Occurrence of Cryptosporidium and Helminthosis in Santa Ines Sheep under Dry and Rainy Season

Cryptosporidiosis is a parasitic disease caused by a protozoan from the genus Cryptosporidium with cosmopolitan distribution and zoonotic potential. The objective of this work was to determine the occurrence of Cryptosporidium and helminthosis in Santa Ines sheep herd during dry and rainy season. This work was developed at the sheep’s breeding sector from the Federal University of Piaui, located in Southern Piauí, Brazil. Fifty sheep kept in a semi-intensive system were used in the experiment using the method according Ritchie, Ziehl-Neelsen and Gordon and Whitlock. Cryptosporidium oocysts were detected in 36% of the animals tested, with 20 males exhibiting a percentage of 50% (10 animals) protozoan in the feces and 50% (10 animals) exhibiting negative results. Among the 30 tested females, 8 (26.6%) were positive and 22 (73.4%) were negative. Concerning the age, 24 animals (48%) were of 0-12 months old, with prevalence of 11 (46%) positive animals showing protozoan in the feces, and 26 animals (52%) between 13-48 months old. The rainy season showed the highest counting of eggs per gram of feces (EPG), reaching a mean value of 2250 thus, a high occurrence of Cryptosporidium was evidenced, with a higher infection degree in young male sheep, predominantly during the rainy season, when a higher EPG was observed.


Introduction
Cryptosporidiosis is a parasitic zoonosis with global distribution, caused by a protozoan from the genus Cryptosporidium (Ederli, Carvalho, & Sales, 2004), which may infect different hosts, including mammals like humans; as well as birds, reptiles, amphibians and fishes (Thompson & Monis, 2012;García-Presedo et al., 2013;Santin, 2013;Bouzid et al., 2013;Slapeta, 2013).This is an opportunistic intracellular obligate parasite that completes its biological cycle in the surface of epithelial cells of the respiratory and gastrointestinal tract, being responsible for the syndrome of the aqueous diarrhea, dehydration, abdominal pain, weight loss, delayed growth and death (Rieux et al., 2013).These are considered not species-specific parasites, thus the transmission from different animal species to humans is possible to occur.The oocyst, the infecting stage, is released with the feces and remains steady for many months; being the fecal-oral considered as the main transmission path.Ingested by the host, the oocyst invades the epithelium, replicates, and through sequential reproductive cycles may result in the release of thousands of parasites per day via feces.The transmission is associated with water intake, food ingestion and contact with infected animals and/or humans (Smith et al., 2007;Dixon et al., 2011;Zucatto et al., 2015).morbidity varying from 10 to 85% due to C. parvum, especially among calves, with high morbidity rates when occurring in association with other infectious agents, deficient nutrition and immunosuppression (Fayer, Morgan, & Upton, 2006;Quadros et al., 2006;Thomaz et al., 2007).
In sheep, Cryptosporidium infection was first described in Australia, in animals from one to three weeks old exhibiting diarrhea (Barker & Carbonell, 1974;Dixon et al., 2011).Its role as a primary agent was confirmed in experiments performed in the beginning of 1980 (Snodgrass, Angus, & Gray, 1984).Causape et al. (2002) reported 59% prevalence of Cryptosporidium spp., in lambs with diarrhea, the prevalence was of 79.4% when compared with animals without diarrhea (22.4%).Santin, Trout, and Fayer (2007) reported prevalence of 25% and 77.4%, respectively, in sheep and lambs.Castro-Hermida et al. (2007) diagnosed 5.3% sheep eliminating oocysts of Cryptosporidium spp.
According Green, Amarante and Mascarini, (2004) the occurrence of Cryptosporidium was associated with rainfall, this authors reported that the highest prevalence of Cryptosporidium oocysts coincided with the rainy months of the year, while studying 184 samples obtained during the period of highest precipitation (more than 150 mm/month), when 102 (55.4%) animals showed Cryptosporidium oocysts, while only 31 (17.3%) of the 179 samples taken during the period with low rainfall (less than 100 mm/month) were positive.
In the State of Rio de Janeiro, Brazil, Santa Inês sheep were positive for Cryptosporidium spp.oocysts (Cosendey et al., 2008b).Vieira et al. (2008) observed that sheep during spring had higher values of EPG due to the increase of rain precipitation and average temperatures of 20 ºC, favorable to the nematodes.
The infection in humans and various animal species constitutes a public health issue, that is why, detection of this parasite in animal feces is important, due to the capacity of ruminants being a source of infection for humans (Ryan, Fayer, & Xiao, 2014;Paz e Silva et al., 2014).Due to the growing significance of cryptosporidiosis as a zoonotic parasitic infection, the present work had the objective to access the occurrence of coccidian of the genus Cryptosporidium and helminthes in fecal samples from sheep.

Study Area and Ethical Issues
The study was performed at the sheep's breeding sector from the Bom Jesus' Technical School (CTBJ) from the Federal University of Piauí (UFPI), Campus Professora Cinobelina Elvas in Bom Jesus, Piauí, Brazil, during a dry and rainy season in December 2011 and March and June 2012.
The region has a mean temperature of 27 o C and mean annual rainfall of 1,000 mm, with a dry season extending from May to November and a rainy season from December to April (Luz et al., 2014).The experiments were approved by the Ethics Committee on Animal Experimentation under the protocol number 034/2011.

Animals and Data Collecting
Fifty sheep from the Santa Ines' breed were randomly selected, being 20 male and 30 female individuals that were properly identified using rings or collars.Within the sample population the age of 24 ovine (10 males and 14 females) varied from 0-12 months old and 26 ovine (10 males and 16 females) from 13-48 months old.Samples were collected in two different periods of the year from all animals: once during the dry season and once during the rainy season.
The animals were growth on pasture under a semi-intensive system with the herd spending the day in paddocks of 50 hectares formed by mixed grazing Brachiaria decumbens and grass Andropogon (Andropogon gayanus cv.Planaltina), late in the afternoon animals were allocated in collective bails and fed with concentrated supplement with 40% bran corn, 15% soybean meal, 10% urea, 30% of salt and 5% limestone, mineral supplementation and water ad libitum.All animals were dewormed every four months with Dectomax.These animals were identified and the date was recorded in individual files.
Individual fecal samples were taken weekly, directly from the ampulla of the rectum and stored in plastic bags, in order to avoid contamination by free-living larvae (Cosendey et al., 2008a;Romero-Salas et al., 2016).Samples were properly conditioned in thermal bags containing ice in order to preserve samples until processing at the Laboratory of Veterinary Parasitology of the Federal University of Piauí, Brazil.

Parasitological Analysis of Faeces Samples
In order to evaluate oocysts of Cryptosporidium spp. the methods according Ritchie (1948), modified for oocyst's concentration and Ziehl-Neelsen (Henriksen & Pohlenz, 1981), modified for oocyst's staining, were used.In addition to identification, the number of eggs per gram of feces (EPG) were evaluated according Gordon and Whitlock (1939).(Ritchie, 1948) For the analysis, 1 g of feces in 4 ml of buffered saline solution was weighed, then this solution was filtered in folded gauze and transferred to 6 ml glass tubes, which were completed with saline solution up to the edge.Tubes were then centrifuged at 500 rpm for 8 minutes, in a Sislab/Tister centrifugue, then the supernatant was eliminated and tubes completed again with buffered saline solution before centrifuged at 500 rpm for 8 minutes.Then the supernatant was discarded and 3 ml of cooled ether was added, then each tube was vigorously homogenized for 30 seconds, and centrifuged again at 500 rpm for 8 minutes.

Formol-Ether Technique
Four layers resulted from this procedure: solvent, saline solution, fecal remains, and sediments containing oocysts of Cryptosporidium ssp., the supernatant was again discarded and fine smears were prepared with the remaining fecal residue from the bottom of the tubes.Smears were air-dried at room temperature and fixed in methanol for 3 minutes, similar to the procedure adopted by Cosendey et al. (2008aCosendey et al. ( , 2008b)).(Henriksen & Pohlenz, 1981) To stain the smears a carbol fuchsine solution was used for 20 minutes, followed by washing in running tap water, until the excess of fuchsine was removed, and then the alcohol-sulfuric acid solution was added for 1 minute until the excess of dye was removed, then the smears were finally washed again in running tap water and dried at room temperature.

Procedure of Ziehl-Neelsen Modified Staining
The smears were counterstained with methylene blue for 5 minutes, washed and air-dried at room temperature.Smears were observed with the aid of light microscope with a 100× objective and immersion oil, oocysts stained by fuchsine were shown with a pink to bright red color against a blue background.

Gordon and Whitlock (1939) Modified Method
In order to evaluate the number of eggs per gram of feces, 2 g of feces were weighted in a plastic cup.Then, with the aid of a graduated cylinder, 58 ml of a saline solution were added gradually to the homogenized feces to facilitate mixing.The homogenized was filtered into another cup using gauze and then vigorously homogenized with the aid of a glass rod.
The results obtained using both techniques were registered in spreadsheets and submitted to a Prevalence analysis using the software Microsoft Excel ® , to verify the results and create graphics.

Results and Discussion
Oocyts of Cryptosporidium spp.were detected in 50 samples from Santa Inês sheep, 20 males (50%) showed Cryptosporidium spp.oocysts and 10 animals (50%) were considered as negative.Among the 30 examined females, 8 (26.6%) had positive results for the protozoa in their feces and 22 (73.4%) had negative results (Table 01).Similar prevalence values were observed in previous studies, as in Paz e Silva et al. ( 2014) found 25 sheep positive for Cryptosporidium (25%) and Cosendey et al. (2008a)  Cryptosporidium spp. is a parasite that may infect humans, therefore an important international public health matter (Zucatto et al., 2015).In a previous research developed in Poland with 159 sheep, 16 animals were found parasitized by oocysts of Cryptosporidium using the Ziehl-Neelsen modified method (Majewska et al., 2000).In a similar study developed in Maryland, USA, with 31 sheep, a prevalence of 77.4% of C. parvum was observed using the PCR method (Santin et al., 2007).
In research accomplished in the Northeastern region of Spain by Díaz et al. (2015), 31.6%pre-weaning lambs were positive form a total of 171 animals tested, showing that Cryptosporidium is a prevalent enteric pathogenic agent widely distributed within small ruminants, similar to the results of 36% positive samples observed in the present study.This is probably resulting from the sanitary conditions of the facilities, especially when associated while working in the micro-region of Campos dos Goytacazes, in the state of Rio de Janeiro, Brazil, where the authors observed oocysts of Cryptosporidium spp. in 47% of the 130 animals examined.Tabela 1. Prevalence of male and female sheep parasited by protozoan of the genus Cryptosporidium and detected by the Ziehl-Neelsen modified technique