Occurrence and risk factors of bovine cryptosporidiosis in Brazil – Systematic Review

ABSTRACT Protozoa of the genus Cryptosporidium are parasites capable of causing animal rearing losses due to diarrhea outbreaks. This review aimed to determine the prevalence of and potential risk factors for bovine cryptosporidiosis in Brazil. The search was carried out in the databases Science Direct, Scopus, Web of Science, and SciELO. The search strategy used the descriptors “Cryptosporidium,” “cryptosporidiosis,” “cattle,” “calves,” and “Brazil.” Nine studies conducted in different Brazilian states between 2008 and 2019 were selected. The age of the evaluated animals ranged from 0 to >36 months. The main risk factors were the low age of the animals, collective pens, presence of diarrhea, contact with other animals, and dairy breed. The average prevalence rate was 30.51%. The species identified were C. ryanae, C. parvum, and C. bovis. Optical microscopy was used as a diagnostic method for oocysts in fecal samples and to determine the species, and PCR was used in three studies. The average prevalence of Cryptosporidium spp. in cattle in Brazil was 30.51%, ranging from 10.2 to 62.5%. The main risk factors found were young animals, the presence of animals with diarrhea, contact with other animals, dairy breeds, and collective pens.


INTRODUCTION
Protozoa of the genus Cryptosporidium are parasites of the gastrointestinal epithelium of vertebrates (Rieux et al., 2013), capable of causing considerable economic losses in animal rearing due to diarrhea outbreaks (Yap et al., 2016).Cattle may be infected with C. parvum, C. andersoni, C. ryanae, and C. bovis; however, only C. parvum is associated with clinical diseases in neonatal calves, whereas C. andersoni is more frequently found in adult cattle, and the clinical signs include a reduction in weight gain and milk production (Thomson et al., 2017).Parasitic diseases cause economic and productive losses (Schmidt et al., 2017) in addition to generating expenses for medicines, electrolyte replacement, fluid therapy, and hygiene measures (Graaf et al., 1999).Several factors may interfere with the occurrence of diarrhea outbreaks caused by Cryptosporidium spp., such as the type and hygiene of the buildings, animal age, breed (Ayele et al., 2018), and season (Kashyap et al., 2021).The prevalence of Cryptosporidium spp. in dairy calves organized in groups was higher (45%) than that in calves in individual pens (20%) (Åberg et al., 2019).In a study in Ethiopia, Ayele et al. (2018) noted a higher prevalence of this parasite in dairy calves under six months of age (28.4%) than in calves aged 6-12 months (15.8%).Cryptosporidium spp.may be found at all ages, and although calves under one month of age are the main carriers of C. parvum, adult cattle also play an essential role in cryptosporidiosis outbreaks, both in calves and humans (Díaz et al., 2021).Cryptosporidium oocysts are robust and have a high survival capacity in soils and watercourses.From the moment oocysts are excreted in the feces, they may move to the ground and along the soil column to reach underground waters (Armon et al., 2016).According to Jenkins et al. (2002), oocysts may survive for months in agricultural soils, and consequently, have the potential to contaminate surface water.The main source of oocysts is cattle grazing in the riverside areas.Oocysts that reach watercourses can be transported over long distances and get disseminated to animals and humans.Sunohara et al. (2012) observed an 88% reduction in oocyst load in the watercourse when the animals were kept with restricted access to water and a 38% reduction when the animals had unrestricted access to water.This systematic review aimed to determine the occurrence and risk factors of bovine cryptosporidiosis in Brazil.

Search strategy
A search was performed in January 2022 to retrieve relevant studies from the Web of Sciences, SciELO, Scopus, and Science Direct databases, using the Descriptors in Health Science (DeCS/MeSH) AND and OR Boolean operators.To identify the search terms, we used the CoCoPop format (Munn et al., 2018)

Eligibility of the studies and inclusion and exclusion criteria
Two researchers evaluated titles and abstracts using the Rayyan platform (Ouzzani et al., 2016).The selected 3apers were evaluated based on the inclusion and exclusion criteria.The inclusion criteria were: (1) crosssectional studies, (2) carried out in Brazil, (3) about the occurrence of Cryptosporidium in cattle, (4) peerreviewed studies, (5) open access, and (6) published between 2011 and 2021.
The exclusion criteria were: (1) thesis and dissertations, (2) case studies, (3) editorials, (4) duplicate studies, and (5) studies published before 2011.Discrepancies in the selection process were resolved by the research team.

Data extraction of the selected studies
Next, the papers were read by two researchers for a new evaluation based on the eligibility criteria, and the following data were extracted: authorship, publication year, state of the country, population (beef or dairy cattle), animal age, risk factors, size of the total samples and of positive samples, identified genus and species, and diagnostic method.

Assessment of the study quality
The risk of bias among the included studies was evaluated using a quality assessment checklist adapted from a study by Ding et al. (2017).The checklist comprised five items that were scored based on a scale system (2 = yes, 0 = no, or 1 = unsure).The studies were ranked as high, moderate, or low based on their analysis.

RESULTS
Nine studies met the inclusion criteria and were included in this systematic review (Table 1).The steps in the selection process are presented in a Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart (Figure 1).
The average prevalence rate was 26.3%.Only three studies identified Cryptosporidium species in feces.C. ryanae (2/9), C parvum (3/9), and C. bovis (1/9), indicating a higher prevalence of C. ryanae and C. parvum infections.It is important to emphasize that these studies involved animals under 24 months of age, and C. parvum was identified in all animals.Optical microscopy was used to diagnose the presence of oocytes in feces in all studies.PCR was used to determine the species in three studies (Table 2).

Assessment quality of eligible studies
The results of the quality assessment of the eligible studies are presented in the Supplementary Table 1.All studies were of acceptable quality, except for the study by Volpato et al. (2017) because the authors neither presented the period of the study nor categorized the animals into subgroups.

Occurrence of bovine cryptosporidiosis
Among Brazilian regions, the southeast region led in milk production in 2019 with 34.35%, followed by the 8hey8 (34%), northeast (14%), midwest (11.6%), and north (6.1%).Bovine cryptosporidiosis occurs in most states in the southeastern and south regions, where higher milk production is concentrated (CNA, 2020).Nine studies reported the occurrence of oocyte excretion in dairy herds, and in one of them (Holsback et al., 2018), the authors reported the occurrence in beef herds, indicating the higher susceptibility of dairy breeds to Cryptosporidium.Dairy cattle are subjected to subpar conditions of welfare compared to beef cattle.Due to the separation of the calves from their mothers, not having free access to the pastures, and the daily milking as an intervention in the animal's life (Silverlås et al., 2009;Mandel et al., 2022).Animals allocated to pens with a considerable number of animals, where hygiene is precarious, are more susceptible to infection because the contamination of the animals is facilitated by quagmire, feces, and urine.In addition, this type of environment favors the presence and survival of oocytes, with possible contamination of drinkers and feeders, causing the animals to be infected when they ingest feed or water (Ayele et al., 2018;Thomson et al., 2019).

Age of the affected animals
We found that young animals are predominantly affected.Among the Cryptosporidium-infected animals, 62.6% were 7-28 days old (Oliveira et al., 2021), 10-15% were between 8-15 days of age (Cruvinel et al., 2020), 26% were 0-30 days of age, 37% were 30-60 days old, and 36% were >60 days old (Conceição et al., 2021), 35% were >46 days of age (Volpato et al., 2017), and 25.3% were 0-2 months old (Toledo et al., 2017), corroborating the findings of Garro et al. (2016Garro et al. ( , 2021) ) and Ayele et al. (2018).The higher occurrence of cryptosporidiosis in younger animals is because they possess a less-developed immune system facilitating the infection (Ayele et al., 2018).In addition, newborns must adapt to a new environment with a diversity of pathogens, adjust the way of receiving nutrients from the umbilical cord to bottles or buckets, and then become ruminants.These changes generate a significant stress load in the animals (Osorio, 2020), reducing their immunity (Hulbert & Moisá, 2016).However, resistance to infection commonly develops with age owing to the maturation of the immune system (Kvac et al., 2006).

Risk factors associated with the occurrence of bovine cryptosporidiosis in Brazil
As mentioned before, young age has also been considered a risk factor for cryptosporidiosis (Toledo et al. (2017);Hoslback et al. (2018);Oliveira et al. (2021).In 33.33% of the studies, animal age was described as a risk factor for cryptosporidiosis.Younger animals are more likely to be infected by Cryptosporidium (Díaz et al., 2021).Silva et al. (2011), Volpato et al. (2017), and Cruvinel et al. (2020) reported collective pens as a risk factor.In collective buildings, the contact among animals is higher, resulting in a greater probability of infection.Couto et al. (2015) reported that only 8.5% of calves reared in individual pens, with high feeders and drinkers, showed oocytes in their feces, compared to the 21.9% of calves reared in collective pens, with low feeders and drinkers, showing oocytes in their feces.Animals with diarrhea contribute to the dissemination of Cryptosporidium in the environment, as mentioned by Toledo et al. (2017) and Cruvinel et al. (2020).This pathogen is strongly associated with diarrhea and economic losses.The incidence of infection is higher in animals with diarrhea than in those with normal feces (Garro et al., 2016(Garro et al., , 2021;;Ayele et al., 2018).This protozoan causes atrophy of the villi, which results in a smaller intestinal absorption surface and causes diarrhea due to the nonabsorption of compounds such as glucose, water, and sodium (Radostits et al., 2007).The contact of calves with other domestic animals is another risk factor, as demonstrated by Volpato et al. (2017) and Conceição et al. (2021).Cryptosporidium may be transmitted from an infected animal to a healthy one, and contact between animals, even from different species, may facilitate parasite dissemination owing to the low specificity of the protozoan (Clode et al., 2015;Ayele et al., 2018).
Dairy breeds are highly susceptible to cryptosporidiosis (Toledo et al., 2017;Holsback et al., 2018).The lower immune response in dairy breeds, mainly in high-yielding dairy cattle, can be either primary, associated with genetic selection for high milk production, or secondary to metabolic stress during the transition period (Bronzo et al., 2020;Vlasova and Saif, 2021).Some researchers have observed that the milk supply, separation of calves from their mothers, consumption of water from rivers, ingestion of colostrum for short periods, co-infections, and poor hygiene in buildings may be risk factors for bovine cryptosporidiosis (Silverlås et al., 2009;Garro et al., 2016;Ayele et al., 2018;Brainard et al., 2020;Ebiyo & Haile, 2022).

The prevalence rate of bovine cryptosporidiosis in Brazil
The prevalence rate varied from 10.2% in the study conducted by Toledo et al. (2017)

Diagnostic methods of cryptosporidiosis
Optical microscopy and PCR were used in nine of the included studies.Cryptosporidium can be diagnosed using several techniques, including optical microscopy of fresh material and smear staining.Immunological and DNA detection methods are also available (Khurana & Chaudhary, 2018).Optical microscopy is the most commonly used method in veterinary medicine owing to its cost-effectiveness.PCR technique has been used (Toledo et al., 2017;Agnol et al., 2021;Oliveira et al., 2021).This method indicates the presence of the parasite, but cannot be quantified (Thomson et al., 2019).The diagnostic method must be appropriately selected because a method with low sensitivity can lead to an underestimation of protozoan presence; however, PCR is useful for identifying Cryptosporidium species.

Conclusions
The average prevalence of cryptosporidiosis in Brazil obtained from the included studies was 30.51%, varying from 10.2 to 52.9%.The main risk factors were young age, the presence of an animal with diarrhea in the herd, contact with another type of animal, dairy breed, and collective pens.

Figure 1 .
Figure 1.Flow diagram of the selection process of studies for the systematic review on bovine cryptosporidiosis in Brazil.
to 52.9% as observed by Oliveira et al. (2021), with an average prevalence rate of 26.3%.Agnol et al. (2021) used a small sample size (n = 8), which may have caused an overestimation of the prevalence rate.Several factors influence the prevalence rate, such as farming type (beef or dairy), animal age, country region, climate, season, management techniques, well-being, and diagnostic methods (Hatam-Nahavandi et al., 2019).The highest prevalence rates were obtained by Cruvinel et al. (2020) and Oliveira et al. (2021), with animals aged 1-135 days (51.5%) and 1-60 days of age (52.9%), respectively, indicating a higher susceptibility of younger animals.

Fagundes
et al. (2014)  verified a prevalence rate of 13.2% in animals up to 79 days of age; however, in this study, the animals were kept in individual pens, which may have contributed to the low Cryptosporidium prevalence.

Table 2 .
Prevalence rate, identified species, and diagnostic methods of Cryptosporidium , 90% of Cryptosporidium infections in cattle are caused by C. parvum and C. bovis.According to Santín et al. (2008), C. parvum was detected in 97% of preweaned calves examined at 2 weeks of age, while the initial detection of C. bovis occurred in calves at 4 weeks of age, with a peak occurrence at 16 weeks of age.C. andersoni was not found until the 19th month of age, representing 25% of infections in this age category.Recently, Díaz et al. (2021) observed the predominance of C. parvum in calves up to one month of age, C. bovis in animals between 1 and 24 months of age, and C. ryanae in calves aged between 2 and 12 months.