Detection of Anaplasma marginale, Babesia bigemina and Babesia bovis by different diagnostic techniques in cattle from the Vassouras municipality, Rio de Janeiro, Brazil

Abstract

This study aimed to determine the presence of Anaplasma marginale, Babesia bigemina, and Babesia bovis in cattle from the Vassouras municipality (Rio de Janeiro, Brazil) using different diagnostic techniques. Blood samples were collected from 226 animals across eight farms using collection tubes with and without anticoagulant. DNA extraction was performed, and molecular analyses were carried out using specific primers targeting the msp5 (A. marginale), sbp-4 (B. bovis), and rap-1a (B. bigemina) genes. In addition, blood smears were prepared, and indirect ELISA serological testing was performed. Nested PCR revealed frequencies of 73.5 % for A. marginale, 27.9 % for B. bovis, and 35 % for B. bigemina. In the blood smear examination, A. marginale was detected in 42.9 % of the animals, whereas Babesia spp. were observed in only 0.88 % of them. Seropositivity rates were 26.5 % for A. marginale, 27.9 % for B. bigemina, and 66.4 % for B. bovis. Animals up to one year of age showed higher infection frequencies by molecular diagnosis, and A. marginale was the pathogen most frequently associated with anaemia. These findings confirm the active circulation of these haemoparasites in cattle from the study area and highlight the importance of using complementary diagnostic approaches to better understand the epidemiology of bovine tick-borne diseases.

Keywords:
bovine tick-borne diseases; PCR; serology; blood smear; epidemiology

Resumo

Este estudo teve como objetivo determinar a presença de Anaplasma marginale, Babesia bigemina e Babesia bovis em bovinos do município de Vassouras (Rio de Janeiro, Brasil) utilizando diferentes técnicas de diagnóstico. Amostras de sangue foram coletadas de 226 animais em oito propriedades rurais, utilizando tubos de coleta com e sem anticoagulante. Realizou-se a extração de DNA e análises moleculares utilizando iniciadores específicos para os genes msp5 (A. marginale), sbp-4 (B. bovis) e rap-1a (B. bigemina). Além disso, foram preparados esfregaços sanguíneos e realizado o teste sorológico ELISA indireto. A técnica de nested PCR revelou frequências de 73,5 % para A. marginale, 27,9 % para B. bovis e 35 % para B. bigemina. No exame de esfregaço sanguíneo, A. marginale foi detectado em 42,9 % dos animais, enquanto Babesia spp. foram observadas em apenas 0,88 % deles. As taxas de soropositividade foram de 26,5 % para A. marginale, 27,9 % para B. bigemina e 66,4 % para B. bovis. Animais com até um ano de idade apresentaram maiores frequências de infecção pelo diagnóstico molecular, e A. marginale foi o patógeno mais frequentemente associado à anemia. Estes resultados confirmam a circulação ativa desses hemoparasitos em bovinos da área de estudo e destacam a importância do uso de abordagens diagnósticas complementares para melhor compreender a epidemiologia das doenças bovinas transmitidas por carrapatos.

Palavras-chave:
Tristeza parasitária bovina; PCR; sorologia; esfregaço sanguíneo; epidemiologia

1. Introduction

Ticks and the pathogens transmitted by them are among the main causes of economic losses to cattle production in Brazil (¹), with bovine tick-borne disease (TBD) standing out. TBD comprises babesiosis, caused by the protozoa Babesia bovis and Babesia bigemina, and anaplasmosis, caused by the bacterium Anaplasma marginale. These pathogens are obligate intracellular parasites that can on their own cause disease in cattle (²). However, co-infection may increase the severity of clinical signs, causing anaemia, bilirubinemia, fever, anorexia, lethargy, ataxia, jaundice, pale mucous membranes, tachypnea, haemoglobinuria, muscle tremors, and teeth grinding (³, ⁴).

The transmission of Babesia spp. occurs mainly through the tick Rhipicephalus microplus, whereas A. marginale can be transmitted both through biological vectors such as haematophagous arthropods and mechanically through fomites (⁵-⁷). Furthermore, transplacental transmission has been demonstrated for all three species (⁸). The environmental conditions of tropical and subtropical regions provide the vector with favourable breeding conditions, influencing the epidemiological dynamics and the endemic areas for these haemoparasites (⁹, ¹⁰).

Given the relevance of TBD to the national cattle industry and the differences among diagnostic methods in their ability to detect active infection and previous exposure, the choice of the diagnostic tool may impact the epidemiological interpretation of the results (¹¹). In this context, the present study aimed to determine the presence of A. marginale, B. bigemina, and B. bovis in cattle from the Vassouras municipality (Rio de Janeiro, Brazil) using molecular, cytological, and serological techniques (nPCR/snPCR, blood smear, and indirect ELISA, respectively).

2. Material and methods

2.1 Ethics Approval

The present study was approved by the Animal Ethics Committee (CEUA) of the University of Vassouras, under protocol Nº. 044/2016.

2.2 Sample collection

The minimum sample size was determined using the following formula: $N=\frac{p.\left(100-p\right),Z^2}{{\left(\frac{d.p}{100}\right)}^2},$, where N designates the number of samples, p the expected prevalence, Z the confidence level and d the margin of error. Assuming an expected prevalence of 50 %, a 95 % confidence interval, and a margin of error of 7 %, the minimum required sample size was estimated to be 196 animals., Visits to the participating farms in the Vassouras municipality (Rio de Janeiro, Brazil; Latitude: 22° 24' 16'' South, Longitude: 43° 39' 48'' West) were carried out from November 2017 to January 2019, and the selection was performed by convenience sampling.

The present study was a cross-sectional, with a total of 226 samples collected from cattle of different breeds raised on dairy farms under extensive and semi-intensive production systems, with ages ranging from 45 days to 13 years. Blood samples were collected from animals of both sexes by puncture of the coccygeal or mammary veins using the Vacutainer® system. The samples were placed in 4 mL tubes containing ethylenediaminetetraacetic acid (EDTA) for haematological and molecular analyses, and in tubes without anticoagulant for serological analysis. After collection, the tubes were identified and placed in an insulated container with reusable ice packs, and kept refrigerated until laboratory processing. The tubes without anticoagulant were centrifuged at 3.000 rpm for five minutes at 22 °C to obtain serum for the serological tests.

The participating farms reported the regular use of acaricides for the control of R. microplus, but no quantification of infestation levels in the animals was performed at the time of sample collection.

2.3 Determination of packed cell volume and detection of haemoparasites in blood smears

Whole blood aliquots were used to determine the packed cell volume (PCV) through microhaematocritas previously described (¹²) and to prepare blood smears, which were stained using a rapid staining method (Diff-Quick^®) for the detection of A. marginale and Babesia spp. Each slide was examined using an L2000-b-pl (Laborcare®) optical microscope at 1000× magnification (oil immersion), with 100 fields observed per slide. Image analysis was performed by an experienced observer blinded to the molecular and serological results.

2.4 Serological analysis

The detection of IgG against A. marginale, B. bigemina, and B. bovis was performed by indirect ELISA. The assay for A. marginale followed the protocol described by Silva (¹³), whereas the assays for B. bigemina and B. bovis were conducted as described by Machado et al (¹⁴). Briefly, microtiter plates (Corning®) were coated with 100 µL per well of antigen diluted to a final concentration of 10 µg/mL in 0.05 M carbonate-bicarbonate buffer (pH 9.6). Control wells received 100 µL of carbonate-bicarbonate buffer without antigen. The plates were sealed and incubated overnight at 4°C in a humid chamber. After incubation, the plates were washed three times with phosphate-buffered saline containing 0.05 % Tween 20 (PBS-Tween; pH 7.2). Blocking was performed with PBS-Tween supplemented with 6 % skim milk powder, followed by incubation at 37°C for 90 min (A. marginale) or 60 min (Babesia spp.).

Bovine sera were diluted 1:200 (A. marginale) or 1:400 (Babesia spp.) in PBS-Tween containing 5 % blocking agent and added in duplicate (100 µL per well). The plates were incubated at 37°C for 90 min and washed again as previously described. Subsequently, anti-bovine IgG conjugated with alkaline phosphatase (Sigma Chemical Co.) diluted 1:25,000 (A. marginale) or 1:10,000 (Babesia spp.) was added to each well, followed by incubation at 37°C for 90 min. After washing, p-nitrophenyl phosphate (pNPP) substrate was added to each well, and the plates were incubated in the dark for 30–40 min at room temperature. The absorbance was measured at 405 nm using a micro-ELISA reader (B.T.-100; Embrabio, São Paulo, Brazil). The cutoff value of the assays was determined based on the mean optical density (OD) of the negative control, plus one standard deviation adjusted by a factor derived from the Student's t distribution, as described by Frey et al. (¹⁵). This factor depends on the number of negative controls (n) and the adopted confidence level. The cutoff was calculated using the following expression: X̄ + SD × √(t / n), where X̄ corresponds to the mean OD of the negative control sera, SD to the standard deviation of the readings; t to the Student's t distribution value corresponding to the confidence level and the number of negative controls, and n to the number of negative controls included. To classify the animals as serologically positive or negative, the OD values were converted into a percentage relative to the cutoff value of each plate using the formula: OD × 100 / cutoff.

2.5 Molecular analysis

DNA extraction from whole blood samples was performed using an adaptation of the phenol-chloroform method, as described by Sambrook (¹⁶). After extraction, the total DNA samples were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific^®). The DNA samples were standardized to a final concentration of 50 ng/µL and stored in 100 µL aliquots at −20°C for subsequent molecular analyses.

The molecular detection of A. marginale was performed using the semi-nested PCR technique targeting the msp5 gene, as described by Singh et al (¹⁷). Nested PCR was employed to amplify fragments of the sbp-4 and rap-1a genes of B. bovis and B. bigemina, respectively, following the method described by Terkawi et al (¹⁸). The primers used in each case as well as the fragment sizes and annealing temperatures are presented in Table 1.

Table 1.
Sequences of the specific primer sets used to amplify the msp-5, sbp-4, and rap-1a genes from A. marginale, B. bovis, and B. bigemina, respectively.

Gene	Assay	Oligonucleotide primers	Product size	Annealing temperature (°C)
msp5	PCR	5’- GCATAGCCTCCGCGTCTTTC-3’ 5’–TCCTCGCCTTGGCCCTCAGA-3’	457 bp	58°C
	snPCR	5’ -TACACGTGCCCTACCGAGTTA-3’ 5’ –TCCTCGCCTTGGCCCTCAGA-3’	344 bp	58°C
sbp-4	PCR	5’-AGTTGTTGGAGGAGGCTAAT-3’ 5’-TCCTTCTCGGCGTCCTTTTC-3’	907 bp	55°C
	nPCR	5’-GAAATCCCTGTTCCAGAG-3’ 5’ -TCGTTGATAACACTGCAA-3’	503 bp	55°C
rap-1a	PCR	5’-GAGTCTGCCAAATCCTTAC-3’ 5’-TCCTCTACAGCTGCTTCG-3’	879 bp	55°C
	nPCR	5’-AGCTTGCTTTCACAACTCGCC-3’ 5’-TTGGTGCTTTGACCGACGACAT-3’	412 bp	55°C

The reaction for A. marginale detection were prepared in a final volume of 25 µL, containing ultrapure water (q.s.p.), reaction buffer at a final concentration of 1X, MgCl₂ (1.5 mM), dNTPs (0.2 mM), forward and reverse primers (0.4 µM each), and Taq DNA polymerase (1 U Promega^®). The cycling conditions for both primary PCR and snPCR consisted of an initial denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min.

The reaction for B. bigemina detection were also prepared in a final volume of 25 µL, containing ultrapure water (q.s.p.), reaction buffer at a final concentration of 1X, MgCl₂ (2.5 mM), dNTPs (0.2 mM), forward and reverse primers (0.8 µM each), and Taq DNA polymerase (1 U). The cycling conditions included an initial denaturation at 94°C for 4 min and 30 s, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min.

The reaction for B. bovis were also prepared in a final volume of 25 µL, containing reagents at the same concentrations used for the reaction used to detect B. bigemina. The cycling protocol comprised an initial denaturation at 94°C for 4 min and 30 s, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 7 min.

One positive control and two negative controls (ultrapure water, HyPure™ Molecular Biology) were included in each run. The positive controls were obtained from blood samples collected from naturally infected cattle from the Seropédica municipality (Rio de Janeiro, Brazil). The PCR products were analysed by agarose gel electrophoresis (UltraPure™ LMP Agarose, Invitrogen^®) at 75 V (5 V/cm) for 45 min in TAE (Tris-acetate-EDTA) buffer. The gels were stained with ethidium bromide (0.5 µg/mL), and visualized using an ultraviolet transilluminator (L-PIX Sti, Loccus).

2.6 Statistical analysis

The chi-square test was used to compare the results of the diagnostic tests and other variables considered, with p < 0.05 considered statistically significant. The analyses were performed using R Core Team (2023) and BioEstat® (¹⁹). Ages were categorized into up to one year, between one and four years, and above four years. Proportions were used to analyse age, whereas the remaining variables were analysed using relative frequencies.

3. Results

During the visits to the eight participating farms, the presence of R. microplus ticks on the sampled herds was observed, but no quantification of the infestation or classification of the degree of parasitism was performed. The detection frequencies for the pathogens varied according to the diagnostic technique employed. In blood smears, A. marginale was identified in 97 out of 226 animals (42.9 %), whereas structures compatible with Babesia spp. were observed in only 2 out of 226 samples (0.88 %). Antibodies against B. bovis were detected by indirect ELISA in 150 out of 226 samples (66.4 %) cattle, whereas those against B. bigemina were detected in 63 out of 226 samples (27.9 %) and those against A. marginale in 60 out of 226 samples (26.5 %). In turn, snPCR revealed positivity for A. marginale in 166 out of 226 samples (73.5 %), for B. bigemina in 79 out of 226 samples (35.0 %), and for B. bovis in 63 out of 226 samples (27.9 %).

Significant differences were observed in the frequency of positive results for each pathogen according to the diagnostic technique (p < 0.05). The frequency of A. marginale infection was significantly higher than those observed for Babesia spp. according to the blood smears and snPCR results, whereas the highest frequency of infection corresponded to B. bovis according to the indirect ELISA results.

Table 2.
Number of samples positive for A. marginale, B. bigemina, and B. bovis discriminated according to analysis technique.

Agent	Blood smear (n=226)	iELISA (n=226)	snPCR/ nPCR (n=226)
Babesia bovis	2 (0,88 %)a	150 (66,4 %)a	63 (27,9 %)a
Babesia bigemina	2 (0,88 %)a	63 (27,9 %)b	79 (35,0 %)a
Anaplasma marginale	97 (42,9 %)b	60 (26,5 %)b	166 (73,5 %)b

Anaplasma marginale showed the highest frequency of infection across all evaluated age groups, with values significantly higher than those observed for Babesia spp. (p < 0.05). Variations among age groups were also observed for all three pathogens, with the highest infection percentages observed in animals of one year of age or younger, and significant differences in the degree of decrease among the remaining age categories (Table 3).

Table 3.
Number of samples positive for A. marginale, B. bigemina, and B. bovis analysed by molecular techniques, discriminated according to age.

Agent	≤ 1 year (n=93)	> 1 to 4 years (n=65)	> 4 years (n=68)	Total
Babesia bovis	36 (38,7 %)a;A	17 (26,2 %)a;B	10 (14,7 %)a;B	63
Babesia bigemina	41 (44,1 %)a;A	25 (38,5 %)a;A	13 (28,9 %)b;B	79
Anaplasma marginale	81 (87,1 %)b;A	40 (61,5 %)b;B	45 (66,2 %)c;B	166

The packed cell volume of the 226 evaluated samples ranged from 14 to 49 %, with a mean of 30 ± 4.2 %. Twenty-three samples (10.2 %) presented values below the reference interval for the species (<24 %) (²⁰). Among these, A. marginale was the most frequently present pathogen, being detected in 18 animals (78.3 %), followed by B. bovis in 7 (30.4 %) and B. bigemina in 4 (17.4 %).

When anaemic animals were stratified by age group, A. marginale was the most frequently detected pathogen in all categories. Among animals aged one year or younger, all anaemic individuals were positive for A. marginale (12/12, 100 %), whereas lower frequencies were observed in animals between one and four years (3/6, 50 %) and those older than four years (3/5, 60 %). For B. bovis, infection frequencies were 25 % (3/12), 33.3 % (2/6), and 40 % (2/5) across the respective age groups. B. bigemina showed the lowest detection frequencies, with 16.7 % (2/12), 16.7 % (1/6), and 20 % (1/5) of positive animals among the respective age categories (Table 4).

Table 4.
Distribution of anaemic animals according to age and positivity for A. marginale, B. bigemina, and B. bovis determined by snPCR/nPCR.

Age group	n*	A. marginale	B. bovis	B. bigemina
≤ 1 year	12	12 (100 %)a	3 (25 %)a	2 (16,7 %)a
1- 4 years	6	3 (50 %)b	2 (33,3 %)a	1 (16,7 %)a
> 4 years	5	3 (60 %)b	2 (40 %)a	1 (20 %)a
Total	23	18 (78,3 %)	7 (30,4 %)	4 (17,4 %)

• ^*
  n: number of anaemic animals. Different lowercase letters in the same column indicate a statistical difference (p<0.05).

According to the results of the molecular techniques, 28 animals (12.4 %) presented co-infection with all three pathogens analysed. Dual co-infection with B. bovis and B. bigemina was observed in 33 animals (14.6 %), whereas 48 (21.2 %) were co-infected with B. bovis and A. marginale, and 66 (29.2 %) with B. bigemina and A. marginale.

4. Discussion

The present study evaluated the occurrence of A. marginale, B. bigemina, and B. bovis infection in cattle from the Vassouras municipality using parasitological, serological, and molecular assays. The application of multiple techniques allowed for the assessment of different aspects of exposure and infection with the pathogens of TBD.

The fact that the blood smear examination detected infection with A. marginale in less than 50 % of the animals and with B. bigemina or B. bovis only in rare cases reflects the low sensitivity of this method, especially for subclinical infections or those with low parasitemia (²¹, ²²). Serology indicated high seropositivity, particularly for B. bovis (66.4 %), demonstrating high levels of exposure to the parasite. Therefore, the discrepancies observed between the methods were expected, and should be interpreted considering the different stages of infection and the host immune response.

Comparing the results with studies conducted in other regions of the state of Rio de Janeiro, the circulation of these pathogens appears to be consistent. In cattle from the Northern Fluminense mesoregion, the seroprevalence of B. bovis was reported to be 90.98 % according to indirect ELISA, whereas A. marginale infection showed high frequencies in all age categories, with a tendency to decrease with increasing age (²³). In Seropédica and surrounding areas, the prevalence for B. bovis and for B. bigemina was approximately 30–35 % by PCR and 70–75 % by ELISA, confirming that serological tests are more suitable for detecting previous exposure, whereas molecular techniques identify the presence of pathogen DNA at the time of sampling (²⁴). In the present study, this pattern was observed for B. bovis, for which the frequency determined by molecular methods was lower than that obtained by serological testing. However, the opposite was observed for B. bigemina and A. marginale.

A lower frequency of infection with A. marginale determined by serological compared to molecular teqchniques is not a common finding but has previously been described (²⁵). This pattern may be related to the stage of infection at the time of sampling, since PCR detects circulating bacterial DNA and is therefore capable of identifying acute and subclinical infections, including in animals with low-level bacteremia (²⁶) that may not have developed a detectable humoral response at the time of sampling. In contrast, serological assays depend on antibody production by the host, which may be delayed during the initial stages of infection or remain below the detection threshold in animals with a mild infection. Furthermore, antigenic variation among A. marginale strains circulating in different geographic regions may reduce the sensitivity of serological tests based on heterologous or non-local antigens (²⁷). Variations in the immune response capacity among individual hosts, as well as potential limitations in assay sensitivity and cutoff values, may further contribute to reduced serological detection. Nevertheless, it was also observed that approximately 49 % of the animals that tested positive for A. marginale were younger than 12 months. This age distribution could be associated with the protection conferred by colostral antibodies and the lower exposure to vectors in young animals, which have less contact with ticks compared to older ones (²⁵).

The molecular analysis revealed the presence of triple and dual co-infections in relevant proportions, indicating that simultaneous exposure to multiple haemoparasites is an important epidemiological phenomenon in the region. Despite this, no clinical outbreaks were observed, suggesting that their circulation occurs predominantly in a subclinical form.

It was observed that younger animals showed higher infection frequencies for all three pathogens determined by molecular techniques, indicating that immunity acquired with age reduces susceptibility. This pattern is consistent with the epidemiology of TBD in endemic areas, where young animals are at greater risk of clinical infection, whereas subclinical infections are more typical for adults (²⁸, ²⁹).

Although the msp5, sbp-4, and rap-1a genes have been widely adopted and validated for the molecular diagnosis of infection with A. marginale, B. bovis, and B. bigemina, respectively, they represent single-copy or low-copy-number targets. Therefore, the diagnostic sensitivity may be lower than that achieved using genes with multiple copies in the genome (³⁰, ³¹, ³², ³³). Thus, it is possible that the occurrence of these agents was underestimated, and the use of targets with multiple copies is recommended in future studies to increase detection sensitivity.

Among anaemic animals, the highest proportion was infected with A. marginale, suggesting a greater haematological impact of this rickettsia, although the infections were frequently subclinical. The extravascular anaemia observed may have resulted both from the destruction of parasitized erythrocytes and from immune mechanisms affecting non-parasitized erythrocytes, explaining the higher frequency of anaemia associated with A. marginale (²⁹, ³⁴).

The results highlight the importance of integrating different diagnostic techniques to understand the epidemiology of TBD, since parasitological, serological, and molecular assays provide complementary information on active infection, previous exposure, and clinical impact. Although the continuous circulation of A. marginale, B. bigemina, and B. bovis in Vassouras, Rio de Janeiro, was confirmed, it is not possible to characterize the enzootic stability based on the results of the present study. For a more robust assessment, it would be necessary to incorporate longitudinal data, outbreak records, measurements of tick infestation intensity, and information on herd management and vector control practices (³⁵) to better understand the balance among exposure and resistance to, and clinical manifestations of hemoparasites in the herd.

Despite the epidemiological relevance of the findings, some limitations should be acknowledged. The sampling strategy was based on convenience, without a formal calculation to define the number of sampled farms or the number of animals sampled per farm, which may limit the representativeness of the results. In addition, no endogenous control was included in the molecular reactions; however, DNA quality and concentration were evaluated by spectrophotometry. Although the presence of ticks was observed on the evaluated animals, the absence of standardized quantification of infestation precluded the analysis of the association between infestation level and presence of the bovine TBD pathogens.

5. Conclusion

The results demonstrate active circulation of haemoparasites in the municipality of Vassouras, Rio de Janeiro, with a predominance of A. marginale (73.5 %), followed by B. bigemina (35.0 %) and B. bovis (27.9 %). PCR demonstrated greater diagnostic sensitivity than blood smear examination, particularly for the detection of Babesia spp. These findings reinforce the importance of continuous monitoring and vector control in the region.

Data availability statement

The complete dataset supporting the findings of this study is available from the corresponding author upon reasonable request.

• Generative AI use statement
  During the preparation of this manuscript, the authors used ChatGPT (version 5.3) for grammatical correction. The authors reviewed and edited the generated text as necessary and assume full responsibility for the content of the published article.

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Edited by

• Editor:
  Luiz Augusto B. Brito

Publication Dates

• Publication in this collection
  14 Sept 2026
• Date of issue
  2026

History

• Received
  12 Mar 2026
• Accepted
  07 July 2026
• Published
  12 Aug 2026