Genome-Based Vaccinology Applied to Bovine Anaplasmosis

Bovine anaplasmosis is an infectious non-contagious disease transmitted mainly by ticks or fomites contaminated with Anaplasma marginale. Once cattle have developed the disease it can be treated with antibiotics or chemotherapy, although with partial success. Still, there is no effective and global prophylactic method available, mainly because of variability and diversity showed by different A. marginale strains distributed worldwide. In this regard, several proteins have been proposed as immunogens, MSPs, OMPs, Type IV Secretion System Proteins and some other hypothetical proteins, which have been chosen either by experimental evidence or more recently by genome-based analysis. So far, the results suggest that a single molecule will not be enough to trigger a protective immune response in the host, so it is necessary to identify other proteins or epitopes with adequate immunological properties, a process in which omics tools have potential. In order to develop a vaccine against bovine anaplasmosis, it has been proposed by the use of combinations of molecules, exposure formats and application protocols to provide an effective control of the disease

Caracterización bioquímica preliminar de clonas de Babesia bovis irradiadas con cobalto 60

Con objeto de caracterizar parcialmente los cambios inducidos por la irradiación gamma en el crecimiento in vitro y bioquímicamente, dos clonas de Babesia bovis fueron expuestas a dosis de 81, 135 Y 189 Greys (Gy) de irradiación usando una fuente de 60 C

Antigen production and standardization of an in-house indirect ELISA for detection of antibodies against Anaplasma marginale

Las pruebas serológicas son importantes para la detección de anticuerpos específicos contra agentes infecciosos. Los ELISA indirectos comerciales son costosos y, por lo general, son tan efectivos como los ELISA caseros. En el presente trabajo fue preparado un lote de antígeno crudo de Anaplasma marginale a partir de sangre infectada, y se probó contra controles oficiales de suero positivos y negativos y comparado con un lote viejo de antígeno. El nuevo lote de antígenos mostró una eficiencia similar a la del lote viejo. La sensibilidad de la prueba entre los lotes nuevo y viejo fue comparable. Tanto el lote de antígeno nuevo como el viejo se están utilizando en exceso. El nuevo lote de antígeno es lo suficientemente grande como para ejecutar miles de pruebas a un precio más asequible que el de los kits comerciales.Serologic tests are important for the detection of specific antibodies against infectious agents. Commercial indirect ELISA are costly and usually as effective as in-house ELISAs. In the present work, it was prepared a batch of Anaplasma marginale crude antigen from infected blood, and tested it against official positive and negative serum controls and compared with an old batch of antigen. The new antigen batch showed an efficiency similar to the old batch. The sensitivity of the test was comparable between the new and old batches. Both, new and old antigen lots are being used at an excess. The new antigen lot is large enough to run thousands of tests at a more affordable price than commercial kits

Identificación de antígenos IgG2 específicos en tres cepas mexicanas de Anaplasma marginale

In order to identify Anaplasma marginale antigens associated with protection, susceptible animals were inoculated on three occasions with inactivated Mex-17 A. marginale initial bodies mixed with saponin or an oil-based adjuvant. Animals were challenge inoculated with strain Mex-30 on d 164. With the exception of two animals which died at challenge, all other immunized animals showed minimum clinical signs compared with control animals. At ELISA, positive antibody titers were observed in immunized whereas no titers were detected in controls. In vitro proliferation indexes (PI) for mononuclear cells cultured in the presence of extracts of the three strains were positive (³2.0) for all immunized bovines, whereas all both vaccinated an control animals developed PI after challenge inoculation. Western blot (WB) analysis of vaccinates sera showed recognition of antigens in a range between 15 to 209 kDa, of which eleven were specifically recognized by IgG2 in Mex-17 and Mex-30 strains. Additionally, a 52 kDa protein was observed in the Mex-15 extract by the sera of immunized and protected bovines. Two- dimension WB analysis of Mex-17 showed an additional 37 kDa protein that co-migrated with a band of similar molecular weight of MSP-2. Our results indicate that these eleven IgG2 specific proteins are associated with an immunoprotective response induced by vaccination. Four of these proteins correspond to previously characterized major-surface proteins and seven are novel antigens previously unreported for their immunoprotective properties.Con objeto de identificar antígenos de Anaplasma marginale asociados a la protección, animales susceptibles se vacunaron tres veces con cuerpos iniciales de la cepa Méx-17, mezclados con saponina o un adyuvante oleoso, y desafiados 164 días después de la primera inmunización con la cepa Méx-30. Los bovinos inmunizados mostraron cambios mínimos de los parámetros clínicos en comparación con los testigos. Al Elisa, los sueros de los bovinos inmunizados mostraron valores positivos y ninguna respuesta para los testigos. Los índices de estimulación (IE) (= == ==2.0) para células mononucleares de sangre periférica cultivadas en presencia de extractos de cada una de las tres cepas de A. marginale fueron positivos sólo en los bovinos inmunizados, al desafío, los IE para los bovinos inmunizados y testigos también fueron positivos. Al inmunoblot, se reconocieron proteínas de entre 15 a 209 kDa de las cuales, 11 fueron específicamente reconocidas por IgG2 en la cepas Méx-17 y Méx-30 y adicionalmente una proteína de 52 kDa en Méx-15 sólo por los sueros de los bovinos inmunizados y protegidos. El inmunoblot de dos dimensiones de la cepa Méx-17 mostró una proteína adicional de 37 kDa que co-migraba con una proteína que corresponde a MSP-2. Los resultados indican que estas 11 proteínas específicamente reconocidas por IgG2 de animales protegidos, participaron en la respuesta protectora, cuatro de éstas corresponden a las MSPs ya caracterizadas, y siete son nuevos antígenos nunca antes publicados por sus propiedades inmunoprotectoras

Immunolocalization of VirB11 protein in the Anaplasma marginale outer membrane and its reaction with bovine immune sera

Type IV secretion system is composed by a group of proteins associated with substrate transfer between bacteria and eukaryotic organisms. VirB11 is a protein of this system and, in A. marginale it induces partial protection in cattle immunized with external membrane fragments of the rickettsia. The objectives of this study were: to determine the conservation rate of VirB11 among different Mexican isolates and to corroborate its cellular localization on the outer membrane of A. marginale. Nucleotide and amino acid sequence alignment of virB11 from nine Mexican isolates showed 100 % identity. Bovine sera from both naturally and experimentally infected cattle reacted against a multiple antigenic peptide (MAP‑VirB11) designed based on a B-type epitope present in VirB11. In addition, MAP‑VirB11‑rabbit immune sera reacted with native VirB11 by ELISA and western blot in rickettsial extracts and on the surface of bovine infected erythrocytes by indirect immunofluorescence and the localization of VirB11 protein was determined on the outer surface of A. marginale initial bodies

Anaplasma marginale: analysis of variable fragment sequences in msp1α and msp4 genes in four new Mexican strains

[ES] La anaplasmosis bovina ocasiona pérdidas cuantiosas a la ganadería en países en desarrollo, y el diseńo de vacunas se ve obstaculizado por la amplia diversidad genética y antigénica que este organismo presenta en cepas de diferentes regiones geográficas. El objetivo del trabajo fue comparar las secuencias de cuatro cepas mexicanas de A. marginale no tipificadas respecto a los genes msp1α y msp4. Se usaron las secuencias de MSP-1a y MSP4, dos de las proteínas del complejo principal de superficie, y que se han usado para estudios filogenéticos. Usando iniciadores específicos, para la región variable del gen msp1α y el gen msp4, se amplificó el ADN de las cepas Pte. de Ixtla, Mor., Aguascalientes, Ags., Pichucalco y Sta. Martha, Chis. El análisis de las secuencias nucleotídicas y de aminoácidos para msp1α reveló una clara similitud entre los aislado Pichucalco y Sta. Martha ambas de Chiapas, al igual que contra los aislados México, Morelos y Veracruz. Los aislados Aguascalientes y Pte. de Ixtla demostraron ser diferentes entre ellos, de las otras cepas mexicanas, y de otros países, mientras que para msp4 no se observó variación en ninguna de las cepas mexicanas hasta el momento reportadas, las cuales contienen 849 pb y coinciden altamente con diversos aislados del mundo. El estudio regional o nacional del grado de conservación de estas proteínas abriría posibilidades para el diseńo de una vacuna con base en este tipo de antígenos y se podría predecir su eficacia.[EN] Bovine anaplasmosis, due to Anaplasma marginale, is responsible for substantial economic losses in livestock production of developing countries. Vaccine development is hampered by wide genetic and antigenic diversity in strains of distant geographical areas. The objective of the present study was to compare sequences of four non typified Mexican A. marginale strains in respect of msp1α and msp4 genes. Sequences of MSP-1a and MSP4 and major surface complex proteins were used, already used for phylogenetic studies. Through specific initiators, for variable areas of both the msp1α and msp4 genes, DNA of the Pte. de Ixtla, Mor.; Aguascalientes, Ags; Pichucalco and Sta. Martha, Chis. strains was amplified. Analyses of nucleotide sequences and amino acids for msp1α showed a strong similitude between Pichucalco and Sta. Martha, both from Chiapas, same as against the isolates Mexico, Morelos and Veracruz. Isolates Pte. de Ixtla and Aguascalientes were different between them and previously identified Mexican strains and from other countries, while for msp4 no variation was reported for Mexican strains previously reported, which contain 849 pb and coincide with different isolates the world over. Studies comprising either regions or the whole country on the conservation rank of these proteins could open possibilities for vaccines based on these antigens and its efficacy could be predicted.Este estudio fue financiado con fondos del proyecto SEP-CONACYT No. 44565.Peer reviewe

Anaplasma marginale: análisis de las secuencias del fragmento variable del gen msp1alpha, y del gen msp4 de cuatro nuevas cepas mexicanas

Bovine anaplasmosis, due to Anaplasma marginale, is responsible for substantial economic losses in livestock production of developing countries. Vaccine development is hampered by wide genetic and antigenic diversity in strains of distant geographical areas. The objective of the present study was to compare sequences of four non typified Mexican A. marginale strains in respect of msp1alpha,,,, and msp4 genes. Sequences of MSP-1a and MSP4 and major surface complex proteins were used, already used for phylogenetic studies. Through specific initiators, for variable areas of both the msp1alpha,,,, and msp4 genes, DNA of the Pte. de Ixtla, Mor., Aguascalientes, Ags, Pichucalco and Sta. Martha, Chis. strains was amplified. Analyses of nucleotide sequences and amino acids for msp1alpha,,,, showed a strong similitude between Pichucalco and Sta. Martha, both from Chiapas, same as against the isolates Mexico, Morelos and Veracruz. Isolates Pte. de Ixtla and Aguascalientes were different between them and previously identified Mexican strains and from other countries, while for msp4 no variation was reported for Mexican strains previously reported, which contain 849 pb and coincide with different isolates the world over. Studies comprising either regions or the whole country on the conservation rank of these proteins could open possibilities for vaccines based on these antigens and its efficacy could be predicted.La anaplasmosis bovina ocasiona pérdidas cuantiosas a la ganaderí­a en paí­ses en desarrollo, y el diseño de vacunas se ve obstaculizado por la amplia diversidad genética y antigénica que este organismo presenta en cepas de diferentes regiones geográficas. El objetivo del trabajo fue comparar las secuencias de cuatro cepas mexicanas de A. marginale no tipificadas respecto a los genes msp1alpha,,,, y msp4. Se usaron las secuencias de MSP-1a y MSP4, dos de las proteí­nas del complejo principal de superficie, y que se han usado para estudios filogenéticos. Usando iniciadores especí­ficos, para la región variable del gen msp1alpha,,,, y el gen msp4, se amplificó el ADN de las cepas Pte. de Ixtla, Mor., Aguascalientes, Ags., Pichucalco y Sta. Martha, Chis. El análisis de las secuencias nucleotí­dicas y de aminoácidos para msp1alpha,,,, reveló una clara similitud entre los aislado Pichucalco y Sta. Martha ambas de Chiapas, al igual que contra los aislados México, Morelos y Veracruz. Los aislados Aguascalientes y Pte. de Ixtla demostraron ser diferentes entre ellos, de las otras cepas mexicanas, y de otros paí­ses, mientras que para msp4 no se observó variación en ninguna de las cepas mexicanas hasta el momento reportadas, las cuales contienen 849 pb y coinciden altamente con diversos aislados del mundo. El estudio regional o nacional del grado de conservación de estas proteí­nas abrirí­a posibilidades para el diseño de una vacuna con base en este tipo de antí­genos y se podrí­a predecir su eficacia

Transmission of Anaplasma marginale by unfed Rhipicephalus microplus tick larvae under experimental conditions

The current description of biological transmission of Anaplasma marginale by Rhipicephalus microplus ticks, includes both intrastadial and transstadial transmission. Both transovarial transmission of Anaplasma from engorged ticks to their progeny and, transmission from infected unfed larvae to the mammalian host are is controversial. In order to demonstrate vertical transmission of A. marginale by R. microplus ticks under experimental conditions, feed-acquisition infected engorged females were incubated at 18° C or 28° C for oviposition. Larvae hatched from these ticks were used to infest two steers for each incubation temperature. None of the four steers infested with either lot of larvae developed clinical disease, yet subclinical infection was observed in the steers infested with larvae from engorged ticks incubated at 28°. DNA from larvae used for the infection of the carrier tick donor, Only DNA from larvae oviposited  at 28 ºC, DNA from blood of positive steers were positive for amplification of msp5 and msp1α variable region by PCR. All other DNA samples from the original stabilate, blood from the donor steer, larvae from ticks incubated at 28° C and steers infested with these same larvae were positive both to msp5 and msp1α PCR. msp1α sequences of all PCR products were the same and coincided with previously reported Tlapacoyan-2 sequence. The present evidence indicates that R. microplus is capable of transmitting A. marginale to its progeny and that these infected larvae can transmit the infection to susceptible hosts.