High prevalence of "Candidatus Rickettsia amblyommii" in Amblyomma ticks from a Spotted Fever Endemic Region in North Argentina.

Ticks from an endemic Spotted Fever region in Argentina were analysed by PCR for Spotted Fever Group Rickettsiae. DNA of "Candidatus Rickettsia amblyommii" was found in 21.3% of Amblyomma hadanii and in 44.0% of A. neumanni. Amblyomma sculptum (formerly A. cajennense) and Haemaphysalis juxtakochi were negative for rickettsial DNA. DNA of Rickettsia rickettsii, the etiological agent of the clinical cases reported within the studied region was not detected in the analysed sample.

Molecular and immunological characterization of three strains of Anaplasma marginale grown in cultured tick cells.

Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.

Detection of genetic diversity of Anaplasma marginale isolates in Minas Gerais, Brazil.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in tropical and subtropical regions of the world and results in economic losses in the cattle industry. Major surface proteins (MSPs) have been used as markers for the genetic characterization of A. marginale strains and demonstrate that many isolates may occur in a given geographic area. However, in Brazil, little is known about the genetic diversity of A. marginale isolates within individual herds. This study was designed to examine the genetic variation among A. marginale infecting calves in a farm in the south of Minas Gerais State, Brazil. Blood samples collected from 100 calves were used to prepare Giemsastained smears that were microscopically examined for the presence of A. marginale. From each blood sample, DNA was extracted and analyzed by a polymerase chain reaction (PCR), followed by sequencing to determine diversity among the isolates. Examination of blood smears showed that 48% of the calves were infected with A. marginale, while the real-time PCR detected 70.2% positivity. Congenital infections were found in four calves. The microsatellite and tandem repeat analyses showed high genetic diversity among the isolates.

Detection of genetic diversity of Anaplasma marginale isolates in Minas Gerais, Brazil / Detecção de diversidade genética de isolados Anaplasma marginale em Minas Gerais, Brasil

Bovine anaplasmosis, caused by the tick-borne rickettsiaAnaplasma marginale, is endemic in tropical and subtropical regions of the world and results in economic losses in the cattle industry. Major surface proteins (MSPs) have been used as markers for the genetic characterization of A. marginale strains and demonstrate that many isolates may occur in a given geographic area. However, in Brazil, little is known about the genetic diversity of A. marginale isolates within individual herds. This study was designed to examine the genetic variation among A. marginale infecting calves in a farm in the south of Minas Gerais State, Brazil. Blood samples collected from 100 calves were used to prepare Giemsastained smears that were microscopically examined for the presence of A. marginale. From each blood sample, DNA was extracted and analyzed by a polymerase chain reaction (PCR), followed by sequencing to determine diversity among the isolates. Examination of blood smears showed that 48% of the calves were infected with A. marginale, while the real-time PCR detected 70.2% positivity. Congenital infections were found in four calves. The microsatellite and tandem repeat analyses showed high genetic diversity among the isolates.

A anaplasmose bovina, causada pela rickettsia Anaplasma marginale e transmitida por carrapatos, é endêmica em regiões tropicais e subtropicais no mundo e causa grandes perdas econômicas na indústria de bovinos. Proteínas principais de superfície (MSPs) foram usados como marcadores para a caracterização genética de amostras de A. marginale, demonstrando que diferentes isolados podem ocorrer numa certa região geográfica. Porém, no Brasil pouco se sabe sobre a variedade genética de isolados de A. marginale em rebanhos individuais. Este estudo teve como objetivo investigar a ocorrência de variação genética entre bezerros infectados com A. marginale numa fazenda do sul de Minas Gerais, Brasil. Amostras de sangue coletadas de 100 bezerros foram utilizadas para o preparo de esfregaços sanguíneos, corados pelo Giemsa, para detecção da infecção por A. marginale. Amostras de DNA extraídas de cada amostra foram analisadas através de PCR seguido de sequenciamento. O exame dos esfregaços demonstrou que 48% dos bezerros estavam infectados com A. marginale, enquanto que o PCR detectou 70,2% de positividade. Infecção congênita foi detectada em quatro bezerros. As análises de microsatélites e 'tandem repeats' comprovaram uma grande diversidade genética entre os isolados.

Detection of genetic diversity of Anaplasma marginale isolates in Minas Gerais, Brazil

Bovine anaplasmosis, caused by the tick-borne rickettsiaAnaplasma marginale, is endemic in tropical and subtropical regions of the world and results in economic losses in the cattle industry. Major surface proteins (MSPs) have been used as markers for the genetic characterization of A. marginale strains and demonstrate that many isolates may occur in a given geographic area. However, in Brazil, little is known about the genetic diversity of A. marginale isolates within individual herds. This study was designed to examine the genetic variation among A. marginale infecting calves in a farm in the south of Minas Gerais State, Brazil. Blood samples collected from 100 calves were used to prepare Giemsastained smears that were microscopically examined for the presence of A. marginale. From each blood sample, DNA was extracted and analyzed by a polymerase chain reaction (PCR), followed by sequencing to determine diversity among the isolates. Examination of blood smears showed that 48% of the calves were infected with A. marginale, while the real-time PCR detected 70.2% positivity. Congenital infections were found in four calves. The microsatellite and tandem repeat analyses showed high genetic diversity among the isolates.

A anaplasmose bovina, causada pela rickettsia Anaplasma marginale e transmitida por carrapatos, é endêmica em regiões tropicais e subtropicais no mundo e causa grandes perdas econômicas na industria de bovinos. Proteínas principais de superfície (MSPs) foram usados como marcadores para a caracterização genética de amostras de A. marginale, demonstrando que diferentes isolados podem ocorrer numa certa região geográfica. Porém, no Brasil pouco se sabe sobre a variedade genética de isolados de A. marginale em rebanhos individuais. Este estudo teve como objetivo investigar a ocorrência de variação genética entre bezerros infectados com A. marginale numa fazenda do sul de Minas Gerais, Brasil. Amostras de sangue coletadas de 100 bezerros foram utilizadas para o preparo de esfregaços sanguíneos, corados pelo Giemsa, para detecção da infecção por A. marginale. Amostras de DNA extraídas de cada amostra foram analisadas através de PCR seguido de sequenciamento. O exame dos esfregaços demonstrou que 48% dos bezerros estavam infectados comA. marginale, enquanto que o PCR detectou 70,2% de positividade. Infecção congênita foi detectada em quatro bezerros. As análises de microsatélites e tandem repeats comprovaram uma grande diversidade genética entre os isolados.

Survey of pyrethroid and organophosphate resistance in Brazilian field populations of Rhipicephalus (Boophilus) microplus: detection of C190A mutation in domain II of the para-type sodium channel gene.

The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2-15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82-100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected.

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.

Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil.

Ehrlichia canis has a worldwide geographic distribution, occurring particularly in tropical and subtropical areas. In Brazil, the main vector in urban areas is believed to be the brown dog tick Rhipicephalus sanguineus, but little is known about the occurrence, transmission and other epidemiological aspects of canine ehrlichiosis in rural areas, where Amblyomma ticks are found more frequently than R. sanguineus. A sero-prevalence study of canine ehrlichiosis was carried out in three distinct rural regions of the State of Minas Gerais, Brazil. Serum samples were collected from 226 dogs living on farms in Lavras (n=85), Belo Horizonte (n=45), and Nanuque (n=96) and were analyzed by an indirect fluorescent antibody test for the detection of anti-Ehrlichia canis antibodies. Age, breed, sex, presence of ticks and packed cell volume were also recorded. There were 65.6% positive dogs in Nanuque, 37.8% in Belo Horizonte, and 24.7% in Lavras. Animals living in Nanuque were 4.6 times more likely to be serologically positive than dogs living in the other two regions and antibody titres were considerable higher in this area. Male dogs, dogs >5 years of age, those infested with ticks, and mongrels all showed higher rates of positivity. The results point to the importance of canine ehrlichiosis in rural areas and indicate the need for further studies on natural transmission and maintenance of the disease.

First molecular detection of Babesia vogeli in dogs from Brazil.

The present work describes the detection and first molecular characterization of Babesia vogeli in dogs, naturally infected in Brazil and even in South America. Microscopic examination of Giemsa-stained peripheral blood smears collected from dogs originating from four different locations in Brazil revealed the presence of large Babesia merozoites and trophozoites (>2.5 microm). DNA was extracted from infected blood samples and PCR amplifications of the 18S rDNA were carried out. As a reference, DNA from an isolate of B. vogeli originated from Egypt was used. PCR products were purified and sequenced. The DNA sequences demonstrated 100% identity among the Brazilian isolates. Comparisons with the 18S rDNA sequence of the B. vogeli isolate from Egypt and with other B. vogeli sequences from Spain, France, Japan, Australia and South Africa confirmed the affiliation of all Brazilian isolates to the species B. vogeli.