Cross-protection between geographically distinct Anaplasma marginale isolates appears to be constrained by limited antibody responses.

The rickettsia Anaplasma marginale causes the haemolytic disease bovine anaplasmosis, an economic problem in tropical and subtropical areas worldwide. The closely related but less pathogenic Anaplasma centrale is commonly used as a live vaccine to prevent anaplasmosis, but it can only be produced from infected blood. UFMG1 is a low pathogenic Brazilian strain of A. marginale, which has been shown to protect cattle against a high pathogenic Brazilian isolate. As UFMG1 can be grown in tick cells, the strain was proposed as a possible cell culture-derived vaccine. We have evaluated whether UFMG1 could protect cattle against a geographically distant heterologous strain, using A. centrale vaccination as a standard for comparison. Trial calves were infected with UFMG1, A. centrale or PBS. UFMG1-infected animals were more symptomatic than those infected with A. centrale, but none required treatment. All calves were then challenged with the Israeli A. marginale Gonen strain (one of the most prevalent strain in Israel). The A. centrale group had the mildest symptoms, while UFMG1 and control groups both had a more severe response. Nevertheless, the challenge did not cause life-threatening disease in any group. Animals infected with A. centrale had a significantly higher IgG response than UFMG1, when measured in an ELISA against initial bodies from their homologous strain or Gonen. The level of cross-reactivity of the response to initial infection correlated significantly with reduced symptoms after challenge. In conclusion, UFMG1 had limited effect in preventing disease by the geographically distant heterologous Gonen strain. While the low pathogenicity of the Gonen strain in this trial makes it impossible to conclusively state that UFMG1 would have given no protective effect against more serious disease, the comparatively low IgG response to UFMG1 suggests it would not have been as effective as A. centrale.

In vitro culture of a novel genotype of Ehrlichia sp. from Brazil.

Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis.

Factors associated with epidemiology of Anaplasma platys in dogs in rural and urban areas of Minas Gerais State, Brazil.

This epidemiological survey of Anaplasma platys was carried out in rural and urban areas of three distinct regions of the State of Minas Gerais, Brazil. EDTA blood samples were collected during the dry season from dogs living on farms with an attempt to resample the same dogs in the subsequent rainy season. Samples were also taken from dogs in urban areas. DNA was extracted from blood samples for real time PCR. Risk factors, such as age, breed, sex, presence of ticks, and packed cell volume were analyzed. During the rainy season, the prevalence of infection by A. platys in dogs in the rural areas was significantly higher (13.9%) than that observed in dogs in the urban areas (5.1%). Dogs in the Nanuque region were 3.74 times (p=0.001) more likely to be real-time PCR positive than dogs in the other two studied regions. Dogs infested with ticks showed higher rates of positivity. The results showed that in rural areas of Minas Gerais A. platys infection is influenced by climatic conditions. In areas of higher temperature and higher humidity, transmission occurs during both the dry and rainy seasons, while in areas with lower temperature and humidity transmission occurs mainly during the dry season.

Use of a Real Time PCR for detecting subspecies of Babesia canis.

This paper reports the development and use of a Real Time PCR for detection of Babesia canis canis, B. canis rossi, and B. canis vogeli in endemic areas of Brazil. The sequences of the internal transcribed spacer (ITS) of several organisms were aligned and five primers and four probes were designed for amplification of a fragment (around 125 bp) which differentiates subspecies of B. canis. Blood samples collected from dogs living in farms in three distinct rural regions within the State of Minas Gerais (Lavras, Belo Horizonte and Nanuque) were tested. Blood samples had been collected during a dry season (Lavras, n=100; Belo Horizonte, n=50; Nanuque, n=102); the dogs were re-sampled in the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=29; Nanuque, n=66). From each sample, DNA was extracted and Giemsa stained smears were microscopically examined for direct detection of Babesia parasites. B. canis vogeli was the only subspecies found, with an overall prevalence of 9.9% during the dry season and 10.8% during the rainy season. Dogs living in Nanuque and Belo Horizonte showed significantly higher prevalence rates than those living in Lavras (13.7%, 12.0% and 5.0%, respectively). The Real Time PCR developed proved to be appropriate to detect B. canis subspecies in endemic areas.

Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil.

Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1∶25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.

Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves.

This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus.

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface.

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.

Manutenção in vitro de células IDE8 em dois tipos de soro bovino / In vitro maintenance of IDE8 cells using two types of bovine serum

The present study had the objective of defining the culture conditions, optimizing the maintenance and expansion of an IDE-8 cell line in Brazil, with the aim to propose its use as a model for in vitro infection and multiplication of Brazilian strains of rickettsia and other hemoparasites. The supplementation of IDE-8 cells with two distinct fetal bovine sera (a Brazilian and an imported) was evaluated. Culture media were changed weekly and subcultures were carried out every 15 days. The development of cultures and subcultures was evaluated by the percentage of viability and cellular morphology. The results indicate that the imported SFB can be replaced by the Brazilian SFB one, as no significant differences (P<0.05) were seen among culture viabilities.

Dopplerfluxometria das artérias renais: valores normais das velocidades sistólica e diastólica e do índice resistivo nas artérias renais principais / Renal arteries Dopplerfluxometry: normal systolic and diastolic flow velocities and resistive index values in the main renal arteries / Renal arteries Dopplerfluxometry: normal systolic and diastolic flow velocities and resistive index values in the main renal arteries

Dopplerfluxometry of renal arteries has been used to estimate renal perfusion in humans. The aim of this study was to use Dopplerfluxometry technique to calculate the resistive index of main renal arteries in dogs, measuring their systolic and diastolic blood flow velocities. Twenty (10 males, 10 females), adult mongrel dogs, were used in this study. The dogs were submitted to Doppler sonographic evaluation of left and right main renal arteries. The systolic and diastolic blood flow velocities, expressed (in centimeters per second) as mean and standard deviation were 79.96± 8.82 and 28.86± 5.11 in the right main renal artery and 80.22± 6.99 and 29.62± 4.14 in the left main renal artery. The value of resistive index expressed as mean ± standard deviation was 0.64± 0.04 for the right main renal artery and 0.63± 0.028 in the left main renal artery.

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

The present work describes a retrospective study of clinical cases of ehrlichiosis in dogs examined from March 1998 to September 2001. From the clinical records with laboratorial confirmation of Ehrlichia canis or E. platys infections, the following parameters were analyzed: demographic aspects (age, race, sex, period of the year and origin), clinical characteristics (body temperature, exposure to ticks and clinical signs), and hematological characteristics (blood cell counts and type of infected cell). A total of 194 clinical records were analyzed, from which 31 animals were infected with E. canis and 21 animals with E. platys. The number of cases of canine ehrlichiosis increased considerably from the year 2000 onwards, and 24.4 percent of the cases occurred in 13- to 24-month-old animals, in different urban and per-urban regions of the municipality of Belo Horizonte. The most frequent symptoms were fever, anorexia, apathy, abdominal pain, lymphadenopathy and dispnea. Regarding hematological alterations, 70.3 percent of the animals presented anemia, 50 percent presented thrombocytopenia and 30 percent leukopenia, and most E. canis morulae were seen in monocytes. The results point to the importance of canine ehrlichiosis, as 35.9 percent of the dogs with suspected hemoparasitic diseases were infected with Ehrlichia canis or E. platys

Uso de inóculo padronizado de Anaplasma marginale e da quimioprofilaxia no controle da anaplasmose bovina / Use of standardized inoculum of Anaplasma marginale and chemoprophylaxis to control bovine anaplasmosis

O uso de inóculo homólogo padronizado de Anaplasma marginale foi comparado à prática de quimioprofilaxia com diidrato de oxitetraciclina na reduçäo da riquetsemia e do volume globular (VG) causada pela anaplasmose bovina. Os animais que receberam o inóculo (10(7) hemácias com Anaplasma marginale)apresentaram, ao serem desafiados em campo, riquetsemia média de 1,2 por cento e reduçäo média de VG de 23,0 por cento, significativamente inferiores às do grupo-controle (P<0,05). No experimento de quimioprofilaxia os animais que receberam três doses de diidrato de oxitetraciclina (20mg/kg), com intervalos de 25 dias, apresentaram riquetsemia de 2,7 por cento e reduçäo de 36,3 por cento no VG, significativamente inferiores às do grupo-controle (P<0,05). Ambas as medidas preventivas testadas foram eficientes na reduçäo da intensidade da riquetsemia e na queda do VG

Infecçäo experimental de cäes com amostras de Babesia canis isoladas em Minas Gerais / Dog experimental infection of Babesia canis isolated in Minas Gerais State

The objective of the present study was to evaluate parasitemia and packed cell volume patterns of dogs experimentally inoculated with two isolates of Babesia canis: one from Belo Horizonte (BH) and the other from Lavras (Lv), Minas Gerais State, Brazil. Both isolates showed similar patterns, with the peak of parasitemia occurring three days post-infection. From the fourth day, parasitemia was detected in low levels (0.01 percent) with small periodical increases. The packed cell volume decreased after the parasitemia beginning, with oscillations during the experimental period. All dogs remained apparently normal, except one, which had been inoculated with the BH isolate and presented classical clinical signs of babesiosis (weakness, anemia, hemoglobinuria and depression). The results suggest that the studied isolates have low pathogenicity, and point to the need for further studies aiming to characterize the subspecies of Brazilian isolates

Quimioprofilaxia da babesiose bovina com diidroxitetraciclina de longa duraçäo / Chemoprophylaxis of bovine babesiosis with long-acting diidroxytetracycline

A açäo da diidroxitetraciclina de longa duraçäo foi testada em bezerros experimentalmente inoculados com Babesia bigemina e em desafio natural em área endêmica. Em uma propriedade de gado de leite, 18 bezerros foram inoculados com 10 elevado a oitava potência hemácias parasitadas por B. bigemina. Dez animais foram tratados com 20 mg/kg de diidroxitetraciclina de longa açäo, sete dias após o inóculo, e oito foram deixados como controle. Todos os animais do grupo controle e apenas três dos tratados apresentaram parasitemia, sendo que dois já estavam infestados no dia do tratamento. Em um segundo experimento, dez bezerros receberam dois tratamentos de 20 mg/kg de diidroxitetraciclina de longa açäo aos 15 e 36 dias após serem liberados em piquetes e oito bezerros foram deixados como testemunhas. Os animais do grupo tratado apresentaram aumento significativo do período pré-patente e menor reduçäo do volume globular, em relaçäo aos do grupo controle

Detecçäo de DNA de herpesvírus eqüino-1 em embriäo e em sêmen eqüino por meio de PCR / Detection of equine herpesvirus 1 DNA in a single embryo and in horses semen by polymerase chain reaction

O genoma das amostras de um embriäo e sêmen de três eqüinos, coletados em uma fazenda brasileira, foram testados para a presença de seqüências específicas dos genes da timidina kinase (TK) de herpesvírus eqüino-1 (HVE-1) e herpesvírus eqüino-4 (HVE-4) por meio de reaçäo em cadeia da polimerase (PCR). A PCR detectou sequências específicas do gene de TK de HVE-1 em todas as amostras testadas. O DNA template extraído de leucócitos periféricos da égua doadora do embriäo também foi amplificado pelos pares de primers designados para TK do HVE-1. Vírus infeccioso näo foi isolado desses espécimes. Os resultados indicaram que o PCR foi mais sensível do que o método de isolamento em cultura de células para a detecçäo do HVE-1 em sêmen de cavalos portadores

Uso de um sistema de ELISA para detecçäo de anticorpos contra herpesvírus eqüino 1 (HVE-1) em éguas gestantes assintomáticas e potros recém-nascidos / Use of an ELISA system for detection of equine herpesvirus 1 (EHV-1) antibodies in non-symptomatic pregnant mares and neonatal foals

Um sistema de ELISA foi utilizado para se estudar a prevalência do herpesvirus eqüino 1 (HVE-1) em plantéis de cavalos brasileiros vacinados e näo vacinados. Amostras de soros foram coletadas de éguas gestantes assintomáticas, antes e após o parto e de seus respectivos potros recém-nascidos. O antígeno foi preparado de uma fraçäo viral purificada (HVE-1 amostra Ab1). O ponto de corte (absorvância de 0,3516) foi determinado considerando o limite máximo para os valores negativos. Esse valor determinou sensibilidade de 100 por cento e especificidade de 94,7 por cento, utilizando um banco de soros de éguas gestantes e fetos. No estudo observou-se alta correlaçäo entre o ELISA e a soro-neutralizaçäo para detecçäo de anticorpos específicos contra o EHV-1

Diagnóstico sorológico de Babesia equi e Babesia caballi em éguas gestantes / Serological diagnosis of Babesia equi and B. caballi in pregnant mares

O objetivo do presente estudo foi determinar a dinâmica da produçäo de anticorpos anti-Babesia equi e B. caballi em éguas gestantes em condiçöes naturais de campo em Minas Gerais e avaliar a eficiência da transferência passiva de anticorpos via colostro. Foram analisadas, pela imunofluorescência indireta, amostras de soros colhidas de 34 fêmeas aos 290, 305 e 320 dias de gestaçäo, e no momento do parto, e de seus respectivos potros até 36 horas após o nascimento. Aos 290 dias de gestaçäo, todas as fêmeas estavam positivas para B. equi e apenas duas estavam negativas para B. caballi. Todos os potros tornaram-se positivos para B. equi após ingestäo de colostro, entretanto 42 por cento permaneceram negativos para B. caballi 36 horas após o nascimento. Näo se observou influência dos níveis de anticorpos durante a gestaçäo na transferência passiva de anticorpos aos potros. A transferência passiva foi menos eficiente para B. caballi, sugerindo que os potros estäo mais sujeitos a adquirirem infecçäo por esse agente

Comparaçäo entre ELISA e reaçäo de imunofluorescência indireta para detecçäo de anticorpos anti-Anaplasma marginale em bovinos / Comparision between ELISA and Indirect Fluorescent Antibody Test in the detection of antibodies against Anaplasma marginale in cattle

Um sistema ELISA foi comparado à reaçäo de imunofluorescência indireta (RIFI) na detecçäo de anticorpos anti-A. marginale em soros de bovinos. Utilizou-se um total de 337 amostras de soros, sendo 298 colhidas de bovinos importados, após terem sido submetidos ao processo de premuniçäo, e 39 amostras colhidas de animais importados, de áreas livres de hemoparasitas. O teste ELISA foi mais específico que a RIFI mostrando-se com potencial utilidade para estudos epidemiológicos da anaplasmose. Com os resultados obtidos determinou-se especificidade de 94,87 por cento e 71,79 por cento, sensibilidade de 100 por cento e 100 por cento, valor preditivo positivo 99,33 por cento e 96,44 por cento e valor preditivo negativo 100 por cento e 100 por cento para o ELISA e a RIFI, respectivamente

Resposta humoral para Babesia bigemina (Smith & Kilborne, 1893) em bovinos submetidos ao processo de premuniçäo / Humoral immune response to Babesia bigemina (Smith & Kilborne, 1893) in cattle submitted to premunition

Trinta e nove animais da raça Limousin, importados dos Estados Unidos, foram submetidos ao processo de premuniçäo, no município de Capitólio (MG). Os animais foram inoculados com sangue contendo 5 x 10 elevado a oitava potência Babesia spp, 5x10 elevado a oitava potência Anaplasma marginale ou infestados com larvas de Boophilus microplus, provenientes de animais naturalmente infectados por Babesia spp. O perfil sorológico para B. bigemina foi determinado através de exames semanais pela reaçäo de imunofluorescência indireta. Todos os animais foram desafiados entre a 8ª e a 10ª semanas após a inoculaçäo/infestaçäo. Todos os animais apresentaram títulos positivos de 1:40 a 1:5120, durante o processo de premuniçäo. A curva de anticorpos apresentou o mesmo comportamento, independente do tipo de inóculo. Títulos máximos de anticorpos anti-B. bigemina foram obtidos entre a 3ª e a 4ª semana após a inoculaçäo inicial, havendo em seguida queda progressiva e posterior aumento, uma semana após o desafio. Nas três últimas semanas do experimento a curva manteve-se constante (AU)999

Influência das helmintoses gastrintestinais sobre o quadro hematológico de bovinos mestiços Holandês x Zebu / Influence of nematode infections on hematological parameters of crossbred calves

Avaliou-se a influência das infecçöes naturais por helmintos gastrintestinais sobre o quadro hematológico de bezerros mestiços Holandês x Zebu, em uma propriedade da Zona Metalúrgica do Estado de Minas Gerais. Foram utilizados 14 animais com idade inicial de três a quatro meses, distribuídos igualmente em dois grupos. Um grupo foi tratado com Ivermectin e o outro foi considerado controle. Hemogramas, contagem de ovos por grama de fezes (OPG) e coprocultura foram realizados mensalmente por um período de 18 meses, totalizando três estaçöes (seca/93, chuva e seca/94). Foram observadas, dentro de cada grupo, diferenças entre médias de contagens de hemácias, concentraçäo de hemoglobina e taxa de hematócrito, entre as estaçöes. Ocorreu aumento dos valores de leucócitos totais, neutrófilos segmentados e eosinófilos e reduçäo de linfócitos, na,s estaçäo chuvosa. Näo foram observadas diferenças entre grupos controle e tratado. Foi observada reduçäo do OPG com o aumento da idade dos animais dentro de cada grupo. Diferenças entre os grupos ocorreram a partir da estaçäo chuvosa. As larvas recuperadas das coproculturas foram Cooperia, Haemonchus, Oesophagostomum, Trichostrongylus e Bunostomum, com predomínio de Cooperia sobre os demais gêneros