Cryptosporidium spp . infection in mares and foals of the northwest region of São Paulo State , Brazil

The present study aimed to analyze the occurrence of infection by Cryptosporidium spp. in mares and their respective foals. This study was carried out in 11 farms located in the municipalities of Araçatuba, Birigui, Guararapes and Santo Antônio do Aracangua, in the northwest region of the State of Sao Paulo, from November 2010 to March 2011. A total of 98 mares and 98 foals of several breeds were analyzed; among foals, 59 were males and 39 females, aged from three to 330 days. Feces were collected directly from the rectal ampulla, purified and processed according to modified Kinyoun stain. Occurrence of Cryptosporidium spp. was 21.4% (21/98) for foals and 18.4% (18/98) for mares. Occurrence of Cryptosporidium spp. had significant association with breeds and age of animals. Results obtained led to the conclusion that foals older than two months and Mangalarga animals are less susceptible to the occurrence of Cryptosporidium spp.

The protozoa Cryptosporidium spp. is included in the Neglected Diseases Initiative of the World Health Organization (WHO) due to their close relationship with deficient sanitation and low income of the population; thus, they are considered responsible for child malnutrition and death (THOMPSON et al., 2008).
Fecal oocysts of Cryptosporidium spp.released by equine may represent a source of human infection and are resistant to the environmental conditions (PLUTZER; KARANIS, 2009).
Evaluation of the occurrence of Cryptosporidium spp. in horses has drawn the attention of research worldwide (BURTON et al., 2010;COLE et al., 1998;JOHNSON et al., 1997;MAJEWSKA et al., 2004;MARQUES, 2010;SOUZA et al., 2009;VERONESI et al., 2009).Positive fecal samples for Cryptosporidium parvum oocysts were significantly greater for foals under six months of age and thoroughbreds compared to other breeds, in Texas (COLE et al., 1998).
Few studies have been carried out in Brazil (MARQUES, 2010;SOUZA et al., 2009).Therefore, data on the occurrence of Cryptosporidium spp. in equine species are still scarce.
The protozoan Cryptosporidium spp.has been associated with the presence of diarrhea in immunocompetent and immunodeficient animals (BJORNEBY et al., 1991;COLEMAN et al., 1989;GIBSON et al., 1983;GRINBERG et al., 2003;MAIR et al., 1990).Nevertheless, some clinical and epidemiological aspects of this infection are still not clear (VERONESI et al., 2009).Meireles (2010) discussed this situation in researches carried out in Brazil on domestic and wild animals infected with Cryptosporidium spp.demonstrating the increasing amount of works on this theme.
The present research aimed to analyze the occurrence of infection by Cryptosporidium spp. in mares and their respective foals in municipalities of the northwest region of the State of Sao Paulo and to correlate the positivity for this parasite with the variables breed, sex, age, degree of fecal hydration, and water source.

Materials and Methods
This study was approved by the Ethics Committee for Animal Use of the Dentistry School of UNESP, Araçatuba Campus; protocol number 2009-002165.
An epidemiological questionnaire was applied to the owners, addressing questions related to age, sex, breed and types of water source for the equines involved in the research.
Fecal samples were collected directly from the rectal ampulla of animals and stored under refrigeration; subsequently, fecal smears were prepared.To standardize the determination of the degree of fecal hydration, the following classification was adopted: solid, semi-solid, pasty and liquid.The following established criterion was used regarding the color: green, mossy green, brown, yellowish green and yellow.Information about fecal conditions was introduced in the data bank to correlate the physical status of feces to the occurrence of the parasite, also including the variables: age, sex, breed, and types of water source (river, pond, millpond, draw-well, water tank) used by the animals involved in this study.
Fecal smears were prepared by centrifugation-sedimentation technique in water and ether (MELONI; THOMPSON, 1996) used in the routine laboratory and, subsequently, stained according to the modified Kinyoun technique (LENNETTE et al., 1985) for observation of Cryptosporidium spp.oocysts.
A light optical microscope, 40× and 100× magnification, was employed for observation.Infection by Cryptosporidium spp. was classified into positive or negative, based on the presence or absence of oocysts in one sample from each animal.

Results and Discussion
Occurrence of Cryptosporidium spp. was detected, using Kinyoun stain, in 21.4% (21/98) of the foals and 18.4% (18/98) of the mares analyzed.Foals presented higher occurrence of Cryptosporidium spp.compared to adult mares, which corroborates the findings by Burton et al. (2010).
There was positivity of 12.2% (12/98) of samples for both foals and their respective mothers; however, there was no significant association.Mature horses are not considered important sources of infection for foals (COLE et al., 1998) and the infection of foals was not associated with that of their mothers (MAJEWSKA et al., 2004).
Compared to the occurrence of infection by Cryptosporidium spp. in the equines detected herein, studies have described lower frequencies: 0% (0/91) by Johnson et al. (1997) in California, USA and 0.75% (3/396) by Souza et al. (2009) in Rio de Janeiro, Brazil.However, a higher frequency of 27.8% (25/90) of Cryptosporidium spp.infection was obtained using Ziehl-Neelsen staining technique by Marques (2010) in Porto Alegre, Brazil.Thus, asymptomatic adult horses can be considered sources of environmental contamination due to the fecal content they produce (MARQUES, 2010).
Foals younger than two months were more susceptible to Cryptosporidium spp.infection (p = 0.0069), with 37,5% (12/32) occurrence, while the group older than 60 days showed 13,6% (9/66) of positive samples (Table 1).Two positive samples from 60 and 90-day-old foals were liquid feces and one positive mother presented feces of pasty consistency.Cole et al. (1998) reported diarrhea related to the presence of the above-mentioned protozoan, and Grinberg et al. (2003) seemed to have evidenced this clinical sign in immunocompetent foals.In the present study, the consistency (p = 0.2119 and p = 0.2403) and color (p = 0.6842 and p = 0.5023) of feces were not associated with the occurrence of this disease in foals and mares, respectively, which agrees with the reports by Veronesi et al. (2009) and Burton et al. (2010).
In this study, there was no significant association between the protozoan occurrence and the sex of foals (p = 0.1838) (Table 1); the same was found for the sources of water drunk by the animals in the farm (p = 0.4877).Souza et al. (2009) noted higher proportion of males naturally infected by Cryptosporidium spp.and concluded that the disease in equines, with different management types, is probably related to the immunological state and sanitary conditions found in the place where they live, since they were already considered sources of environmental infection (VERONESI et al., 2009).Concerning this aspect, it must be highlighted that, in previous epidemiological studies, Cryptosporidium spp.infection was concentrated on one farm or site, and this may be due to the characteristics of the management adopted in the farm (VERONESI et al., 2009;BURTON et al., 2010).

Conclusion
The results obtained led to the conclusion that foals older than two months and the breed Mangalarga were less susceptible to the occurrence of Cryptosporidium spp.

Table 1 .
Occurrence of infection by Cryptosporidium spp.by modified Kinyoun stain according to the variables breed, sex and age from 98 foals in the northwest region of the State of Sao Paulo, 2012.