Alfano, Flora (2014) Molecular characterization of genes of immune response in water buffalo (Bubalus bubalis) and major bubaline infectious agents. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: Molecular characterization of genes of immune response in water buffalo (Bubalus bubalis) and major bubaline infectious agents
Date: 30 March 2014
Number of Pages: 118
Institution: Università degli Studi di Napoli Federico II
Department: Agraria
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
Galiero, GiorgioUNSPECIFIED
Date: 30 March 2014
Number of Pages: 118
Keywords: Bubalus bubalis, TLRs
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Date Deposited: 08 Apr 2014 11:06
Last Modified: 11 Apr 2017 01:00

Collection description

The water buffalo (Bubalus bubalis) is one of the most important livestock species, its economic importance is due to the production of the worldwide famous mozzarella cheese (Mozzarella di Bufala Campana). Among the major pathogens with zoonotic potential affecting water buffalo species a crucial role is played by Escherichia coli and Salmonella spp., responsible for gastrointestinal diseases and Mycobacterium bovis causing tuberculosis. The prophylaxis for gastrointestinal pathogens allows the use of specific vaccines. By contrast, for Mycobacterium bovis the Italian National Program for the tuberculosis control and eradication, prohibits vaccination. In this context the gastrointestinal pathogens were studied to well characterize them and allow the production of effective autogenous vaccines, while for tuberculosis a molecular characterization of host genes (Bubalus bubalis toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) has been performed. The role of genes in protection also against bacterial infections prompted the search for polymorphisms conferring resistance to mycobacterial infection in this species.The genetic selection, as a mean to increase the frequency of genes providing resistance to infection diseases, may have a positive impact on the economics of dairy industry. In the current study were performed: (I) a molecular characterization of the virulence factors of E. coli and Salmonella spp. and (II) a molecular characterization of genes involved in the immune response of water buffalo. Escherichia coli isolates from water buffalo (Bubalus bubalis) calves affected by severe diarrhoea with a lethal outcome were characterized for the presence of the virulence factors LT, ST, Stx1, Stx2, haemolysins, intimin, CNF, CDT, and F17-related fimbriae. The prevalence of ETEC, STEC and NTEC were 1.8%, 6.8% and 20.9%, respectively. The ETEC isolates were all LT-positive and ST-negative. The STEC isolates were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%). The NTEC isolates were all CNF and Hly-positive. Susceptibility assays to antimicrobials displayed high rates of resistance (>30%) to antimicrobials tested. These data show that the most prevalent strains in diarrhoeic water buffalo calves were NTEC, mostly CNF and hly positive, with strong associations CNF/CDT and CNF/F17. Salmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a characterization of isolated strains of S. Typhimurium was performed. The microbiological analysis of the intestinal contents obtained from water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp., characterized by different serovars, most frequently Typhimurium. The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis. The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. These results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes. Toll-like receptors play a key role in innate immunity by recognizing pathogens and activating appropriate responses. Pathogens express several signal molecules (pathogen-associated molecular patterns, PAMPs) essential for survival and pathogenicity. Recognition of PAMPs triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines. A case-control study was performed to characterize the distribution of polymorphisms in three candidate genes (toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) and to test their role as potential risk factors for tuberculosis infection in water buffalo (Bubalus bubalis). The case-control study included 174 subjects, 54 of which resulted positive to both intradermal TB test and Mycobacterium bovis isolation (cases) and 120 resulted negative to at least three consecutive intradermal TB tests. The statistical analysis indicated that six polymorphisms exhibited significant differences in allelic frequencies between cases and controls. Indeed, the GG and TT genotypes at TLR2 381 A>G and TLR2 2064 T>C sites, respectively, resulted significantly associated with susceptibility to bovine tuberculosis (P<0.001, OR=52.25, 95% CI=6.75÷404.57, and P <0.001, OR= 48.5, 95% CI=10.88÷216.26, respectively). Four polymorphisms resulted significantly associated with resistance to the disease, and included the AG and CC genotypes, at the TLR2 381 A>G and TLR2 2064 T>C sites, respectively, (P <0.001, OR= 0.06, 95% CI= 0.01 to 0.25, and P <0.001, OR=0.04, 95% CI=0.01÷0.13, respectively); the CC genotype at the TLR4 672 A>C site (P = 0.01, OR= 0.28, 95% CI=0.10÷0.76), and the C/C genotype at the TLR9 2340 C>T site (P = 0.04, OR=0.33, 95% CI=0.11÷0.92). Haplotype reconstruction of the TLR2 gene revealed an haplotype (CTTACCAGCGGCCAGTCCC) associated with disease resistance (P=0.04, OR=0.51, 95% CI=0.27÷0.96), including both allelic variants associated with disease resistance. The work describes novel mutations in bubaline TLR2, TLR4 and TLR9 genes and presents their association with M. bovis infection. These results will enhance our ability to determine the risk of developing the disease by improving the knowledge of the immune mechanisms involved in host response to mycobacterial infection, and will allow the creation of multiple layers of disease resistance in herds by selective breeding.


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