RESUMO
BACKGROUND AND METHODS: This study described the detection, prevalence and phylogeny of Anaplasma marginale in the bovine (cattle and buffaloes) and Rhipicephalus (Boophilus) microplus tick belonged to the tribal area of coastal South Gujarat, India, by amplifying 576 bp of major surface protein (msp) 5 gene using custom designed primers in the polymerase chain reaction (PCR). RESULTS: The PCR detection limit was up to 20 parasites/µl of blood in sensitivity experiment, and observed 100% specificity against Trypanosoma evansi, Babesia bigemina and Theileria annulata. Prevalence rate of the A. marginale in the bovine (n = 211)) was 18.48% and 6.64% (p < 0.05) as per the PCR and Giemsa stained blood smear, respectively. Febrile animals (35%) observed significantly (p < 0.05) higher incidence rate than the non-febrile (14.62%). The amplified msp5 had single cut site for the EcoR1 enzyme, upon digestion yielded two fragments of 365 and 211 bp on 1.0% agarose gel. The current sequence (KC811329) showed 100% homology and 1064 total score with the published nucleotide sequences of msp5 of A. marginale in the NCBI-BLAST study. Monophyletic relationship was observed with high bootstrap proportion (> 76% in Neighbor-Joining/ Maximum Likelihood) between the current and published nucleotide sequences in the phylogeny. Twenty out of 39 A. marginale infected bovine recorded R. (B.) microplus on their body surface, out of which 18 had detected the infection. The rickettsia was in 55%, 65% and 25% of anterior half, posterior half and egg of tick, respectively. CONCLUSION: The test detected A. marginale in a carrier, pre-symptomatic and symptomatic vertebrate hosts (cattle and buffalo) and different body parts of the starved R. (B.) microplus including its egg. The current genotype could be an explanation for the frequent outbreaks of bovine anaplasmosis in the targeted areas.
Assuntos
Anaplasma marginale/genética , Anaplasmose/microbiologia , Búfalos/microbiologia , Doenças dos Bovinos/epidemiologia , Filogenia , Rhipicephalus/microbiologia , Anaplasmose/sangue , Anaplasmose/epidemiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Bovinos/microbiologia , Doenças dos Bovinos/microbiologia , Doenças Endêmicas , Feminino , Variação Genética , Genótipo , Índia/epidemiologia , Limite de Detecção , Reação em Cadeia da Polimerase/veterinária , Prevalência , Análise de Sequência de DNARESUMO
Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/diagnóstico , DNA Bacteriano/sangue , Ehrlichia canis/isolamento & purificação , Ehrlichiose/veterinária , Reação em Cadeia da Polimerase/métodos , Anaplasma/genética , Anaplasmose/sangue , Anaplasmose/microbiologia , Animais , Brasil , Tamanho Celular , Coinfecção/sangue , Coinfecção/microbiologia , Coinfecção/veterinária , DNA Bacteriano/genética , Doenças do Cão/sangue , Doenças do Cão/microbiologia , Cães , Ehrlichia canis/genética , Ehrlichiose/sangue , Ehrlichiose/diagnóstico , Ehrlichiose/microbiologia , Genes Bacterianos , Genes de RNAr , Granulócitos/microbiologia , Testes Hematológicos/métodos , Leucócitos Mononucleares/microbiologia , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Sensibilidade e EspecificidadeRESUMO
Between June and September 2002, a preliminary study was conducted to assess the prevalence of blood parasites of cattle in eastern Algeria. Fifty-four bovines of different genotypes were submitted to clinical examination. From each animal, blood smears were made and stained by Giemsa. Four species of parasites, namely Theileria annulata, T. orientalis, Babesia bovis and Anaplasma marginale were encountered. Fifty animals carried single or multiple infections with blood parasites and four were found negative. The rate of single infections (72.3%, n = 39) was almost three times higher than multiple infections (20.3%, n = 11). The high percentage of single infections was recorded with T. annulata (53.7%). However single infection with Anaplasma marginale (7.4 %), B. bovis (5.6%) and T orientalis (5.6%) were very low compared to T. annulata infection. The rates of mixed infection were as follows: T. annulata/A. marginale 9.3%, T. annulata/T. orientalis 5.6%, A. marginale/T. orientalis 3.7% and B. bovis/A. marginale 1.9%.
