High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil

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Introduction
Anaplasma marginale is an obligate intracellular Gram-negative alpha-proteobacterium of the Anaplasmataceae family that parasites erythrocytes of ruminants. Because it is the etiological agent of bovine anaplasmosis, A. marginale stands out as one of the major causes of diminished bovine beef and milk production. It is also associated with high herd mortality. This bacterium parasites bovines in tropical, subtropical and temperate regions (Kocan et al., 2010;Aubry & Geale, 2011). In Brazil, bovine anaplasmosis is considered endemic (Pohl et al., 2013). A. marginale is transmitted biologically by vector ticks of the species Rhipicephalus (Boophilus) microplus and also mechanically by both hematophagous flies and contaminated fomites, may pathogen establish a persistent infection in cattle (Palmer et al., 2000). Persistent infection arises from the agent's ability to perform antigenic variation, resulting in structural changes, which make it difficult for the host's immune system to recognize it. Transplacental transmission has also been reported previously Silva et al., 2015Silva et al., , 2016.
The Médio Mearim microregion of Maranhão comprises 20 municipalities, occupying an area of 11,023 km 2 with 411,884 inhabitants, with a population density of 37.4 inhab/km 2 . The area has an average altitude of 61 m (IBGE, 2018). The Microregion has a semi-humid climate, with two well-defined seasons, rainy (January to June) and dry (July to December), Cerrado and Amazon biomes, and average annual temperature ranging from 24 to 26°C (Neto et al., 2016). These characteristics contribute to the occurrence of a great diversity of ticks in the region, including R. microplus, which is the main vector of A. marginale (Costa et al., 2020). According to Medlock et al. (2013), climatic and demographic characteristics can influence the distribution and density of the R. microplus ticks.
Brazil, despite being considered enzootic for bovine anaplasmosis, has a variable prevalence according to the climatic conditions of each region (Dantas-Torres & Otranto, 2016). The molecular prevalence in the country ranges from 39.8% in semi-arid of Pernambuco (Santos et al., 2017) to 100% in São Paulo . Where there is a high prevalence of anaplasmosis, several A. marginale strains have been detected, and some animals may be infected with more than one strain Pohl et al., 2013;Machado et al., 2015;Silva et al., 2015).
The genetic diversity of A. marginale has been characterized based on sequencing major surface proteins (MSPs), such as MSP1α, MSP1β, MSP4 and MSP5 (Palmer et al., 1999). The last two were considered conserved and used for phylogenetic and molecular studies, along with serological diagnoses, whereas MSP1α is considered for a marker of the bacterium's genetic diversity and strain variations (Cabezas-Cruz & de la Fuente, 2015). The MSP1α protein has a conserved C-terminal region and a variable N-terminal region, where sequence repetitions follow a pattern according to each existing strain of the bacterium (de la Fuente et al., 2001). Therefore, pattern variations regarding molecular weight and changing amino acids (de la Fuente et al., , 2006 allow identification of different strains of A. marginale.
Thus, considering there are no studies reporting the occurrence of A. marginale in Maranhão, and that the Middle region of Mearim, Maranhão has characteristics that favor the spread of the tick R. microplus, the aim of the present study was to investigate the serological and molecular prevalence and genetic diversity of A. marginale in naturally infected cattle in dairy herds in six municipalities in the Médio Mearim microregion, Maranhão, northeastern Brazil.
The farms contained an average herd of 40 to 60 animals, and approximately 10 animals of each were randomly collected. The microregion's climate is hot with high relative humidity in the rainy season. Habitually there is a high flow of animals between farms located in the region, due to the replacement of dams/breeders and disposals due to age or death. To determine the sample size, the Simple random sampling method was used, assuming an expected prevalence of 70% (Ramos et al., 2019), considering that the prevalence of A. marginale is unknown in the state of Maranhão, with an absolute precision of 5% and a 95% confidence interval as indicated by the formula below: (1) 2 2 z (1 Py)Py n d − = z = confidence coefficient (z = 1.96) n = sample size Py = expected prevalence (70%) d2 = Absolute precision (5%) (Thrusfield, 2006) For convenience, the blood samples were taken from 179 calves (89 males and 90 females between 1 and 12 months old) and 164 cows (> 2 years old), thus totaling 343 animals.
Two blood samples were drawn from the jugular vein of each animal. The first was collected in tubes with EDTA anticoagulant (ethylenediaminetetraacetic acid) in order to estimate the presence of rickettsemia by means of absolute quantification using qPCR and through obtaining msp1α amplicons using semi-nested PCR (snPCR). The second blood sample was collected in tubes without anticoagulant, to obtain serum for detecting IgG antibodies against A. marginale by means of the indirect enzyme-linked immunosorbent assay (iELISA). iELISA for detecting IgG antibodies against Anaplasma marginale iELISA was performed to detect IgG antibodies against A. marginale, using recombinant major surface protein (rMSP5) of A. marginale (Imunodot ® Diagnostics, Jaboticabal-SP, Brazil), in 343 cattle serum samples, according to the manufacturer's instructions.
The plates used in the assay (Maxisorp ® ; Nunc, Thermo Scientific, Brazil) were coated with recombinant protein MSP5 (protein concentration was adjusted to 2.5 μg/mL) in 100 μL of sodium bicarbonate buffer 0.05 M (pH 9.6) at 4°C for 18 hours. Blockade was then performed using PBS-Tween 20 (pH 7.2) plus 6% skimmed milk powder (Molico ® , Nestlé, Brazil). The plates were incubated in a humid chamber at 37°C for 90 min. After three washes with PBS-Tween 20, 100 µL of the diluted positive and negative reference serum samples, as well as the test samples, were tested in a block titration at dilutions of 1: 100 in PBS-Tween 20 and added to the ELISA plate. The plates were incubated at 37°C for 60 minutes. After three washes with PBS-Tween 20 buffer, 100 µL of anti-bovine IgG antibodies (cat. no. A0705, Sigma®, St. Louis, USA) conjugated to alkaline phosphatase were added to the ELISA plate (at a dilution of 1:30,000 for cattle, in PBS-Tween 20 solution), with subsequent incubation at 37°C for 60 minutes. After three washing steps, 100 µL of the alkaline phosphatase substrate, p-nitrophenyl phosphate (Sigma ® , St. Louis, USA), was diluted to 1 mg/mL in diethanolamine buffer, pH 9.8 (Sigma ® , St. Louis, USA). The plates were sealed with aluminum foil and incubated at room temperature for 45 minutes.
Readings were done on a spectrophotometer (B.T.-100; Embrabio, São Paulo, Brazil), with a 405 nm filter. The discriminant absorbance value (cutoff) was determined to be 2.5 times the average absorbance value of the negative controls (Machado et al., 1997).
The negative controls used were bovine/cattle serum samples obtained from newborn animals before colostrum intake that had been found to be negative for A. marginale through qPCR and serological assays. Serum samples positive for A. marginale through serological tests and qPCR were obtained from naturally infected animals. These serum samples had been stored in the Animal Serum Bank of the Laboratory of Immunoparasitology of the Department of Pathology, Theriogenology and One Health, UNESP Jaboticabal, SP, Brazil.

DNA extraction from blood samples
Out of 343 animals, 235 blood samples were randomly chosen and submitted to molecular analysis. DNA from 235 bovine blood samples was extracted using the InstaGene Matrix kit (Biorad™), following the manufacturer's recommendations. The DNA concentration obtained from each sample, along with their purity ratios, were measured using a spectrophotometer (NanoDrop; Thermo Scientific). The DNA samples for further PCR were stored at -20ºC.

Endogenous control PCR
To avoid false-negative results caused by the presence of inhibitors, and to check for amplifiable DNA, the DNA samples were subjected to a conventional polymerase chain reaction (cPCR) to amplify the endogenous glyceraldehyde-3-phosphate dehydrogenase gene (gapdh) of mammals, following the established protocol (Birkenheuer et al., 2003).
qPCR targeting the Anaplasma marginale msp1β gene The samples that were positive for the gapdh gene through conventional PCR were subjected to a quantitative real-time PCR assay (qPCR) to detect the A. marginale msp1β gene (95 bp), following the protocol previously described by Carelli et al. (2007). The amplification reactions were conducted in a CFX96 ® thermal cycler (BioRad, Hercules, CA, United States) using the primers AM-For (5'-TTGGCAAGGCAGCAGCTT-3') and AM-Rev (5'-TTCCGCGAGCATGTGCAT-3') and probe Am-pb (6FAM-TCGGTCTAACATCTCCAGGCTTTCAT-BHQ1). All samples were tested in triplicates. The number of DNA copies/µL was quantified using the plasmid pSMART (Integrated DNA Technologies, Coralville, Iowa, USA), which contains the target sequence for amplifying the A. marginale DNA. The serial dilutions performed made it possible to obtain standards with different concentrations of plasmid DNA containing the target sequence (2.0 x 10 7 to 2.0 x 10 0 copies/µL), determined as (X g/µL DNA/[plasmid size (bp) x 660]) x 6.022 x 10 23 x copies of the plasmid/µL. Ultrapure sterile water (Qiagen ® , Madison, United States) and A. marginale DNA  were used as negative and positive controls, respectively, in all (q)PCR assay. snPCR targeting the A. marginale msp1α gene The samples that were positive for the A. marginale msp1β gene through qPCR were further subjected to snPCR targeting the msp1α gene, following the protocol previously described by Castañeda-Ortiz et al. (2015). All products from the PCR assays underwent horizontal electrophoresis and the results were visualized and analyzed using an ultraviolet light transilluminator coupled to computer software for image analysis (Chemi-Doc MP Imaging System, Bio-Rad ® ).

Purifying and sequencing the amplified products
The snPCR products based on the msp1α gene were purified using the ExoSAP-IT™ PCR product cleanup reagent kit (Thermo Scientific, San Jose, CA, USA), following the manufacturer's recommendations. The amplified products were sequenced using an automated technique based on the dideoxynucleotide chain termination method (Sanger et al., 1977) in an ABI PRISM 3700 DNA Analyzer sequencer (Applied Biosystem, Foster City, CA, USA). The amplimers were sequenced at the Center for Biological Resources and Genomic Biology (CREBIO) at FCAV/UNESP.

msp1α sequence analysis
The nucleotide sequences were inserted in the Phred Phrap software (Ewing et al., 1998) to screen and evaluate their quality, on electropherograms. Bases with Phred quality above 20 were considered reliable. Consensus sequences were also generated in the same software. The identities of the nucleotides obtained were compared with those in sequences deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (Benson et al., 2002), using the BLASTn software (Altschul et al., 1990).
Classifying A. marginale strains and assessing genetic diversity The nucleotide sequences of the present study and sequences previously deposited in GenBank were subject to strains classification according to Cabezas-Cruz et al. (2013), including the microsatellite genotypes and the composition of tandem repeats (TR). The 5′-UTR microsatellite is positioned between the putative Shine-Dalgarno (SD) sequence and the translation initiation codon (ATG). The structure of microsatelites was determined by GTAGG (G/A TTT)m (GT)n T ATG (Estrada-Peña et al., 2009). The analysis of the genotypes was performed according to the qPCR assays for the A. marginale msp1β gene All the 235 DNA samples were positive for the endogenous mammalian gene (gapdh) in the cPCR assay, with an average DNA concentration of 11.1 ng/µl. The 260/280 and 260/230 parameters exhibited means of 1.08 and 0.15, respectively. The qPCR assays targeting the msp1β gene indicated that 224 samples (95.32%) were positive (Table 1), with 97.12% (101/104) from calves and 93.89% (123/131) from cows (P = 0.24; X 2 = 1.34). The quantity of copies of a fragment of the msp1β/μL gene ranged from 1.07 x 10 1 to 9.46 x 10 9 copies, with an average of 9.72 x 10 7 in calves and 3.19 x 10 6 in cows. The qPCR efficiency ranged from 91.6% to 108.6%; the coefficient of determination ranged from 0.952 to 0.99; the slope ranged from -3.13 to -2.86; and the y-axis intercept ranged from 37.13 to 43.42. nomenclature proposed by de la Fuente et al. (2007). The SD-ATG distance was calculated according to the formula (4 x m) + (2 x n) +1 previously described (Estrada-Peña et al., 2009).
Using the Repeat Analyzer software, we performed the identification and analysis of tandem repeats of A. marginale msp1α (Catanese et al., 2016). Genetic diversity was calculated by metric indices that measure the percentage of single repeats in a region as well as the regularity with which repeats are distributed. Additionally, the software was used to calculate the frequency of each short-sequence repeats (SSRs) in the site under study (by the number of genotypes) and to list the sequences that were exclusive to the area under study.
Finally, the genetic diversity was calculated using the Repeat Analyzer software and was divided into two categories. The first category of metrics (GDM1) measures the amount of unique repeats in a region; while the second (GDM2) measures how uniformly the repeat occurrences in a region are distributed. GDM1 and GDM2 were presented in two variants, local and global, depending on whether the metric calculation was an average of the values of each genotype or of its region, respectively (Catanese et al., 2016). The expected values range from 0 to 1.

Statistical analysis
We used the statistical test Chi-Square (χ 2 ) to distribute the prevalence of the disease by age group and by sampled municipalities. All statistical analyses were performed using the statistical program Graphpad Prism. 8 -Windows, with a confidence interval (CI) of 95%.

Anaplasma marginale seropositivity via iELISA
The iELISA serological test using the recombinant protein MSP5 revealed the presence of IgG antibodies against A. marginale in 81.34% (279/343) of the bovine serum samples. Table 1 shows the percentage of seropositive animals in each municipality studied in the Médio Mearim macroregion, Maranhão, northeastern Brazil. Of the 279 seropositive animals, 59.50% were cows and 40.50% calves. In the prevalence of seropositivity analysis by animal age, the cows presented more seropositive animals (92.74%) when compared to the calves (68.90%), but there was no statistical difference (P = 0.15; X 2 = 32.04). cPCR for the A. marginale based on msp1α gene and BLASTn analysis In the qPCR assays, among the 224 samples that were positive for the msp1β gene, 67.4% (151/224) were also positive for the msp1α gene fragment. Among the positive samples, it was possible to obtain 50 sequences of quality regarding the msp1α gene, through quality analysis carried out in the Phred Phrap software. The BLASTn analysis on the sequences obtained revealed percentage similarities ranging from 95% to 100%, in relation to A. marginale sequences previously deposited in the GenBank, with coverage of between 96% and 100%. The sequences of A. marginale generated in this study were deposited in the GenBank database under accession numbers: OL629247-OL629255.
Genetic diversity analysis of A. marginale based on msp1α gene The RepeatAnalyzer software, which was used for alphanumeric genotypic classification and strain identification, detected 50 distinct strains. Among these, 21 had not previously been reported in the literature. These 21 strains are highlighted in red in Table 2. The metric indexes of A. marginale genetic diversity obtained from the dairy cattle in the Médio Mearim Region, Maranhão, were determined as GDM1-local (0.81) and GDM1-global (0.312). GDM1-local was considered high, given that this index is measured on a scale from 0 to 1. This indicates that the repetition (SSRs) was quite diverse among each other in the region studied. However, the GDM2-local (0.07) and GDM2-global (0.057) indices were low, which indicated that the SSRs were present in equal amounts, i.e. they occurred approximately the same number of times, with uniform distribution among the animals. The low GDM2 values indicated that the SSRs were sparsely dispersed. The metric indexes of genetic diversity in this study and in other Brazilian regions are shown in Table 3.
Regarding the frequencies of strains, TR Q, 15 and EV8 occurred only once; 18 and 22-2, twice; 27, τ and β, three times; α four times; and 13 five times (Figure 2). Figure 3 shows the relationship between the number of TR and the number of amino acids. The correlation between the number of genotypes and the number of SSRs found is shown in Figure 4.

Discussion
Herein, the prevalence and the genetic diversity of A. marginale was assessed in northeastern Brazil. In the current study, iELISA using the recombinant protein MSP5 revealed high seropositivity rates (81.34% [279/343]) for A. marginale among the animals sampled. The high titer of IgG antibodies against A. marginale detected in the The most common genotypes in the region studied were H (26/50) and E (18/50), followed far behind by F (2/50) and C (2/50). Genotypes B and G were found only once each. Figure 5 shows the distribution of the most common genotypes according to the municipality where the samples originated from.  present study is corroborated by previous studies carried out in Brazil (72.2% [289/400] Ramos et al., 2019 and 91.25% [73/80] Garcia et al., 2021). Similar to seroprevalence results, the molecular prevalence found (95.32% [224/235]) is corroborated by data previously reported in different Brazilian states, such as Minas Gerais (70% -66/94 [Pohl et al., 2013), São Paulo (94% -47/50 ) and Santa Catarina (79% -248/311 [Casa et al., 2020]), emphasizing the endemic nature of this agent in Brazil.
The prevalence of A. marginale has been associated with different risk factors i.e., cattle breeds, management system, climate, presence of ticks, production type and others (Kocan et al., 2010;Jaimes-Dueñez et al., 2018;Ola-Fadunsin et al., 2018). The climate in the Médio Mearim microregion where the present study was carried out is hot with high relative humidity in the rainy season. These environmental conditions are favorable for ticks and blood-sucking flies development and, therefore, biological and mechanical transmission of A. marginale. During sample collection, high infestation by ticks and the presence of blood-sucking insects (Haematobia irritans, Stomoxys calcitrans and tabanids) was observed (data not shown). Thus, the high prevalence found may be partially associated to presence of arthropod vectors. However, future studies are much needed to assess the epidemiological factors -i.g., iatrogenic transmission and animal trade -coupled to the high prevalence herein found.
The high seroprevalence for A. marginale in cows (92.74%) observed in this study can be also partially explained by the time these animals remain in the herd exposed to infection (Ramos et al., 2019). In calves, it is possible that immunological immaturity is related to a lower rate of seropositivity, as this age group had high levels of parasitemia (9.72 x 10 7 ). Other possible causes are failure to transfer antibodies from the mother, the natural decline of antibodies during the first months of life, high infestations by ticks and flies and also transplacental transmission .
High diversity was observed in the microsatellite analyses and tandem repeats among A. marginale sequences obtained from sampled animals in Maranhão, northeastern Brazil. Out of 50 strains identified, 21 had never been reported in the literature, according to the RepeatAnalyzer software. The high genetic diversity of MSP1a of A. marginale seems to occur in tropical regions, as evidenced in previous studies carried out in Brazil Ramos et al., 2019;Bahia et al., 2021). In addition to impacting the prevalence of bovine anaplasmosis, the presence of vectors may have direct effect on the diversity of A. marginale (Bahia et al., 2021), mainly in endemic areas (Estrada-Peña et al., 2009). Therefore, the high genetic diversity observed in the current study could be attributed to the presence of high infestation by arthropod vectors observed during the blood sampling. However, the trade of infected animals as well as herds infected for long periods may also affect the A. marginale diversity (de la Fuente et al., 2007). Thus, the factors associated with genetic diversity herein observed must be further evaluated.
Among the A. marginale strains found circulating in the sampled animals, the α-β3-Γ strain has been associated with acute anaplasmosis and high mortality in dairy herd calves on a farm located in the state of São Paulo, southeastern Brazil . In addition, the same strain was predominantly associated with mortality of calves, heifers, and lactating cows in Mambaí, Goiás state, central-western, Brazil . This A. marginale strain seems to be highly transmissible and has been previously isolated from cattle in Mexico (Almazán et al., 2008) and Argentina (Ruybal et al., 2009). Moreover, according to Cabezas-Cruz et al. (2013), this strain constitutes one of the most common A. marginale strains worldwide.
The obtained strain τ-10 2 -15 has also been previously recorded by Silva et al. (2015) in Rio de Janeiro, southeastern Brazil. The authors suggested the probability of transplacental transmission for this strain since three animals were born infected with A. marginale presenting the MSP1a tandem repeat τ-10 2 -15.
The genetic diversity recorded also reflected in the A. marginale genotypes observed in cattle herds from Médio Mearim, according to the classification described by Estrada-Peña et al. (2009). In the present study, H was the most frequent genotype, but genotypes E, F, C, B, and G were also found. In contrast, previous studies performed in Brazil have reported E as the most frequent genotype Ramos et al., 2019;Bahia et al., 2021). According to Estrada-Peña et al. (2009), the genotype H was present across different ecoregion clusters (ecoregion clusters 2 and 3). Usually, this A. marginale genotype is related to regions where R. microplus ticks are common or in sites where R. microplus has been prevalent in the past but has been eradicated (Estrada-Peña et al., 2009).
Regarding GDM1 indexes of diversity by the RepeatAnalyzer, high genetic diversity was observed among the A. marginale genotypes analyzed, as well as in the region where the current study was performed, suggesting the prevalence of new A. marginale genotypes in Brazil. Additionally, the values of the GDM2 indices obtained suggest a low dispersion rate of these new A. marginale genotype. Similar fidings has been recorded in Brazil (Ramos et al., 2019).