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      Bovine babesiosis and anaplasmosis complex: diagnosis and evaluation of the risk factors from Bahia, Brazil Translated title: Complexo tristeza parasitária bovina: diagnóstico e avaliação dos fatores de risco na Bahia, Brasil

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          Abstract

          Direct diagnoses were made by using - blood smears and nested PCR (nPCR) tests on 309 blood samples from crossbred dairy cattle in the municipality of Ibicaraí, Bahia. From diagnostic blood smear slides, the observed parasitic frequencies were 31.1% for Anaplasma marginale and 20.4% for Babesia sp. From nPCR diagnoses, they were 63% for A. marginale, 34% for Babesia bigemina and 20.4% for Babesia bovis. There were significant differences (P <0.01) between the two diagnostic methods (nPCR and blood smear slides). The compliance obtained from the kappa test was 0.41 and 0.48 for A. marginale and Babesia sp., respectively. The tick samples from the six farms analyzed using nPCR were only positive for A. marginale. Evaluation of the risk factors relating to the presence of ticks and the age of the animals showed that there was a significant association (P <0.01) with the frequency of animals infected with both pathogens. Therefore, under the conditions studied, nPCR proved to be a good tool for diagnosing the agents of the bovine babesiosis and anaplasmosis complex because of its sensitivity and specificity in comparison with blood smears. The municipality of Ibicaraí is an area with endemic prevalence of bovine babesiosis and anaplasmosis confirmed by nPCR and A. marginale is the main agent of the disease.

          Translated abstract

          Realizou-se o diagnóstico direto por esfregaço sanguíneo e nested PCR (nPCR) em 309 amostras de sangue de bovinos mestiços leiteiros provenientes do município de Ibicaraí, Bahia. A frequência observada no diagnóstico por lâminas de esfregaço sanguíneo foi 31,1% para Anaplasma marginale e 20,4% para Babesia sp. Enquanto que no diagnóstico por nPCR foi 63% para A. marginale, 34% para Babesia bigemina e 20,4% Babesia bovis. Verificaram-se diferenças significativas (P<0,01) na comparação entre os dois métodos de diagnósticos (nPCR e esfregaço sanguíneo). A concordância ao teste KAPPA obtida foi de 0,41 e 0,48 para A. marginale e Babesia sp., respectivamente. As amostras de carrapatos das seis propriedades analisadas por nPCR foram positivas apenas para A. marginale. Na avaliação dos fatores de risco verificou-se que a presença de carrapato e idade dos animais apresentaram associação significativa (P<0,01) com a frequência de animais infectados por ambos os patógenos analisados por nPCR. Portanto, nas condições estudadas, a nPCR revelou-se uma boa ferramenta para diagnóstico dos agentes do complexo tristeza parasitária bovina (TPB) devido a sensibilidade e especificidade, quando comparado ao esfregaço sanguíneo. O município de Ibicaraí apresenta-se como uma área endêmica para TPB com prevalência comprovada através de nPCR e, A. marginale o principal agente encontrado.

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          Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

          The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.
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            Veterinary epidemiology

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              Current state and future trends in the diagnosis of babesiosis.

              An overview is given of the currently available methods to diagnose babesiosis in livestock. Microscopic techniques are still the only appropriate techniques to diagnose acute disease. Thin or thick blood films stained with Giemsa's stain are sufficient. The sensitivity ranges from 10(-5) to 10(-6), i.e. one parasite per 10(5)-10(6) erythrocytes can be detected. Thick films stained with acridine orange (sensitivity approximately 10(-7)) and the Quantitative Buffy Coat (QBC) analysis tube system (sensitivity approximately 10(-7)-10(-8)) are applicable for diagnosis in the laboratory. DNA probes are very specific tools to identify haemoparasites in organs post mortem and in ticks. For the identification of carrier animals the sensitivity (approximately 10(-5)-10(-6)) is generally not sufficient. For the latter the polymerase chain reaction (PCR) technique is a very powerful tool (sensitivity approximately 10(-9)). Many different serodiagnostic tests have been described; however, the immunofluorescence antibody test is the most widely used, while the enzyme-linked immunosorbent assay (ELISA) is the test system which holds the greatest promise for the future. Thus far, improvements to the ELISA have been limited as the quality of antigen preparations made from infected blood is generally poor with a few exceptions (Babesia bovis, Babesia caballi). Potentially, most of the problems associated with crude antigens can be overcome by the production of recombinant antigens. Several ELISAs based on highly defined recombinant antigens have been described and show promise. None of these tests has been validated to the extent that it could be applied globally. Future research requirements as well as the need for coordination of the research effort and collaboration between institutions involved in the diagnosis of babesiosis are discussed.
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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                rbpv
                Revista Brasileira de Parasitologia Veterinária
                Rev. Bras. Parasitol. Vet.
                Colégio Brasileiro de Parasitologia Veterinária (Jaboticabal )
                1984-2961
                September 2014
                : 23
                : 3
                : 328-336
                Affiliations
                [1 ] Universidade Estadual de Santa Cruz Brazil
                [2 ] Universidade Estadual de Santa Cruz Brazil
                [3 ] Universidade Estadual do Sudoeste da Bahia Brazil
                Article
                S1984-29612014000300328
                10.1590/S1984-29612014064
                25271452
                fb0ce318-81e4-4267-8c1c-b4900717a640

                http://creativecommons.org/licenses/by/4.0/

                History
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                SciELO Brazil

                Self URI (journal page): http://www.scielo.br/scielo.php?script=sci_serial&pid=1984-2961&lng=en
                Categories
                PARASITOLOGY
                VETERINARY SCIENCES

                Parasitology,General veterinary medicine
                Bovine Babesiosis and Anaplasmosis Complex,blood smear,nPCR,risk factors,Complexo TPB,esfregaço sanguíneo,fatores de risco

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