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.

Potential effects on Rhipicephalus microplus tick larvae fed on calves immunized with a Subolesin peptide predicted by epitope analysis.

The cattle tick Rhipicephalus microplus economically impacts cattle production in tropical and subtropical regions of the world. The recombinant R. microplus Subolesin antigen has been shown to protect cattle against tick infestations. In this study, we searched in silico protective epitopes in the subolesin gene and a recombinant peptide containing the predicted epitopes was expressed, and evaluated against a tick challenge. Two different epitope types, linear B-cells and conformational discontinuous epitopes, were predicted using bioinformatics strategies, in order to synthesize the recombinant peptide. Two Eightmonth-old calves European crossbred were immunized with two subcutaneous doses of the subolesin recombinant peptide, emulsified with Montanide ISA 50 V as an adjuvant, at 30-day intervals. The tick challenge was conducted with 5 000 R. microplus larvae/animal. ELISA test was used to evaluate the IgG immune response elicited against the peptide. After tick challenge, reduction in the number of engorged females (79%), and reduction in egg hatching (30%) was observed in tick population fed on immunized calves with regards to an untreated control group. The results showed a potential higher effect on tick reproduction for the recombinant peptide compared to other studies reported with Subolesin protein, demonstrating that the use of bioinformatics strategies to predict protective epitopes may lead to improve the immune response elicited against tick recombinant peptides and therefore to prevent cattle tick infestations.

Adulticidal and oviposition- and hatching-altering activities of essential oil from Mexican oregano leaves (Lippia graveolens H.B.K.) against the cattle tick Rhipicephalus microplus (Acari: Ixodidae).

Adulticidal and oviposition- and hatching-altering activities of essential oil extracted from Mexican oregano leaves (Lippia graveolens H.B.K.) (OEO) were evaluated on engorged adult female Rhipicephalus microplus ticks using the adult immersion test bioassay. Twofold dilutions of OEO were tested from a starting dilution of 10% down to 1.25%. Results showed 100% adulticidal activity at 10% OEO concentration and oviposition inhibition of 65.8% and 40.9% at 5.0% and 2.5% OEO concentration, respectively. Egg hatching inhibition was achieved by 26.0% and 11.5% at 5.0% and 2.5% OEO concentration, respectively. These effects could be attributed to OEO major components: thymol, carvacrol and p-cymene, which together account for more than 60.0% of the OEO chemical composition. Mexican oregano could represent a potential source for development of alternative tick control agents.

Adulticidal and oviposition- and hatching-altering activities of essential oil from Mexican oregano leaves (Lippia graveolens H.B.K.) against the cattle tick Rhipicephalus microplus (Acari: Ixodidae)

Adulticidal and oviposition- and hatching-altering activities of essential oil extracted from Mexican oregano leaves (Lippia graveolens H.B.K.) (OEO) were evaluated on engorged adult female Rhipicephalus microplus ticks using the adult immersion test bioassay. Twofold dilutions of OEO were tested from a starting dilution of 10% down to 1.25%. Results showed 100% adulticidal activity at 10% OEO concentration and oviposition inhibition of 65.8% and 40.9% at 5.0% and 2.5% OEO concentration, respectively. Egg hatching inhibition was achieved by 26.0% and 11.5% at 5.0% and 2.5% OEO concentration, respectively. These effects could be attributed to OEO major components: thymol, carvacrol and p-cymene, which together account for more than 60.0% of the OEO chemical composition. Mexican oregano could represent a potential source for development of alternative tick control agents.

Alpha 2HS-glycoprotein, a tumor-associated antigen (TAA) detected in Mexican patients with early-stage breast cancer.

Several studies have demonstrated that the serum of patients with cancer contains antibodies that react with a group of autoantigens denominated tumor-associated antigens (TAA). TAA can be detected prior to clinical diagnosis; thus, they would be ideal biomarkers for early detection of cancer, using only a few microliters of a patient's serum. In the current study, we used an immune proteomic approach, combining two-dimensional (2D) electrophoresis, Western blot, and matrix-associated laser desorption/ionization-mass spectrometry (MALDI-MS) methods to identify TAA in the sera of patients diagnosed with breast cancer. Sera were obtained from 36 newly diagnosed patients with stage II breast cancer and those from 36 healthy volunteers were evaluated for the presence of the TAA. Alpha 2HS-glycoprotein (AHSG) antibodies were detected in 33 of 36 patients with breast cancer (91.7%) and in only 3 of 36 healthy patients (controls, 8.3%). Sensitivity of detection of autoantibodies against AHSG in patients with breast cancer was 91.7%. AHSG was detected in cancer tissue by immunohistochemistry. Our results strongly suggest that the presence of serum autoantibodies against AHSG protein may be useful as serum biomarkers for early-stage breast cancer screening and minimally invasive diagnosis in Mexican populations. BIOLOGICAL SIGNIFICANCE: In the present study, 2D immunoblot analysis was used to make a screening in samples of sera from patients with a diagnosis of early-stage breast cancer, in order to identify some autoantibodies that react against TAA. Proteins identified in the present study, particularly alpha 2HS-glycoprotein (AHSG), might be useful as potential biomarkers for breast cancer in early stages for Mexican populations.

Expression of non-replicating persistence associated genes of Mycobacterium bovis in lymph nodes from skin test-reactor cattle.

Upon oxygen shift-down, Mycobacterium tuberculosis complex bacteria can induce a genetic program characterized by halted duplication, which is called Non-replicating persistence (NRP). During this phase, at least 48 genes, collectively named Dormancy survival regulator (DosR) regulon, are important for the long-term survival of bacilli under a non-respiring state, a condition that bacilli encounter inside granulomatous lesions. It remains unclear whether expression of NRP genes occurs within the tissue of Mycobacterium bovis naturally infected cattle. In order to start dissecting this question, total RNA from bovine lymph node tissues of sacrificed tuberculin reacting animals was isolated and transcription of genes required for in vivo duplication (esxB and fbpB) and in vitro NRP (hspX, pfkB, and mb2660c) were analyzed by RT-PCR approaches. Detection of transcripts was positive in bovine tissue samples for genes hspX, pfkB, and mb2660c in 84, 32, and 21%, respectively. NRP genes were upregulated even in animals with a negative IFN-γ in vitro test, and the expression of NRP genes occurred more often than expression of the esxB gene.

Acaricidal effect of essential oils from Lippia graveolens (Lamiales: Verbenaceae), Rosmarinus officinalis (Lamiales: Lamiaceae), and Allium sativum (Liliales: Liliaceae) against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Acaricidal effects of three essential oils extracted from Mexican oregano leaves (Lippia graveolens Kunth), rosemary leaves (Rosmarinus officinalis L.), and garlic bulbs (Allium sativum L.) on 10-d-old Rhipicephalus (Boophilus) microplus (Canestrini) tick larvae were evaluated by using the larval packet test bioassay. Serial dilutions of the three essential oils were tested from a starting concentration of 20 to 1.25%. Results showed that both Mexican oregano and garlic essential oils had very similar activity, producing high mortality (90-100%) in all tested concentrations on 10-d-old R. microplus tick larvae. Rosemary essential oil produced >85% larval mortality at the higher concentrations (10 and 20%), but the effect decreased noticeably to 40% at an oil concentration of 5%, and mortality was absent at 2.5 and 1.25% of the essential oil concentration. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. Mexican oregano essential oil included thymol (24.59%), carvacrol (24.54%), p-cymene (13.6%), and y-terpinene (7.43%) as its main compounds, whereas rosemary essential oil was rich in a-pinene (31.07%), verbenone (15.26%), and 1,8-cineol (14.2%), and garlic essential oil was rich in diallyl trisulfide (33.57%), diallyl disulfide (30.93%), and methyl allyl trisulfide (11.28%). These results suggest that Mexican oregano and garlic essential oils merit further investigation as components of alternative approaches for R. microplus tick control.

Benzene metabolites induce apoptosis in lymphocytes.

Benzene is an important environmental pollutant with important health implications. Exposure to this aromatic hydrocarbon is associated with hematotoxicity, and bone marrow carcinogenic effects. It has been shown that benzene induces oxidative stress, cell cycle alterations, and programmed cell death in cultured cells. Hepatic metabolism of benzene is thought to be a prerequisite for its bone marrow toxicity. Nevertheless, there are no reports on the cellular effects of reactive intermediates derived from hepatic metabolism of benzene. Thus, the goal of this project was to determine the cellular alterations of benzene metabolites produced by the cultured hepatic cell line HepG2. Supernatants collected from these cells were applied to a culture of freshly isolated lymphocytes. A higher decrease in cell viability was found in cells exposed to these supernatants than to unmetabolized benzene. This viability decrease was due to apoptosis, as determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and internucleosomal fragmentation of DNA. When supernatants were analyzed by HPLC, we found that not all the hydrocarbon was biotransformed, since a 28 microM concentration (37%) remained. The only metabolite found in the culture medium was muconic acid. The present results show that muconic acid derived from benzene metabolism is able to cooperate with the pollutant for the induction of apoptosis in rat lymphocytes.