001-, MOST 110-2634-F-020-001), Taiwan, R.O.C.