High-yield production of major T-cell ESAT6-CFP10 fusion antigen of M. tuberculosis complex employing codon-optimized synthetic gene.

Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 µg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.

Whole genome sequence phylogenetic analysis of four Mexican rabies viruses isolated from cattle.

Phylogenetic analysis of the rabies virus in molecular epidemiology has been traditionally performed on partial sequences of the genome, such as the N, G, and P genes; however, that approach raises concerns about the discriminatory power compared to whole genome sequencing. In this study we characterized four strains of the rabies virus isolated from cattle in Querétaro, Mexico by comparing the whole genome sequence to that of strains from the American, European and Asian continents. Four cattle brain samples positive to rabies and characterized as AgV11, genotype 1, were used in the study. A cDNA sequence was generated by reverse transcription PCR (RT-PCR) using oligo dT. cDNA samples were sequenced in an Illumina NextSeq 500 platform. The phylogenetic analysis was performed with MEGA 6.0. Minimum evolution phylogenetic trees were constructed with the Neighbor-Joining method and bootstrapped with 1000 replicates. Three large and seven small clusters were formed with the 26 sequences used. The largest cluster grouped strains from different species in South America: Brazil, and the French Guyana. The second cluster grouped five strains from Mexico. A Mexican strain reported in a different study was highly related to our four strains, suggesting common source of infection. The phylogenetic analysis shows that the type of host is different for the different regions in the American Continent; rabies is more related to bats. It was concluded that the rabies virus in central Mexico is genetically stable and that it is transmitted by the vampire bat Desmodus rotundus.

Assessment of antigenic supplementation of bovine purified protein derivative for diagnosis of subclinical infection with Mycobacterium bovis in cattle.

Bovine tuberculosis (bTB) is usually diagnosed in vivo and ex vivo on the basis of delayed hypersensitivity reactions with a complex pool of antigens named bovine tuberculin (PPDB). The IFN-γ release assay (IGRA) for bTB is a blood-based assay that improves detection of infected cattle at early stages that escape skin testing. Improvements to IFN-γ testing with specific proteins have been performed to increase sensitivity. DosR regulon-related antigens are well known mycobacterial proteins expressed during the non-replicative phases of infection, this has been useful to improve the diagnosis of subclinical forms of TB in suspected individuals. Transcripts of DosR genes mb2054c, mb2057c, and mb2660c have been identified by our group in lymph nodes of IFN-γ test negative cattle. This led us to hypothesize that DosR-related proteins may potentiate the IFN-γ response to PPDB in animals with a false negative IFN-γ test, making evident subclinical infection. Three hundred animals were evaluated by means of IGRA and post-mortem microbiological analysis of tissue samples to validate M. bovis infection. We found that 176 out of 300 animals showed an overall increased OD in complemented IGRA with two purified protein cocktails in comparison to PPDB alone, and were scrutinized for a subclinical infection; thirty percent when PPDB was supplemented with a cocktail of four DosR antigens, and 70% when PPDB was supplemented with a cocktail of six antigens (four DosR and two RD1 antigens). Forty five animals showed a substantial IFN-γ overproduction but remained negative, and 40 animals changed the result to a positive test. Only 18 out of 176 IFN-γ high producing animals were also positive to M. bovis isolation. Fifty seven animals with no visible lesions at slaughter and with a negative IGRA test result contained M. bovis DNA in tissue samples. In conclusion, Mb1762c, Mb2054c, Mb2057c, and Mb2660c have the potential to increase sensitivity of the IFN-γ in vitro test for bTB diagnosis when supplemented to PPDB.

Understanding the relationship between Mycobacterium bovis spoligotypes from cattle in Latin American countries.

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.

IFN-g response to vaccination against tuberculosis in dairy heifers under commercial settings.

The purpose was to determine IFN-g release as a response to vaccination against tuberculosis in dairy heifers under commercial settings. Four-hundred pregnant heifers from ten herds were randomly allocated into four groups: (1) unvaccinated, (2) BCG vaccinated, (3) BCG vaccinated plus a CFPP400 µg+polygen boost, and (4) BCG vaccinated plus a CFP200 µg+polygen boost, under a completely randomized blocks design. A dose of 106CFU of BCG was delivered SC in the neck, then blood samples were taken at days 0, 30, 120, 210, 300 and 720 to estimate IFN-g release in response to bovine-PPD antigen. No significant difference (P > 0.05) was observed in IFN-g release between groups at days 0 and 120. At days 30 and 210, vaccinated groups show higher IFN-g release than the control group but only difference of group 3 was significant (P < 0.05). At day 300, group 1 showed significantly higher IFN-g release. No significant difference was observed at day 720. Using IFN-g release as a surrogate for vaccine efficacy, BCG plus a boost with CFP or CFPP combined with an adjuvant that improves cellular immune response has the potential to protect cattle against tuberculosis for moderate periods of time in vaccinated cattle under commercial settings.

[Prevalence of diabetes mellitus and hyperlipidemia among Otomi indians]. / Prevalencia de diabetes mellitus e hiperlipidemias en indígenas otomíes.

OBJECTIVE: To determine prevalence and risk factors for diabetes mellitus (DM) and hyperlipidemias in a population of Otomi Indians. MATERIAL AND METHODS: A cross-sectional study was conducted between 1996 and 1997, in a convenience sample of 91 Otomi Indians, aged 15 to 77 years, in the communities of Yosphi and El Rincon, Queretaro, Mexico. Fasting blood samples were obtained to measure glucose, cholesterol and triglyceride levels. RESULTS: DM was found in 4.4% of subjects; hypercholesterolemia in 7.6%; and hypertriglyceridemia (HTG) in 26%. Mean concentrations of glucose -(81.0 +/- 24.4 mg/dl) and triglycerides (157.4 +/- 88.9 mg/dl) increased significantly with age, p = 0.0279 and p < 0.0001, respectively; as well as the prevalence of HTG (p < 0.0001). CONCLUSIONS: Results suggest that drastic changes in the diet of Otomi Indians may cause severe problems related to high concentration of lipids in blood.

Identification of tuberculosis in cattle slaughtered in Mexico.

OBJECTIVES: To determine epidemiologic factors associated with tuberculosis (TB) in dairy cattle slaughtered in 6 important regions for milk production in Mexico. ANIMALS: 2,500 cattle. PROCEDURE: Tissue specimens with lesions typical of TB were obtained during routine inspection of carcasses at abbatoirs between July 1996 and January 1997. Infection with Mycobacterium organisms was confirmed by histologic examination and bacteriologic culture. Species identification was made by use of selective growth medium, conventional biochemical tests, and radiometric procedures. Epidemiologic information for affected cattle was obtained by personal interviews with cattle dealers and owners. RESULTS: 400 (16%) of 2,500 cattle carcasses had gross lesions typical of TB. Of the 400 infected cattle, 336 (84%) had lesions in > or = 1 lymph node. Infection was confirmed in 87% of cattle with gross lesions by histologic examination, in 77% by bacteriologic culture at a laboratory in the United States, and in 59% by bacteriologic culture at a laboratory in Mexico. Most cattle were adult females in fair to good body condition that came from large herds (> 500 cattle) and were not included in the Mexican TB control program. CONCLUSIONS AND CLINICAL RELEVANCE: Mean prevalence of lesions typical of TB in dairy cattle at 6 locations in Mexico was 16%. Mycobacterium infection was confirmed by various techniques in most lesions. Recognition of typical gross lesions at slaughter may expedite TB control procedures.

Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico.

OBJECTIVE: To assess phylogenetic relationships among Mycobacterium bovis isolates by use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting and to relate genetic profiles of isolates to epidemiologic characteristics. ANIMALS: 400 cattle with tuberculosis. PROCEDURE: Mycobacterium bovis was isolated from various organs of cattle slaughtered in 6 geographic regions of Mexico. Most cattle were adult Holsteins from large herds that did not participate in a tuberculosis control program. Four random primers and 2 selected primers were used in RAPD-PCR fingerprinting of 88 isolates. Pairwise genetic distance between isolates was obtained and subjected to cluster analysis with bootstrapping to test for levels of support. RESULTS: 98 different fragments were obtained; there was broad genetic diversity among isolates, and each isolate had a unique RAPD-genotype, including those originating from the same herd. Clustering by geographic location, affected organ, or severity of lesion was not detected. Linkage disequilibrium analysis suggested that M. bovis was highly clonal and that mutations develop at a rapid rate among isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Use of RAPD-PCR could not differentiate M. bovis isolates by epidemiologic characteristics or identify common sources of infection.