Prevalence and risk factors of coccidiosis in calves from Veracruz, México

The objectives of the present study were: (1) to determine the prevalence of Eimeria spp. sporulated oocysts in calves from 26 Municipalities in the Central Zone of the State of Veracruz, Mexico, (2) to identify the Eimeria spp. infecting calves, and (3) to identify the risk factors associated with the presence of Eimeria spp. in tropical cattle. A total of 930 individual fecal samples were analyzed by using the McMaster technique; then, oocysts were maintained in 2.5% potassium dichromate to allow sporulation. The general prevalence of calves with Eimeria spp. oocysts was 39.7% (370/930). Of a total of 10 identified species, Eimeria canadensis was the most observed, followed by Eimeria bovis and Eimeria zuernii . The statistical analysis showed an association between the age of the calves (5 to 9 months), the presence of other animals, the physiographic regions and the restricted type of husbandry with the presence of Eimeria spp. in calves (P<0.05). Protective risk factors, such as: routine coprological analysis was also associated with a decrease in infection. These data demonstrate the presence of coccidia in cattle from the State of Veracruz, additionally to the necessary measures that must be taken to control this parasitosis.


Introduction
Bovine coccidiosis is a gastrointestinal disease associated with apicomplexans of the genus Eimeria, where at least 21 species have been reported in cattle around the world (Lee et al., 2018). Coccidiosis is one of the most economically important among diseases in the livestock industry that causes both clinical and subclinical losses, especially in young cattle (Lopez-Osorio et al., 2018). Clinical infection may result in diarrhea, anorexia, weakness, dehydration, and occasional deaths (Tomczuk et al., 2015); whereas subclinical diseases are defined by weight loss, reduced weight gain, and loss of appetite, resulting in potentially long-lasting effects (Fitzgerald, 1980). Subclinical cases are more common and are proposed to quietly disrupt intestinal physiology, thereby resulting in high feed conversion (Ekawasti et al., 2022). The control of coccidiosis in the herd consists of reducing the parasitic load by improving the hygienic measures of the cattle habitat, as well as stress management and colostrum feeding, among other activities (Bangoura & Bardsley, 2020); and/or the use of chemical drugs to inhibit the life cycle as well as eliminate the etiological agents responsible for the disease (Philippe et al., 2014).
The presence of coccidiosis on farms and in individual animals can be influenced by a wide group of intrinsic and extrinsic factors (Tomczuk et al., 2015). Perhaps the risk factor most associated with coccidiosis in cattle has been the age of the host. Young animals (less than 1 year old) are more susceptible to coccidiosis, as well as the presence of animals of different ages that are housed in the same place (Hermosilla et al., 2012). Alcala-Canto et al. (2020) mentioned that macroenvironmental variables such as temperature and rainfall influence the presence of Eimeria spp. in cattle and, for some species, different factors such as: rearing system, type of installation, farm size, seasonality and altitude affect the occurrence of coccidiosis. The presence of Eimeria spp. is attributed to subtropical zones with humid -sub-humid climates, the size of the UPB, as well as the feeding type of the animals. Although in Mexico, the State of Veracruz has climatic and management factors that favor the occurrence of Eimeria spp. in cattle, studies at farm level are scarce (INEGI, 2018). The presence of coccidiosis in livestock production units in Mexico represents economic losses of up to $23.7 million dollars per year (Rodríguez-Vivas et al., 2017).
Epidemiological information on the presence of Eimeria spp. in tropical livestock farms is a basic step in building an efficient coccidiosis control program and improving health and well-being. Likewise, updated information is necessary on the species of Eimeria that are circulating in Veracruz. Therefore, the objectives of the present study were: (1) to determine the prevalence of Eimeria spp. oocysts in calves from 26 Municipalities in the central zone of the State of Veracruz, Mexico, (2) to identify Eimeria spp. infecting calves, and (3) to identify the risk factors associated with their presence.

Ethical considerations
The work was approved by the Bioethics Committee of Faculty of Veterinary Medicine and Zootechnics, University of Veracruz, under the protocol n. 007/21.

Study area and sample size
A cross-sectional study was carried out on 62 production units through 26 Municipalities of the physiographic region of Sotavento, Las Montañas, Papaloapan, Capital and Nautla (from August 2020 to April 2021). These areas belong to the central region of the State of Veracruz, where there is a humid tropical climate with an average annual temperature of 23.4 ± 0.5, an annual rainfall of 1,991 ± 392 mm, and a mean relative humidity (RH) of 85% (INEGI, 2018).
The sample size (n = 367) was calculated using an expected prevalence of Eimeria spp. of 60% (Alcala-Canto et al., 2020), an animal census of 91,589 calves (México, 2018), a confidence level of 95% and statistical error of five (Thrusfield, 1995). The selection of cattle farms and animals were for convenience (Thrusfield, 1995). Due to a greater participation of cattle producers in the study, as well as a greater number of animals within each cattle production unit, the sample size (n) increased to 930 calves.

Animals and sample collection
For each livestock farm, young animals and calves were selected and assigned to three age groups: ≥1 -4, 5 -9 and 10 -13 months (with or without the presence of clinical signs of coccidiosis). Calves dewormed 15 days before the sample collection were excluded from the study.
At least five grams of feces were collected directly from the rectum of each calve in order to perform the parasitological tests mentioned below. The feces were identified by number or name, age, and sex and transported in a plastic cooler (4-5•C) to the Parasitology Laboratory of the University of Veracruz, where they were kept in refrigeration at 4º C for 24 hours before analysis.

Laboratory analysis
The fecal oocyst count per gram of feces (OoPG) was determined using McMaster technique according to Taylor et al. (2016). Positive samples ≥ 500 OoPG were subjected to a sporulation process, using 2.5% potassium dichromate in Petri plates and incubated at room temperature, followed by oxygenation of the samples every 24 hours for ten days (Cruvinel et al., 2018). The oocysts were isolated by flotation in sugar solution (specific gravity 1.27) (Florião et al., 2016), and approximately 10 oocyst per pool from each farm were identified under an optical microscope using a Velab VE-87 (Puebla, Mexico) x 1000 magnification. This identification was done according to the phenotypic characteristics of the sporulated oocysts according to Taylor et al. (2016) (Table 1). Questionnaire of the Bovine Production Unit A survey was applied to the managers of the Bovine Production Unit, which contained dichotomous and multiplechoice answers, to obtain information by animal and management practice in the unit. Table 2 shows the variables studied for the analysis of risk factors.

Statistical analysis
Prevalence of cattle parasitized with Eimeria spp. was calculated using the following Formula 1: . / Prevalence No of animals withcoccidiosis total number of sampled animals = Associations between independent and dependent variables were, first, analyzed using univariate analysis with Chi-square (χ 2 ) test with Fisher correction. Posteriorly, all the variables with a significance value of (p) ≤ 0.20 with χ 2 test, were entered in a logistic regression model (Stata program Version 14.0, Texas, USA), which provides exact regression estimates, 95% confidence intervals (CI 95% ), odd ratios (OR: a measure of association which quantifies the relationship between exposure variables and outcomes), p-values (P), standard error (SE) and a beta value (regression coefficient).

Results
Prevalence of Eimeria spp.  Table 3). Images on sporulated Eimeria spp. oocysts are presented in Figure 1. The image of E. bukidnonensis was not displayed due to technical problems; but it was identified through the size and characteristics according to Taylor et al. (2016).    Table 4 shows the univariate analysis of the presence of Eimeria spp. with the type of variable responses. Risk factors that showed greater association with the occurrence of Eimeria spp. in calves were the physiographic region, the age, the presence of other animal species and the type of calf rearing (P<0.05; Table 5). Calves from the region of Nautla had 1.55 greater probability of being infected with Eimeria (OR1.55; CI 95% 1.07-2.26) compared with the Papaloapan region. The 5 -9-month-old group had a higher number of oocysts compared to 10-13-month-old calves (OR 1.49 (CI 95% 1.08-2.04; P=0.013). Furthermore, the presence of other animal species [OR of 1.86 (CI 95% 1.31-2.55)] and calves with restricted suckling [OR of 2.31 (CI 95% 1.37-2.39)] had higher probabilities of being infected with Eimeria spp. These data are summarized in Table 5.

Protective factors
In this study, we determine that the physiographic region of Papaloapan and the performance of coprological studies in the animal unit should be considered as protective factors, since they obtained OR values of 0.50 (CI 95% 0.26-0.99) and 0.29(CI 95% 0.19-0.45), respectively.

Discussion
The first objective of the present study was to determine the prevalence of Eimeria spp. oocysts in calves from 26 Municipalities in the center area of the State of Veracruz, México. The overall prevalence of calves with coccidiosis was 39.7%. These results were consistent with the previous ones acquired by Quiroz & Casillas (1971) concerning the prevalence of 38% of the parasite in the south of Veracruz. The State of Veracruz has the ideal conditions that facilitated the presence of Eimeria spp. such as: the climate, physiography, and tropical areas, which are considered the best means for the parasite to survive and reproduce (Lucas et al., 2014). This State has tropical and subtropical climates, which facilitate the survival of Eimeria spp. in the yards of young animals (INEGI, 2018). However, the prevalence shown in this analysis is below compared with other studies made in México with cattle (60.2%) (Alcala-Canto et al., 2020) and other countries such as Brazil (66%) (Florião et al., 2016), Indonesia (65.4%), (Ekawasti et al., 2022), Iran (63%) (Adinehbeigi et al., 2018) and Colombia (75.6%) (Lopez-Osorio et al., 2020). This variation is most likely attributed to the differences in study design, biotic and abiotic factors and husbandry practices of the study animals in different countries (Tamrat et al., 2020), a high animal density, accumulation of organic material and mix of animals of different ages (Florião et al., 2016). On the other hand, in this study, a high prevalence was observed in the physiographic region of "Nautla" with 48.8%. Nautla represents 3.2% of the cattle volume in the state of Veracruz. It has 12 regions with hydrological basins where a warm humid climate prevails with rains all year round. In addition, it has an annual rainfall of 1,383.00 and a maximum temperature of 40 o C. The Eimeria spp. shows a significant association with humid and sub-humid areas and high temperatures, in addition to high rainfall that allows the development and abundance of the parasite (Das et al., 2015).
Bovine coccidiosis is a gastrointestinal disease associated with apicomplexans of the genus Eimeria, where at least 21 species have been reported in cattle around the world (Lee et al., 2018). The second objective of this study was to be observed Eimeria spp. natural infecting calves. From a total of 10 species of Eimeria were identified, E. canadensis was the most observed followed by E. bovis and E. zuernii. Of the 12 Eimeria spp. previously reported for Mexico by Alcala-Canto et al. (2020), E. cylindrica and E. ellipsoidalis were the most observed in cattle. However, in the same spatial distribution of Eimeria species of cattle in Mexico, E. canadensis was the most encountered species with a frequency of 77.58% in temperate humid climates, but also had a high prevalence in warm semi humid and humid climates with 68.13% and 67.08%, respectively. The presence of E. canadensis does not cause significant damage to the intestine of the infected animals, since this species is not considered pathogenic within the pathogenicity classification (Bangoura & Bardsley, 2020), however, their high frequency in this study deserves future epidemiological and clinical studies throughout the year. It is mentioned that economical losses due to subclinical disease even are higher than clinical coccidiosis (Fitzgerald, 1980) because it is more common and can affect the productive and reproductive parameters in cattle (Larsson et al., 2006).
In our study, E. bovis and E. zuernii were detected with an important frequency of 24.2% and 15.7%, respectively. This finding agrees with Quiroz & Casillas (1971) who described E. bovis as the most frequent in cattle. These results are also consistent with several authors reporting the species E. bovis and E. zuernii identified more frequently in the fecal samples analyzed (Bruhn et al., 2012;Lee et al., 2018;Hamid et al., 2019;Ola-Fadunsin et al., 2020;Cruvinel et al., 2021). Although mixed infections are commonly observed under natural conditions, only two highly pathogenic species E. zuernii and E. bovis are generally involved in the highest number of clinical cases in calves and can cause moderate to severe enteritis (Daugschies & Najdrowski, 2005). These animals have abdominal pain, fever, anemia, dehydration, weakness, anorexia (Li et al., 2021) and tenesmus (Daugschies & Najdrowski, 2005). Authors also reports that mortality might reach 7-20% (Pilarczyk et al., 1999). Eimeria bovis is one of the main species that produce clinical signals in animals, among which are weight loss, damage to the intestinal endothelium and intermittent hemorrhagic diarrhea (Lopez-Osorio et al., 2020). In the case of E. bovis and E. zuernii predominate in animals from 4 and 3 months of age (Cruvinel et al., 2018). However, the presence of mixed infections of E. zuernii and E. bovis has been observed in different ages of animals (Bangoura et al., 2012). This implies that it is not only to observe the presence of Coccidia in the feces, but also to make an identification by species to establish control measures to reduce economic losses in the UPB.
The identification of species and genus through morphological characteristics is controversial because can be subjective and ambiguous between intraspecies variations (Kawahara et al., 2010;Florião et al., 2016). It is mentioned that the size, color and surface of the wall can vary in the ocysts of a given species (Dubey, 2019); therefore, the morphological study is not conclusive and further confirmation by molecular techniques such as PCR is recommended. The presence of specific primers allows molecular identification to be a rapid technique for the diagnosis of pathogenic Eimeria spp. (Kawahara et al., 2010). The classification through six specific primers for the pathogenic Eimeria species by PCR has been reported (Kawahara et al., 2010;Al-Jubory & Al-Rubaie, 2016;Lee et al., 2018;Ekawasti et al., 2022). Therefore, this aspect requires further research to confirm by PCR Eimeria spp. circulating at the UPB in the State of Veracruz, Mexico.
The third objective was to identify the risk factors associated with the presence of Eimeria spp. in calves. In this study, different risk factors related to intrinsic (age) and extrinsic factors (e.g., physiographic region, presence of other animal species, type of calf rearing and coprological studies) were identified in livestock production units. The 5 -9-month-old group had a higher number of oocysts compared to 10-13-month-old calves. The prevalence of Eimeria natural infection in cattle is highly age dependent (Cornelissen et al., 1995). Young animals up to 1 year of life are usually the most susceptible; also, some studies indicate that this disease is manifested in animals less than 18 months, on average (Makau et al., 2017). As a result, when they grow into adults, they usually become asymptomatic hosts after recurrent reinfections, serving as a source of infection for younger animals (Matjila & Penzhorn, 2002). Lopez-Osorio et al. (2020) found that the most susceptible age to Eimeria spp. was between 3 and 6 months, concurring with our findings, where risk was observed in calves between 5 and 9 months of age. The logistic regression analysis revealed an OR of 1.49 (CI 95% 1.08-2.54; P = 0.01), associated with a possible lack of immune response or low immunity (Yun et al., 2000). Therefore, there is a concordance with what has been observed by other authors regarding this factor (Daugschies & Najdrowski, 2005;Almeida et al., 2011;Rehman et al., 2011;Tomczuk et al., 2015). It should be mentioned that although calves obtain immunoglobulins from the colostrum, these are not enough to prevent an infection of the apicomplexans (Faber et al., 2002).
Additionally, the presence of other animal species in the bovine production unit was considered a potential risk factor with an OR of 1.86 (CI 95% 1.31-2.65; P= 0.001) since they facilitate the accumulation of organic matter. Lopez-Osorio et al. (2020) and Rehman et al. (2011) suggest that the lack of cleaning and sanitation, followed by poor sanitation, optimal humidity, concentration of organic matter and temperature are important factors for oocyst sporulation and survival in contaminated soils. Furthermore, the restricted calf suckling was also considered as a risk factor with an OR of 2.31 (CI 95% 1.37-3.89 and P=0.002). In this research, the type of rearing is defined by the way the calves consume the milk, either artificially or directly from the mother (restricted or continuous). Tomczuk et al. (2015) described the highest extent of invasions in those farms where calves were raised with their mothers. Mother ewes have also been found to be the main source of infection for lambs cohabiting with them. Therefore, there is a strong association between possible infected mothers and calves with low immunity who are susceptible to the parasite infection. On the other hand, low Eimeria spp. infections were observed in animals that undergo routine coprological studies for the control and treatment of parasites in livestock management. These results agree with the findings of Philippe et al. (2014) who demonstrated that metaphylactic treatment with diclazuril and toltrazuril reduce the impact of coccidiosis by previously identifying the specific parasite to attack. The Papalopan region had an OR of 0.50 (CI 95% 0.26-0.99; P=0.05), suggesting a decrease of the parasite with this variable. The three Municipalities that belong to this physiographic region have dry seasons from September to May approximately, which coincides with the months in which the samples were collected. For an oocyst to be infectious, it must sporulate and mature. An adequate amount of oxygen and moisture is required for oocyst sporulation to occur. If there are periods of drought with constant high temperatures and a decrease in relative humidity, the cycle is interrupted, so the oocyst dies and ceases to be an infectious agent for the calf (Rodríguez-Hawkins, 2011).

Conclusion
This work revealed the diversity of Eimeria species in calves in central region of Veracruz, Mexico. The risk of infection was observed in intrinsic and extrinsic factors, such as age and type of calf rearing. This study will help to implement adequate antiparasitic therapies and management strategies for Eimeria spp. infection control, but it also highlights the need to monitor Eimeria species considered non-pathogenic in UPBs over time.