Dynamics of natural infection by Babesia bovis and Babesia bigemina in dairy cattle from an enzootic instability area in Northeastern Brazil

This study aimed to determine the dynamics of natural infection in the transmission of Babesia spp. to cattle in an enzootic instability area in Northeastern Brazil. Blood samples were collected from 30 calves located on two dairy farms to determine the packed cell volume (PCV) and the timing of the primo-infection using polymerase chain reaction (PCR) and their association with climatic factors and management practices. On Farm A, the determination of primo-infection was observed on average at 249.4 (±24.42) days of age for B. bigemina and at 252.6 (±17.07) days of age for B. bovis ; there was no significant difference between the times of infection (P> 0.05). The infection coincided with a period of high rainfall in the region. On Farm B, primo-infection infection was not observed. There was no infection by Babesia spp. on Farm B due to the intensive use of acaricides that led to an absence of ticks. There was no significant difference between the average PCV of animals from Farms A and B (P> 0.05). The management practices on the properties, in addition to the weather conditions influenced the exposure of the animals to disease vectors and may have contributed to the maintenance of this enzootic area in Northeastern Brazil.


Introduction
Bovine babesiosis is caused by Babesia bovis and Babesia bigemina, which are cattle parasites that exist in almost all Brazilian territories; the Riphicephalus (Boophilus) microplus tick is a vector for these parasites (ESTRADA-PENÃ et al., 2006;RÍOS-TOBÓN et al., 2014). In Brazil, the seroprevalence rates of babesiosis vary from 27.9% to 100%, and the epidemiological condition for this disease is related to the climatic conditions and management systems that directly affect the vector cycle (ALVES, 1987;SANTOS et al., 2001;RÍOS-TOBÓN et al., 2014). In this panorama, three different areas are observed: disease-free areas, areas of enzootic stability and areas of enzootic instability . In the latter area, there is a risk of bovine babesiosis outbreaks due to high rates of adult bovine mortality; moreover, bovine babesiosis may occur because many animals in the area have not developed a specific immune response because they were not infected during their first months of life (GOFF et al., 2001;BERTO et al., 2008).
In some situations, enzootic instability may be related to unsatisfactory passive immunity, stress, nutritional status, time of year, management, type of pasture or high infestation rates of pastures and cattle by a vector (BARROS et al., 2005;AMORIM et al., 2014). The primo-infection usually occurs between four and six months of life, with the peak of the parasitemia coinciding with the fall of the packed cell volume; in old age, primo-infection is usually severe and can be fatal (CARRIQUE MAS et al., 2000;SMITH et al., 2000).
In Northeastern Brazil, predominantly endemic areas unstable for babesiosis include cities such as Garanhuns-PE (ALVES, 1987) and Uauá-BA (BARROS et al., 2005); the semi-arid region of Paraíba ; and the middle-north area of Brazil (SOUZA et al., 2013). In the latter area, babesiosis is certainly one of the main diseases that affect cattle, which may facilitate outbreaks (SOUZA et al., 2013).
Knowledge of the factors that favor enzootic instability is fundamental in developing efficient measures to control babesiosis in Brazil. Thus, the present research aims to determine the dynamics of primo-infection by B. bovis and B. bigemina and to examine the relationship between primo-infections and climatic factors and management in the cattle residing in an enzootic instability area in Northeastern Brazil.

Ethical principles
This study was conducted under the terms and conditions of the Ethics Committee for Animal Experimentation of the Universidade Federal do Piauí, Brazil, approved under number 028/13.

Location and climatic conditions
This study was conducted from June 2013 to June 2014 on two farms located 15 km apart; each farm had different management practices for dairy farming (Table 1); and the farms were located in the Litoral Piauiense micro region of Northeastern Brazil in an area of enzootic instability for babesiosis, as described by Souza et al. (2013). Farm A, which is in the municipality of Buriti dos Lopes-PI, is located at latitude 3° 6 '9 "S and longitude 41 ° 53' 38" W, has a total area of 850 ha, has a seroprevalence of 55.5% for B.bigemina and has a seroprevalence of 33.3% for B. bovis. Farm B is in the municipality of Parnaíba-PI, is located at latitude 2° 58 '56 "S and longitude 41° 47' 36" W, has an area of 95 ha, has a seroprevalence of 23.1% for B. bigemina and has a seroprevalence of 30.7% for B. bovis (SOUZA et al., 2013).
Monthly data, including the daily average rainfall, temperature and humidity, were obtained from the agrometeorological station of the Instituto Nacional de Meteorologia (INMET), located in the Embrapa Meio-Norte experimental area, Parnaíba-PI ( Figure 1).

Collection procedures and laboratory tests
Fifteen Gir x Holstein crossbred females from each farm were sampled from birth (occurring during the months of June and July) until 12 months of age, totaling 30 heifers. The animals were naturally exposed to infestation by vectors under the specific management practices of each farm (Table 1). Every 15 days, individual clinical examinations were performed, including an inspection for ectoparasites as well as whole blood collection (with EDTA), to determine PCV using the microhematocrit technique and to extract DNA. DNA was extracted from aliquots of 300 µL of whole blood using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, WI, USA) according to the manufacturer's instructions. The ectoparasites were placed in tubes containing 70% alcohol and were identified according to Aragão & Fonseca (1961).
The molecular detection of B. bigemina and B. bovis to determine primo-infection was performed by PCR using GAU7/GAU6 and GAU9/GAU10, respectively, which are primers previously described by Linhares et al. (2002)

Statistical analysis
All statistical analyses were performed using a commercial statistical package, GraphPad Prism, version 6.0 (GraphPad Software, CA, USA). A descriptive statistical analysis was performed for each variable. The monthly averages for temperature, humidity and rainfall, as well as for primo-infection, were compared between the months of the year using Student's t-tests (95% confidence). The mean and standard deviations were estimated for PCV values. Mean PCV values in the different age groups (0 to 60 days, 60 to 120 days, 120 to 180 days, 180 to 240 days and 240 to 300 days) of the heifers were compared between the farms using unpaired t-test with Welch's correction, with a significance level of 0.05.

Results and Discussion
The average period of primo-infection on Farm A, determined by PCR, was 249.4 (± 24.42) and 252.6 (±17.07) days of age for B. bigemina and B. bovis, respectively; there were no significant differences (P> 0.05). The highest percentage of infection was observed in animals between 240 and 300 days of age, but the first animals that emerged infected with B. bigemina and B. bovis were from 180 to 240 days of age, with a higher percentage of infected by B. bigemina (Figure 2). Only 20% (3/15) of the animals on Farm A did not become infected with B. bovis. All animals on this farm were diagnosed with B. bigemina until day 291; 13.3% (2/15) of the animals showed clinical signs of babesiosis, such as inappetence, anemia, anorexia and hemoglobinuria, all of which emerged in the third month after the onset of rains (Figure 1).
The highest rates of infection and clinical symptomatology on Farm A coincided with the highest rates of R. (B.) microplus infestations for the animals. The onset of animal infestation was observed in September, but the highest rate of infestation (46.6%) was observed in January, when the rainy season began; at that point, only the R. (B.) microplus species was found infesting the animals (Figure 1).  Little information is available on the epidemiology of the R. (B.) microplus tick in Northern and Northeastern Brazil. Although the presence of this tick in these regions was recorded, the regions were not considered preference areas due to their climatological and vegetation characteristics (ESTRADA-PEÑA et al., 2006;SANTOS et al., 2017). However, in other Brazilian regions, there is an increase in the R. (B.) microplus population during the rainy season FERRAZ-DA-COSTA et al., 2014).
We consider that the late infection date of the animals may have been due to the dry period in this region being adverse to ticks, contributing to vector reduction in the environment and interfering with Babesia spp. infection in the animals. Thus, according to with Brown et al. (2006), Correia (2006) and Amorim et al. (2014) with the absence of the etiological agent, the production of antibodies is limited, leaving the animals susceptible to clinical disease and even to death.
On Farm B, no infection by Babesia spp. was observed due to breeding systems intensive, where the animals were confined 24 hours a day from birth, without access to pasture, consequently without contact with ticks. As well as due to the intensive and indiscriminate control of R. (B.) microplus through the use of doramectin every three months (Table 1) in all animals up to one year old. Then, after this period, the use of doramectin was every six months. All this practice kept the farm free of the Babesia spp. infection. This type of control is used by some producers in Brazil who are unaware of the factors that lead to enzootic instability, which ultimately hinders the implementation of control (AMARAL et al., 2011).
The absence of observed vectors on Farm B is directly related to health management measures. According to Santos-Júnior et al. (2000), Rocha et al. (2006) and Amaral et al. (2011), the lack of knowledge among the producers about the biology of the tick and the importance of the Babesia spp. infection in calves at an early age further aggravates the productivity losses caused by the enzootic instability. Moreover, the producers' lack of knowledge exposes the animals to a high risk because the babesiose agents do not circulate in the environment; therefore, the animals do not develop protective antibodies and the matrices cannot transmit the passive immunity to the calves (SANTOS-JÚNIOR et al., 2000;BROWN et al., 2006;AMORIM et al., 2014).
The primo-infection of the calves in the first six weeks of life was not observed on either property. According to Pereira et al. (2009) this is worrisome because the first six weeks of life would be the ideal time to contract the parasite and to develop antibodies since, at that stage, the clinical and hematological manifestations are less severe. This reduced severity is due to the increased erythropoietic activity of the bone marrow, the protective function of fetal hemoglobin and the rapid activity of innate immunity through macrophages and Natural Killer (NK) cells; macrophages process and present the Babesia spp. antigens for CD4 + cells, in addition to producing cytokines, such as INF-γ, TNF-α and interleukins 1 and 12 (BOCK et al., 2004;BENAVIDES et al., 2006;RÍOS-TOBÓN et al., 2014).
The average PCV in the different age groups (0 to 60 days, 60 to 120 days, 120 to 180 days, 180 to 240 days and 240 to 300 days) of the heifers ranged from 25.0% to 32.2% (29.5 ± 2.7%) on Farm A and ranged from 29.2% to 30.6% (29.2 ± 1.1%) on Farm B; there was no significant difference (P> 0.05) between the properties. Our results indicated that on Farm A, the PCV average had significantly declined to 240-300 days of age (25.0 ± 5.88%), coinciding with the primo-infection by Babesia spp. This reduction in the PCV indicates that anemia is proportional to parasitemia due to intravascular hemolysis as well as sequestration and erythrocyte lysis mechanisms (VIEIRA et al., 2001;BOCK et al., 2004).
The analysis of the climatological data showed that the region had a rainy season (January to June), not exceeding 180 mm/month, and a dry season (August to December), with average temperatures ranging between 28 to 30 °C, a maximum temperature of 35 °C and relative air humidity below 70% (Figure 1).
These conditions, with high temperatures, are not very favorable for the development of the biological cycle of R. (B.) microplus, influencing the mortality of larvae in the pastures due to caloric stress (SANTOS-JÚNIOR et al., 2000;GUIMARÃES et al., 2011) and the intensity of infection of these ticks by Babesia spp. (QUINTÃO-SILVA & RIBEIRO, 2003). In this region, an imbalance in the parasite/host relationship probably occurred, facilitating the appearance of new clinical cases and maintaining the region as an area of enzootic instability, which had already been observed in other studies (QUINTÃO-SILVA & RIBEIRO, 2003;SOUZA et al., 2013).
The dynamics of primo-infection by B. bovis and B. bigemina are directly related to the type of management adopted and the climate of Northeastern Brazil. The climate of this region considerably decreases the population of ticks during the dry period, causing infection by Babesia spp. to occur only during the rainy season. Thus, excessive use of acaricides and the climate of the region interfere with the tick population, facilitating the maintenance of an area of enzootic instability for Babesia spp. in Northeastern Brazil.