The dynamics of erythrocyte infection in bovine anaplasmosis: a flow cytometry-based analysis.

Anaplasma marginale (A. marginale) is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia. In the present work, we combine SYTO16 labeling of parasitized cells with the statistical power of flow cytometry to study the evolution of erythrocyte infection during bovine anaplasmosis.

A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves.

Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine.

Detection of bovine tuberculosis in herds with different disease prevalence and influence of paratuberculosis infection on PPDB and ESAT-6/CFP10 specificity.

Bovine tuberculosis (BTB) is a major animal health problem with zoonotic implications. Current control programs are based on test and slaughter strategies utilizing skin tests with tuberculins as antigens. The low specificity and associated operative difficulties of these tests have driven the search for new antigens and diagnostic assays. In this multicenter study, using herds from Argentina, Mexico and Northern Ireland, we selected skin test positive and negative animals from herds with different prevalence's of BTB and compared tuberculin (PPDB) and ESAT-6+CFP10 as antigens ex vivo. In low prevalence herds, crossreactivity of PPDB was apparent since up to 60% of the PPDB skin test and ex vivo positive animals did not responded to ESAT-6+CFP10 ex vivo. The superior specificity of ESAT-6+CFP10 was confirmed in a Mycobacterium avium sp. paratuberculosis infected herd where several of the animals had strong crossreactivity to PPDB and PPDA but not to ESAT-6+CFP10. In high prevalence herds 85% of the skin test-positive animals, were confirmed ex vivo using either PPDB or ESAT-6+CFP10 as antigen. However, within this group 60% of the skin test negative animals were PPDB and ESAT-6+CFP10 positive ex vivo indicating that the skin test can in some herds yield a significant number of false negative results. In conclusion, the ex vivo test is recommended as an ancillary test to accelerate BTB eradication. In high prevalence herds, PPDB or ESAT-6+CFP10 can be used as antigen whereas in low and medium prevalence herds ESAT-6+CFP10 is the preferred choice.

Identification of novel Mycobacterium bovis antigens by dissection of crude protein fractions.

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-gamma) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-gamma release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.

Individual animals of a cattle herd infected with the same Mycobacterium bovis genotype shows important variations in bacteriological, histopathological and immune response parameters.

Cattle are the host and main reservoir of the etiologic agent of bovine tuberculosis, Mycobacterium bovis; although other mammalian species, including humans, are susceptible. The tuberculin test and/or slaughterhouse surveillance is the diagnostic method used by control programs all around the world to control and eradicate the disease. In order to compare different tuberculosis diagnostic tests and to reach disease confirmation, a study was performed in a group of 14 steers of Friesian breed, reacting positively to tuberculin test. Three ante-mortem assays were performed according to the type of sample: the gamma interferon (IFN-gamma) test (which quantifies the release of this cytokine by sensitized lymphocytes in whole blood in response to purified protein derivative (PPD) and recombinant ESAT-6 and CFP10 proteins); PCR and bacteriologic culture from nasal swab and intradermal tuberculin test. These assays were taken at different times to assess the evolution of clinical parameters. Post-mortem examination showed macroscopic and microscopic tuberculosis lesions with acid-fast bacillus and positive cultures. By spoligotyping, we observed that all the isolates showed the same pattern. The positive results based on comparison to lesions observed ranged from 58% to 75% for the IFN-gamma assays, to 72% for cultures, and ranged from 50% to 90% for PCR in nasal swabs. In conclusion, in a herd infected by the same strain, ante-mortem direct and immune-diagnostic parameters change, suggesting that several tests are needed for a faster control of infection at herd level.

Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis: ESAT6-CFP10 mixture shows optimal sensitivity and specificity.

Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.

Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.

A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.