INTRODUCTION

The milk industry is an important component of agribusiness in Western Santa Catarina, Brazil. From this activity, the state reached an important contribution to gross domestic product (GDP) of the state of Santa Catarina, where dairy agribusiness still plays an extremely important role for the state, responsible for the survival of thousands of families in the countryside and in the generation of numerous direct and indirect jobs. Sanitary control herds are generally inefficient and can cause problems in production system, since they may be caused by various etiological agents, such as viruses, bacteria, fungi, and parasites ^1,2. Helminths are among the diseases that most affect livestock productivity in many regions of the world. In the 70s, it was estimated that each year about 10 million cattle and buffaloes died from consequences direct or indirect in presence of helminths ¹. At present, it is believed that the number of animals with a fatal affection by parasites was reduced, due to strategic programs and new drugs; although significantly increased the diagnosis of treatment-resistant helminth ³. The damage caused by endoparasites can occur directly and indirectly reflected in weight gain, milk production, costs with antiparasitic, and mortality ^1-3.

One of the main parasite that affects the Brazilian herd is the cattle tick Rhipicephalus (Boophilus) microplus, an ectoparasite that causes important economic losses in domestic cattle. Because of its ease way of reproduction, causes discomfort to the animals, as well as affect the development of the herd and the production of meat and milk. Furthermore, it can transmit diseases such as babesiosis and anaplasmosis, which can occur simultaneously and thus referred to as cattle tick fever ³. Besides the damage caused directly to the animal, the tick causes damage to the producer due to indirect spending with acaridae, labor and equipment necessities for application of such products ⁴. Another problem caused by tick relates to the environmental impact caused by the indiscriminate use of chemotherapeutic products. Over time, the difficulties in controlling the parasites increased significantly due to increasing resistance to chemotherapeutics in your control and treatment, often uncontrollable. Similarly, have occurred with flies control, among these stands out the Musca domestica (often related to mastitis) and Haematobia irritans, a hematophagous insect that has caused serious damage to livestock ^3,5.

In this study we provide information about the Neospora caninum, the etiological agent responsible for neosporosis in cattle mainly. Recent studies have shown that several species of domestic animals can be infected, such as sheep, horses and goats ^3,6. The disease in cattle have been associated with clinical signs such as abortion, absorption, mummification or birth of weak calves ⁷, but usually the calves are born healthy, but infected, because the N. caninum can be transmitted congenitally. Horizontal transmission, i.e. ingestion of oocysts shed in the feces of the dog has been mentioned as a risk factor for disease ⁸.

Ticks, helminths and protozoa are responsible for diseases that cause economic problems for producers of milk will globally due to treatment costs and reduction in milk production (often unnoticed). In Santa Catarina Western, where you have a large milk cows stock, researches are limited. Therefore, the aim of this study was to investigate the relation between type and size of farms with production management and especially the presence of parasites, beyond the identification of risk factors related to infection by N. caninum.

MATERIAL AND METHODS

Local: Investigated properties. In this study 65 properties classified as specialized or semi specialized were surveyed ⁹ sampled from 20 municipalities in the western of Santa Catarina, Brazil (Figure 1). Of these, 56 semi-extensive system (cows in pastures), classified in this study as small (Class 1: n=15; up to 15 cows), medium (Class 2: n=20; between 16 and 30 cows) and large (Class 3: n=21; over 31 cows). Also as part of the study nine properties freestall system (Class 4: n=9; confinement, over 70 cows). The survey was conducted between November of 2013 and February of 2014, being applied a questionnaire for data collection, in order to verify the characteristics of the properties, as well as production and health management (Table 1).

Figure 1: Map of epidemiological distribution of positive samples derived from milk from the farm tank, i.e. collective samples (n=36) in a total of 65samples in Santa Catarina state, Brazil. 

Sample collection. During the visit at the farm was performed of collection of feces of 10 lactating cows, as well as a bulk milk tank. Feces were collected directly from the rectum, allocated into plastic containers, identified and stored in insulated cooler bags until analysis in the laboratory.

Parasitological analysis of feces. In the laboratory, the feces were processed by the McMaster technique, using supersaturated sugar solution to quantify the number of eggs per gram (EPG) of feces helminth ³. Of each farm was performed a pool of feces of the animals with positive EPG to carry out the culture of larval, kept in an incubator with constant temperature and humidity to identify helminths present on the farm.

Serologic assay for Neospora caninum in bulk milk tank. Milk collected from the tank of each farm was centrifuged at 7000 g for 10 minutes to obtain milk serum. So, the presence of antibodies to N. caninum in milk was verified by indirect immunofluorescence assay (IFA) using blades made with the protozoan tachyzoites ¹⁰. The samples considered positive were those in which full fluorescence from tachyzoites of N caninum was observed, as described by Camillo et al ¹¹.To validate the tests, samples known as positive and negative were used as reaction control.

Data analysis. The relationship between the size of the properties for reproductive management and presence of parasites was performed only in a descriptive manner, seeking to identify management practices and sanitary control measurements of every classified type of farm.

Antibodies to Neospora caninum in milk. The data generated by the questioner applied records and by the test were recorded and analyzed in statistical software R, version 3.1 (R core team, 2012) and map was produced using Arc Map ^(r) version 3.2.1 (ESRI, Redlands, CA, USA). Cross tabulation and descriptive statistics were performed. Variables were first screened based on the response variable (positive or negative for Neosporosis), variables with large amounts of missing data (>10%) and limited variability (<20%) were not included in the multivariable model. The remaining variables were entered individually into a univariable logistic regression model and selected for inclusion in the multivariable model if p<0.25. Subsequently, all the screened variables were submitted to correlation analysis. The non-collinear variable was <0.7. Interactions between all pairwise variables suitable for the final model were examined and, if significant (p<0.05), were submitted to further analysis. Subsequently, the selected variables (n=5), (Table 2) were included in the multivariable model. Multivariable models were built in a manual forward method; each remaining variable was added to the best previous model, selected by the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). A backward elimination step was finally used, resulting in a final model in which only variables with p<0.05 were retained. Confounding effects were investigated by checking changes in the point estimates of the variables that were kept in the model. Changes in parameter estimates >25% were considered as a confounding factor, age was found to be a potential confounding factor and it was controlled during the regression. The goodness-of-fit of the final model was tested using Hosmer-Lemeshow (¹²).

Parasitological feces. For the output of EPG variables were first screened based on the response variable count of Helminthes and Eimeria by descriptive statistics for contingency of the information and for further assumptions and what is presented as descriptive measurement is the mean and standard deviation. All variables were tested for normality of variance by Shapiro-Wilk test ¹³, skewness, kurtosis and homogeneity by Levene's test ¹⁴ previously to analysis by linear regression. Wald statistics was used to assess the significance of association (farm class production; Helminthes control and EPG count) and variability of the model was expressed by R².

RESULTS

Characterization of the farm as productive management. The data relating to production management of the four classes divided by size and management system were presented in Table 1. Considering the average among the surveyed properties without considering size and production system, it was found that the time working in the dairy business was 17.7 years, as well as the frequency of the total number of cows and lactating cows were 53.3 and 38.2%, respectively. The average milk production of the surveyed properties was 18.2 liters/day. From the properties it was found that the labor used in dairy cattle as familiar was 84.6%, outsourced (6.15%) and mixed (9.23% - family and outsourced). In percentages, other important data relating to the farms studied were: receivable amount (83.7%) and milk quality (85.4%), performing pre-dipping (70.8%), and post-dipping (88.8%), rapid test for mastitis (66.3%), reproductive control (95%), use of artificial insemination (86.2%), use of sexed semen (18.4%), providing commercial feed (86%) and silage (91.2%).

Relationship between farm and sanitary control with emphasis on parasites. Considering the class 1, 2, 3 and 4 of the farms (size and system) for the sanitary handling and parasites were presented in Table 1. If we consider the 65 farms, without evaluating class, we found that 62.8% of the farms have shown reproductive problems. In percentages, was also identified farms investigated had composter for feces (58.8%), presence of dogs (86.7%), dogs with access to feeders and food (60.5%), use of acaridae to control ticks (73.2%), use of anthelmintic (67%), rotation of anti-parasitic in the control of parasites (57.4%), parasitological of feces with frequency (13.2%), and resistance testing (6.1%). This study also identified the occurrence of cranial injury to the udder of the cow, caused by the nematode Stefenofilaria sp. (53.9%), and bovine babesiosis and anaplasmosis (55.1%). On the farm, on the day of the visit, it was also found the presence of domestic fly (Musca domestica) and horn fly (Haematobia irritans) in 68.6 and 59.7%, respectively.

Parasitological of faces. The results of EPG and OOPG (oocyst per gram) according to classification of the farms were presented in Table 1. Between farms investigated, only 9.23% were negative for parasites in feces, i.e., none of the cows showed helminthes eggs and coccidia oocysts in the feces. Among animals positive for helminths, was observed variation between cows 50-3650 EPG, but in general, and average of 25.6 EPG/animal. However, 80% of animals had positive EPG less than 300. Among the animals positive for coccidia, was detected variation between cows 50-3050 OOPG, but the overall average was 16.7 OOPG/animal.

No significant association was found between EPG counts and (farm class production; Helminthes control and OOPG count), as well as no significant association was found between OOPG counts and (farm class production; Helminthes control and EPG count).The oocysts in the faeces were identified as the genus Eimeria. The helminth eggs identified in the feces of cattle were of Trichostrongylus spp. (35.28%), Haemonchus spp. (6.15%), Teladorsagia spp. (1.53%), Oesophagostomun spp. (1.53%). In 33.84% of the farms was found mixed infection by two or three helminths.

Antibodies for N. caninum in milk. The antibodies in milk farms were detected in 36 farms (55%; CI₉₅% 42-67) (Figure 1).When we consider the classification of the farm, was found higher occurrence in farms from class 1 (Table 2). In this study, a significant association was observed between following factors: classification of the farms (small, medium, large; freestall); providing concentrated to the cattle and access of dogs to the local storage of inputs supplied to animals with positive farms for neosporosis. Medium and large farms had higher risks of infection by N. caninum (PR=5.86-IC_95%:1.33-25.72) and (PR=8.32-IC_95%:1.95-35.40), respectively. It was also identified that the risk of infection by N. caninum is related to the supply of concentrate and access to the dogs feed stocks (PR=6.67-IC_95%:1.35-32.88) and (PR=4.51-IC_95%:1.56-13.00), respectively (Table 3).

DISCUSSION

The classification of the farm by number of dairy cows provided to verify numerical difference regarding managements, production and sanitary control, something expected due to the different degrees of technological improvement. For example, levels of protein and fat in milk are smaller farms that have a higher level of production technology, but in return these farms have lower number of somatic cell count (SCC) and total bacterial count (TBC)⁹. The explanation for this behavior may be due to improved nutrition of animals and use of specialized breeds with the increase in production levels. Another point related to the production this hygiene practices at farm, observed in this study in a good sizeable percentage (pre-dipping, post-dipping and mastitis test); however, these practices were lower on farms of small and medium size (class 1 and 2) the extensive system. According of Simioni et al ⁹ and Martins et al ¹⁵, in specialized farms, the dairy cattle shows greater economic importance, encouraging the farmers to adopt appropriate hygiene practices during milking, and reproduction of the herd; similar to that observed in this study.

Ectoparasites are also factors that can cause loss in milk production, and the farms visited the main ectoparasites were identified to flies (Musca domestica and Haematobia irritans) and ticks (Rhipicephalus microplus). These flies can cause chronic dermatitis in cattle (horn fly), as well as being vectors of mastitis-causing bacteria ¹⁶, and nematode that causes stefanofilariosis, characterized by skin lesions, cranial to the mammary gland of the cow, caused by the genus Stephanofilaria ¹⁷, observed in this study, in a high percentage (56%) farms investigated. Approximately 62% of farms are facing serious problems with outbreaks of flies during the year, independent of the class 1 to 4 in this study. The presence of composters can help control flies ³, but only 60% used on farms in the west of Santa Catarina State.

According to the literature, ticks are the most important ectoparasites for dairy farming, and approximately 75% of the cattle population is affected by this parasite. Tropical and subtropical regions are favorable for the development of ticks ⁴, target region this study. In addition to direct damage to the animal, the tick transmits Babesia spp. and Anaplasma spp. ³, responsible for causing one of the most common diseases (bovine parasitic sadness) in dairy cows in the Brazilian states. This study was no different, as the interview was found that 58% of farms have annual cases of babesiosis and anaplasmosis, regardless the classification given in this study. The disease is a sanitary problems greatest economic loss in cattle due to treatment costs, high rates of mortality and morbidity, as well as reduction in the production of meat and/or milk ^3,18. Most farms investigated make use of antiparasitic to control ticks, and only 16% of farms do not tick control, because according to the producers the degree of infestation is low. This can be explained because these farms are rotational grazing system or animals are in confinement (freestall), which reduces the chance of development of the parasite, and contact with animals.

The EPG generally considered low, as observed by Pasetti et al ², however, the number of infected animals was superior in study current. Helminthes were identified Strongylida order, being the Trichostrongylus spp the most frequent (35.28%), similar of observed in Minas Gerais state by Araujo ¹⁹ in dairy cows. In this study, it was found that about 72% of farmers perform routinely the anthelmintic treatment, usually in the dry cow period, having a preventive effect. Despite the presence of Eimeria spp. oocysts in the feces of cattle, no treatment for coccidiosis is done, but according to the literature infection cause destruction of intestinal cells, and consequently interferes with the absorption of nutrients ³

Our model was able to identify the presence of dogs as a risk factor for N. caninum, this finding is well reported on many studies and it's a very important epidemiological factor since dogs become N. caninum definitive hosts ²⁰, but what we found provide more detail to this issue. It was found that the possibility of a dog having contact with cattle fed, what includes stocked food represent a challenging risk factor for N. caninum infectious. Our finds are in accordance with others that have shown that farm dogs may defect on feeding alleys and on stored grass or corn silage what may cause evidence of postnatal bovine infection than by those of herds with no such evidence ^21,22. Based on these results, it may be justified to assume that contaminations of the feeding area are more closely related to N. caninum infection ²³. Complementary to the factor above it was also find that farms how provide concentrate to the cattle were more likely to be positive for N. caninum, this knowledge is in accordance to Topazio et al ⁶ that have shown that goats farms that provide rich diet what included concentrate have more chances of the infections, and a possible explanation for these may due to inadequate food storage what may make possible a direct contact between fed and infected dogs. Finally, larger farms were identify on the study as class 2 and 3 were associated to risk of N. caninum infection, in a study from Italy, the risk of individual cattle becoming seropositive increased with the size of the herd. When the analysis was restricted to data from northern Italy, the number of dogs per farm interacted significantly with herd size; i.e., the risk of being seropositive increased in larger herds with an increasing number of dogs per farm ²⁴ in addition a study conducted in Germany, larger herds had an increased risk of being bulk milk positive ²⁵. An explanation for herd size as a risk factor could be that hygienic measures to prevent dogs from feeding on placentas or other infectious material are more difficult to follow with large herds than with small herds ²⁵.

In summary, we can conclude that there are technical differences between the farms, which is reflected in handling and production, i.e. the largest farms (Class 3 and 4) can produce a greater volume of milk, as is best compensated, reflecting to increased investment in the farm (technification).The contamination by gastrointestinal parasites had no difference between production systems. Also concluded that over 50% of farms in western Santa Catarina has infected cows by N. caninum, which may be related to reproductive problems reported by producers. The detection of antibodies to N. caninum in collective samples of milk proved to be a great diagnostic tool to herd, reducing costs with individual tests in a research first.