Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis.

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.

Characterisation of quinolone-resistant Escherichia coli of 1997 and 2005 isolates from poultry in Mexico.

Forty-two enrofloxacin-resistant Escherichia coli strains isolated from eggs and first-week mortality associated with yolk sac infection of two vertically integrated poultry companies of Central Mexico in 1997 and 2005 were characterised. E. coli resistance to 19 antibiotics was determined, as well as the minimum inhibitory concentrations (broth dilution) for ciprofloxacin. The presence of gyrA,B, parC,E chromosomal point mutations, qnrA,B,S plasmid genes and the aminoglycoside acetyltransferase aac(6')-Ib-cr were determined by PCR and sequencing. Resistance to ampicillin (95%), piperacillin (95%), gatifloxacin (95%), levofloxacin (95%), ampicillin/sulbactam (90%), cefazolin (85%), trimethoprim/sulfamethoxazole (80%), amoxicillin/clavulanic acid (80%), aztreonam (80%), cefepime (80%), cefotaxime (80%), ceftazidime (80%), ceftriaxone (80%) and cefoxitin (75%) was high in the 2005 strains and 19 (95%) strains were resistant to 7 or more antimicrobials. The strains from 1997 expressed high rates of resistance only to the fluoroquinolones and 4 strains (18%) expressed resistance to 7 or more antimicrobials. All strains had a gyrA mutation (Ser83Leu) and a parC mutation (Ser80Ile or Ser80Arg) and 41 (97.6%) strains had a second gyrA mutation (Asp87Asn, Asp87Tyr or Asp87Gly). Only two (4.7%) strains had a parE mutation (Ser458Ala). A total of 10 strains were positive for the aac(6')-Ib wild-type gene, 6 strains for the aac(6')-Ib-cr variant and 6 strains possessed both the wild type and the variant. No gyrB mutations or qnrA,B,S genes were detected. This is the first report in Latin America of chromosomal and plasmid quinolone resistance genes in E. coli strains recovered from poultry.

Resistencia a antimicrobianos en cepas de Mannheimia haemolytica aisladas de exudado nasal de bovinos productores de leche / Antimicrobial resistance in Mannheimia haemolytica strains isolated from dairy cattle nasal exudate

Two hundred and one strains of M. haemolytica isolated from nasal exudate of dairy cattle were used, 123 strains from clinically healthy (CH) bovines and 78 from clinically ill (CI) bovines affected by pneumonia, obtained from a dairy complex in the Tizayuca region of the state of Hidalgo, Mexico. Strains were previously identified by conventional culture and biochemical tests, and serotyped by indirect haemagglutination. Disk diffusion test was performed to determine antimicrobial resistance to antibiotics, such as: ampicillin, gentamicin, ceftiofiur, penicillin, streptomycin, trimethoprim-sulfamethoxazole, tetracycline and erythromycin. Frequencies of higher antimicrobial resistance were: streptomycin (81.6%) and gentamicin (24.4%), all strains were susceptible to ampicillin and penicillin. Because of the high resistant strain frequency (81.6%) of M. haemolytica to streptomycin, obtained by Kirby-Bauer test, presence of the strA gene, which encodes the enzyme aminoglycoside-3-phosphotransferase that provides resistance to streptomycin, PCR was performed by testing the presence of the strA gene. Of the 201 strains tested, 42.7% showed the gene sfrA, 17.4% of which was serotype A1, 1.4% serotype A6 and 23.8% non-typeable strains. Of the 78 CI strains and 123 CH strains, 80% and 18.7%, had the gene sfrA, respectively.

Se emplearon 201 cepas de Mannheimia haemolytica provenientes de muestras de exudado nasal de bovinos productores de leche, 123 de bovinos clínicamente sanos (CS) y 78 de bovinos enfermos de neumonía (CE), obtenidas de un complejo lechero en la región de Tizayuca, Hidalgo, México, las cuales fueron identificadas previamente mediante pruebas convencionales de cultivo y bioquímicas, y serotipificadas mediante la prueba de hemaglutinación indirecta. Se les realizó la prueba de difusión en placa para determinar la resistencia a diversos antimicrobianos como ampicilina, gentamicina, ceftiofiur, penicilina, estreptomicina, trimetoprim con sulfametoxazol, tetraciclina y eritromicina. Las frecuencias más altas en la resistencia a antimicrobianos se presentaron a la estreptomicina (81.6%) y gentamicina (24.4%), todas las cepas fueron susceptibles a la ampicilina y penicilina. Debido a la alta frecuencia (81.6%) de cepas de M. haemolyfica resistentes a St con la técnica de Kirby-Bauer, se buscó la presencia del gen sfrA. Se realizó la técnica de PCR para comprobar la presencia del gen sfrA que codifica para la enzima aminoglycoside-3-phosphofransferase que proporciona resistencia contra la estreptomicina. Del total de cepas estudiadas (n = 201), 42.7% presentaron el gen sfrA, del cual 17.4% pertenecía al serotipo A1, 1.4% al A6 y 23.8% a cepas no tipificables. De las 78 cepas de CE y las 123 de CS, 80.0% y 18.7% respectivamente, presentaron el gen sfrA.

Specificity of the tuberculin skin test is modified by use of a protein cocktail containing ESAT-6 and CFP-10 in cattle naturally infected with Mycobacterium bovis.

The mycobacterial immunodominant ESAT-6 and CFP-10 antigens are strongly recognizable in tuberculosis-infected cattle, and they do not elicit a response in cattle without infection. In addition, they are absent in most environmental mycobacterial species, and therefore, their use can be an alternative to purified protein derivative (PPD) tuberculin in the development of a more specific skin diagnostic test in cattle. The aim of the current study was to assess the potential of an ESAT-6 and CFP-10 (E6-C10) protein cocktail in a skin test format in naturally tuberculosis-infected and paratuberculosis-infected cattle. We also included MPB83 as a third component in one of the protein cocktail preparations. The protein cocktail was tested at different dose concentrations (5, 10, and 15 µg per protein). The best skin response to the E6-C10 protein cocktail was obtained with 10 µg. Subsequently, this concentration was tested in 2 herds with high and low bovine tuberculosis prevalence, the latter with paratuberculosis coinfection. Our data show that the E6-C10 cocktail allows identification of an important proportion of animals that PPDB is not able to recognize, especially in low-prevalence herds. The protein cocktail did not induce reactions in tuberculosis-free cattle or in paratuberculosis-infected cattle. Addition of MPB83 to the protein cocktail did not make any difference in the skin reaction.

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.

Characterisation of Mannheimia spp. strains isolated from bovine nasal exudate and factors associated to isolates, in dairy farms in the Central Valley of Mexico.

Mannheimia spp. strains obtained from bovine nasal exudates of either clinically healthy or clinically affected by respiratory tract disease animals were isolated and characterised to estimate the prevalence of isolated serotypes in dairy farms in Mexico, by means of a trans-sectional descriptive study. Strains were isolated and typified through biochemical and immunological tests. chi(2) or Fisher statistical tests were applied, as well as odds ratio calculation and logistic regression analysis to evaluate the association and effect of some variables on Mannheimia spp. isolation. The apparent prevalence rates of Mannheimia haemolytica was significantly higher in diseased bovines (OR = 1.94; p < 0.05), as well as in bovines younger than 1 year of age (OR = 23.98; p < 0.05), and in bovines not vaccinated against bovine pasteurellosis (OR = 1.52; p < 0.05). Age was the variable that remained in the logistic regression model. Serotype A1 showed the highest prevalence, even when most isolates were not-typable. Bovines younger than one year of age and those with disease were the groups with the highest frequency of M. haemolytica and Mannheimia glucosida isolates.

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.

Subcellular fractions of Brucella ovis distinctively induce the production of interleukin-2, interleukin-4, and interferon-gamma in mice.

The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-gamma production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine.

Protection against brucellosis in goats, five years after vaccination with reduced-dose Brucella melitensis Rev 1 vaccine.

The protection conferred by the reduced-dose Rev 1 Brucella melitensis vaccine in goats that had been immunized 5 years previously was evaluated. Sixteen goats vaccinated 5 years before with Rev 1 (1 x 10(5) cfu) and 5 non-vaccinated goats were challenged with B. melitensis 16M (4 x 10(5) cfu) using the conjunctival route. After giving birth or aborting, the goats were sacrificed and tissue samples were taken for bacteriological study. The challenge strain was recovered in 12%, of the animals from the vaccinated group, and in (80% of the control group. It is concluded, therefore that the use of reduced-dose Rev 1 protects goats vaccinated in endemic areas for at least 5 years after immunization.

Absence of shedding of two B. abortus strains in goats after vaccination with live vaccines.

The objective of this study was to determine if B. abortus rough mutant strains RB51 and rfbK are eliminated in goat milk. Thirty milk goats were divided into two groups. Group I was inoculated with 4x10(10) cfu/ml of B.abortus RB51 strain and Group II with 1x10(9) cfu/ml of B. abortus rfbK strain by subcutaneous route in the right axilary region. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on Farrell's media and incubated under 5-10% CO(2) at 37 degrees C for 10 days. The suspicious colonies were recultured in Farrell's media. There were no isolations of bacteria with characteristics of Brucella from any of the milk samples collected during 90 days of the study. It was concluded that neither of the strains used at these doses were eliminated by milk in goats inoculated during lactation.

Isolation of Chlamydia psittaci involved in abortion of goats in Mexico: first report.

A group of 35 healthy adult goats ranging from two to six years old were bought from a chlamydiosis and brucellosis-free flock; they were vaccinated against Brucella melitensis with Rev1 vaccine at reduced doses, and one month later placed in isolation units. The animals were one month pregnant at the moment of purchase, and during the third month of pregnancy 10 out of the 35 dams aborted. Necropsy of the aborted fetus and examination of the foetal membranes was performed where no macroscopic lesions were observed. Abomasal liquid, foetal lung and liver, and placenta samples were taken for bacteriological analysis while sera from the goats that aborted was collected for serological investigation. Chlamydia psittaci was isolated in all cases, while no Brucella was detected. All sera reacted positive to anti-Chlamydia antibodies by the indirect immunofluorescence test. This represents the first report of Chlamydia psittaci isolation from cases of goat abortion in Mexico.

Sensitivity and specificity of an ELISA as a screening test for the diagnosis of Brucella ovis in sheep.

A hot saline extract of Brucella ovis strain REO 198 at a concentration of 5 micrograms/ml in phosphate buffer pH 7.2 was used to adsorb onto Maxisorb plates and incubated at 37 degrees C during 12 h; unadsorbed excess antigen was washed off thrice with phosphate buffer containing 0.5% Tween 20. As blocking agent 1% skim milk was used. The conjugate used was protein G bound to peroxidase diluted 1:100. Thirty three sheep sera from bacteriologically confirmed infected animals and 39 sheep sera from healthy animals from disease-free zones were used. Sera were diluted 1:200. ELISA's sensitivity was 97% and specificity 84%. The cut-off value was chosen for a high sensitivity (100%) despite some loss of specificity in order to diminish false negative results rendering thus a suitable screening test for sheep epididimitis caused by Brucella ovis.

Molecular epidemiology of Mycobacterium bovis in Texas and Mexico.

Seventy-nine Mycobacterium bovis isolates recovered from Mexican and Texas cattle were categorized into 16 and 25 distinct types on the basis of IS6110 and direct-repeat fingerprint patterns, respectively. By using a combination of both fingerprint patterns, 30 distinct restriction fragment length polymorphism types were defined. Fifty-eight of 79 isolates (73%) were distributed among nine clusters. Clustered isolates were identified within herds, as well as in geographically disperse herds in Texas and Mexico. This observation is consistent with active transmission within herds and among herds, presumably as a result of active or historical cattle movements. The majority of bovine isolates (64 of 79) exhibited a single copy of IS6110. Interestingly, in contrast to previous studies, a high percentage of bovine isolates (15 of 79) exhibited multiple IS6110 copies (two to five) distributed among 11 different restriction fragment length polymorphism types. It is speculated that transmission from noncattle sources may be responsible. Continued fingerprinting of isolates originating from nonbovine sources and herd surveys is expected to provide useful information regarding the epidemiology of tuberculosis in this region.