Prevalence of Cryptosporidium in small ruminants from Veracruz, Mexico

Background Cryptosporidiosis is a zoonotic disease caused by the protozoan parasite Cryptosporidium spp. that can affect domestic animal and human populations. In newborn ruminants, cryptosporidiosis is characterized by outbreaks of diarrhea, which can result in high morbidity and economic impact. The aim of the present study was to determine the prevalence of Cryptosporidium spp. in small ruminants from the Perote municipality in Veracruz State, Mexico. One hundred and sixty small ruminants (80 sheep and 80 goats) from eight farms located in four towns of the Perote municipality were examined following a cross-sectional study design. Stool samples were analyzed by a modification of the Faust centrifugation method, and the presence of Cryptosporidium spp. oocysts was examined using a modification of the Ziehl-Neelsen staining procedure. Bivariate and multivariate analyses were used to assess the association of Cryptosporidium infection and the general characteristics of the animals studied. Results Overall, 112 (70 %, 95 % CI: 62.3–76.9) of the 160 small ruminants sampled were infected with Cryptosporidium spp. The prevalence of Cryptosporidium spp. infection in goats was 72.5 % (95 % CI: 61.4–81.9) and in sheep 67.5 % (95 % CI: 56.1–77.6). Small ruminants aged 1 month old had the highest (88.2 %; 95 % CI: 63.6–98.5) prevalence of infection. Prevalence varied from 60 % to 85 % among herds. Animal species, age, sex, breed, farm, town or cohabitation with cattle did not influence the prevalence of Cryptosporidium infection. Conclusions A high prevalence of infection with Cryptosporidium spp. was observed in small ruminants from the Perote municipality in Veracruz, Mexico. Infection was widely distributed among sheep and goats regardless of their age, breed or farm location. Further research is required to identify risk factors for, and to assess the veterinary public health significance of Cryptosporidium infection among sheep and goats in the Mexican state of Veracruz.


Background
Cryptosporidium is a zoonotic protozoan parasite of the Apicomplexa phylum and the Cryptosporidiidae family [1]. Cryptosporidium is an important pathogen in cattle and humans [2], and infection with this parasite in cattle may lead to economic losses due to morbidity and mortality [3,4]. Cryptosporidium spp. infecting sheep and goats have been found to be of public health significance [5,6].
Cryptosporidiosis, the disease caused by Cryptosporidium, is characterized by diarrhea, dehydration, and weight loss [7][8][9]. Cryptosporidium affects epithelial cells of the small intestine and occasionally stomach, gall bladder, liver, trachea, and lungs in a number of mammals including humans [10,11]. Cryptosporidium is a widely distributed parasite and can infect more than 170 species of vertebrates [1]. Transmission of Cryptosporidium occurs mainly by the fecal-oral route [7]. In sheep and goats, cryptosporidiosis is an important enteric disease resulting in diarrhea and inefficient weight gains [12], and is more severe in young than in adult animals [13,14]. Animals infected with Cryptosporidium shed a large (10 8 -10 9 /g) number of oocysts [15]. These oocysts are source of infection for animals and humans [16]. In humans, infection with Cryptosporidium causes diarrheal disease [17], and chronic and fatal disease in immunocompromised individuals [18]. Additionally, Cryptosporidium infection has been linked to cancer in humans [4,19].
Infection with Cryptosporidium in young sheep and goats may lead to morbidity and even mortality due to diarrhea [20][21][22]. Effective disease prevention requires an understanding of the environmental factors predisposing animal and human populations to infectious causes of diarrhea like Cryptosporidium spp. [23,24]. However, very little is known about the infection with Cryptosporidium spp. in sheep and goats in Mexico in general and there is a lack of information about this infection in the southern Mexican state of Veracruz in particular. Therefore, we sought to determine the prevalence of infection with Cryptosporidium spp. in sheep and goats in the municipality of Perote in Veracruz State, Mexico.

Study design
This cross-sectional study was performed in the municipality of Perote in Veracruz, Mexico from July to December 2014. Perote, the capital of the municipality with the same name, is located in the central-west region of Veracruz State (19°34'N 97°15'W) at an altitude of 2400 meters above sea level and has a cold and dry climate with a mean annual temperature of 12°C, and a mean annual rainfall of 493.6 mm.

Sheep and goats studied
Eighty sheep and 80 goats from the Perote municipality in Veracruz State were included in the study. Convenience sampling was applied. The small ruminant population sampled was raised in eight farms where sheep and goats herds were kept. Farms were located in four towns: 1) Perote; 2) San Antonio Limón; 3) Los Molinos; and 4) La Gloria. Ten sheep and 10 goats were sampled from each farm. Sheep and goats were young (up to 3 months old). A fecal sample from the rectum was obtained from each animal. Fecal samples were placed in a plastic bag and analyzed a day after sampling. General data of the agroecosystem and animals studied were obtained with the aid of a questionnaire. Animal data included species, age, sex, breed, presence of diarrhea, and history of deworming. Agroecosystem data collected included farm location, nearest town, sharing water with other animals, and cohabitation with dogs or cattle.

Laboratory tests
Small ruminant fecal samples were analyzed using a modification of the centrifuge-flotation method of Faust [25]. Briefly, three grams of fecal sample were homogenized in distilled water and centrifuged at 1500 rpm for 1 min. Then supernatant was removed and the homogenization and centrifugation processes were repeated. After removing the supernatant, the sediment was resuspended in ZnSO 4 (44 %) and centrifuged. Finally, the supernatant was removed and sediment was examined. Cryptosporidium oocysts were detected by a modification of the Ziehl-Neelsen staining method [26]. Samples were examined under oil immersion objective (100x) and oocysts were identified based on morphological features.

Statistical analysis
Results were analyzed with the aid of the software Microsoft Excel, SPSS version 15.0 (SPSS Inc. Chicago, Illinois) and STATA version 11.0. Descriptive statistics were used to determine the prevalence of Cryptosporidium infection in sheep and goats. We used the Pearson's chisquared test for comparison of infection frequencies among groups. The association between Cryptosporidium infection, animal characteristics, and agroecosystem features was assessed by bivariate and multivariate analyses. The dependent variable was positivity to Cryptosporidium by Ziehl-Neelsen staining per individual animal. Independent variables included in the multivariable analysis were those with a P value ≤0.10 in the bivariate analysis: age, town, and farm. Odds ratio (OR) and 95 % confidence interval (CI) were calculated and statistical significance was set at a P value of < 0.05.

Ethics statement
The Bioethics and Animal Welfare Commission of the Facultad de Medicina Veterinaria y Zootecnia of Universidad Veracruzana approved this project. Owners of the sheep and goats gave their consent to study their animals. Adequate veterinary care was provided to all animals studied. Information about the prevalence of infection with Cryptosporidium in animals in Mexico is scant. The prevalence of Cryptosporidium infection in sheep reported here is higher than the 34.3 % prevalence of Cryptosporidium infection in sheep in northern Mexico [27]. We are not aware of reports about the prevalence of Cryptosporidium infection in goats in Mexico. Therefore, we cannot compare our results with those in other region in the country. In an international context, the prevalence of Cryptosporidium infection found in sheep in Veracruz is higher than the prevalences found in Table 1 Correlation of Cryptosporidium infection with the general characteristics of the animals(sheep and goats together) and the agroecosystem where they were raised sheep in Spain (31-59 %) [28,29], Poland (10.1 %) [30], Egypt (2.5 %) [31], Iran (11.3 %) [32], Iraq (13.3 %) [33], Tunisia (11.2 %) [34], India (1.8 %) [35], and Papua New Guinea (2.2 %) [36]. Similarly, the prevalence of Cryptosporidium infection found in goats in Veracruz is higher than prevalences found in goats in Spain (30 %) [29], Poland (0 %) [30], Iraq (17.7 %) [33], and Papua New Guinea (4.4 %) [36]. However, comparison of prevalences of Cryptosporidium infection in animals among countries should be interpreted with care because matching for characteristics of animals and their raising conditions is challenging.

Results and discussion
Sheep and goats may serve as reservoirs of Cryptosporidium for human infections [36]. Outbreaks of infection with Cryptosporidium in schoolchildren associated with contact with lamb/goat kids have been reported [37]. The occurrence of these outbreaks 3 years apart, with the same parasite in the same farm suggested that Cryptosporidium might establish in the environment [37]. In addition, cryptosporidiosis has been associated with consumption of unpasteurized goat milk in children [38]. Therefore, our results should alert for the risk of infection in humans in Veracruz through contact with infected sheep and goats, and consumption of unpasteurized milk from infected goats. Cryptosporidium infection has been reported in animals other than sheep and goat in Veracruz, Mexico. Prevalence of Cryptosporidium infection in cattle in several municipalities in Veracruz varied from 14.3 % to 75 % [39,40]. Sheep, goats, and cattle are frequently raised together in Veracruz, and this interaction along with poor hygienic sanitary conditions in the agroecosystems where they are raised might account for transmission of Cryptosporidium infection among animal species [41]. A number of Cryptosporidium species/genotypes infecting sheep has been described including C. parvum, C. bovis, C. cervine [42][43][44], and C. hominis [42]. Whereas some Cryptosporidium species/ genotypes infecting goats include C. xiaoi [36,45], C. parvum, and C. hominis [36].
We sought to determine the association between the general characteristics of animals, their environment, and Cryptosporidium infection. The prevalence of Cryptosporidium infection did not vary significantly with small ruminant species, age, sex, or breed of the animals studied. Neonatal diarrhea in small ruminants is one of the most frequent syndromes during the first week of age and leads to important economic losses due to mortality and delayed growth. Cryptosporidium is a well-known cause of neonatal diarrhea in sheep [46]. However, in our study the history of diarrhea in the animals studied was not associated with Cryptosporidium infection. This finding agrees with that found in a study of lambs and adult sheep in Poland [30]. In the present study, the effect of dogs on Cryptosporidium prevalence could not be determined because all farms sampled had dogs present. We found no association between Cryptosporidium prevalence and a history of deworming or cohabitation with cattle. All sheep and goats studied shared water with other animals of the same farms. Table 1 shows a correlation between Cryptosporidium infection and general characteristics of sheep and goats and the agroecosystem where they were raised. Bivariate analysis of these characteristics showed three variables with a P < 0.10: age, towns and farms. Multivariate analysis of the three variables with a P < 0.10 (age, towns and farms) obtained in bivariate analysis showed no association with Cryptosporidium infection. The failure to find an association between the presence of infection and characteristics of the animals may have been due to the small sample size. Further studies with a larger sample size to determine the risk factors associated with Cryptosporidium infection in sheep and goats should be conducted to assess the veterinary public health burden of cryptosporidiosis in Mexico. In addition, further research with molecular methods to determine the Cryptosporidium genotypes, and subtyping of C. parvum or C. ubiquitum at the gp60 locus [47,48] in sheep and goats in Mexico are needed.

Conclusions
The prevalence of infection with Cryptosporidium spp among sheep and goats in the Perote municipality in Veracruz, Mexico is relatively high. Infection was widely distributed among small ruminants regardless of their age, breed or the agroecosystem where they were raised. Further research to identify risk factors for Cryptosporidium infection in sheep and goats and the zoonotic potential of cryptosporidiosis in Veracruz State is needed.