L.M.F. Passos

Phylogeography of New World isolates of Anaplasma marginale based on major surface protein sequences

Gene and protein sequences of major surface proteins (MSP) 1a and 4 of Anaplasma marginale (Rickettsiales: Anaplasmataceae) were used to infer phylogenetic relationships between New World isolates from Argentina, Brazil, Mexico and the United States. Seventeen isolates of A. marginale plus two outgroup taxa (A. centrale and A. ovis) were used for maximum-parsimony analysis of MSP4, while 20 isolates were used for phylogenetic analysis of MSP1a. msp4 analysis provided strong bootstrap support for a Latin American clade and, within this clade, support was detected for Mexican and South American clades. Isolates of A. marginale from the United States also grouped into two clades from the southern (isolates from Florida, Mississippi, and Virginia) and west-central (isolates from California, Idaho, Illinois, Oklahoma, and Texas) states. Although little phylogeographic resolution was detected within these higher clades, msp4 sequences appear to be a good genetic marker for inferring phylogeographic patterns of A. marginale isolates. In contrast to the phylogeographic resolution provided by msp4, MSP1a DNA and protein sequence were quite variable and did not provide phylogeographic resolution. Most variation in MSP1a sequences appeared unique to a given isolate and similar DNA sequence variation in msp1alpha was detected within isolates from Idaho and Florida and from Idaho and Argentina. The results of these studies demonstrated that msp4 provided phylogenetic information on the evolution of A. marginale isolates. In contrast MSP1a sequences appeared to be rapidly evolving and these sequences may provide phylogeographic information only when numerous isolate MSP1a sequences are analyzed from a geographic area.

Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil

Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PCR were sequenced and compared with each other and with known sequences. The serological results showed a total prevalence of 91.0% for T. equi and 83.0% for B. caballi, while by PCR, prevalences of 59.7% for T. equi and 12.5% for B. caballi were observed. However, no correlations were seen between positivity (neither by serology nor by PCR) and PCV values. As expected, the microscopic examination of blood smears showed low sensitivity in detecting the infections when compared to the PCR. Only 35 out of 570 blood smears were positive, with parasitemias below 0.1%. No congenital transmission was detectable. As far as sequencing is concerned, no differences were seen among the isolates of each species nor among them and known sequences available. These results confirm, by molecular methods, the high prevalence rates of T. equi and B. caballi infections in carrier horses in Brazil. However, no diversity was observed among the isolates within the studied regions.

Genetic Variants of Anaplasma phagocytophilum in Wild Caprine and Cervid Ungulates from the Alps in Tyrol, Austria

The occurrence of genetic variants of Anaplasma phagocytophilum was studied in wild ungulates from the northern and central eastern Alps in Tyrol, Austria. For this purpose, spleen samples collected from 53 game animals during the hunting season 2008/2009 (16 roe deer [Capreolus capreolus], 10 red deer [Cervus elaphus], 16 Alpine chamois [Rupicapra r. rupicapra], 7 Alpine ibex [Capra i. ibex], and 4 European mouflons [Ovis orientalis musimon]) were analyzed. Thirty-five animals originated from the Karwendel mountains, 12 from the Kaunertal area (Ötztal Alps), and the remaining from other mountainous areas in Tyrol. DNA extracts were screened with a real-time polymerase chain reaction targeting the msp2 gene of A. phagocytophilum. A total of 23 (43.4%) samples, from all ungulate species studied, were A. phagocytophilum positive. As of the date of this article, A. phagocytophilum has not been reported in the Alpine ibex. The positive samples were investigated further with polymerase chain reactions for amplification of the partial 16S rRNA, groEL, and msp4 genes. Sequence analysis using forward and reverse primers revealed seven different 16S rRNA gene variants. No variant could be attributed to any particular ungulate species. The groEL gene revealed 11 different variants, which grouped in the phylogenetic analysis into two distinct clusters: one cluster contained the sequences from roe deer, whereas the sequences of the other species formed the second cluster. The msp4 gene showed a high degree of variability in the amplified part with a total of 10 different sequence types. The results show that the wild mountain ungulates were infected to a considerable extent with various variants of A. phagocytophilum. The pathogenicity of the variants and the reservoir competence of the species investigated in this study deserve further attention in future studies.

Retrospective study (1998-2001) on canine ehrlichiosis in Belo Horizonte, MG, Brazil

O presente trabalho descreve um estudo retrospectivo da casuistica clinica de erliquiose em caes atendidos entre marco de 1998 e setembro de 2001. Foram analisadas 194 fichas clinicas de animais com suspeita de hemoparasitoses, nas quais 31 caes foram diagnosticados com Ehrlichia canis e 21 com Ehrlichia platys, por meio de exame parasitologico direto de esfregacos sanguineos. Foram considerados alguns aspectos demograficos (idade, raca, sexo, epoca do ano e regiao de origem), caracteristicas clinicas (temperatura corporal, presenca e/ou historico de carrapatos e sinais clinicos) e hematologicas (hemograma completo e celula parasitada). O numero de diagnosticos de erliquiose aumentou consideravelmente a partir de 2000, sendo que 24,4% dos casos ocorreram em animais entre 13 e 24 meses de idade na cidade de Belo Horizonte e areas peri-urbanas, verificando-se maior risco de infeccao em animais que residiam em casas. Os sintomas mais frequentes foram febre, anorexia, apatia, dor abdominal, linfadenopatia e dispneia. Quanto as alteracoes hematologicas, 70,3% apresentaram anemia, 50% trombocitopenia e 30% leucopenia e a maioria das infeccoes por E. canis foi visualizada em monocitos. Os resultados apontam para a necessidade de maior atencao por parte dos clinicos e tecnicos para a erliquiose canina uma vez que 35,9% dos caes atendidos com suspeita de hemoparasitoses apresentavam infeccoes por E. canis ou E. platys.

Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction.

Summary. In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens.

Rickettsia felis in fleas, Germany.

Among 310 fleas collected from dogs and cats in Germany, Rickettsia felis was detected in all specimens (34) of Archaeopsylla erinacei (hedgehog flea) and in 9% (24/226) of Ctenocephalides felis felis (cat flea). R. helvetica was detected in 1 Ceratophyllus gallinae (hen flea).

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.

Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina.

Cross-reactivity between Babesia bovis and B. bigemina becomes a problem in discrimination of the two infections in endemic areas where the two species usually occur in association. With the aim of identifying candidate proteins for use as specific diagnostic tools, culture-derived components of three geographically different stocks of B. bovis (Lismore, Kwanyanga and Mexico) and one of B. bigemina (Mexico) were analyzed by immunoprecipitation using acrylamide gel electrophoresis. The approach taken was based on the analysis of 35S-methionine-labelled parasite antigens released into culture supernatant. A variety of serum samples were tested, including a panel of calf sera experimentally produced against the different stocks of Babesia, serum samples from cattle naturally infected in the field in Brazil, and a panel of anti-B. bovis monoclonal antibodies, previously characterized by the indirect fluorescent antibody test, ELISA and Western immuno-blotting. Approximately 28 and 23 bands (with molecular weights ranging from 200 to 14 kDa) were detected in total protein profiles of B. bovis and B. bigemina culture supernatants, respectively, whereas no bands were seen in the uninfected red blood cell culture supernatant (negative control). The immunoprecipitation analysis showed antigenic diversity amongst the stocks of B. bovis and resulted in identification of at least five B. bovis specific antigens common to the three stocks (molecular weights of 80, 72, 58, 38 and 24 kDa) and four B. bigemina specific antigens (molecular weights of 240, 112, 50 and 29 kDa).

Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.

New species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus shows an ortholog of the E. canis major immunogenic glycoprotein gp36 with a new sequence of tandem repeats

BackgroundEhrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil.MethodsThe agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees.ResultsThe phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium.ConclusionsBased on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts.