Introduction

Babesiosis is a disease transmitted by ticks whose causative agent are intracellular haemoparasites of the Apicomplexa phylum, Piroplasmorida order, Babesiidae family and Babesia genus (^{Montes- Farah, De la Vega-del Risco, Bello-Espinosa, & Fortich-Salvador, 2012}). Within this genus the most important species associated with cattle infections are Babesia bovis and B. bigemina (^{Mosqueda, Olvera-Ramirez, Aguilar-Tipacamu, & Canto, 2012}), and their transmission is carried out naturally by the bite of the tick species Rhipicephalus (Boophilus) microplus (Canestrini, 1888) (Ixodida: Ixodidae). This species has a development related to the climatic and ecological conditions characteristic of the tropical and subtropical zones (^{Mosqueda et al., 2012}), where they are abundant and parasitize domestic animals. Therefore, the presence of the parasite and its vector, makes it difficult to eradicate the disease (^{Polanco & Rios, 2016}).

Babesiosis is considered an emerging zoonosis, which affects domestic and wild animals (^{Becker, 2011}). When outbreaks of the disease occur in cattle, it causes haemoglobinuria, fever, jaundice, anemia, weight loss, dehydration, muscle tremor and weakness (^{Benavides & Sacco, 2007}; ^{Rios, Zapata, Reyes, Mejia, & Baena, 2010}). Furthermore, there is also a decrease in the production of milk and meat, a reduction in the reproductive rate due to alterations in the quality of the semen in males, abortions in females, and finally, the death of the individual (^{Rios et al., 2010}). This in turn causes an increase in handling costs of bovine exploitation system due to pharmacological treatments, veterinary attention and control over the haemoparasitic vector (^{Polanco & Rios, 2016}).

In Colombia, the livestock industry is the main cattle production modality with great importance in the national economy. This economic resource is affected by haemoparasitic infections such as bovine babesiosis, which has an incidence of two cases per 100 cattle heads in the country according to the Animal Health Bulletin [Boletín de Sanidad Animal] of Instituto Colombiano Agropecuario (ica) for 2014 (^{Diaz-Martinez et al., 2017}). Its transmission is determined by the vector-parasitehost relationship, which is conditioned by the biotic and abiotic factors of the environment (^{Rios et al., 2010}). In Colombia, the first report of the disease was described by Lleras in 1908, who cataloged the etiological agent as Piroplasma bigeminum (currently known as Babesia bigemina) by microscopic observation of erythrocytes from infected cattle (^{Mosqueda et al., 2012}), and later was recognized as a widely distributed disease (^{Lleras, 1908}). Colombia has the right geoclimatic characteristics for babesiosis to manifest in cattle, by massive infestation of ticks distributed in most of the geographical areas of the country, recorded initially below 2,100 m.a.s.l. Nonetheless, although these have been reported at altitudes above 2,800 m.a.s.l., it has yet not been proven whether the vector reproduces under these conditions (^{Benavides, Polanco, Vizcaino, & Betancur, 2012}).

Current records published in the country indicate the presence of the species Babesia bovis and B. bigemina. In several regions of Bajo Cauca (Antioquia) and Alto San Jorge (Cordoba), the prevalence of the Babesia sp. parasite is 3.1 % (^{Herrera et al., 2008}). In Puerto Berrio (Antioquia) the seroreactivity before specific IgG antibodies against B. bovis and B. bigemina was 65 % in cattle between three and nine months of age (^{Rios et al., 2010}); in the municipality of Gomez Plata (Antioquia) the seroprevalence found in cattle was 89.8 % for at least one species of Babesia spp. (^{Zapata et al., 2011}). Moreover, in Cordoba the seroprevalence of Babesia spp. in purebred Gyr cattle was 3.05 % (^{Blanco-Martinez, CardonaAlvarez, & Vargas-Viloria, 2015}).

However, the data is mostly restricted to the departments of Antioquia and Cordoba, whereas, the department of Sucre is an area without registration, control or published studies of haemoparasites of the genus Babesia that affects cattle. However, in 2014 a study carried out in Ovejas (Sucre) found an infection frequency of 17.34 % for B. bigemina in bovines (^{Barboza, 2018}), however, this disease is not found within the zoonoses monitored by ica (^{Osorio et al., 2013}). From the above, and taking into account that the northern coast of Colombia meets the conditions that contributes to the preservation of the biological cycle of many diseases transmitted by arthropods (^{Buelvas-Alvis, Buelvas, Miranda, & Mattar, 2008}) as Babesiosis, the aim of this study was to detect Babesia spp. that are present in bovines in the municipality of Los Palmitos (Sucre) employing duplex PCR.

Materials and methods

Duplex PCR

The reaction mixture was made in a volume per sample of 25 μL which included buffer Taq (1X), MgCl₂ (1.5 mM), dNTP Mix (0.2 mM), primers (10 pmol) (table 1) and DNA Taq Polymerase (1 U/ μL). The thermal amplification profile started with a denaturation at 90 °C for 3 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, alignment at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. In addition, a final extensión was made at 72 °C for 5 min. Finally, the products were kept at 4 °C, and modifications were carried out as proposed by Smeenk et al. (²⁰⁰⁰).

Table 1. Primers used for duplex PCR of Babesia bovis and B. bigemina 

Source: Elaborated by the authors

Statistical análisis

A univariate and bivariate analysis of the data recorded in the survey was made, including origin, race, age and sex. These were contrasted with the results of microscopic observation and the PCR. For this, an exact Fisher's test was used (table 2) in order to establish the relationship between the variables and analyzes carried out. The statistical test was performed using the software R version 2.9.1.

Table 2. Fisher's F-test for positive bovine by PCR 

Source: Elaborated by the authors

Results

From the sample of 218 bovines, 215 individuals were analyzed, of which 69.77 % were females (150), and 30.23 % were males (65), distributed according to age in ranges, as follows. Between 0 and 2 years of age (161 animals; 74.88 %); between 3 and 4 years old (23 animals; 10.70 %), between 5 and 6 years old (23 animals; 10.70 %), and between 7 and 9 years of age (8 animals; 3.72 %) (figure 1). In relation to the breed, all the bovines studied belong to the Cebu breed. The positive samples obtained (5/215) for Babesia bigemina were found in the same field of 12 respondents during the field phase, which are also asymptomatic animals carrying the parasite.

Source: Elaborated by the authors

Figure 1. Percentage distribution of different cattle age groups involved in the study. 

Under direct observation, the samples that allowed the evaluation of intracellular parasitic forms morphologically compatible with Babesia spp., were considered positive. The visualization of the smears was carried out in the form of a labyrinth, observing between 100 and 200 fields of the blood smears, whose result indicated that 1.4 % (3/215) of the bovines contained parasites in their red blood cells, which also corresponded to male individuals younger than two years old (figure 2).

Fuente: Elaboracion propia

Figura 2. Observacion microscopica de Babesia bigemina en frotis sanguineo de bovinos (La flecha indica el merozoito de B. bigemina observado a traves de microscopia optica con objetivo 100x). 

When using the duplex PCR technique for the molecular determination of Babesia in cattle, five samples were positive for the species B. bigemina, which corresponds to the 278 bp amplified fragment (figure 3), corresponding to 2.32 % of the total number of animals studied. These samples also coincide with the three positive results found in blood smear. In no animal, DNA amplification for B. bovis was found.

Source: Elaborated by the authors

Figure 3. PCR of the positive samples for Babesia bigemina (Numbers 11b, 12b, 13b, 14b and 42b correspond to the positive samples found by PCR, C+b is the positive control, C-b the negative control, and mpm is the molecular weight marker). 

The statistical F-test reflected in table 2 indicates that there is a significant difference between the sexes (gender) of the bovines that were detected as positive by PCR. The bovines of the age group between 0 and 2 years old (2.48 % of the 161 individuals sampled with this age range), are the most susceptible to infection with the protozoan B. bigemina, being these four individuals males.

Only one bovine of the age group between three and four years (4.34 % of a 23 individuals sampled) was positive by using the PCR technique for the species B. bigemina, being the only female animal infected.

In addition, these results agree with the three positive ones found by direct search in blood smear.

Discussion

In the 12 livestock farms assessed, babesiosis was found to be caused by the haemoparasite B. bigemina, identified by duplex PCR in five infected animals, which were located in the same field.

In these, tick treatments and manual removal of arthropods are carried out. However, ticks were not found in infected cattle. In the area, tick-vector control measures are the application of tickicide or other substances such as poison or natural extracts every four months, in addition, also the manual removal of the arthropods during the milking process in the mornings. This interrupts the transmission cycle of Babesia spp. (^{Mtshali & Mtshali, 2013}), since female ticks can become infected at the end of the wet phase of the infection, when there is a manifestation of a parasitaemia in the bovine host. However, this parasitaemia is rare and the infestation time is limited. Therefore, according to Rios et al. (²⁰¹⁰), only a small number of arthropods contract the parasite. Moreover, these authors state that the high frequency of tick treatments generates a lower degree of infection of R. microplus, and the low frequency of treatment results in a higher degree of infection by ticks.

The microscopic identification of Babesia bigemina in blood smears resulted in three bovines infected with the parasite, however, this method requires trained personnel to identify the Babesia species that infect the erythrocytes (^{Benavides et al., 2012}; ^{Homer, Aguilar-Delfin, Telford, Krause, & Persing, 2000}), since, its morphological difference lies in their size, i.e. B. bigemina is larger than B. bovis (^{Benavides et al., 2012}; ^{Smeenk et al., 2000}). Although microscopic detection is the reference technique in the identification of babesiosis (^{Homer et al., 2000}), particularly during the acute stage of the disease, the number of intraerythrocytic parasites increases in such a way that they can be detected microscopically (^{Mosqueda et al., 2012}). This technique is not very useful in chronically infected animals in which the disease occurs subclinically or asymptomatically. For this reason, it is necessary to resort to molecular methods such as pcr to detect nucleic acids, due to their high sensitivity and specificity to identify Babesia species in hosts and vectors (^{Mosqueda et al., 2012}; ^{Mtshali & Mtshali, 2013}). Employing this technique, results showed that B. bigemina is the haemoparasite that can cause babesiosis in Los Palmitos, finding it in five of the 215 animals assessed in this study. By amplifying a 278 bp fragment (figure 3) corresponding to the gp45 gene specific for B. bigemina (merozoite surface glycoprotein with 45 kD) (^{Figueroa et al., 1993}; ^{Mtshali & Mtshali, 2013}), it was established that the species B. bigemina coincides with this species registered in the GenBank database (figure 4). Moreover, it also corresponds to the species found in a study carried out in the municipality of Ovejas (Sucre). Furthermore, it also corresponds to the recent publications that register the presence of the species Babesia spp., B. bovis and B. bigemina as the etiological agents of bovine babesiosis in the region of Bajo Cauca and Alto San Jorge (^{Herrera et al., 2008}), in Puerto Berrio (Antioquia) (^{Rios et al., 2010}), in the municipality of Gomez Plata de Antioquia (^{Zapata et al., 2011}); in Magdalena Medio (^{Lopez, Florez, Munera, Rios, & Rios, 2012}) and in Cordoba (^{Blanco et al., 2015}).

Source: Elaborated by the authors

Figure 4. Comparison of the amplified nucleotide sequences with those registered in the GenBank database employing the BLAST tool. 

In the municipality of Los Palmitos (Sucre) the frequency of infection found was 2.32 % (5/215), and is considered low in relation to a study carried out in sheep from the same department (17.34 %) and in Cordoba, where the Babesia spp. frequency was 3.05 % in pure Gyr cattle (^{Blanco et al., 2015}).

The animals studied were asymptomatic, which indicates that, even though they are positive for Babesia, the globular damage by the protozoan is minimal. This shows that in these animals there are no signs of anemia (^{Blanco et al., 2015}), due to the use of tickicides that interrupts the Babesia spp. transmission cycle, and thus, decreases the number of infected cattle in the area.

In addition, the haemoparasite occurs mostly in cattle between zero and two years of age (4/5), which are less susceptible to the clinical effects of the disease than adult cattle (1/5) (^{Benavides et al., 201}2; ^{Benavides & Sacco, 2007}). Individuals that become infected with the parasite at an early age acquire passive immunity provided by the mother through the colostrum that lasts until the age of 9 months (^{Blanco et al., 2015}; ^{Rios et al., 2010}), although it may persist for four more years (^{Bock, Jackson, De Vos, & Jorgensen, 2004}), making them asymptomatic.

From the above, the individual animal develops an acquired immunity that is conditioned by the inoculation of parasites by the vector, which allows the immunity acquired in the hosts to be maintained, and therefore, the absence of clinical signs or symptoms that causes the disease (^{Rios et al., 2010}). Similarly, in an endemic region for the vector and the haemoparasite such as the northern coast of Colombia, the infection can remain unnoticed when it occurs at an early age, a situation known as enzootic stability (^{Benavides et al., 2012}; ^{Rios et al., 2010}). Together, these mechanisms contribute to the natural control of babesiosis.

Conclusions

In the municipality of Los Palmitos (Sucre, Colombia), bovines under two years old are carriers of Babesia bigemina, detected by duplex PCR. The absence of clinical symptoms in infected cattle may be because they act as reservoirs of microorganisms, which awaits the optimal physiological conditions to cause the pathology or the transmission by the vector to other cattle.

Disclaimer

All authors made significant contributions to this study and all agree with its publication, stating that there are no conflicts of interest in this study.