Presence of Cryptosporidium spp. in calves from dairy herds in Northern Antioquia, Colombia

ABSTRACT Cryptosporidium spp. are important enteropathogen protozoan parasites that infect humans and other animals throughout the world. Cryptosporidiosis in cattle industry leads to considerable economic losses due to diarrhea, dehydration, growth retardation, weight loss, and possibly death, however, data on the occurrence ofCryptosporidiumspp. in cattle in Colombia are limited. Therefore, this study aimed to investigate the occurrence and possible factors associated to the excretion of Cryptosporidium spp. oocyst in pre-weaned calves from dairy farms in Northern Antioquia, Colombia. In addition, Sheather’s sugar floatation (SSF), and Modified Ziehl-Neelsen staining (MZN) methods were compared. A total of 41 fecal samples were collected from calves between 1 and 92 days of age of which 23 were positive (56.1%). Crossbreed calves were nine times less susceptible than purebred dairy cattle to excretion of Cryptosporidium spp. oocysts (OR=0.10). MZN was the best technique for the detection of oocysts in fecal samples, however, the mean number of days to detect cryptosporidial oocysts was lower for the SSF method. Cryptosporidium is widespread among calves under 2 months of age in dairy herds of Northern Antioquia, although further investigations considering a greater number of farms and animals are necessary.


INTRODUCTION
Cryptosporidium spp.are important protozoan parasites that infect a wide variety of vertebrates including humans and farm animals (Feng et al., 2018).Bovine cryptosporidiosis is caused by Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni, but only C. parvum is associated with clinical disease in neonatal calves (Thomson et al., 2017).The latter is widely endemic and is considered to be the major zoonotic reservoir for humans (Ryan et al., 2021).
A diagnosis of cryptosporidiosis is based on the identification of Cryptosporidium spp.oocysts in the fecal sample by conventional, immunodiagnostic, and molecular methods.Several conventional techniques such as formal ether concentration-sedimentation followed by floatation on Sheather's solution (SSF), and unconcentrated stools by microscopy of smears stained with either modified Ziehl-Neelsen (MZN) are routinely used for the diagnosis of cryptosporidiosis (Pena et al., 1997;Chalmers et al., 2011).
In Colombia (South America), there are a lack of knowledge about the epidemiology of Cryptosporidium in dairy calves.C. parvum has been reported in the major dairy regions of Colombia's provinces.In central Cundinamarca, Avendaño (2010) reported a prevalence of 22% (33/151) with MZN, and Pardo and Oliver (2012) reported a 38.6% (51/132) of prevalence with ELISA.In north Cundiboyacencian dairy herds a 7% (14/190) of prevalence with Heine stain was reported by (Montaño and Avendaño, 2012).Prevalence in the province of Caldas was 7.2% (11/80) with PCR (Ocampo et al., 2012).In Antioquia, the presence of C. parvum has been reported by Chaparro et al., (2014) who evaluated a cohort of 60 calves during the first month of life and found prevalence rates of 89.5% (51/57).
In general, data regarding the epidemiology of Cryptosporidiosis in dairy cattle in the province of Antioquia (Colombia) are still limited and only explored in the last 10 years, therefore, the aim of this study was to investigate the occurrence and potential risk factors associated to the excretion of Cryptosporidium spp.oocyst in pre-weaned calves from two dairy farms in Northern Antioquia, Colombia.In addition, Sheather's sugar floatation (SSF), and Modified Ziehl-Neelsen staining (MZN) methods were compared.

MATERIALS AND METHODS
All fecal samples used in this study were collected from cattle by veterinary surgeons with the permission of farm owners.The Bioethics Committee for Animal Investigation (CIBA) of the Corporación Universitaria Remington, Acta 08-2022, approved the procedures performed on the animals.
For the detection of Cryptosporidium oocysts in fecal samples, two techniques were used: 1) Sheather's sugar floatation method (SSF), and 2) Modified Ziehl-Neelsen staining technique (MZN).For the SSF technique, two grams of feces was taken and mixed in 10 ml of 10% formalin, and the suspension passed through a sieve (425 µm aperture size).To the filtrate was added 3 ml diethyl ether and the solution was shaken vigorously for 30 s and centrifuged at 500 x g for 3 min.The fluid and the fatty plug were discarded, and the pellet retained.To the sediment was added 10 ml of Sheather's sugar solution of 1.20 specific gravity; centrifugation was done at 500 × g for 3 min.Cryptosporidium Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.800-806, 2023 oocysts were observed under magnification (40x) for observation of internal morphology (Ferreira et al., 1962;Pena et al., 1997).The oocysts appeared as round or oval, refractile bodies with a thin cytoplasmic membrane and finely as pink granular cytoplasm (Figure 1A).
For the modified Ziehl-Neelsen staining (MZN), the protocol established by Casemore et al., (1984) was considered and some modifications were made.A smear of fecal material was made on each of the samples, dried at room temperature.Fixation and staining were done as follows: 1) fixation with methanol for 2 min (drying at room temperature), 2) staining with carbol-fuchsin for 5 min, (rinsing with tap water), 3) decolorization with alcohol acid 30 s (rinse with tap water), 4) counterstaining with methylene blue 1 min (rinse with tap water).The plate was allowed to dry at room temperature and was subsequently visualized under 100X objective with immersion oil.The oocysts appeared as round or oval, stained pink color (Figure 1B).For Statistical analysis, farm and animal information was obtained throughout the application of a questionnaire to the farmers.The data generated from the interviews (independent variables) and the oocyst presence was the (dependent variable) were recorded and analyzed by R-language.A univariate analysis was performed using Fisher's exact test (F²).The multivariate analysis was based on the logistic regressions in a model including the variables with p ≤ 0.25 identified in the association test.An alpha error probability of less than 5% (p < 0.05) was considered statistically significant.The measure of agreement between the conducted tests was determined by kappa and McNemar's test.Interpretation of the κ value was based on the following categories of agreement: <0.2 (slight), 0.2-0.4(fair), 0.4-0.6 (moderate), 0.6-0.8(substantial), >0.8 (excellent) (Munoz and Bangdiwala, 1997).

RESULTS
A sample was considered positive for Cryptosporidium if an oocyst was detected through the SSF or MZN techniques (Figure 1A, and 1B).Overall prevalence of Cryptosporidium spp.oocysts was 56.1% (23/41), with specific rates of 47% (8/17) at San Pedro, and 62.5% (15/24) at Entrerríos (Table 1).In the F 2 univariate analysis, age results exhibited values in which calves >2 mo were less prone to likely to be positive to Cryptosporidium when compared to younger animals, however these differences were not statistically significant (p > 0.05; Table 1).In relation to breed, the multivariate analysis verified that crossbred (Jersey x Holstein, Holstein x Gyr) animals were nine times less susceptible than purebred dairy cattle to acquiring infection (OR = 0.10; Table 2).In addition, statistical differences were not found in farm location and fecal consistency.The mean number of days to detection of cryptosporidial oocysts was 30 for the SSF method and 41.3 for the MZN method.
Varying numbers of samples were positive by each of the diagnostic Cryptosporidium technique (Table 3).Of the 43 samples, 10 were positive by SSF method, and 20 samples were positive by MZN.There was 56% overall agreement between the SSF and MZN methods of oocyst detection with a kappa statistic of 0.11.The McNemar test indicated the proportion of tests determined positive by the two detection methods was not equal (P<0.05)showed that MZN was the most effective technique for the detection of oocysts.(Chen et al., 2023).Likewise, high presence of C. parvum was found compared with previous studies in South America such as Argentina (Lombardelli et al., 2019), Uruguay (Caffarena et al., 2021) and northeastern Brazil (Conceição et al., 2021).
More recent studies carried out on calves in Colombia indicated less prevalence of C. parvum.From 74 dairy cattle farms located in four departments of central Colombia (including 2 farms in Antioquia), Cryptosporidium prevalence was found to be 26.6%(115/432).Oocyst shedding was recorded in calves aged 3day-old onwards, although the infection rate peaked at 8-14 days (40.7%) (Avendaño et al., 2018).In addition, our results indicated that dairy calves less than two months old are at more risk of a positive result compared with older calves.Age-related infection dependence of Cryptosporidium infection of calves has been recognized as one of the main risk factors (Santín et al., 2004;Kvác et al., 2006;Tiranti et al., 2011;Smith et al., 2014;Delafosse et al., 2015;Avendaño et al., 2018), however, this variable did not persist in the multivariable analysis.
In this study, purebreds were more susceptible to excreting Cryptosporidium oocysts when compared to crossbreed.Nevertheless, these results must be analyzed with caution, since many of the sampled animals belonged to the Holstein breed (79%), therefore, it would be inappropriate to compare whether there is a breed predisposition to the presence of Cryptosporidium.However, some studies consider that purebreds are more vulnerable to infection when compared to crossbreeds (Imre et al., 2015;Brainard et al., 2020).More studies with a larger number of samples from different breeds are necessary.
Diagnosis of Cryptosporidium spp.include different laboratories approaches.Although the detection and diagnosis of Cryptosporidiosis has many challenges including performance, cost, and time, microscopy techniques can boost reliance in the diagnosis (Abdou et al., 2022).The SSF method has shown good performance for Cryptosporidium spp.diagnosis in dairy calves (Rekha et al., 2016), small ruminants (Pavlović et al., 2020), even in humans (Manthalkar et al., 2017).Nevertheless, Danišová et al. (2016) considered PCR the gold standard reference test because PCR has high accuracy, however, a full screening of Cryptosporidium infection in fecal samples include the use of composite reference standard as a gold standard (Abdou et al., 2022).Although the SSF method requires reduced labor time when compared to MZN technique, the kappa agreement between MZN and SSF in this study was low.It could be explained because there is not a standard recommendation for the recovery of Cryptosporidium oocysts from samples with low infection (Weber et al., 1991).However, future studies may contemplate repeated examination of more than one fecal sample on three consecutive days enhancing the detection of Cryptosporidium oocysts.SSF technique is a rapid method of oocysts detection.Oocysts are easily identified and present as pink spherical bodies inside.This technique could be applied with other molecular and immunological assays for future studies in this dairy population.
Limitations of this study include the use of a convenience sample (selection bias).Nevertheless, due the lack of information around C. parvum infection in calves from the dairy region of Northern of Antioquia, our exploratory study demonstrates the first effort for determine the presence of Cryptosporidiosis in two municipalities in this dairy region.Further research is needed to understand the epidemiology of Cryptosporidiosis in calves in the northern of Antioquia, linking the role of C. parvum with animal and human health.

CONCLUSION
In conclusion, the high rate of presence of Cryptosporidium in two farms in northern Antioquia demonstrates the persistence of this protozoan in the country's dairy industries.This study identified that purebreds are more susceptible to crossbreeds when compared, however, studies involving a larger number of farms, and other potential risk factors are needed to determine appropriate measures that farmers and veterinary practitioners should take to reduce the prevalence of Cryptosporidium.

Table 1 .
Univariate analysis of risk factor for Cryptosporidium infection in calves

Table 3 .
A 2 x 2 contingency table for overall comparisons of Sheather's sugar floatation method (SSF) with modified Zielh-Neelsen technique (MZN) for detection of Cryptosporidium spp.