Occurrence of Cryptosporidium spp. and its association with ponderal development and diarrhea episodes in nellore mixed breed cattle

Article history Due to the increasing importance of cryptosporidiosis in animal productivity, this study was carried out to investigate the occurrence of Cryptosporidium spp. and its association with animal weight development and fecal consistency in crossbred Nellore cattle. Thus, fecal samples of 30 cattle were collected biweekly, and their growth was accompanied by weighing animals from birth to 210 days of age (June 2014 to May 2015). The modified Ritchie and Ziehl-Neelsen techniques were used to screen for the presence of Cryptosporidium spp. oocysts. The chi-square, Fisher exact, Tukey and Spearman’s correlation tests were used for statistical analysis. Oocysts of Cryptosporidium spp. were found in 69 (16.43%) of the 420 samples of cattle feces, and these parasitized animals presented a mean weight gain (4.76 kg) lower than that of non-parasitized individuals (10.58 kg) (p<0.05). The presence of this protozoan was detected in diarrhea and pasty stools (81.78%), indicating an association of the parasite with persistent diarrheal episodes (p<0.001). Among the bovines examined in this study, a higher occurrence of Cryptosporidium spp. was observed in lean diarrheic calves, which presented lower weight gain and poor productive performance. Received 19 September 2018 Received in revised form 12 December 2018 Accepted 17 December 2018


INTRODUCTION
Beef cattle ranching is an economic activity of fundamental importance for Brazil.Among the sanitary problems, parasitoses act directly lowering sector indexes, directly reflecting animal production.FEITOSA et al., 2008).
Among domestic animals, it occurs more frequently in cattle, especially in animals in the growing phase, whose morbidity cases by C. parvum varies from 10 to 85%, causing episodes of diarrhea and anorexia, resulting in low growth rates.These low growth rates are expressive when there is an association with other infectious agents, or in animals with deficient nutritional status and compliant immunosuppression (THOMAS et al., 2007).
Due to the increasing importance of cryptosporidiosis as a zoonotic parasitic infection and its association with animal productivity impairment, the objective of this study was to investigate the occurrence of Cryptosporidium spp.and its association with ponderal development in crossbred Nellore cattle, reared in the municipality of Alvorada do Gurguéia, Piauí state, Brazil.

Animals, study location, and ethical aspects
The present work was developed at the experimental farm of Alvorada do Gurgueia, which belongs to the Federal University of Piauí (UFPI), Campus Professora Cinobelina Elvas (CPCE) (08°22'39'' South and 43°51'34'' West), located in the municipality of Alvorada do Gurguéia, in the Sudoeste Piauiense mesoregion.This study was approved by the animal experiment ethics committee of the UFPI, under protocol number 034/11.

Data collection and experimental group selection
Data were collected from June 2014 to May 2015.A total of 14 samples were collected, eight in the dry period and six in the rainy season, in a 15-day interval between collections, totaling 420 samples.Seasonal variation between dry and rainy periods was set by the data from the meteorological station (INMET, 2018).
In this study, 30 crossbred Nellore cattle from a single herd were used, all born from multiparous cows.Data were collected from birth to 210 days of age.Prior to live-weight assessment, the animals were fasted at least 12 hours, in order to empty the digestive tract.
Data such as sex, age, weight, and stool consistency were observed and recorded in individual records at the time of collection.The latter variable was characterized into three classes: normal, pasty, and diarrheal.Nutritional status was also estimated through phenotypic observations of animal carcass conformation, attributing qualitative classifications as lean, medium, and fat, besides factors predisposing to infection.
Individual stool specimens were collected directly from the rectal bulb of animals, using plastic bags to avoid contamination with free-living larvae or other pathogens presumably in the environment.After collection, the specimens were identified and conditioned in an isothermal box at 4 °C.Simultaneously, the animals were weighed for ponderal development monitoring, during the 210 days of age, aiming to associate growth rates with parasitic infections.
The breeding system used in the farm was semiintensive, with herds spending the day grazing in 50hectare pastures, formed by Brachiaria decumbens mixed with Andropogon grass (Andropogon gayanus cultivar Planaltina).In the late afternoon, the animals were allocated in collective stalls with dirt floors, in which they were given concentrate supplementation containing 40% maize meal, 15% soybean meal, 10% urea, 30% common salt, and 5% calcitic limestone, as well as mineral supplementation and water ad libitum

Fecal parasitological analysis
Samples were maintained at a temperature of 4°C until processing.The concentration of Cryptosporidium spp.oocysts was measured by the modified technique of Ritchie (1948), and the staining of slides was made in duplicate by the modified Ziehl-Neelsen technique (HENRICKSEN;POHLENZ, 1981).

Statistical analysis
The results were submitted to the analysis of variance (ANOVA) by nonparametric statistics using PROC NPAR1WAY procedure (SAS, 1999) 2013), using molecular characterization, observed these parasites in only 5.3% (16/300) of the parasitized calves reared in the city of Formiga (Minas Gerais), which is smaller than that found in this study.
Figure 1 shows the climate data for the study location and period, including environmental temperature and monthly precipitation.During the experimental period, a clear dry season was observed from May to November and another rainy one from December to April.These data were gathered from the website of the meteorological station belonging to the National Meteorological Institute (INMET, 2018) (Figure 1).et al., 2013).
In this study, non-parasitized animals had a daily mean weight gain of 0.705 kg, similar to the finding of Cappelle et al. (2001) of 0.740 kg.However, our finding was superior to that obtained by Perotto et al. (2001), who observed a weight gain of only 0.339 kg.Such variation may be due to the rearing conditions and climatic differences between the regions.
We also found oocysts to be more frequent in diarrheal feces (55.55%) followed by pasty stools (26.23), indicating that this infection is associated with diarrheal episodes (p < 0.001) (Table 1).Safavi et al. (2011) found 36.6% (82/224) of fecal samples from calves to be positive for Cryptosporidium spp.These infected animals were less than one month old and, of these, 51.8% of them had diarrhea.The authors also highlighted that the highest infection rates were diagnosed in calves between 8 and 14 days of age.In short, these findings reveal a higher infection risk within the second week of life and that the peak oocyst excretion in calves with diarrhea is six times larger than that in non-diarrheic animals.Likewise, Asadpour et al. (2013) observed by microscopy that 28.3% (85/300) of calf fecal samples were positive for Cryptosporidium spp., and when analyzing fecal consistency, they found the all animals presenting positive samples were diarrheic.
Oliveira Filho et al. (2007) pointed out the protozoan Cryptosporidium spp. as one of the most worrisome enteropathogens of domestic ruminants.This organism leads to malabsorption syndrome, impairing food digestion and causing host diarrhea, as well as atrophy of microvilli and loss of digestive enzymes (LI et al., 2013).Feitosa et al. (2008) evaluated 57 positive animals for Cryptosporidium spp.and observed that 26.3% had or developed diarrhea during clinical follow-up, while 73.7% of the animals were asymptomatic carriers, demonstrating their importance as a primary source of environmental contamination and Cryptosporidium infection.
Nutritional status was compared between animals with and without parasite presence.Among the parasitized animals, 41% showed a poor nutritional status, 15.14% a medium nutritional status, and 14.81% were fat, showing a larger parasitic infection in lean animals (p < 0.001).This outcome might have occurred due to the negative influence of the parasite on the gastrointestinal tract of animals (SILVERLÂS et al., 2010).Conversely, Lima et al. (2013) diagnosed the occurrence of Cryptosporidium spp.infection, but found no significant effect when comparing positive animals and their body scores.
Several factors can affect animal nutritional status (such as weight development and gain).Among these factors are animal management type, climatic conditions, forage availability, and parasitic diseases (e.g., cryptosporidiosis).As previously reported, the action of these parasites causes food malabsorption, which can slow down growth, increase weight loss and, in some cases, lead to death (RIEUX et al., 2013;VARGAS JÚNIOR et al., 2014).
The occurrence of Cryptosporidium spp. was higher in younger animals, aged 01 to 90 days, among which a total of 21.66% (180) of positive samples were found.Moreover, the percentage of positive samples decreased (p < 0.05) as animal age increased (Table 2).In young calves up to three months of age, cryptosporidiosis can lead to severe diarrhea and cause morbidity and even death (CHAKO et al., 2010).infection and stool consistency, with a correlation coefficient of 0.50.This justifies the fact that pasty and diarrhea feces showed a higher presence of oocysts.Thus, it is extremely important to obtain data on the occurrence and distribution of Cryptosporidium spp.by region, as well as to gain knowledge about its seasonal occurrence, so that its epidemiology can be defined for future planning and establishment of preventive measures.

Figure 1 -
Figure 1 -Monthly averages of temperature and precipitation from June 2014 to May 2015 in Alvorada do Gurguéia, Piauí, Brazil.

Figure 2 -
Figure 2 -Microscopic appearance of Cryptosporidium spp.oocysts in fecal smears of crossbred Nellore calves (arrows) diagnosed by light microscopy (objective lens of 100x), using the modified Ziehl-Neelsen technique.
Rodrigues et al. (2016)s of bovines, 69 (16.43%) had oocysts of Cryptosporidium spp.A similar result was observed byRodrigues et al. (2016), working under the same conditions, these authors diagnosed parasites from this genus in 17.1% (26/152) of the fecal samples taken from cattle up to three months of age.Likewise, Silva  Júnior et al. (2011)found oocysts in 21.62% (77/356) of the calf stool samples collected from ten farms in the south of Minas Gerais.By contrast,Lima et al. (

Table 1 -
Number of fecal samples, total number and percentage of positive samples for Cryptosporidium spp., fecal consistency and nutritional status of Nellore cattle, diagnosed by modified Ritchie and Ziehl-Neelsen techniques.

Table 2 -
Total number of studied samples, positive samples for Cryptosporidium spp., and the relationship among age, sex, and sampling station of Nellore cattle diagnosed by modified Ritchie and Ziehl-Neelsen techniques.Cryptosporidium spp.presence, stool consistency, and animal nutritional status.A positive correlation (p < 0.05) was observed between Cryptosporidium spp.

Table 3 -
(CHAKO et al., 2010) coefficients among the evaluated variables in crossbred Nellore cattle from birth to 210 days of age.EC), contemporary group (GC), and nutritional status (EN).*Significantcorrelation(p<0.05).Diarrhea remains as one of the leading causes of morbidity and mortality in calves(CHAKO et al., 2010)since it is associated with dehydration, weakness, and eventual death.Vargas Júnior et al. (2014) warned of the importance of cryptosporidiosis as a primary agent of diarrhea in calves.Regarding management, preventive measures are needed to reduce economic losses, environmental contamination, and risks to public health.