Introduction

Borrelia theileri is a cosmopolitan microorganism identified in cattle, goats, sheep, and solipeds (Callow 1967). This species belongs to the relapsing fever group Borreliae (RFGB) from the Spirochaetaceae family, which includes gram-negative spirochetes (McCoy et al. 2014; Cordeiro et al. 2018). Species in this group differ from others in the order Spirochaetales in that they have a greater number of periplasmic flagella, few turns, and are microaerophiles (Barbour and Hayes 1986).

Borreliosis, also known as spirochetosis, caused by B. theileri is still poorly understood regarding its pathogenicity in ruminants but is initially associated with fever (Koch et al. 1990) and anemia (Abanda et al. 2019). Furthermore, co-infection by B. theileri and other pathogens, such as piroplasmid protozoa (Koch et al. 1990; Sharma et al. 2000), may worsen clinical signs and, therefore, require an integrated treatment.

Biological transmission by either transovarial or transstadial perpetuation of B. theileri was described for the first time in Brazil in Rhipicephalus (Boophilus) australis ticks (probably Rhipicephalus (Boophilus) microplus) by Brumpt (1919), who also reported the first finding of B. theileri in a bovine in the country. Transmission has also been identified between populations of Rhipicephalus decoloratus and Rhipicephalus evertsi ticks in South Africa (McCoy et al. 2014). Experimental transmission of B. theileri by R. microplus in splenectomized calves was reported by Smith et al. (1985), who confirmed the presence of the spirochete after 15 to 17 days of tick infestation. The only change observed in the animals in the experiment was an increase in rectal temperature, reaching 40.2 °C.

Recently, Morel et al. (2019) characterized a new bovine isolate in Argentina. In Brazil, characterization has been performed only in R. microplus ticks (Cordeiro et al. 2018), and subclinical infection in a cow (Paula et al. 2022). No other studies have been conducted to determine the molecular frequency of B. theileri in herd of cattle in Latin America. To address some of these data gaps, the objective of this study was to describe the occurrence of Borrelia sp. in blood samples from cattle in two different Brazilian regions.

Materials and methods

A cross-sectional study was carried out from 2019 to 2020 on nine dairy farms in the cities of Unaí, Cabeceira Grande, and Arinos, located in the micro-region of Unaí in the northwestern region of the state of Minas Gerais (MG), Brazil (16°21′6″S, 46°54′43″W). This area is located in Cerrado biome. Its climate, according to Köppen (1948), is type Aw, which corresponds to a tropical rainy climate, a savannah climate, with a predominance of dry winters. Average annual temperatures range between 31 °C, maximum, and 15 °C, minimum. The average annual rainfall is between 1400 and 1500 mm (Naime et al. 2014). Additionally, an incidental finding of infection by spirochete on calf in the city of Marabá, in southeastern Pará, Brazil (5°33′44.5″S and 49°06′01.1″W) was sampled. This area is located in the Amazon biome. Its climate, according to Köppen (1948), is type Aw, which corresponds to a tropical, hot and rainy climate, with predominant rains in the summer. Average annual temperatures vary between 35 °C, maximum, and 22 °C, minimum. The average annual precipitation is 2200 mm (Junior et al. 2017).

All animals in the study showed nonspecific signs, such as a drop in milk production in cows and reduced food consumption.

Blood was collected from Holstein cows between 3 and 9 years of age in the state of Minas Gerais and from a crossbred calf (Holstein x Gyr) in the state of Pará by puncture of the jugular vein using Vacutainer® tubes containing ethylenediaminetetraacetic acid (EDTA), which were kept at a temperature of 2–8 °C. In the laboratory, blood smear slides were made, and the hematocrit centrifugation technique for diagnosis of African trypanosomiasis (Woo’s test) was used to detect hemoparasites with motility (primarily Trypanosoma vivax). The Woo’s test was performed by centrifuging whole blood with anticoagulant EDTA in microhematocrit tubes and identifying motility by direct observation of the capillary under a microscope at × 40 magnification (Woo 1970).

The slides were fixed in methyl alcohol for 3 min, stained with a 10% Giemsa stain for 30 min, and viewed under the 100 × objective of an optical microscope.

A 300-µl aliquot of blood from each animal was used for total DNA extraction using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The extracted DNA was amplified by polymerase chain reaction (PCR) to detect the flagellin B (flaB) gene of Borrelia spp., Samples from animals positive for Borrelia spp. were also tested for the 16SrRNA genes of bacteria of the Anaplasmataceae family, a genomic region that encodes the 8 kDa antigen of the protozoan T. vivax, the sbp gene of the protozoan Babesia bovis, and the rap gene that characterizes Babesia bigemina. The primers used are shown in Table 1. All reactions and thermocycling protocols were performed as described in the original articles. The products of the PCR reactions were analyzed using an electrophoresis gel. For each reaction, a positive control of Borrelia anserina strain AL (culture), Anaplasma marginale strain AmRio 2 (culture), B. bovis, B. bigemina and T. vivax (cattle positives) and two negative controls (water) were included.

Table 1 Sequences of the primers used, together with the respective target genes and the size of the amplified fragment
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The amplification products from samples of positive animals were sent for sequencing, having been previously treated with Exo-Sap-IT (GE Healthcare®) following manufacturer’s guidelines, and were sequenced through the capillary-type Sanger platform in an ABI 3730 DNA Analyzer (Applied Biosystems, Life Technologies®). The generated sequences were compared with published data using the NCBI nucleotide BLAST program. Phylogenetic analysis was performed using MEGA version 11.0 software.

Results and discussion

Among a total of 132 samples taken from dairy cows from Minas Gerais, one blood smear sample was positive for Borrelia sp. (Fig. 2), reflecting a positivity rate of 0.75% (1/132) by this technique. Additionally, a spirochete was detected in a blood smear from the calf from Pará.

Molecular analysis revealed two positive animals from Minas Gerais (2/132, 1.52%) and confirmed the diagnosis in the calf from Pará. The positivity of the three animals was confirmed by the presence of spirochetes on the blood smear slides (Fig. 1) and/or by molecular analysis (Table 2).

Fig. 1
figure 1

Photomicrograph of two specimens of Borrelia theileri observed in blood smear from a lactating cow (A) and Calf from Pará (B). Giemsa, × 1000 magnification

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Table 2 List of tests performed on animals positive for Borrelia theileri in northern and southeastern Brazil
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Blood smear and molecular analyses for detecting bacteria of the Anaplasmataceae family and the protozoa Babesia spp. and T. vivax in animals positive for Borrelia spp. found bacteria of the Anaplasmataceae family in the calf and only one of the cows, as shown in Table 2.

Sequencing of the amplified flagellin B fragments revealed a shared identity of over 99% with B. theileri isolate C5 (MG601737). Analysis of the phylogenetic relationship based on the flaB gene sequences obtained in this study showed that they formed a clade highly related to the B. theileri strains deposited in GenBank. This clade includes the B. theileri strain C5 detected in R. microplus in Brazil (Fig. 2). The three spirochetes detected in this study are herein referred to as B. theileri isolate MG7, B. theileri isolate MG8, and B. theileri isolate Pará. The novel sequences were deposited in the GenBank with the following accession numbers OQ344268 (B. theileri isolate MG7), OQ344269 (B. theileri isolate MG8), and OQ344270 (B. theileri isolate Pará).

Fig. 2
figure 2

Sequences from the study and GenBank aligned using MAFFT in JALVIEW 2.11 software. The best-fit evolutionary model determined with Bayesian Information Criterion. Phylogenetic relationships estimated using Maximum Likelihood (ML) phylogenetic inference with PhyML and Bayesian Markov Chain Monte Carlo (MCMC) method with MrBayes v.3.2.6. MCMC settings included two independent runs with 4 chains each, run for 10 million generations with a sampling frequency of 100th generation, resulting in 100,000 trees. After removing 25% of the samples as burn-in, a consensus tree was built. Statistical support for clades was measured using a heuristic search with 1000 bootstrap replicates and Bayesian posterior probabilities. Numbers (≥ 70/ ≥ 0.7) above branches indicate node bootstrap or probabilities values (ML/ MrBayes). Asterisks indicate values below 70/0.7

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Since it was first described in 1902, this spirochete has been identified in Africa, Australia, and both North and South America (McCoy et al. 2014). Although B. theileri is a tick-borne agent of cosmopolitan occurrence, its molecular description in South America has been reported infrequently. Ours is the first study showing the molecular frequency of Borrelia sp. in bovine in South America and the only study based on such a large number of animals.

The first molecular description of B. theileri was performed on homogenized samples of R. microplus by Yparraguirre et al. (2007) in the southeastern region of Brazil. Subsequently, Cordeiro et al. (2018) described the morphological and molecular characteristics of this spirochete in the hemolymph of this same tick species collected from a calf in the state of Rio de Janeiro, Brazil. Recently, B. theileri was incidentally detected in one of the cows during the examination of stained blood smears of 10 cows from Goiás State, Brazil (Paula et al. 2022). Morel et al. (2019) detected the presence of B. theileri in a bovine in Argentina, indicating that the agent is present in much of South America.

It is noteworthy that positive animals showed infestation with ticks of the species R. microplus. This suggests that infestation with R. microplus may be a determining factor for the presence of Borrelia sp. in cattle. However, other tick species, including R. decoloratus and R. evertsi in South Africa, have also been reported to transmit this spirochete (McCoy et al. 2014).

Two important features of RFGB are a high concentration of bacteria in the blood of competent hosts and the presence of transovarial transmission in ticks (Dworkin et al. 1998). The high concentration of the B. theileri bacteria has been observed in the hemolymph of R. microplus, as well as transovarial transmission (Cordeiro et al. 2018). However, parasitemia appears to be low in cattle. Thus, the spirochete is rarely found circulating in the blood and almost always with the presence of a single specimen in the blood smear (Fig. 1).

Although little studied, infection by B. theileri alone is known to be of low pathogenicity (Abanda et al. 2019). However, infection with other arthropod-borne agents may potentiate symptoms generated by these agents in co-infected animals.

Despite a negative test for T. vivax in the blood smear and in the molecular analysis, occasionally, Woo’s test was positive in one of the dairy cows, demonstrating that this technique, possible can be used to detect the presence of Borrelia spp. Woo’s technique, described more than 50 years ago, is widely used in the diagnosis of bovine trypanosomiasis and has a high sensitivity compared to other techniques (Woo 1970). However, although further studies are needed, the results of the present study suggest that both Borrelia spp. and trypanosomiasis should be included in the differential diagnosis for positive cattle using Woo’s technique.

The frequency of 1.52% for B. theileri-related relapsing fever group spirochete in cattle from northwestern Minas Gerais suggests a low prevalence of this agent in this region. Among the few previous studies on B. theileri as a disease agent, none address the actual molecular prevalence of this microorganism or elucidate the host-parasite relationship. Abanda et al. (2019) detected a frequency of 17.9% (255/1260) but confirmed 100% shared sequence identity with B. theileri in only 42 samples (3.33%). Serological diagnoses of Borrelia spp. in bovids have been carried out in several regions of Brazil (Guedes-Junior et al. 2008; Silva et al. 2013). Using Borrelia burgdorferi crude antigens, these studies obtained frequencies ranging between 30 and 90%. As these were crude antigens, the possibility of cross-reaction cannot be ruled out; nonetheless, we hypothesize that the serological prevalence of B. theileri is within this range. This hypothesis can only be tested, however, by obtaining an isolate of the agent in pure culture to develop a standardized serological technique taking into account cross reactions with other pathogens like Leptospira and Treponema. Thus, it can be used in future seroepidemiological studies.

Therefore, our results present novel data for B. theileri-related relapsing fever group spirochete in bovine from two Brazilian regions, contributing to the expansion of knowledge about this agent in the country.