Joaquín H. Patarroyo

Polymorphism of the bm86 gene in South American strains of the cattle tick Boophilus microplus.

Thirty Boophilus microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the bm86 and bm95 gene. A fragment of cDNA of 794 base pairs of the parasite larvae, included between nucleotides 278–1071s, was amplified and cloned on the pGEM-T vector. Two random clones were sequenced for each population and the nucleotides 278–1071 and predicted amino acid sequences compared with the bm86 and bm95 genes. Variations from 1.76 to 3.65% were detected in the nucleotides sequence when compared with the homologous sequence of the bm86 gene and a 3.4–6.08% in the homologous amino acid sequence of the Bm86 protein. When the sequences obtained were compared with the bm95 gene, variations from 0.50 to 3.15% were detected. Variations from 1.14 to 4.56% were detected for the Bm95 protein homologous sequences in the deduced amino acid sequence. Only five of the 30 strains analyzed presented two different types of alleles expressed and the two alleles of the Alegre population and allele 1 of the Betim population were the most divergent of all those analyzed.

Synthetic vaccine (SBm7462) against the cattle tick Rhipicephalus (Boophilus) microplus: Preservation of immunogenic determinants in different strains from South America

The synthetic vaccine SBm7462 is based on three immunogenic epitopes (4822, 4823 and 4824) contained within protein Bm86 derived from the Australian Yeerongpilly strain of the tick Rhipicephalus (Boophilus) microplus. Twenty strains of the tick originating from Brazil, Argentina, Colombia and Uruguay were analysed in order to identify differences compared with sequences present in components of vaccine SBm7462. For each parasite population, three cDNA fragments containing the nucleotides coding for the epitopes 4822, 4824 and 4823 were sequenced, and the amino acid sequences were deduced and compared with those of the homologous bm86 gene. The results indicate that the epitope sequences of vaccine SBm7462 are conserved in the South American populations of the tick. The conservation of such sequences is very important for the immunological response of different populations of R. (B.) microplus.

Exoantigens of an attenuated strain of Babesia bovis used as a vaccine against bovine babesiosis

Bovine babesiosis caused by Babesia bovis remains a significant constraint to beef and milk cattle production throughout the world. Exoantigens released by the parasites in culture supernatants are a potential source of antigen to induce protective immunity. An attenuated strain of B. bovis from Brazil, catalogued as BbUFV1, was maintained in vitro by the MASP method, and exoantigen-containing supernatant fluids were collected daily to form a pool representing a 72-h culture cycle for preparation of the vaccine. Exoantigen concentration was estimated using a two-site EIA. Three groups of susceptible non-splenectomised male Bos taurus cattle, 14 months old, were used. Group A (vaccinated) received two subcutaneous immunizations with a 21-day interval of B. bovis supernatant, content 6500 EIA units of exoantigens plus 1.5 mg saponin, and Group B (adjuvant control) received two injections of adjuvant alone. Four weeks after the second immunization, Groups A, B and C (control) were challenged intravenously with 10(8) virulent parasites of a heterologous B. bovis strain. The results showed that exoantigens present in in vitro cultures can induce a high degree of protection against virulent heterologous challenge exposure. In Group A only one animal showed discrete parasitaemia; all developed a fever and slight decreases in PCV, with a rapid return to normal values. One animal of Group B died; the survivors showed fever, anaemia and parasitaemia. All animals of Group C died between 7 and 13 days after challenge. Vaccination elicited both humoral and cell-mediated immune responses. In Group A, after the challenge, the maximum antibody titer was 12,800. When vaccinated, cattle were tested at the moment of challenge for B. bovis-specific cell-mediated immunity by the monocytemigration inhibition test. A mean inhibition index of 60 +/- 0.33 was observed. Preliminary Western blot analysis of the immunogen revealed at least four proteins of molecular weight ranging between 30 and 160 kDa.

Immune response of bovines stimulated by synthetic vaccine SBm7462 against Rhipicephalus (Boophilus) microplus.

Ten-month-old calves Bos taurus taurus were immunized with three doses of SBm7462 with saponin as an adjuvant at 30-day intervals and were evaluated for IgG isotypes, phenotype circulating lymphocytes and changes in the lymph nodes (LN). SBm7462 stimulated the production of predominantly IgG1-isotype IgG antibodies. The lymph nodes exhibited activation at the seventh day after the first immunization, with areas of paracortical and interfollicular hyperplasia and the early formation of germinal centers (GC). Fifteen days after the first immunization, the GC exhibited compartmentalization of cellular populations, a light zone (LZ), a dark zone (DZ) and a mantle. At the same time, hyperplasia of the medullary cords was observed with cells associating with DC cells. Seven days after the first immunization, apoptosis in the DZ and in the paracortical region became evident. By day 15, there was an increase in the medullary cords, which became more numerous at days 35 and 42. PAP-positive cells were found in the paracortical region, medullary cords and GC 7 days after the first immunization. At day 35, there were further strongly PAP-positive cells in the medullary cords. By comparison, none of these changes were observed in the lymph nodes of control groups at any of the days analyzed. The number of CD21(+) lymphocytes increased in the immunized groups after the first inoculation, with a maximum number observed at 15 and 10 days after the first and third immunizations, respectively. Compared to pre-immunization counts, the percentage of WC1(+) gammadelta T-lymphocytes displayed more variation, increasing 5 days after the second immunization but decreasing over the following days. According to the results, the synthetic anti Rhipicephalus microplus vaccine elicits a complete immune response being T-dependant.

Cattle tick vaccine researchers join forces in CATVAC

A meeting sponsored by the Bill & Melinda Gates Foundation was held at the Avanti Hotel, Mohammedia, Morocco, July 14–15, 2015. The meeting resulted in the formation of the Cattle Tick Vaccine Consortium (CATVAC).

Antigenic profile of a pure isolate of Anaplasma marginale of Brazilian origin, using a Western blot technique

Anaplasma marginale initial bodies of the Brazilian isolate AUFV1 were purified from infected erythrocytes using a combination of lysis, ultrasonic disruption and differential centrifugation. Initial bodies were solubilised with a buffer containing protease inhibitors and non-ionic detergents. Immunochemical analysis by the Western blot technique revealed at least five proteins with apparent molecular weights (MW) of 105, 100, 97, 87 and 38 kDa when homologous sera were used as primary antibodies. Sera from cattle from Mato Grosso do Sul State in Brazil revealed five proteins of 105, 100, 87, 38 and 25 kDa; other heterologous sera obtained from Illinois, USA, bound to four antigens with MW of 105, 100, 87 and 38 kDa, the latter being stronger and broader than the others. No bands were observed in the non-infected erythrocyte control when the different A. marginale sera or antibodies against Babesia bovis and Babesia bigemina were used. Antibodies from cattle infected with the A. marginale isolates bound to proteins of 105, 100, 87 and 38 kDa, indicating that there are at least four peptides common to the isolates. The major surface proteins, designated MSP-1, MSP-2 and MSP-3, are also present in the Brazilian isolate AUFV1 and the sample from the National Research Centre of Beef Cattle in Mato Grosso do Sul State. One practical consideration of this study is the possibility of cross-protection between different Anaplasma isolates including some from Brazil.

Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies

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.

Babesia bovis: infectivity of an attenuated strain of Brazilian origin for the tick vector, Boophilus microplus

In order to verify the infectivity and transovarian transmission of one strain of Babesia bovis of Brazilian origin, attenuated by repeated passages in splenectomized calves to the biological tick vector, Boophilus microplus, an inoculation of that strain was performed in hemoparasite-free calves infested with hemoparasite-free ticks. Subsequently, engorged female ticks were collected to examine hemolymph and progeny for presence of developing stages of Babesia bovis. Using an optical microscope, intrahemocytic merozoites were observed in female hemolymph on the second and fourth days, respectively, after ticks dropped from calves. By histological observation of F1 larvae, after more than 24 h of adherence on calves, sporokinetes and small merozoites were present in the samples of hemolymph. These results confirmed the maintenance of infectivity by the vaccine strain attenuated after 26 passages for tick Boophilus microplus.

Ocorrência de anticorpos anti-Neospora spp. em jumentos (Equus asinus) no estado de Sucre - Colômbia

The neosporosis has great importance in cattle, and its pathogenesis in this host has been generally clarified, however, in horses, neosporosis is poorly known, and in Colombia anti-Neospora spp antibodies have not been reported. Therefore, the main objective in the present study was to evaluate the occurrence of this parasite in donkeys (Equus asinus) from farms that presented a risk of disease in these animals, as well as no health plan for them. Were used 56 animals randomly chosen inside selected farms in the state of Sucre (Colombia). A recombinant peptide originated from Neospora caninum (NcGRA1) was used for the diagnosis with Dot-ELISA and serum was diluted 1:200. This study is the first to report the presence of anti-Neospora spp. in donkeys (Equus asinus) in the state of Sucre, and in Colombia. The occurrence was in 19.7% of the animals sampled (11/56).

Efeito do estresse moderado crônico sobre parâmetros de avaliação óssea em ratos adultos machos e fêmeas

A osteoporose e uma doenca multifatorial, de alta prevalencia e que tem um grande impacto na qualidade de vida, principalmente porque os efeitos sobre a estrutura do osso aumentam o risco de fraturas, que podem ser muito debilitantes. Com base na observacao de que pacientes com depressao tem menor densidade mineral ossea que individuos saudaveis​​, muitos estudos tem indicado que o estresse pode ser um fator agravante para a perda ossea. Este estudo avalia o efeito de um protocolo de estresse moderado cronico (EMC) em parâmetros de avaliacao ossea em ratos machos e femeas. Cinco animais de cada sexo, com cinco meses de idade, foram submetidos a um cronograma de aplicacao de estressores durante 28 dias. Os estressores incluidos foram: frio, calor, contencao, inclinacao da gaiola, isolamento, iluminacao durante a noite e privacao de agua e racao. Cinco animais de cada sexo foram mantidos com um minimo de intervencao como grupo controle. Os animais foram pesador no inicio e no final do periodo, e apos eutanasia tiveram seus ossos coletados. Femur, tibia e vertebra lombar foram analisados por densitometria ossea. Testes biomecânicos foram realizados na cabeca e na diafise do femur. Volume trabecular osseo foi obtido a partir de analise histomorfometricas da cabeca do femur e do corpo vertebral, bem como medidas da secao transversal diafise femoral. Nem todos os parâmetros avaliados sofreram efeito do protocolo de EMC. No entanto, a densidade mineral ossea da tibia e da vertebra L4 e a razao osso cortical/medula da secao transversal da diafise femoral foram menores nas femeas submetidas ao protocolo. Entre os ratos machos, as diferencas foram significativas no volume trabecular osseo da cabeca femoral e na carga maxima obtida no teste biomecânico. Assim, confirma-se que o protocolo de EMC pode afetar o equilibrio da homeostase ossea em ratos, o que pode contribuir para o estabelecimento de osteopenia ou osteoporose.