Important frequency of Anaplasma phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil / Importante frequência da infecção por Anaplasma phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil

Anaplasma phagocytophilum is responsible for granulocytic anaplasmosis in humans and various animal species. The aim of the present study was to determine the prevalence of A. phagocytophilum-infected dogs in a residential area of Belo Horizonte, Minas Gerais state, Brazil. A total of 62 dogs were submitted to serological (indirect fluorescent-antibody -IFI) and molecular (PCR) tests. Anti-A. phagocytophilum antibodies were detected in 43.8% of the dogs. Seven dogs (10.9%) were PCR-positive for the msp4 gene, six and four of these were positive for the for the msp2/p44 gene of A. phagocytophilum and 16S rRNA region of granulocytic Anaplasmataceae respectively. This study confirms a relatively high frequency of A. phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil and highlights the need for further studies on the role of Rhipicephalus sanguineus sensu lato ticks in the transmission of this bacterium to dogs in urban Brazilian areas.(AU)

Anaplasma phagocytophilum é responsável pela anaplasmose granulocítica, doença que acomete seres-humanos e várias espécies de animais. O objetivo do presente estudo foi determinar a prevalência de cães acometidos por A. phagocytophlium em uma área residencial de Belo Horizonte, MG, Brasil. Sessenta e dois cães foram submetidos a testes sorológicos (reação de imunofluorescência indireta - IFAT) e moleculares (PCR). Anticorpos anti-A. phagocytophilum foram detectados em 43,8% dos cães. Sete cães (10,9%) foram positivos no PCR para o gene msp4 de A. phagocytophilum, seis para o gene msp2/p44 A. phagocytophilum e quatro para a região 16S rRNA de Anaplasmataceae granulocíticas. Esse estudo confirma a frequência relativamente alta da infecção por A. phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil e destaca a necessidade de pesquisas para determinar o papel do carrapato Rhipicephalus sanguineus sensu lato na transmissão desse microrganismo para cães de áreas urbanas brasileiras.(AU)

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.

Complete Genome Sequence of Ehrlichia mineirensis, a Novel Organism Closely Related to Ehrlichia canis with a New Host Association.

We report here the complete genome sequencing of Ehrlichia mineirensis, an Ehrlichia organism that was isolated from the hemolymph of Rhipicephalus microplus-engorged females. E. mineirensis is the best characterized Ehrlichia isolate from a novel cattle-related clade closely related to the monocytotropic pathogen E. canis.

Characterization of two strains of Anaplasma marginale isolated from cattle in Rio de Janeiro, Brazil, after propagation in tick cell culture.

IDE8 tick cell cultures have been used for the isolation and propagation of several isolates of Anaplasma marginale. The genetic heterogeneity of A. marginale strains in cattle is diverse in endemic regions worldwide and the analyses of msp1α (major surface protein 1 alpha) gene sequences have allowed the identification of different strains. This study reports the isolation and propagation of two new isolates of A. marginale in IDE8 cells from blood of two cattle and their morphological and molecular characterization using light microscopy and the msp1α gene, respectively. Small colonies were observed in cytospin smears of each of the isolates 60 days after culture initiation. Based on msp1α sequence variation, the two isolates were found to be separate strains and were named AmRio1 and AmRio2. Analysis of msp1α microsatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of one MSP1α tandem repeat from the strain AmRio1 resulted in a new sequence (named 162) with one amino acid change. The results of these phylogenetic analyses demonstrated that A. marginale strains from Brazil and Argentina formed two large clusters of which one was less divergent that the other.

Use of Percoll gradients to purify Anaplasma marginale (Rickettsiales: Anaplasmataceae) from tick cell cultures.

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is an obligate intracellular bacterium that multiplies exclusively within membrane-bound vacuoles in the cytoplasm of host cells. A number of A. marginale isolates can be propagated in the Ixodes scapularis IDE8 tick cell line, which provides a reliable source of antigens for a wide variety of studies. However, because of its intracellular nature, separation of bacteria from host cell materials remains an important constraint for researchers. In the present study, we evaluated the use of Percoll gradients for purification of two Brazilian strains of A. marginale grown in IDE8 tick cells. The purified A. marginale monitored in Giemsa-stained smears contained only minimal amounts of IDE8 cell stroma. The total protein yields were 1.2mg and 1.7mg, while the DNA titers quantified with real-time PCR were 6.4×10(9) for UFMG1 and 4.87×10(9) for UFMG2 copies in the purified material, respectively. Additionally, we confirmed the viability of purified bacteria by infecting tick cells after being freshly purified and after retrieval from long-term storage. Importantly, the viability of the organisms is preserved after use of this separation method, and therefore the purified organisms can be used in enzymatic assays and other research approaches where live organisms would be preferred.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.

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.

Dynamics of tick infestations in foxes in Thuringia, Germany.

This study aimed to provide up-to-date information on the dynamics of tick infestations on foxes in Thuringia, as the most recent information available was published in 1997. Fox carcasses that had been sent to the Thuringian State Authority for Food Safety and Consumer Protection (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz - TLLV), between January 1st and December 31st, 2009, were examined for the presence of ticks. All ticks collected were stored at -20 °C before being identified and classified according to their developmental stage and sex. Out of a total of 1286 foxes examined, 989 (76.9%) were infested with ticks. A total of 13,227 ticks were collected from the foxes. The stage most frequently found was the larva (48.1%), followed by the adult (34.1%), and the nymphal stage (17.8%). Regarding the adult stage, Ixodes ricinus was the most frequent tick species detected (82.2%), followed by I. canisuga (10.8%) and I. hexagonus (6.7%). Dermacentor reticulatus ticks were very rare (0.3%). With regard to nymphs, I. canisuga and I. hexagonus were the most frequent tick species found, and this was also assumed for the larval stage. The results indicate the occurrence of tick infestations in foxes throughout the year, mainly by I. ricinus, I. canisuga, and I. hexagonus, with seasonal variations. Foxes were infested by I. ricinus ticks significantly more frequently from April to September. This applied to all tick developmental stages, but especially to adults. In contrast to I. ricinus, the infestation of foxes with I. canisuga and I. hexagonus was significantly higher from January to March and from October to December, especially with the immature developmental stages.

The European hedgehog (Erinaceus europaeus)--a suitable reservoir for variants of Anaplasma phagocytophilum?

The European hedgehog (Erinaceus europaeus) is a common insectivore in most parts of Europe and is frequently infested by the ticks Ixodes ricinus and I. hexagonus. I. ricinus ticks have been found infected with Anaplasma phagocytophilum, an obligate intracellular bacterium, but little is known about the potential of the hedgehog as a reservoir host. In this study, the infection with A. phagocytophilum and the genetic variants involved were investigated in a captive hedgehog population which was kept in a fenced, natural grass and bush garden habitat, and also in its ticks. Additionally hedgehogs from hedgehog caretaking stations were investigated. EDTA blood and ticks were collected from the captive hedgehog population once a month from March to October 2007 and in March and April 2008. All 3 developmental stages of I. ricinus and I. hexagonus occurred on the hedgehogs. After DNA extraction, the samples were screened for A. phagocytophilum with a real-time PCR, and selected samples were further investigated with a nested PCR targeting the partial 16S rRNA gene, followed by sequencing. One hundred thirty-six out of 220 hedgehog blood samples (61.8%) from altogether 48 individuals, 413 out of 563 I. ricinus samples and 90 out of 338 I. hexagonus samples were PCR-positive. Thirty-two hedgehogs were positive more than once, most frequently twice or 3 times, but also up to 9 times. Sequencing of the partial 16S rRNA gene resulted in 6 variants, but one variant ('A') was the most frequent which appeared in 93.8% of the positive hedgehogs. This variant (equaling Frankonia II, GenBank AF136712) has recently been reported from human, equine, and canine granulocytic anaplasmosis cases and thus, its specific association with hedgehogs is an important finding in the epidemiology of A. phagocytophilum in Europe. The high infection rate of both hedgehogs and ticks with A. phagocytophilum and the simultaneous infestation with 2 tick species of all developmental stages suggest that the hedgehog may be a suitable reservoir for at least some variants of A. phagocytophilum.

Isolation, propagation and preliminary characterisation of Anaplasma phagocytophilum from roe deer (Capreolus capreolus) in the tick cell line IDE8.

Anaplasma phagocytophilum is an obligate intracellular bacterium causing granulocytic anaplasmosis in dogs, horses, and humans and tick-borne fever of ruminants. The bacterium has been detected in a variety of other mammals including wild ruminants without overt clinical signs of disease. Isolates in cell culture have been obtained from humans, dogs, horses, sheep, and ticks, but no strain from wild ruminants exists in cell culture in Europe. From September to November 2010, EDTA blood samples were collected from the jugular vein of 19 shot roe deer from a forest in southern Germany. The presence of specific A. phagocytophilum DNA was demonstrated with a real-time PCR targeting the msp2 gene in all 19 animals. Subsequently, blood cells were used to inoculate the tick cell line IDE8. The first infected IDE8 cells were detected in Giemsa-stained smears 57 days post inoculation. Only one roe deer yielded a positive culture which has been propagated for 9 consecutive passages thus far representing 228 days in culture. Further analysis of the A. phagocytophilum strain was performed by PCR followed by sequencing for the partial 16S rRNA, groEL, msp2, and msp4 genes. Phylogenetic topology of groEL and msp4 sequences placed the roe deer isolate in close proximity to sequences available from roe deer and goats from the neighbouring Alpine regions of Austria and Switzerland, and of msp2 with other ruminant species. This represents the first isolation of A. phagocytophilum in a tick cell line directly from an infected wild ruminant reservoir host, Capreolus capreolus, in Europe. The availability of a cultured A. phagocytophilum strain isolated from roe deer will allow us to study the biological characteristics and the pathogenic potential of this strain as well as to compare its host tropism and its genetic and antigenetic properties with those of other A. phagocytophilum strains from other animal species.

In vitro establishment and propagation of a Brazilian strain ofAnaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells / Estabelecimento e propagação in vitro de uma amostra brasileira de Anaplasma marginale com apêndice em células IDE8 (Ixodes scapularis)

A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.

Uma amostra brasileira de Anaplasma marginale com apêndice foi estabelecida e mantida in vitro em uma linhagem de células de carrapatos (IDE8). A infecção foi confirmada através de microscopia ótica e eletrônica de transmissão. Além disso, os primers MSP1aNF2 e MSP1aNR2 amplificaram produtos do DNA extraído das células infectadas. Comparações de sequências parciais do gene msp1α e do genoma completo de A. marginale confirmaram que as sequências dos fragmentos amplificados pertenciam ao genoma de A. marginale. Este é o primeiro estabelecimento in vitro de uma amostra brasileira de A. marginale em células de carrapatos, representando um novo sistema para estudos biológicos e moleculares, além de ser uma nova fonte de material para o desenvolvimento de testes diagnósticos e de vacinas.

Population dynamics of Rhipicephalus sanguineus (Latrielle, 1806) in Belo Horizonte, Minas Gerais state, Brazil.

Rhipicephalus sanguineus ticks are distributed throughout the world, especially in those areas in which dogs are in close contact with humans. R. sanguineus and fleas are regarded as the main ectoparasites infesting dogs in Brazil. Besides causing direct damage during the blood feeding process, this tick species can also transmit pathogens to dogs and humans. Despite its importance in Brazil, data regarding the seasonality of R. sanguineus are limited, especially with regard to natural infestations of dogs. The present study aimed to evaluate the seasonality of R. sanguineus on dogs living in Belo Horizonte, state of Minas Gerais. From August 2006 to July 2007, ticks were collected monthly from 12 adult dogs in nine houses, which were located in two districts in the north region of the city. In parallel, canine clients of a pet care department of the small animal veterinary clinic were examined for the presence of ticks before bathing and/or clipping. The climatic data recorded for Belo Horizonte during the experimental period were: mean temperature 18.6 degrees C; relative air humidity 56.5%; rainfall 37mm. The only species of ticks identified from all infested dogs was R. sanguineus, which was found in all its development stages. Among dogs living in houses, three tick population peaks were observed (August, February, and June), suggesting the occurrence of three generations per year in Belo Horizonte. A total of 7318 ticks were collected, of which 5422 were adult ticks and 1896 represented immature stages (744 larvae and 1152 nymphs). The monthly inspection of dogs living in houses demonstrated significantly higher parasitism during the dry season (p<0.05). A total of 2848 dogs from the pet care department of the small animal veterinary clinic were examined, of which 222 (7.8%) were infested with ticks and the percentage of infested dogs in the dry season was higher (p<0.05) than in the hot wet. The percentage of male dogs infested with ticks was significantly higher (58.29%) than the percentage of infested female dogs (41.70%). This study of the dynamics of R. sanguineus infestations in Belo Horizonte will contribute to establishing appropriate measures to control tick infestations in dogs.

Babesia bigemina: in vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells.

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.

In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells.

A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.

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.

Epidemiological aspects of canine babesiosis in the semiarid area of the state of Minas Gerais, Brazil.

Epidemiological aspects of Babesia vogeli infection were studied in the canine population of a rural town located in the Brazilian "Drought Polygon" of the state of Minas Gerais, Brazil. The survey was carried out in March 2003, when 505 dogs were identified and their characteristics registered on appropriate forms. Blood samples were collected at this time and again in June, September and December 2003. Serum samples were tested by the indirect fluorescent antibody test (IFAT) to detect antibodies against B. vogeli. The prevalence of anti-B. vogeli antibodies was 18.8%; however, no correlations were found between prevalence of infection and the age or gender of the animals. Cross-bred dogs presented a higher chance of acquiring infection in comparison to pure-bred dogs. Significant differences concerning the incidence of the disease were found during the period April-June in comparison to other months, demonstrating that transmission of B. vogeli is related to seasonal variations of tick infestations. The results indicate that climatic factors within the semiarid area interfere directly in the epidemiology of canine babesiosis.

Natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens (Acari: Ixodidae).

The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.

Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world, and many isolates of A. marginale may occur in a given geographic area. Phylogenetic relationships have been reported for A. marginale isolates from the US using gene and protein sequences of MSP1a and msp4. These studies demonstrated that msp4 sequences, but not MSP1a DNA or protein sequences, provide phylogeographic information and also that MSP1a sequences are highly heterogeneous among A. marginale populations. However, little information is available on the genetic diversity of A. marginale isolates from other regions of the world. The present study was undertaken to examine genetic variation among 10 isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, where A. marginale is endemic. Neighbor-joining analysis of msp4 sequences of Brazilian and New World isolates of A. marginale from Argentina, Mexico and the US provided bootstrap support for a Latin American clade. The sequences of the MSP1a repeats of four Brazilian isolates of A. marginale were compared to sequences of Latin American and US isolates. The MSP1a repeated sequences of Latin American isolates of A. marginale had nine repeat forms, alpha-phi, which have not been reported previously in North American isolates of A. marginale. Furthermore, the repeated forms tau, sigma and mu were only present in the Brazilian isolates. The results demonstrated that the genetic heterogeneity observed among isolates of A. marginale is common in endemic areas, independent of the predominant tick vector and is consistent with previous studies in which msp4 provided phylogeographic information about A. marginale isolates, while MSP1a was found not to be a useful marker for phylogeographic characterization of A. marginale isolates.

Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies.

The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.

Natural infection of calves by Anaplasma marginale in dairy herds of the Metalúrgica Region, Minas Gerais

The dynamic of natural infections by Anaplasma marginale in calves was evaluated during a period of one year on two farms located in the Metalúrgica Region, State of Minas Gerais, Brazil. Blood samples were collected weekly for rickettsemia and packed cell volume (PCV) determination. The animals born from March to July suffered the infection in October and November, independently of their age, whilst calves born from September to December acquired the infection during the first days of life. These animals presented patent rickettsemia from 30 days of life. During the patent period PCV decreased after one week of infection, ranging from 20 to 23 (per cent). It was concluded, that in the region studied, the transmission of A. marginale is influenced by climatic conditions, and that calves born during the dry season are more likely to acquire the infection when they are exposed to high transmission levels during the subsequent raining season