IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Soroprevalência de Anaplasma marginale em bovinos na mesorregiäo norte fluminense / Seroprevalence of Anaplasma marginale in cattle in the \"norte fluminense\" mesoregion

Avaliou-se a soroprevalência de Anaplasma marginale em bovinos de nove municípios na mesorregião Norte Fluminense do estado do Rio de Janeiro através do ensaio de imunoadsorção enzimática (ELISA) indireto. 532 amostras de soro de bovinos foram analisadas; dos quais, 497 eram fêmeas e 35 eram machos; e destes, 444 animais com aptidão zootécnica para corte e 88 com aptidão para leite. A avaliação sorológica revelou que 485 (91,16%) foram positivas, dos quais: 55,45% com título de 1:500, 22,18% com título de 1:1000, 6,77% com título de 1:2000, 3,01% com título de 1:4000, 1,50% com título de 1:8000, 0,94% com título de 1:16000, 0,75% com título de 1:32000, 0,56% com título de 1:64000 e 8,84% foram negativos. A análise da prevalência segundo a faixa etária foi realizada dividindo-se em três grupos etários: 1 a 3 anos (n= 110), 3 a 6 anos (n= 241) e > 6 anos (n= 181), onde 91,82%, 92,95% e 88,95% dos animais foram positivos, respectivamente. Segundo a aptidão zootécnica, 91,22% dos bovinos com aptidão para corte e 90,91% dos bovinos com aptidão para leite foram positivos. Em relação ao sexo, 91,35% das fêmeas e 88,57% dos machos foram positivos. Não houve diferença significativa (P>0,05) entre os grupos etários, entre os sexos e entre as aptidões zootécnicas. A prevalência entre os municípios não diferiu significativamente (P>0,000), demonstrando que a infecção por A. marginale em bovinos é alta e homogênea entre os municípios. A mesorregião estudada foi caracterizada como uma área de estabilidade enzoótica.

Evaluación en Condiciones de Campo de la Vacuna inactivada de Anaplasma Marginale Denominada Plazvax / Evaluation in field conditions of inactivated vaccines of anaplasma marginale named Plazvax

Para determinar la efectividad de la vacuna Plazvax© contra la anaplasmosis bovina, se utilizaron 32 novillos susceptibles a la enfermedad. Un grupo de 24 novillos fue inmunizado contra Anaplasma marginale, inoculando subcutáneamente 1 ml (dos veces, 21 días aparte) de la vacuna. Ocho animales fungieron como testigos sin vacunar. Un mes después de la segunda inmunización, los animales fueron trasladados a una zona endémica de anaplasmosis para ser desafiados naturalmente. A partir del día ocho posintroducción al potrero (PIP) y hasta el día 105 se realizó un seguimiento que incluyó observación de manifestaciones clínicas, temperatura rectal (TR), hematocrito (HT) y parasitemia (PP). Tres bovinos murieron de babesiosis y los 29 animales restantes tuvieron seguimiento dos veces por semana a partir del día 42 PIP. El día 52 PIP se detectaron los primeros animales infectados con Anaplasma marginale (6, todos del grupo vacunado), y cuatro fueron tratados con oxitetraciclinas (días 52-64 PIP). A partir del día 84 PIP los animales testigo presentaron anaplasmosis severa con TR promedio de 40.50C, HT promedio de 13.2 por ciento y PP promedio de 9.4 por ciento hacia el día de tratamiento (días 87-95 PIP). Los animales vacunados presentaron los siguientes valores promedios de TR, 40.2ºC; HT, 16.3 por ciento; y PP 3.3 por ciento en el día de tratamiento (días 93-105 PIP). A pesar de existir diferencia en PP entre los animales vacunados y testigos, todos padecieron anaplasmosis clínica, requiriendo tratamiento. En conclusión, en su forma actual la vacuna no es recomendable para usarse en México; se sugiere el empleo o introducción en la vacuna Plazvax, de material antigénico de uno o más aislados de A. marginale provenientes de distintas regiones geográficas de México y realizar estudios sobre su capacidad inmunoprotectora en bovinos

Avaliaçäo da resposta humoral para anaplasmose em bovinos submetidos à premuniçäo / Humoral response to anaplasmose in cattle submitted to premunition

Trinta e nove animais, de ambos os sexos, da raça Limousin, importados dos Estados Unidos, foram submetidos ao processo de premuniçäo, no Município de Capitólio (MG). Os animais, divididos em cinco grupos, foram inoculados com sangue contendo 5x10 elevado à oitava potência Babesia spp. ou infestados com larvas de Boophilus microplus, provenientes de animais naturalmente infectados por Babesia spp. e receberam inoculaçäo de 5x10 elevado à oitava potência Anaplasma marginale simultaneamente ou 17 dias após a inoculaçäo da Babesia. O perfil sorológico para A. marginale foi observado através de exames semanais pela reaçäo de imunofluorescência indireta. Todos os animais foram desafiados, em relaçäo ao A. marginale, 56 (grupos 3 e 5) e 73 (grupos 1, 2 e 4) dias após a inoculaçäo. Títulos máximos de anticorpos anti-A. marginale foram obtidos 49 dias após a inoculaçäo. Após o desafio os títulos foram crescentes até o 48§ dia

Anaplasmosis in Uganda. III. Parasitological and serological evidence of Anaplasma infection in Ugandan goats

Randomly selected goat sera from north-western; central; and south-western regions of Uganda were analyzed parasitologically and serologically for evidence of anaplasmosis. Prevalence rates of 3.2pc by parasitemia; 4.8pc by card-agglutination test; and 12.9pc by DOT-ELISA combined with western blotting were established. Parasitologically positive samples were consistently serologically positive. Positive samples were all from either the north-western or south-western regions of the country. Goats in these regions graze with cattle and are presumable exposed to the same tick species. There was no evidence of clinical caprine anaplasmosis; whereas bovine anaplasmosis cases are very common. Rhipicephalus evertsi was frequently observed on goats which cograze with cattle