Kajian ketahanan ayam kampong terhadap Salmonella enteritidis menggunakan gen TLR4 sebagai penciri genetik

Abstract

Kampung chicken is a kind of Indonesian local chicken that do not have special characteristic and spread out in various regions of Indonesia. Kampung chicken eggs is trusted by the majority of Indonesian people to increase stamina and to heal diseases. Therefore Kampung chicken eggs was mostly used by Indonesian people as ‘jamu’ (a potion) or as an ingredient of potion that it was directly consumed without cooking. Salmonella free eggs become significant in producing the safe ‘jamu preparation’ so that the users of Kampung chicken eggs be spared from salmonellosis. Salmonella free eggs might be produced by chickens which have high resistancy to this bacteria. One of excellent markers showing resistance of chicken against Salmonella is an active Toll-like Receptor 4 (TLR4) gene. TLR4 gene was associated with non-specific immune response, because it transcribes TLR4 protein, which is a phagocytes cell surface receptor that plays a role to recognize lipopolysaccaride of gram negative bacteria including Salmonella sp. The aim of the research was to prove resistance of Kampung chicken against S. enteritidis, using TLR4 gene as marker. The evidence was done by analyzing the association between genotype TLR4 gene with the factors that indicate the resistance to these bacteria that was obtained from molecular and biological assays. To identify the genotype TLR4 gene, 50 Kampung chickens was genotyped using PCR-RFLP technique (the first stage of these study). The second stage was conducted tests on several chickens resistance indicators against S. enteritidis from natural infection. The third stage in these research was testing resistance of chickens when was challenged with S. enteritidis (ID50 dose : 105 cfu ml-1), through molecular and biological assays. Then the test results were associated with genotype of TLR4 gene, and it was analyzed by ANOVA (analysis of variance). The results showed that the PCR-RFLP technique in exon 2 of TLR4 gene was identified that this gene on the Kampung chicken was polymorphic. A presence of mutation was detected on site 3924. This mutation caused bases change from guanine to adenine (GA). From this genotyped was found three kinds of genotype TLR4 gene. They were AA, AG and GG. GG genotype dominated the genotype frequencies of TLR4 gene of Kampung chickens. AA genotype was only found on one individual Kampung chicken, so in subsequent association analysis was not included in the statistical test. The second stage of this study showed that in all of TLR4 gene genotypes of the Kampung chicken was not found S. enteritidis in the blood and eggs produced. In the blood serum of Kampung chicken positive was found IgY specific S. enteritidis (in all genotypes). Egg yolk that produced by Kampung chicken was found specific IgY of S. enteritidis in high concentrations (2.47-3.58 mg ml-1). On all genotypes, the concentration of leucocytes and differentiation of leucocytes were in the range of physiological normal. Kampung chickens which were challenged with S. enteritidis not impaired physiological, it is seen from the results the concentration of leucocytes and differentiation of leucocytes assays that were still in the normal range. Although the Kampung chicken was challenged with S. enteritidis, in all genotypes were not found S. enteritidis in the blood and eggs were produced. It happened because the results of phagocytic activity assays (on all genotypes of TLR4 gene) was obtained a very high value. The value of macrophage activity of AA, AG and GG genotypes respectively 74.00, 74.40 ± 4.39, and 71.86 ± 3.72%. The value of macrophage capacity of these genotypes were 40.96, 40.33 ± 1.26 dan 42.13 ± 3.29 bacteria macrophage-1. The value of macrophage activity and capacity from AG and GG genotypes were not statistically different. The high value of the phagocytic activity of macrophage was caused by the all of TLR4 gene genotypes include an active category. The results assay on the TLR4 gene expression of AA, AG, and GG genotypes that obtained from the intestine and kidney tissues after being exposed with S. enteritidis produces a high copy number of mRNA. The number of mRNA copies from AG and GG genotypes were not statistically different. The overall of the mRNA copies number were ranged 1.12-3.92 x 107. Feed consumption on AA, AG, and GG genotypes was obtained at 91.70, 92.19 ± 4.28, and 91.20 ± 2.67 g bird-1 day-1. Eggs weight that was produced almost the same and it ranged between 40.47-41.00 g egg-1. Egg production (hen day) was obtained at 60.00, 62.23 ± 13.72, and 34.84 ± 11.86%. From a series of the observations in this study were obtained that the TLR4 gene of Kampung chicken was polymorphic with three kinds of genotypes (AA, AG and GG) found. The three kinds of TLR4 gene genotypes in this study did not associate with resistance to S. enteritidis infection, either natural infection or artificial infection through a challenge test. All of TLR4 gene genotypes on Kampung chicken were category resistant to S. enteritidis bacteria. Eggs were produced by the Kampung chicken with AA, AG, and GG genotypes contained specific antibodies to S. enteritidis with high concentrations.