Bruno Lopes Bastos

Corynebacterium pseudotuberculosis: Immunological Responses in Animal Models and Zoonotic Potential

Corynebacterium pseudotuberculosis is a member of the Corynebacterium, Mycobacterium and Nocardia (CMN) group that comprises species of medical, veterinary and biotechnological interest. This pathogen mainly affects small ruminants, causing caseous lymphadenitis (CLA), but it also infects bovines, equines, pigs, deer, camels and humans, showing its zoonotic relevance. Phospholipase D (PLD) and the toxic lipid cell wall are the two most wellstudied virulence factors of this bacterium. They are responsible, in part, for the establishment of disease in the host. Current knowledge on the immunity induced by C. pseudotuberculosis indicates that the resistance to infection is a complex process involving components of both the non-specific and specific host responses, in which humoral and cellular immune responses are both operative. Despite this knowledge and the importance of the disease, a satisfactory vaccine model for sheep and goats has not been developed. Moreover, a control program that includes an efficient diagnostic method in addition to vaccination is crucial for avoiding the spread of bacteria inside flocks. Further, because of its zoonotic potential, C. pseudotuberculosis infection of animals can contaminate meat and milk, putting consumers at risk. The ability of C. pseudotuberculosis to infect both animals and humans makes studies on prevention and diagnosis of this pathogen important.

Corynebacterium pseudotuberculosis secreted antigen-induced specific gamma-interferon production by peripheral blood leukocytes: potential diagnostic marker for caseous lymphadenitis in sheep and goats

In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis—secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.

Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseous lymphadenitis in sheep

BACKGROUNDnCorynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides.nnnOBJECTIVEnThe goal of this study was to determine if serum haptoglobin and plasma fibrinogen concentrations and peripheral blood leukocyte counts are biologic markers of CLA in sheep.nnnMETHODSnBlood from 38 clinically healthy Santa-Inês ewes selected and segregated from a commercial flock of 2500 sheep in an area endemic for C. pseudotuberculosis was collected every 30 days for 6 months. An indirect ELISA was used to detect IgM and IgG antibodies against C. pseudotuberculosis. Serum haptoglobin concentration was measured using a hemoglobin-binding assay and plasma fibrinogen concentration by refractometry following heat precipitation. Total leukocyte counts were determined using a hemocytometer, and differential leukocyte counts were performed on smears of peripheral blood.nnnRESULTSnTwenty-one sheep were seropositive at the start of the study; 15 became seropositive during the study. Only 2 sheep were seronegative at the conclusion of the study. Haptoglobin and fibrinogen concentrations and WBC counts were not significantly different for seropositive and seronegative animals. Nine sheep, 5 that were seropositive positive at the start and 4 that became seropositive during the study period, developed abscesses in peripheral lymph nodes. There were 15 animals that became seropositive during the study, and their values did not differ significantly among the 3 phases--seronegative, acute (IgM+/IgG±), and chronic (IgM-/IgG+)--of infection. However, 11 of these sheep did not develop peripheral abscesses and had significantly higher haptoglobin concentrations and lower monocyte counts during the acute phase of the disease than did the 4 sheep that later developed abscesses.nnnCONCLUSIONnSerum haptoglobin concentration and monocyte counts may be potential markers for progression of CLA in sheep.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.

Avaliação da eficiência de acaricidas sobre Rhipicephalus (Boophilus) microplus no estado da Bahia

The Rhipicephalus (Boophilus) microplus tick is responsible for large losses in the national livestock, mainly due to the reduction in the productive performance of cattle, such damages estimated in billions of dollars per year are proved by the direct action of the hematophagia of the parasite that in addition is transmitter of other pathogens. The development of resistance to the main classes of acaricides currently employed has been widely reported, as well as the occurrence of residues in animal products and ecological problems. The present work had the objective of evaluating the efficiency of carrapaticides on R. (B.) microplus collected in properties in several regions of the state of Bahia. In order to do so, we were able to collect engorged telegraphs in several production units in the region. In vitro tests were performed with seven chemical groups of diluted acaricides as recommended by the manufacturers. For the tests, the ticks were divided into homogeneous groups, where it was later quantified the reduction oviposture and hatching. The results obtained in the experiments indicated varying degrees of efficacy of the several bases

Determinação de valores de referência séricos para os eletrólitos magnésio, cloretos, cálcio e fósforo em ovinos das raças Dorper e Santa Inês

The sheep industry in Brazil is an important economic activity, and with the increasing global demand for sheep meat there is a great interest in the monitoring of the herd health, and serum reference ranges are basic tools for veterinary clinical pathology assays. Mineral elements correspond to 2-5.5% of the body of vertebrates, holding different functions in their physiology. The objective of this study was to obtain reference intervals of the electrolytes magnesium, phosphorus, chloride and calcium for the Dorper and Saint Ines sheep breeds. Sera samples were collected from 487 clinically healthy sheep, 146 from Dorper and 341 from Santa Ines breed. Electrolytes were measured using commercial kits. Data were analyzed taking the race, sex and age variables in account, and reference ranges were established. The results revealed significant statistical differences in reference ranges obtained for the electrolytes calcium and magnesium concerning the variable race, and for the electrolyte phosphorus in the variable age and, when compared with reference values already published, proved the existence of significant differences.