Transovarial Transmission of Anaplasma marginale in Rhipicephalus (Boophilus) microplus Ticks Results in a Bottleneck for Strain Diversity.

Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.

Molecular Prevalence and Genetic Diversity Based on Msp1a Gene of Anaplasma ovis in Goats from Türkiye.

Anaplasma ovis is a tick-borne obligated intraerythrocytic bacterium that infects domestic sheep, goats, and wild ruminants. Recently, several studies have been carried out using 16S rRNA and msp4 genes to identify the genetic diversity of A. ovis. Instead of these genes, which are known to be highly stable among heterologous strains, Msp1a, which is accepted as a stable molecular marker to classify A. marginale strains, was used in A. ovis genetic diversity studies. The genetic diversity of A. ovis strains according to the Msp1a gene has not been extensively reported. Therefore, the purpose of this study was to examine the genetic diversity of A. ovis in goats specifically using analysis of the Msp1a gene. Blood samples were taken from the vena jugularis to the EDTA tubes from 293 randomly selected goats (apparently healthy) in the Antalya and Mersin provinces of Mediterranean region of Türkiye. The Msp1a gene of A. ovis was amplified in all DNA samples through the use of PCR, using a specific set of primers named AoMsp1aF and AoMsp1aR. Among the amplified products, well-defined bands with different band sizes were subjected to sequence analysis. The obtained sequence data were converted into amino acid sequences using an online bioinformatics program and the tandem regions were examined. The Msp1a gene of A. ovis was amplified in 46.1% (135 out of 293) of the goats. Through tandem analysis, five distinct tandems (Ao8, Ao18, Tr15-16-17) were identified, and it was found that three of these tandems (Tr15-16-17) were previously unknown and were therefore defined as new tandems. The study also involved examination of ticks from goats. It was observed that the goats in the area were infested with several tick species, including Rhipicephalus bursa (888/1091, 81.4%), R. turanicus (96/1091, 8.8%), Dermacentor raskemensis (92/1091, 8.4%), Hyalomma marginatum (9/1091, 0.8%), and R. sanguineus s.l. (6/1091, 0.5%). This study provides important data for understanding the genetic diversity and evolution of A. ovis based on tandem repeats in the Msp1a protein.

Characterization of strains of Anaplasma marginale from clinical cases in bovine using major surface protein 1a in Uruguay.

The major surface protein 1a (MSP1a) gene has been used to characterize Anaplasma marginale genetic diversity. This pathogen causes significant productivity and economic losses to the cattle industry. The objective of the present study was to report the first characterization of A. marginale genetic diversity in Uruguay based on MSP1a genotypes and their putative relationship with Rhipicephalus microplus. This cross-sectional study was conducted between 2016 and 2020. The study included whole blood samples from clinical cases of bovine anaplasmosis obtained from 30 outbreaks located in six Uruguay territorial departments. Diagnosis was performed using Giemsa-stained smears and confirmed by nested Polymerase Chance Reaction (nPCR) targeting the A. marginale major surface protein 5 gene. The genetic diversity of A. marginale strains was characterized by analyzing the microsatellite and tandem repeats of MSP1a. Based on the microsatellite structure, four genotypes were identified. Genotype E was the most prevalent. Analysis of MSP1a tandem repeats showed 28 different strains from the combination of 31 repeats, with τ-10-15 and α-ß-ß-ß-Γ being the most common. Repeats Γ, ß, α, and γ were associated with the absence of R. microplus with statistical significance (p < 0.05). Molecular observations showed that 46.7% of the strains identified in our samples lacked the ability to bind to tick cells; therefore, they were probably transmitted by other vectors. Strain genetic diversity provides valuable information for understanding the epidemiological behavior of A. marginale and could contribute to the development of effective vaccines for the control of this disease.

Genetic diversity of Anaplasma marginale in calves with anaplasmosis on farms in Minas Gerais, Brazil.

The objective of this study was to identify Anaplasma marginale strains in dairy heifers from farms with a history of anaplasmosis in the northwest region of the State of Minas Gerais, Brazil. Among the examined animals of the four farms, the overall prevalence total of A. marginale was 55.7 % for gene msp5 and 36.7 % for blood smear. Thirty DNA samples (from 24 asymptomatic and six symptomatic animals) positive for A. marginale msp1α were sequenced to study genotype and strain diversity. The majority (28/30) were the E genotype, followed by C (1/30) and G (1/30). Thirteen different strains were found: α-ß-F-F-F (nine animals), 13-27-27 (three animals), τ-27-18 (three animals), α-ß-ß- BRA1-31 (three animals), α- 22-1318 (three animals), 80-F-F- F-F (three animals), and α -22-13-13, α-ß-ß-Г, M-φ-φ-φ-φ-F, 42-25- 25-31, Q-Q-Q-M, B-Q-B-Q-B-M, and 16-17-F-F (one animal each). A new structure repeated in tandem was described and named BRA 1 (TDSSSASGVLSQSGQASTSSQLG). The α-ß-F-F-F strain was present in all animals with acute anaplasmosis and in three animals asymptomatic. Thus, although 13 strains were observed in the animals evaluated, only the α-ß-F-F-F strain was identified during occurrence of acute disease and mortality, we suggest that this strain has important pathogenicity for calves in northeastern Minas Gerais.

Epidemiology and genetic diversity of Anaplasma marginale in Zamora-Chinchipe, Ecuador.

Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is a hemolytic disease that constitutes a major constraint to cattle production in tropical and subtropical regions including Ecuador. However, the epidemiological situation of this hemoparasitosis in Ecuador is poorly characterized. The present study was aimed to determine the prevalence and genetic diversity of A. marginale in cattle of Ecuador. A cross-sectional study was carried out covering several farms from six out nine cantons of the Zamora-Chinchipe province. A total of 185 cattle were randomly selected and blood samples were collected from the animals. The studied group of animals included six breeds, three age groups, and both sexes. The molecular diagnostic was performed based on a nPCR assay targeting the A. marginale msp5 gene. Anaplasma marginale prevalence was 63.8 % and the bacteria were detected in all the cantons studied. Thirteen representative strains were selected and genetically characterized based on the msp1α gene. Genetic diversity analysis revealed that different strains circulate in the bovine herds studied. The results suggest that cattle movement may contribute to the circulation of common strains in the area. The results demonstrate a high prevalence of A. marginale in the region which should be considered by the sanitary authorities. The epidemiological surveillance for this disease should increase to anticipate acute disease outbreaks with high mortality. Bovine anaplasmosis outbreaks can cause economic losses and the death of several animals; therefore, measures for the prevention and control of this disease are required.

Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%-97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5'UTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

Balanced Th1/Th2 immune response induced by MSP1a functional motif coupled to multiwalled carbon nanotubes as anti-anaplasmosis vaccine in murine model.

Anaplasmosis is one of the most prevalent tick-borne diseases of cattle caused by Anaplasma marginale. MSP1a surface protein has been shown to be involved in eliciting immunity to infected cattle. Carbon nanotubes (CNTs) has been increasingly highlighted due to their needle like structure, which contain multiple attachment sites for biomolecules and may interact with or cross biological membranes, increasing antigen availability to immune system. Here, we have successfully designed a nanocomplex of a synthetic peptide noncovalently attached to multiwalled CNT (MWCNT). Peptide comprising the core motif of the MSP1a was efficiently adsorb onto the nanoparticle surface. The MWCNT-Am1 nanocomplex exhibited high stability and good dispersibility and in vivo immunization showed high levels of IgG1 and IgG2a, followed by increased expression of pro-inflammatory and anti-inflammatory cytokines. This is a proof-of-concept of a nanovaccine that was able to generate a strong immune response compared to the common antigen-adjuvant vaccine without the nanoparticles.

Molecular characterization of the msp1 α AmRio1 strain of Anaplasma marginale in calves and experimentally infected ticks.

This study aims to evaluate the infection's clinical parameters and genetic diversity of msp1α of the AmRio1 strain in acute and chronic infections in cattle and ticks. A calf experimentally infected with the A. marginale AmRio1 strain was monitored during acute infection, and the presence of the msp1α gene was verified in the semi-nested polymerase chain reaction (PCR) during the acute and persistent phases of infection. From day seven after inoculation of the pathogen, the calf showed an increase in body temperature, decrease in hematocrit and increase in the percentage of cells infected by the agent, as well as clinical signs. Blood samples from the experimentally infected calf were positive during the acute infection and the persistent PCR infection for the msp1α gene. During the acute phase, infestation with Rhipicephalus microplus was performed. To evaluate the chronic phase, a blood sample was collected at 90 days post-infection. There was no variation of the MSP1a protein in this study. The AmRio1 strain was pathogenic as it caused severe changes in the clinical parameters of the monitored cattle. The positivity of this strain in organs and saliva of the analyzed ticks indicates a probable biological transmission.

A nearest-neighbors network model for sequence data reveals new insight into genotype distribution of a pathogen.

BACKGROUND: Sequence similarity networks are useful for classifying and characterizing biologically important proteins. Threshold-based approaches to similarity network construction using exact distance measures are prohibitively slow to compute and rely on the difficult task of selecting an appropriate threshold, while similarity networks based on approximate distance calculations compromise useful structural information. RESULTS: We present an alternative network representation for a set of sequence data that overcomes these drawbacks. In our model, called the Directed Weighted All Nearest Neighbors (DiWANN) network, each sequence is represented by a node and is connected via a directed edge to only the closest sequence, or sequences in the case of ties, in the dataset. Our contributions span several aspects. Specifically, we: (i) Apply an all nearest neighbors network model to protein sequence data from three different applications and examine the structural properties of the networks; (ii) Compare the model against threshold-based networks to validate their semantic equivalence, and demonstrate the relative advantages the model offers; (iii) Demonstrate the model's resilience to missing sequences; and (iv) Develop an efficient algorithm for constructing a DiWANN network from a set of sequences. We find that the DiWANN network representation attains similar semantic properties to threshold-based graphs, while avoiding weaknesses of both high and low threshold graphs. Additionally, we find that approximate distance networks, using BLAST bitscores in place of exact edit distances, can cause significant loss of structural information. We show that the proposed DiWANN network construction algorithm provides a fourfold speedup over a standard threshold based approach to network construction. We also identify a relationship between the centrality of a sequence in a similarity network of an Anaplasma marginale short sequence repeat dataset and how broadly that sequence is dispersed geographically. CONCLUSION: We demonstrate that using approximate distance measures to rapidly construct similarity networks may lead to significant deficiencies in the structure of that network in terms centrality and clustering analyses. We present a new network representation that maintains the structural semantics of threshold-based networks while increasing connectedness, and an algorithm for constructing the network using exact distance measures in a fraction of the time it would take to build a threshold-based equivalent.

Immune response and biochemistry of calves immunized with rMSP1a ( Anaplasma marginale) using carbon nanotubes as carrier molecules / Resposta imunológica e bioquímica de bezerros imunizados com rMSP1 a (Anaplasma marginale) utilizando nanotubos de carbono como moléculas carreadoras

Abstract Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.

Resumo Vacinação contra Anaplasma marginale tem sido considerada uma importante estratégia de controle da anaplasmose bovina. Recentemente, camundongos imunizados com rMSP1a funcionalizada à nanotubos de carbono (MWNT) apresentaram resposta imune significante, gerando nova possibilidade para o uso da vacina inativada. O objetivo desse estudo foi investigar a resposta celular e humoral em bezerros imunizados com MWNT+rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, e avaliar os efeitos tóxicos dos MWNT nas funções hepática e renal. rMSP1 a foi ligada covalentemente aos MWNT. Vacina inativada (AmUFMG2) foi produzida através do cultivo de A. marginale em células IDE8. Vinte e quatro bezerros Holandeses foram divididos (quatro grupos) e imunizados subcutaneamente com: PBS e MWNT não-carboxilados (controle, G1), AmUFMG2 (G2), MWNT+rMSP1 a (G3), e AmUFMG2 com MWNT+rMSP1a (G4). Amostras de sangue foram coletadas para contagem de leucócitos, perfil bioquímico e avaliação da resposta celular e humoral. Imunização com MWNT+rMSP1a induziu aumento dos leucócitos totais, células NK, na população de linfócitos e altos níveis de anticorpos comparado com animais imunizados apenas com AmUFMG2. Além disso, MWNT não induziu alterações no perfil bioquímico. Esses dados indicam que MWNT+rMSP1a foram capazes de induzir eficientemente a resposta imune comparado com AmUFMG2 sozinho, sem gerar toxicidade.

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.

Insight into the genetic diversity of Anaplasma marginale in cattle from ten provinces of China.

BACKGROUND: Anaplasma marginale is an important tick-transmitted rickettsial pathogen of cattle, with worldwide distribution and an important economic impact. The genetic diversity of A. marginale strains has been extensively characterized in different geographical regions throughout the world, while information is limited on studies in China. This study was carried out to determine the prevalence and genetic diversity of A. marginale strains in cattle from ten provinces of China. METHODS: A total of 557 blood samples from cattle were collected and screened for the occurrence of A. marginale by PCR based on the msp4 gene. The partial msp1a gene containing tandem repeat sequences was further amplified from msp4 positive samples. The Msp1a amino acid repeats were identified, and genetic variation of A. marginale strains was characterized based on the variation in the repeated portion of Msp1a. RESULTS: Our results showed that 31.6% of 557 cattle were positive for A. marginale. The infection rates of A. marginale varied considerably from 0 to 96.9% in different sampling regions. Sequence analysis revealed that two msp4 sequence variants of A. marginale exist in cattle. One hundred and three msp1a sequences were obtained and permitted to identify 42 Msp1a tandem repeats, 21 of which were not previously published for A. marginale. Moreover, 61 A. marginale genotypes were identified based on the structure of Msp1a tandem repeats. CONCLUSIONS: Anaplasma marginale is widely distributed in China and a high prevalence of infection was observed in cattle. The geographical strains of A. marginale were molecularly characterized based on the structure of Msp1a tandem repeats. Forty-two Msp1a tandem repeats and 61 genotypes of A. marginale were identified. This study, for the first time, revealed the genetic diversity of A. marginale strains in cattle in China.

RepeatAnalyzer: a tool for analysing and managing short-sequence repeat data.

BACKGROUND: Short-sequence repeats (SSRs) occur in both prokaryotic and eukaryotic DNA, inter- and intragenically, and may be exact or inexact copies. When heterogeneous SSRs are present in a given locus, we can take advantage of the pattern of different repeats to genotype strains based on the SSRs. Cataloguing and tracking these repeats can be difficult as diverse groups of researchers are involved in the identification of the repeats. Additionally, the task is error-prone when done manually. RESULTS: We developed RepeatAnalyzer, a new software tool capable of tracking, managing, analysing and cataloguing SSRs and genotypes using Anaplasma marginale as a model species. RepeatAnalyzer's analysis capability includes novel metrics for measuring regional genetic diversity (corresponding to variety and regularity of SSR occurrence). As a part of its visualization capabilities, RepeatAnalyzer produces high quality maps of the geographic distribution of genotypes or SSRs over a region of interest. RepeatAnalyzer's repeat identification functionality was validated for all SSRs and genotypes reported in 21 publications, using 380 A. marginale isolates gathered from the five publications within that list that provided access to their isolates. The tool produced accurate genotyping results in every case. In addition, it uncovered a number of errors in the published literature: 11 cases where SSRs were misreported, 5 cases where two different SSRs had been given the same name, and 16 cases where two or more names had been given to a single SSR. The analysis and visualization functionalities of the tool are demonstrated using several examples. CONCLUSIONS: RepeatAnalyzer is a robust software tool that can be used for storing, managing, and analysing short-sequence repeats for the purpose of strain identification. The tool can be used for any set of SSRs regardless of species. When applied to A. marginale, our test case, we show that genotype lengths for a given region follow a normal distribution, while SSR frequencies follow a power-law-like distribution. Further, we find that over 90 % of repeats are 28 to 29 amino acids long, which is in agreement with conventional wisdom. Lastly, our analysis reveals that the most common edit distance is five or six, which is counter-intuitive since we expected that result to be closer to one, resulting from the simplest change from one repeat to another.

Low genetic diversity of Anaplasma marginale in calves in an endemic area for bovine anaplasmosis in the state of São Paulo, Brazil.

Major surface protein 1a has been used as a marker for genetic stability in identifying geographical isolates of Anaplasma marginale. We conducted a longitudinal study to ascertain the genetic diversity of A. marginale in a dairy cattle herd with a history of clinical anaplasmosis. A total of 20 calves were evaluated every 3 months, from birth to 1 year of age. They were evaluated using blood smears, IFAT, ELISA, and qPCR. Additionally, samples positive for the msp1a gene using nPCR were sequenced. The detection of A. marginale ranged from 20 to 90% using blood smears, 20-80% using ELISA/IFAT, and 15-100% using qPCR. We found evidence that suggested transplacental transmission of A. marginale in 15% (3/20) of the calves based on qPCR and 20% (4/20) based on blood smears. Additionally, these four animals were A. marginale ELISA/IFAT-positive. The A. marginale strains found were α-ß(3)-Γ (n=7), α-ß(2) (n=1), and α-ß(2)-190-Γ (n=1). The new MSP1a tandem repeat 190 was described. The results showed that the genetic diversity of A. marginale in a group of calves up to 1 year of age from Taiaçu (SP) was low, with only three different strains identified, showing the microsatellite genotype E. Eighty percent of the animals evaluated had clinical signs of bovine anaplasmosis and were treated using oxytetracycline and imidocarb dipropionate. Additionally, it was found that 30% (6/20) and 10% (2/20) of the animals required a second and third treatment, respectively, based on clinical signs. Four animals infected with the α-ß(3)-Γ strain died, showing that this strain is also involved in the clinical anaplasmosis cases in Brazil.

Outbreak of anaplasmosis associated with the presence of different Anaplasma marginale strains in dairy cattle in the states of São Paulo and Goiás, Brazil / Surto de anaplasmose associado com a presença de diferentes cepas de Anaplasma marginale em gado leiteiro dos estados de São Paulo e Goiás, Brasil

Abstract The present study reports the genetic diversity of Anaplasma marginale during anaplasmosis outbreaks in rural properties of the states of Goiás and São Paulo, Brazil. Mortality rates of 3.5% (37/1,050) in calves, 4.7% (45/954) in heifers and 1.1% (25/2,200) in lactating cows were observed in a cattle herd of the municipality of Mambaí, state of Goiás, central-western Brazil. In a cattle herd from the municipality of Lins, state of São Paulo, in southeastern Brazil, none of the animals died, despite presenting clinical signs suggestive of bovine anaplasmosis and exhibiting a drastic decrease in milk production. Thus, blood samples were collected from 100 animals with clinical signs suggestive of bovine anaplasmosis in the municipalities of Mambaí and Lins. Based on the microsatellite structure of the MSP1a of A. marginale, the genotypes E and H were observed in Lins, and the C, D and E genotypes were found in Mambaí. The analysis of the tandem repeat structures of the MSP1a showed nine different strains (τ-10 -15, α-β2, α-β3-13, α-β2 192, τ-β-100, α-β2-Γ, 193-β-100, 191-13-Γ and 191-13-18) in Lins and two (α-β3-Γ and E-F-φ2-F2) in Mambaí. Three new tandem repeats of MSP1a (191, 192 and 193) were described. The τ-10-15 and α-β3-Γ strains were predominantly associated with the occurrence of clinical anaplasmosis and mortality in calves, heifers and lactating cows.

Resumo O presente estudo relata a diversidade genética de Anaplasma marginale durante surtos de anaplasmose bovina no Brasil em propriedades localizadas nos Estados de Goiás e São Paulo. No rebanho bovino de Mambaí, Estado de Goiás, Centro-oeste do Brasil, observaram-se taxas de mortalidade de 3,5% (37/1050) nos bezerros; 4,7% (45/954) nas novilhas e 1,1% (25/2200) nas vacas em lactação. No rebanho bovino de Lins, Estado de São Paulo, Sudeste do Brasil, embora os animais tenham apresentado sinais clínicos sugestivos de anaplasmose bovina, culminando em redução drástica da produção leiteira, nenhum animal veio a óbito. Assim, amostras de sangue de 100 bovinos com sinais clínicos sugestivos de anaplasmose foram coletadas em Mambaí-GO e Lins-SP. Baseando-se na estrutura do microssatélite da MSP1a de A. marginale, observou-se a presença dos genótipos E e H em Lins e C, D e E em Mambaí. A análise da estrutura em “tandem repeats” da MSP1a mostrou nove diferentes estirpes (τ-10 -15, α-β2, α-β3-13, α-β2 192, τ-β-100, α-β2-Γ, 193-β-100, 191-13-Γ e 191-13-18) em Lins e duas (α-β3-Γ e E-F-φ2-F2) em Mambaí. Três novos “tandem repeats” da MSP1a (191, 192 e 193) foram descritos. Foi observado predomínio das estirpes τ-10-15 e α-β3-Γ associado à ocorrência de anaplasmose clínica e mortalidade em bezerras, novilhas e vacas em lactação.

Bacterial membranes enhance the immunogenicity and protective capacity of the surface exposed tick Subolesin-Anaplasma marginale MSP1a chimeric antigen.

Ticks are vectors of diseases that affect humans and animals worldwide. Tick vaccines have been proposed as a cost-effective and environmentally sound alternative for tick control. Recently, the Rhipicephalus microplus Subolesin (SUB)-Anaplasma marginale MSP1a chimeric antigen was produced in Escherichia coli as membrane-bound and exposed protein and used to protect vaccinated cattle against tick infestations. In this research, lipidomics and proteomics characterization of the E. coli membrane-bound SUB-MSP1a antigen showed the presence of components with potential adjuvant effect. Furthermore, vaccination with membrane-free SUB-MSP1a and bacterial membranes containing SUB-MSP1a showed that bacterial membranes enhance the immunogenicity of the SUB-MSP1a antigen in animal models. R. microplus female ticks were capillary-fed with sera from pigs orally immunized with membrane-free SUB, membrane bound SUB-MSP1a and saline control. Ticks ingested antibodies added to the blood meal and the effect of these antibodies on reduction of tick weight was shown for membrane bound SUB-MSP1a but not SUB when compared to control. Using the simple and cost-effective process developed for the purification of membrane-bound SUB-MSP1a, endotoxin levels were within limits accepted for recombinant vaccines. These results provide further support for the development of tick vaccines using E. coli membranes exposing chimeric antigens such as SUB-MSP1a.

Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil.

Anaplasma marginale is the most prevalent tick-borne pathogen in cattle in tropical and subtropical regions of the world. Major Surface Protein 1a (MSP1a) has been found to be a stable genetic marker for identifying A. marginale isolates within geographical regions. It is conserved in cattle during infection and tick-borne transmission of the pathogen. The aim of the present longitudinal study was to determine occurrences of genetic diversity associated with high prevalence of A. marginale under natural transmission conditions. Twenty calves were evaluated every 3 months during the first year of life. Rickettsemia levels due to A. marginale, measured as the number of msp1αcopies/ml in the blood of positive calves, ranged from 2.06×10(3) to 4.36×10(12). The numbers of MSP1a tandem repeats among MSP1a tandem repeats were 3 and 6. The predominant msp1α microsatellite was E, and another MSP1a tandem repeat was found that presented genotype G. Nineteen different MSP1a tandem repeats of A. marginale were found circulating in animals. The MSP1a tandem repeats 4-63-27 (27.5%), 78-24(2)-25-31 (n=21.6%) and τ-10(2)-15 (n=17.6%) were the ones most commonly observed. Twenty-two MSP1a tandem repeats resulted in new sequences with amino acid changes, as shown in this study. Thirty sequences were found for the first time in Brazil. Glycine, glutamate, serine and alanine amino acids were found at position 20. During the study, 80% (16/20) of the animals were infected by more than one genotype. Three animals were born infected, with MSP1a tandem repeats 4-63-27, 78-24(2)-25-31 and τ-10(2)-15, thus indicating occurrence of transplacental transmission. In the phylogenetic analysis, 19 different MSP1a tandem repeats of A. marginale were found in the cattle, which suggested that many MSP1a tandem repeats and high variation in MSP1a were occurring.

Low genetic diversity associated with low prevalence of Anaplasma marginale in water buffaloes in Marajó Island, Brazil.

The rickettsia Anaplasma marginale is the etiologic agent of bovine anaplasmosis, an important tick-borne disease affecting cattle in tropical and subtropical regions of the world. In endemic regions, the genetic diversity of this pathogen is usually related to the high prevalence of the disease in cattle. The major surface protein 1 alpha (MSP1a) has been used as a marker to characterize the genetic diversity and for geographical identification of A. marginale strains. The present study reports the characterization of A. marginale MSP1a diversity in water buffaloes. Blood samples were collected from 200 water buffaloes on Marajó Island, Brazil where the largest buffalo herd is located in the Western hemisphere. Fifteen buffaloes (7.5%) were positive for A. marginale msp1α by PCR. Four different strains of A. marginale with MSP1a tandem repeat structures (4-63-27), (162-63-27), (78-24-24-25-31) and (τ-10-10-15) were found, being (4-63-27) the most common. MSP1a tandem repeats composition in buffalos and phylogenetic analysis using msp1α gene showed that the A. marginale strains identified in buffaloes are closely related to A. marginale strains from cattle. The results demonstrated low genetic diversity of A. marginale associated with low bacterial prevalence in buffaloes and suggested that buffaloes may be reservoirs of this pathogen for cattle living in the same area. The results also suggested that mechanical transmission and not biological transmission by ticks might be playing the major role for pathogen circulation among water buffaloes in Marajó Island, Brazil.

Epidemiology and evolution of the genetic variability of Anaplasma marginale in South Africa.

Bovine anaplasmosis caused by infection of cattle with Anaplasma marginale has been considered to be endemic in South Africa, an assumption based primarily on the distribution of the tick vectors of A. marginale and serological studies on the prevalence of anaplasmosis in Limpopo, Free State, and North West. However, molecular evidence of the distribution of anaplasmosis has only been reported in the Free State province. In order to establish effective control measures for anaplasmosis, epidemiological surveys are needed to define the prevalence and distribution of A. marginale in South Africa. In addition, a proposed control strategy for anaplasmosis is the development of an A. marginale major surface protein 1a (MSP1a)-based vaccine. Nevertheless, regional variations of this gene would need to be characterized prior to vaccine development for South Africa. The objectives of the present study were therefore to conduct a national survey of the prevalence of A. marginale in South Africa, followed by an evaluation of the diversity and evolution of msp1a in South African strains of A. marginale. To accomplish these objectives, species-specific PCR was used to test 250 blood samples from cattle collected from all South African provinces (including 26 districts and municipalities), except the Free State province where similar studies were reported previously. The prevalence of A. marginale ranged from 65% to 100%, except in Northern Cape province where A. marginale was not detected. A correlation was found between the prevalence and genetic diversity of A. marginale MSP1a. Additionally, the genetic diversity of the A. marginale MSP1a was found to evolve under negative and positive selection, and 23 new tandem repeats in South Africa were shown to have evolved from the extant tandem repeat 4. Despite the MSP1a genetic variability, some types of tandem repeats were found to be conserved among the A. marginale strains, and low-variable peptides in MSP1a tandem repeats were subsequently identified. The results of this research confirmed that anaplasmosis is endemic in South Africa. The results of the molecular characterization of the MSP1a can then be used as the basis for development of new and novel vaccines for anaplasmosis control in South Africa.