Arquivo Brasileiro de Medicina Veterinária e Zootecnia
Dopplerfluxometria das artérias renais: valores normais das velocidades sistólica e diastólica e ... more 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] ABSTRACT 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.
The present work describes the detection and first molecular characterization of Babesia vogeli i... more 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.
This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a ce... more 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.
Epidemiological aspects of Babesia vogeli infection were studied in the canine population of a ru... more 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 va...
a b s t r a c t IDE8 tick cell cultures have been used for the isolation and propagation of sever... more a b s t r a c t 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 msp1˛ (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 msp1˛ gene, respectively. Small colonies were observed in cytospin smears of each of the isolates 60 days after culture initiation. Based on msp1˛ sequence variation, the two isolates were found to be separate strains and were named AmRio1 and AmRio2. Analysis of msp1m icrosatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of one MSP1␣ 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.
Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, lit... more Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, little is known about A. phagocytophilum in goats. In the present study, 72 animals from six goat flocks (373 EDTA blood-samples) in Central Switzerland were analysed for A. phagocytophilum DNA. A real-time PCR targeting the msp2 gene of A. phagocytophilum was performed and in positive samples the partial 16S rRNA, groEL and msp4 gene were amplified for sequence analysis. Four DNA extracts were positive. Different sequence types on basis of the amplified genes were found. For comparison, sequences of A. phagocytophilum from 12 cattle (originating from Switzerland and Southern Germany) were analysed. The 16S rRNA gene sequences from cattle were all identical amongst each other, but the groEL and msp4 gene differed depending on the origin of the cattle samples and differed from the variants from goats. This study clearly provides molecular evidence for the presence of different types of A. phagocytophilum in goat flocks in Switzerland, a fact which deserves more thorough attention in clinical studies.
The present work describes the detection of infected cells in the bone marrow aspirates of dogs e... more The present work describes the detection of infected cells in the bone marrow aspirates of dogs experimentally infected with a Brazilian isolate of Ehrlichia canis. Dogs were monitored twice a day by clinical evaluation and peripheral blood smear examination. Every three days, blood samples were collected for cell counts. Weekly, aspirates from the bone marrow were examined and serum samples were tested by IFAT. The clinical signs observed were fever, pallid membranes, lymphadenopathy, serous nasal secretions, and pronounced weight loss. Hematological alterations included normocytic normochromic anemia, decrease of neutrophils and lymphocytes, and thrombocytopenia. Few E. canis infected cells were seen in blood smears. However, stages of E. canis were visualized in bone marrow aspirates 15 days post infection.
The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Braz... more 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.
The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma m... more 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.
Species: Canine One of the main goals for researchers working on canine monocytic ehrlichiosis ha... more Species: Canine One of the main goals for researchers working on canine monocytic ehrlichiosis has been to clarify the clinical and hematological alterations, as well as the immunological mechanisms involved on the pathogenesis of the disease. Usually, treatments instituted during the acute phase promote fast improvement of the animal's clinical conditions. In the present study, nine dogs were experimentally infected with a Brazilian strain of Ehrlichia canis and were treated with tetracycline hydrochloride (22 mg/kg/per day) for 21 days, beginning at the thirtieth day after infection. Some clinical, hematological and immunological parameters were evaluated during the acute phase of the disease and after the treatment. In addition, their immune responses were evaluated before and after the treatment by cytometric analysis and cell phenotype (FACS) using monoclonal antibodies to cell surface markers for CD5, Thy-1, CD4, CD14, MHC II, CD45R, and IgM. The acute phase was clinically characterized by fever, pale mucous membranes, progressive weight loss and moderate lymphadenopathy. The biochemical-hematological analysis revealed decrease of creatinine and total protein concentrations along with anemia, thrombocytopenia, monocytosis and increased ALT enzyme concentration in the infected animals. The ex vivo immune response evaluation of peripheral blood leucocytes showed a percentile reduction of Thy 1+ lymphocytes during a short period of time at the beginning of the acute phase, accompanied by a progressive percentile reduction of T CD4+ lymphocytes, which was significantly reduced at the end of the acute phase. At this time, a decreased percentile of blood MHC-II+ and CD45R+ lymphocytes was observed. On the other hand, the percentage of B (IgM+) lymphocytes and CD14+ cells increased during the acute phase, corroborating with seroconversion, observed by the indirect fluorescent antibody test (IFAT), and monocytosis observed in blood examinations. A higher percentile of CD4+ neutrophils was observed in the granulocytic series after experimental infections. The initial response to the treatment was characterized by fast clinical recovery with weight gain and increased physical activity. There was an evident tendency to reestablish biochemical and hematological parameters, with monocytes responding immediately to the antibiotic treatment. Most parameters of immunophenotyping that were altered during the acute phase returned to normal, demonstrating the favorable response after treatment. These results indicate that the E. canis infection promotes important alterations, which seem to modulate the immune response, allowing persistence of the agent and the establishment of the acute phase. The 21-day treatment regime, on the other hand, results in restoration of immune condition.
This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an append... more 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. marginaleinfected 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.
This epidemiological survey of Anaplasma platys was carried out in rural and urban areas of three... more 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.
Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma margin... more 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 59-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 59-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.
Pesq. Vet. Bras. 21(4):146-150, out./dez. 2001 146 RESUMO. RESUMO.-[Infecção natural por Infecção... more Pesq. Vet. Bras. 21(4):146-150, out./dez. 2001 146 RESUMO. RESUMO.-[Infecção natural por Infecção natural por Anaplasma marginale Anaplasma marginale em em bezerras de fazendas leiteiras da região Metalúrgica de bezerras de fazendas leiteiras da região Metalúrgica de Minas Gerais Minas Gerais.] Realizou-se um estudo da dinâmica de infecções naturais por Anaplasma marginale, durante o período de um ano, em duas propriedades localizadas na Região Metalúrgica do Estado de Minas Gerais. Foram realizados esfregaços sangüíneos e determinação do volume globular (VG).
Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterho... more 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.
Background: Tick cell lines are now available from fifteen ixodid and argasid species of medical ... more Background: Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short-and longterm low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For shortterm cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.
A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection ... more A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.
Arquivo Brasileiro de Medicina Veterinária e Zootecnia
Dopplerfluxometria das artérias renais: valores normais das velocidades sistólica e diastólica e ... more 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] ABSTRACT 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.
The present work describes the detection and first molecular characterization of Babesia vogeli i... more 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.
This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a ce... more 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.
Epidemiological aspects of Babesia vogeli infection were studied in the canine population of a ru... more 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 va...
a b s t r a c t IDE8 tick cell cultures have been used for the isolation and propagation of sever... more a b s t r a c t 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 msp1˛ (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 msp1˛ gene, respectively. Small colonies were observed in cytospin smears of each of the isolates 60 days after culture initiation. Based on msp1˛ sequence variation, the two isolates were found to be separate strains and were named AmRio1 and AmRio2. Analysis of msp1m icrosatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of one MSP1␣ 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.
Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, lit... more Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, little is known about A. phagocytophilum in goats. In the present study, 72 animals from six goat flocks (373 EDTA blood-samples) in Central Switzerland were analysed for A. phagocytophilum DNA. A real-time PCR targeting the msp2 gene of A. phagocytophilum was performed and in positive samples the partial 16S rRNA, groEL and msp4 gene were amplified for sequence analysis. Four DNA extracts were positive. Different sequence types on basis of the amplified genes were found. For comparison, sequences of A. phagocytophilum from 12 cattle (originating from Switzerland and Southern Germany) were analysed. The 16S rRNA gene sequences from cattle were all identical amongst each other, but the groEL and msp4 gene differed depending on the origin of the cattle samples and differed from the variants from goats. This study clearly provides molecular evidence for the presence of different types of A. phagocytophilum in goat flocks in Switzerland, a fact which deserves more thorough attention in clinical studies.
The present work describes the detection of infected cells in the bone marrow aspirates of dogs e... more The present work describes the detection of infected cells in the bone marrow aspirates of dogs experimentally infected with a Brazilian isolate of Ehrlichia canis. Dogs were monitored twice a day by clinical evaluation and peripheral blood smear examination. Every three days, blood samples were collected for cell counts. Weekly, aspirates from the bone marrow were examined and serum samples were tested by IFAT. The clinical signs observed were fever, pallid membranes, lymphadenopathy, serous nasal secretions, and pronounced weight loss. Hematological alterations included normocytic normochromic anemia, decrease of neutrophils and lymphocytes, and thrombocytopenia. Few E. canis infected cells were seen in blood smears. However, stages of E. canis were visualized in bone marrow aspirates 15 days post infection.
The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Braz... more 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.
The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma m... more 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.
Species: Canine One of the main goals for researchers working on canine monocytic ehrlichiosis ha... more Species: Canine One of the main goals for researchers working on canine monocytic ehrlichiosis has been to clarify the clinical and hematological alterations, as well as the immunological mechanisms involved on the pathogenesis of the disease. Usually, treatments instituted during the acute phase promote fast improvement of the animal's clinical conditions. In the present study, nine dogs were experimentally infected with a Brazilian strain of Ehrlichia canis and were treated with tetracycline hydrochloride (22 mg/kg/per day) for 21 days, beginning at the thirtieth day after infection. Some clinical, hematological and immunological parameters were evaluated during the acute phase of the disease and after the treatment. In addition, their immune responses were evaluated before and after the treatment by cytometric analysis and cell phenotype (FACS) using monoclonal antibodies to cell surface markers for CD5, Thy-1, CD4, CD14, MHC II, CD45R, and IgM. The acute phase was clinically characterized by fever, pale mucous membranes, progressive weight loss and moderate lymphadenopathy. The biochemical-hematological analysis revealed decrease of creatinine and total protein concentrations along with anemia, thrombocytopenia, monocytosis and increased ALT enzyme concentration in the infected animals. The ex vivo immune response evaluation of peripheral blood leucocytes showed a percentile reduction of Thy 1+ lymphocytes during a short period of time at the beginning of the acute phase, accompanied by a progressive percentile reduction of T CD4+ lymphocytes, which was significantly reduced at the end of the acute phase. At this time, a decreased percentile of blood MHC-II+ and CD45R+ lymphocytes was observed. On the other hand, the percentage of B (IgM+) lymphocytes and CD14+ cells increased during the acute phase, corroborating with seroconversion, observed by the indirect fluorescent antibody test (IFAT), and monocytosis observed in blood examinations. A higher percentile of CD4+ neutrophils was observed in the granulocytic series after experimental infections. The initial response to the treatment was characterized by fast clinical recovery with weight gain and increased physical activity. There was an evident tendency to reestablish biochemical and hematological parameters, with monocytes responding immediately to the antibiotic treatment. Most parameters of immunophenotyping that were altered during the acute phase returned to normal, demonstrating the favorable response after treatment. These results indicate that the E. canis infection promotes important alterations, which seem to modulate the immune response, allowing persistence of the agent and the establishment of the acute phase. The 21-day treatment regime, on the other hand, results in restoration of immune condition.
This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an append... more 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. marginaleinfected 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.
This epidemiological survey of Anaplasma platys was carried out in rural and urban areas of three... more 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.
Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma margin... more 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 59-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 59-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.
Pesq. Vet. Bras. 21(4):146-150, out./dez. 2001 146 RESUMO. RESUMO.-[Infecção natural por Infecção... more Pesq. Vet. Bras. 21(4):146-150, out./dez. 2001 146 RESUMO. RESUMO.-[Infecção natural por Infecção natural por Anaplasma marginale Anaplasma marginale em em bezerras de fazendas leiteiras da região Metalúrgica de bezerras de fazendas leiteiras da região Metalúrgica de Minas Gerais Minas Gerais.] Realizou-se um estudo da dinâmica de infecções naturais por Anaplasma marginale, durante o período de um ano, em duas propriedades localizadas na Região Metalúrgica do Estado de Minas Gerais. Foram realizados esfregaços sangüíneos e determinação do volume globular (VG).
Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterho... more 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.
Background: Tick cell lines are now available from fifteen ixodid and argasid species of medical ... more Background: Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short-and longterm low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For shortterm cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.
A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection ... more A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.
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