Mangesh Bhide

Animal board invited review: advances in proteomics for animal and food sciences

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid – i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 – Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East–West and North–South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle.

Toll like receptors (TLRs) are a class of pattern recognition receptors belonging to the innate immune system. Mutations in the protein coding region of TLRs are associated with altered responsiveness to pathogen-associated molecular patterns (PAMPs). A search was performed for novel mutations in bovine TLR1, TLR2 and TLR4 genes associated with the Mycobacterium avium subsp. paratuberculosis (MAP) infection. The work was also focused on the assessment of linkage between well known mutations in TLR genes (TLR2: Arg677Trp, Pro681His and Arg753Gln; TLR4: Asp299Gly and Thr399Ile), and the susceptibility of cattle to MAP infection. Detection of MAP infection in cattle population (n=711) was based on IS900 PCR, which revealed 22.50% (n=160) MAP positivity. Known mutations in TLR2 and TLR4 genes were not found in cattle population. A novel mutation Val220Met was associated (Odds ratio, OR-3.459) with increased susceptibility to MAP infection. Toll/interleukin-1 receptor (TIR) domain of TLR2 was screened for the presence of mutations, wherein a novel Ile680Val mutation was linked with MAP infection. In silico analysis of the bovine TLR4 ectodomain (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. LRR11 of the TLR4 showed five missense mutations possibly linked with the increased susceptibility to MAP infection. The most critical position that may alter the pathogen recognition of TLR molecule was 4th residue downstream to LRR domain. Two such missense mutations in TLR4 (Asp299Asn downstream to LRR11, and Gly389Ser downstream to LRR15) were associated with MAP infection. Briefly, the work describes novel mutations in the bovine TLRs and presents their association with the MAP infection.

Molecular Method for Bartonella Species Identification in Clinical and Environmental Samples

ABSTRACT A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection.

BackgroundToll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.ResultsThe study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.ConclusionThe most critical positions that may alter the pathogen recognition ability of TLR were: the 9th amino acid position in LRR motif (TLR1–LRR10) and 4th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.

Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

BackgroundBorreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH) and factor H like-1 (FHL1)], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diversity. Here we show the differential binding of borrelial proteins to fH from human and animal sera.FindingsAffinity ligand binding experiments, 2-D electrophoresis, and protein identification and peptide de novo sequencing based on mass spectrometry, revealed novel fH putative binding proteins of Lyme- and relapsing fever Borrelia. An OspA serotype-associated differential human and animal fH binding by B. garinii was also observed, which could be related with the ability of some strains from serotypes 4 and 7 to invade non-nervous system tissues. Also, the variable affinity of binding proteins expressed by different Borrelia to animal fH correlated with their host selectivity.ConclusionThe novel animal and human putative fH binding proteins (FHBPs) in this study underscore the importance of evasion of complement in the pathogenesis of Borrelia infections.

Presensitization of microorganisms by acid treatments to low dose gamma irradiation with special reference to Bacillus cereus

Effect of acid presensitization to low dose gamma irradiation on Bacillus cereus in sheep/goat meat stored at refrigeration temperature was assessed. Food grade organic acids (viz. propionic, lactic and acetic acids) were used as presensitizers followed by treatment with 1, 2 and 3 kGy irradiation doses (Co(60)). Two percent acetic acid plus 3 kGy irradiation elicited most effective to lower the total viable count and B. cereus count. Combination of treatments availed to reduce the dose required for elimination of radioresistant B. cereus and also masked the irradiation odour developed due to radiolysis. The study also revealed substantial increase in shelf life of mutton after combination treatment than single treatment without any adverse effect on acceptability of meat.

Pathogen translocation across the blood-brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial-host interactions (receptor-ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo. This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.

Molecular Method for Discrimination between Francisella tularensis and Francisella-Like Endosymbionts

ABSTRACT Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5′-3′ exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA–related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Beyond the Rat Models of Human Neurodegenerative Disorders

The rat is a model of choice in biomedical research for over a century. Currently, the rat presents the best “functionally” characterized mammalian model system. Despite this fact, the transgenic rats have lagged behind the transgenic mice as an experimental model of human neurodegenerative disorders. The number of transgenic rat models recapitulating key pathological hallmarks of Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, or human tauopathies is still limited. The reason is that the transgenic rats remain more difficult to produce than transgenic mice. The gene targeting technology is not yet established in rats due to the lack of truly totipotent embryonic stem cells and cloning technology. This extremely powerful technique has given the mouse a clear advantage over the rat in generation of new transgenic models. Despite these limitations, transgenic rats have greatly expanded the range of potential experimental approaches. The large size of rats permits intrathecal administration of drugs, stem cell transplantation, serial sampling of the cerebrospinal fluid, microsurgical techniques, in vivo nerve recordings, and neuroimaging procedures. Moreover, the rat is routinely employed to demonstrate therapeutic efficacy and to assess toxicity of novel therapeutic compounds in drug development. Here we suggest that the rat constitutes a slightly underestimated but perspective animal model well-suited for understanding the mechanisms and pathways underlying the human neurodegenerative disorders.