Introduction

Cryptosporidium spp. are protozoan parasites that cause gastrointestinal infections in a wide variety of vertebrate hosts, including domestic and wild animals as well as humans (^{Ramírez et al., 2004}; ^{Cacciò and Pozio, 2006}; ^{Ng et al., 2012}). In cattle, at least four species have been identified, including C. parvum, C. andersoni, C. bovis and C. ryanae; the first species listed is found most frequently in suckling animals, while the others are more common in weaned and adult animals (^{Lindsay et al., 2000}; ^{Fayer et al., 2005}, ²⁰⁰⁷, ²⁰⁰⁸). Cryptosporidiosis is particularly important in suckling animals that are younger than 30 days of age, in which abundant diarrhea, fever, anorexia, weight loss, electrolyte imbalance and eventually death can be observed (^{Ramírez et al., 2004}; ^{Cacciò and Pozio, 2006}). The group with the highest risk to contract an infection are calves 8 to 14 days of age (^{Castro-Hermida et al., 2002a}; ^{Santín et al., 2004}; ^{Feitosa et al., 2004}; ^{Trotz-Williams et al., 2005}), because the prevalence and parasitosis intensity tend to be reduced when the animals are older (^{Castro-Hermida et al., 2002b}; ^{Ortolani and Soares, 2003}; ^{Starkey et al., 2006}; ^{Castro-Hermida et al., 2006}; ^{Bhat et al., 2013}). However, infection is very rare in animals younger than four days (^{Uga et al., 2000}).

Animals infected with Cryptosporidium spp. may excrete feces with a high number of oocysts that may be easily disseminated in the environment in such a manner that different management factors have been associated with infection by Cryptosporidium spp. These factors should be considered in prevention programs for this parasite, particularly because there is a lack of effective pharmacological treatments (^{Cacciò and Pozio, 2006}). Nevertheless, some of these factors vary across regions and in their production systems and geographical characteristics, while others may be present in any farm. Among the most frequently cited factors are age, multiple birthing areas, a low frequency of cleaning of the premise, the materials or types of bedding, the use of contaminated water for the cattle to drink, the technical level of the production system, sand-floor pens and the rainy season (^{Maldonado et al., 1998}; ^{De la Fuente et al., 1999}; ^{Mohammed et al., 1999}; ^{Castro-Hermida et al., 2002b}; ^{Trotz-Williams et al., 2007}; ^{Brook et al., 2008}; ^{Almeida et al., 2010}; ^{Muhid et al., 2011}; ^{Silverlås and Blanco-Penedo, 2013}; ^{Bhat et al., 2013}). In Mexico, cryptosporidiosis has been reported in several cattle raising regions (^{Maldonado et al., 1998}; ^{Castillo-García et al., 2009}; ^{Castelan-Hernández et al., 2011}; ^{Cano-Romero et al., 2011}), but numerous topics still required research regarding its epidemiology in the different production systems.

The aim of this study was to identify the prevalence and some of the risk factors associated with infection by Cryptosporidium spp. in suckling calves maintained in confinement in three dairy farms in Aguascalientes, México.

Materials and methods

Survey

To establish the hypothetical risk factors associated with infection by Cryptosporidium spp., data were obtained from a survey given to the people responsible for the rearing areas in the studied dairy farms; this survey asked questions regarding the characteristics of the facilities and management practices. Five groups were formed with the hypothetical risk factors, and each group was integrated with different variables and their respective categories; this was used as a basis for the risk analysis (Table 1).

Table 1 Cryptosporidium spp. infection prevalence for the different categories of each variable and the group of characteristics under study. 

Results and Discussion

The general prevalence of Cryptosporidium spp. infection in the studied population was 40% (669/1658). Table 1 shows the prevalence for each category within the different variables.

The risk analysis identified 5 variables as risk factors for infection by Cryptosporidium spp., with the variable of the age of the calves being the most significant, particularly in the group of calves aged 8 to 14 days (OR=15.2; 95% CI 11.2 - 20.6; p<0.000), followed by the group of calves aged 15 to 21 days (OR= 2.5; 95% CI 1.9 - 3.2, p<0.000). The other variables that were identified as risk factors were related to the person in charge of the care of calving, the level of immunoglobulins, the type and hygiene of the bedding and/or floor of the lodging area, and the type of container in which the milk or milk substitute was given. Table 2 shows the categories identified as risk factors. None of the variables related to the operation and administration of the dairy farms were shown to be risk factors. In the variable related to the person in charge of the care of calving, the technician on guard proved to be a protective factor (OR= 0.73; 95% CI 0.5828 - 9237, p<0.008).

Table 2 Risk factors for infection by Cryptosporidium spp. identified in three dairy farms in Aguascalientes, Mexico. 

In this study, the general prevalence of infection by Cryptosporidium spp, in suckling calves (≤28 days of age) was found to be 40%, while in a study conducted in the same state that included animals from 8 to 14 days old, the prevalence of infection was found to be 75% (^{Castillo-García et al., 2009}). In the dairy farms of Aguascalientes, the abundant presence of the parasite is clearly evident. A similar situation has been observed in the central region of Mexico (Hidalgo, Jalisco and the state of Mexico) and in the state of Veracruz, where other authors have reported general prevalences of 25, 35.7 and 73.6%, respectively; they have also found positive animals in at least 90% of the studied production units (^{Maldonado et al., 1998}; ^{Castelan-Hernández et al., 2011}; ^{Cano-Romero et al., 2011}). However, the public health implications of zoonotic transmission in Mexico are uncertain because very few studies regarding human criptosporidiosis have been conducted (^{Valenzuela et al., 2014}). Several studies on the cryptosporidiosis prevalence in suckling calves have been carried out in cattle raising regions of other countries using different diagnostic techniques, with a wide variety of results ranging from a prevalence of 17 to 47.9% (^{Lefay et al., 2000}; ^{Castro-Hermida et al., 2002a Joachim et al., 2003}; ^{Ortolani and Soares, 2003}; ^{Santín et al., 2004}; ^{Kváč et al, 2006}; ^{Brook et al., 2008}; ^{Bhat et al., 2013}); this testifies to the ample geographic distribution and elevated frequency of this parasite.

Infection by Cryptosporidium spp. in dairy cattle is more frequent and has a greater impact in suckling calves; additionally, the magnitude of the disease is influenced by diverse factors such as age and the immunological status of the calves as well as the climate and other factors related to management practices during rearing. Therefore, the epidemiology of this parasite has specific characteristics for each unit and production system (^{Fayer et al., 2000}). In this study, calves that were 8 to 14 days of age had a Cryptosporidium spp. infection prevalence of 81%; this was the highest of the 4 age groups investigated in this study. This result is similar to a previous report in Aguascalientes, in which the same age group had a 75% prevalence rate of infection (^{Castillo-García et al., 2009}); in the other 3 age groups, the parasite prevalence rate ranged from 21 to 40%. In Mexico, a higher prevalence has been reported in calves under 4 months of age (^{Cano-Romero et al., 2011}; ^{Castelan-Hernández et al., 2011}), with a higher rate of oocyst excretion between days 15 to 19 after birth (^{Maldonado et al., 1998}). The prevalence according to age group is variable in suckling calves; nevertheless, it has been shown that the calves are the most affected between 8 and 15 days of age (^{De la Fuente et al., 1999}; ^{Ortolani and Soares 2003}; ^{Santín et al., 2004}, ²⁰⁰⁸; ^{Feitosa et al., 2004}; ^{Avendaño et al., 2010}). This coincides with the fact that in this study, the 8- to 14-day-old age group was identified as the most important risk factor for infection by Cryptosporidium spp. (OR = 15.2), followed by the 15- to 21-day-old age (OR = 2.5). Other authors have found similar results (^{Maldonado et al., 1998}; ^{De la Fuente et al., 1999}; ^{Santín et al., 2004}; ^{Trotz-Williams et al., 2007}; ^{Brook et al., 2008}), which indicates that the age range of 8 to 21 days has the highest risk of infection and excretion of parasite oocysts, although this risk is reduced as the age increases (^{Maldonado et al., 1998}; ^{Santin et al., 2004}, ²⁰⁰⁸), as observed in our study. It is inferred that contamination with Cryptosporidium spp. occurs after the calves come into contact with other animals, the initiation feeder and drinking water. As ^{Maldonado et al. stated (1998)}, the indicated initiation rations that are contaminated by rodents or birds act as fomites and become risk factors; similarly, drinking water has been referred to as a contamination risk due to contamination of either the liquid ducts (^{Sischo et al., 2000}) or the drink containers (^{Almeida et al., 2010}). Thus, age should be considered a risk indicator because it is an individual trait (^{Silva-Ayçaguer, 2005}).

The variable represented by the person who cares for the births of the off spring, was identified as a risk factor when the births were overviewed by a night watchman or when calving was overseen by the same worker who took care of the nursing area. In the instances in which the calving was overseen by the technician on guard (OR = 0.73), this variable was not a risk factor but served as protection because the main job of the technician on guard is precisely to care for night births and calving on holidays. Similarly, when births under similar circumstances were cared for by the area worker or the custodial staff, it is inferred that the care is not as appropriate because in addition to caring for births, these individuals have other chores to perform; information on this topic was not found in the literature.Concerning the type and hygiene of the bedding and/or floor of the lodging area, the sawdust bedding category that was changed every 5 days was identified as a risk factor (OR = 1.80), perhaps because of the physical characteristics of the material: it tends to accumulate humidity and excrement, which is ideal for the parasite oocysts to survive and contaminate the material. In contrast, ^{Maldonado et al. (1998)} and ^{Mohammed et al. (1999)} did not find significant relationships between parasitosis and wood shavings serving as bedding. In this context, it seems that independent of the type of bedding, the cleaning frequency has a greater influence because longer time periods between cleanings promote the development of the parasite in the feces and the possibility that the calves or their feed have contact with that material. Similarly, floor slat lodgings and pens with sand bedding were reported as risk factors (^{Muhid et al., 2011}). In both cases, the presence of discarded fecal matter, as occurred in our study, represented a source of oocyst contamination.

Regarding the type of container used to provide the calves with milk or milk substitute, plastic materials were found to be a risk factor in this study (OR = 1.4). This could be related to deficient washing because the plastic materials may be splashed with diarrheic feces that are most likely contaminated with the parasite; the containers are then licked by the calves, thereby causing infection. This is feasi ble because even though the calf lodgings in the studied dairy farms are individual, they do not offer total isolation; therefore the calves may lick the containers of their neighbors at any time. Further, ^{Almeida et al. (2010)} reported a significant association of the water supply tray with the risk of infection by C. parvum; in this case, they noted that liquid was an important reservoir of protozoan oocysts when contaminated with fecal matter. However, ^{Maldonado et al. (1998)} demonstrated that no type of container or feeding bottle presented a risk of infection by ryptosporidium.

The prevalence of infection was higher in animals that were vaccinated against diarrheic diseases; ^{Trotz-Williams et al (2008)} found a similar result in both cows and calves that had received such prophylaxis. Therefore, vaccination has been perceived as a risk factor for infection by Cryptosporidum. However, ^{Trotz-Williams et al (2008)} noted that vaccination is not as much a risk factor as it is a measure taken in response to a high frequency of calf scours because vaccines marketed for calf scours have no known impact on the risk of cryptosporidiosis. In fact, vaccines would be ineffective against scouring caused by Cryptosporidium infection.

In this study, the analysis of the level of immunoglobulins as a risk factor was conducted separately from the general model because the test was performed only in calves that were 1 to 7 days of age. Of these calves 61.7 % had insufficient levels of immunoglobulins and the immunoglobulin levels were therefore considered a risk factor (OR = 1.85). It was not possible in this study to establish a relationship between the level of immunoglobulins in the calves and the birth number of the mother, though there is some evidence that this has an influence on the concentration of G immunoglobulin in colostrums (^{Kehoe et al., 2011}). Another study has evaluated the level of immunoglobulins in nursing calves and found serum protein values below 5.2 g/dL; these values were significantly related to infection by Cryptosporidium spp. (^{Trotz-Williams et al., 2007}). Because cryptosporidiosis is associated with young and immunosuppressed animals, these results suggest that for the calves examined in this study, the colostrum feeding process was not sufficiently effective because newborns require the ingestion of colostrums in the first 6 hours of life to guarantee the intestinal absorption of immunoglobulins; additionally, serum amounts of antibodies below 10 mg/mL may cause up to 60% mortality caused by gastrointestinal infections (^{Weaver et al., 2000}). In the dairy farms included in this study, the offspring were reported to consume 4 or more liters of the colostrums, but there were no data proving that the first take of colostrums occurred within the first 6 hours after birth; therefore, deficiencies in the postnatal feeding process are inferred.

Despite the present findings it must be noted that this study was undertaken in only 3 dairy farms; therefore, these results may not be generalizable to other farms in Mexico.

Conclusions

In this study, the age of the calves is reported to be a risk factor for acquiring infection by Cryptosporidium spp.; in particular, there was an association between infection and the age group of 8 to 14 days that was higher than the results reported in other studies. Therefore, the calving and the care of calves during birth and nursing should be performed by personnel who are dedicated exclusively to those chores to ensure optimum feeding with colostrums; this would also ensure an appropriate immunoglobulin level that may protect calves from infection by this parasite and other diseases. The results of this study suggest that cleaning the lodgings of the calves should be more stringent, particularly when using sawdust, and that feces and humidity should be removed constantly to avoid parasite proliferation. Additionally, containers for drinking milk or milk substitute should be cleaned perfectly to avoid the risk of infection because it seems that type and frequency of cleaning the premises and equipment is more important than the materials that are used.