RESUMO
The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (-Preg, n = 4), and seronegative non-pregnant cows (-Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg (p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio (p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting (p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg (p <0.05). Avidity correlated with total IgG and IgG2 (p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle.
RESUMO
The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg-1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg-1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas.
Assuntos
Anaplasmose , Doenças dos Bovinos , Imidocarbo/análogos & derivados , Oxitetraciclina , Anaplasma marginale , Anaplasmose/tratamento farmacológico , Animais , Argentina , Bovinos/parasitologia , Doenças dos Bovinos/tratamento farmacológico , Imidocarbo/uso terapêutico , Oxitetraciclina/uso terapêuticoRESUMO
N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.
RESUMO
Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.
RESUMO
Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 µg of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p<0.05), requiring treatment with oxytetracycline to prevent death. The levels of IgG2 induced in the immunized groups did not correlate with the observed lack of protection. Additional strategies are required to evaluate the role of these proteins and their potential utility in the development of effective vaccines.
Assuntos
Anaplasma marginale/imunologia , Anaplasma marginale/patogenicidade , Vacinas Bacterianas/imunologia , Proteínas Recombinantes/imunologia , Sistemas de Secreção Tipo IV/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/genética , Bovinos , Imunização , Proteínas Recombinantes/genética , Sistemas de Secreção Tipo IV/genética , Virulência/imunologiaRESUMO
The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doenças dos Bovinos/imunologia , Coccidiose/veterinária , Neospora/imunologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/imunologia , Coccidiose/parasitologia , Citocinas/metabolismo , Feminino , Imunização/veterinária , Neospora/patogenicidade , Distribuição AleatóriaRESUMO
Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.
Assuntos
Babesia bovis/imunologia , Babesiose/prevenção & controle , Doenças dos Bovinos/prevenção & controle , Vacinas Protozoárias/imunologia , Vacinação/veterinária , Animais , Anticorpos Neutralizantes/imunologia , Babesiose/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Masculino , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Vacinas Atenuadas/imunologia , Vaccinia virus/imunologiaRESUMO
BACKGROUND: Tritrichomonas foetus is the etiologic agent of the sexually transmitted disease Bovine Trichomonosis (BT). In Argentina, BT is endemic and represents a relevant health problem that causes reproductive inefficiency in cattle and large economic losses. Metronidazole is the drug of choice in the treatment of BT. Treatment has been associated with a temporary resolution of the clinical signs but is not able to control the disease. In recent years, the apparition of in vivo and in vitro aerobic and anaerobic resistance leading to ineffective treatments has been reported. AIMS: Thus, the aim of the present study was to explore the susceptibility of six different isolates of T. foetus under aerobic (AC) and anaerobic (ANC) conditions. RESULTS AND DISCUSSION: Six isolates of T. foetus were obtained from samples of preputial smegma of bovine origin. Values of minimum lethal concentration and minimum inhibitory concentration were higher than those observed in other works and represent current data in Argentina and provide information to establish new treatment protocols.
Assuntos
Antiprotozoários/farmacologia , Metronidazol/farmacologia , Tritrichomonas foetus/efeitos dos fármacos , Aerobiose , Anaerobiose , Animais , Argentina , Bovinos , Doenças dos Bovinos/parasitologia , Infecções Protozoárias em Animais , Tricomoníase/veterinária , Tritrichomonas foetus/isolamento & purificaçãoRESUMO
Water buffaloes (Bubalus bubalis) are raised in tropical and subtropical regions of the world, and act as hosts of Babesia bovis parasites and the tick vector Rhipicephalus microplus. As no clinical cases of B. bovis-infection have been reported, we hypothesized that, unlike bovines, water buffaloes respond asymptomatically to an acute infection. To test this hypothesis, we inoculated two groups of 24-month-old Mediterranean breed water buffaloes with 108 erythrocytes infected with two Argentine B. bovis isolates: BboM2P (nâ¯=â¯5) or BboS2P (nâ¯=â¯5). These strains displayed mild (BboM2P) or high (BboS2P) pathogenicity in Bos taurus calves of the same age (nâ¯=â¯5 and nâ¯=â¯1, respectively), when tested in parallel. In water buffaloes, no changes in body temperature were observed with both strains, and no hematocrit changes were detected in BboM2P-inoculated animals. In contrast, in the BboS2P-inoculated water buffalo group significant but relatively minor reductions in haematocrit values were noted compared to the infected bovine. The parasitemia attained in water buffaloes was considerably lower than in bovines and could only be detected by nested PCR, or indirectly via serology, whereas in most bovines, it could also be detected in Giemsa-stained smears under the light microscope. Our results show that water buffaloes present no or significantly mitigated clinical symptoms to B. bovis infections and suggest that they are able to substantially reduce and/or eliminate B. bovis parasites from circulation by an efficient innate immune mechanism.
Assuntos
Babesia bovis/isolamento & purificação , Babesiose/parasitologia , Doenças dos Bovinos/diagnóstico , Parasitemia/diagnóstico , Animais , Babesia bovis/genética , Babesia bovis/imunologia , Babesia bovis/patogenicidade , Babesiose/diagnóstico , Babesiose/imunologia , Búfalos/imunologia , Búfalos/parasitologia , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/parasitologia , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Eritrócitos/parasitologia , Hematócrito , Imunidade Inata , Masculino , Parasitemia/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Rhipicephalus/microbiologia , Testes SorológicosRESUMO
BACKGROUND & OBJECTIVES: : The presence of Babesia spp in humans, bovine cattle and ticks (the transmitting vector) has not been well characterized in Colombia. Babesia infection in humans can be overlooked due to similarity of the disease symptoms with malaria specially in the regions where malaria is endemic. The aim of the present work was to study the frequency of Babesia infection in humans, bovines and ticks in a malaria endemic region of Colombia, and explore the possible relationship of infection with host and the environmental factors. METHODS: : A cross-sectional study was carried out between August 2014 and March 2015 to determine the frequency of B. bovis and B. bigemina infection in a sample of 300 humans involved in cattle raising, in 202 bovines; and in 515 ticks obtained from these subjects, using molecular (PCR), microscopic and serological methods. In addition, the demographic, ecological and zootechnical factors associated with the presence of Babesia, were explored. RESULTS: : In the bovine population, the prevalence of infection was 14.4% (29/202); the highest risk of infection was found in cattle under nine months of age (OR = 23.9, CI 8.10-94.30, p = 0.0). In humans, a prevalence of 2% (6/300) was found; four of these six cases were positive for B. bovis. Self-report of fever in the last seven days in the positive cases was found to be associated with Babesia infection (Incidence rate ratio = 9.08; CI 1.34-61.10, p = 0.02). The frequency of B. bigemina infection in the collected ticks was 18.5% (30/162). INTERPRETATION & CONCLUSION: : The study established the presence of Babesia spp in humans, bovines and ticks. The most prevalent species responsible for babesiosis in humans and bovines was B. bovis, while B. bigemina was the species most frequently found in the tick population. The results contribute to the knowledge of the epidemiology of babesiosis in the country and can provide guidelines for the epidemiological surveillance of this non-malarial febrile illness in humans as well as cattle.