Development And Application Of A Simple Latex Agglutination Test And ERIC-PCR Method For Detection Of Haemophilus Parasuis

The dissertation describes two experiments.1. Development of a simple and rapid Latex Agglutination Test to detectHaemophilus parasuisHaemophilus parasuis is a potentially pathogenic gram-negative organism that colonizes the upper respiratory tract of pigs. It is also the causative agent of porcine polyserositis and arthritis (Gl(a|¨)sser's disease). With immunity of the herds decreased, outbreaks of Gl(a|¨)sser's disease occurred. Therefore, rapid diagnosis of Haemophilus parasuis and effective vaccination are essential to the prevention and control of this disease. TbpB protein was supposed to be one of the outer membrane protein of Haemophilus parasuis and a potential antigen which could be applied to surveillance and diagnosis of Glasser's disease. The N-terminal domain of TbpB protein has higher immunogenicity, antibodies against the N-terminus domain were sufficient to develop an efficient immune response against the infection by H.parasuis.In our study, TbpB N-terminal domain containing 280 amino acids was selected and the recombinant GST-TbpB fusion protein was generated. In addition, a unique 105-bp fragment within TbpB N-terminal domain from position -226 to -330 was man-made synthesized, which is highly homologous among different serovars of H.parasuis but is distinct from other bacteria except H.parasuis. Then two groups of six to eight-week-old female BALB/c mice each was immunized with the recombinant TbpB fusion protein and the peptide which is conjugated with KLH (keyhole limpet hemocyanin) and two individual monoclonal antibodies were produced for covalently coupling with carboxylated latex bead. Sensitized latex particles (SLPs) were agglutinated by 15 serovar reference strains culture of Haemophilus parasuis except for serotype 9, 11, 13 and 14, but not by heterologous strains of Pasteurella multocida, Actinobacillus Pleuropneumoniae, Escherichia coli, Staphylococcus aureus or Streptococcus expect Bordetella bronchiseptica. 148 field strains isolated from lungs, spleens, arcula cordis, articular cavities and brains were detected by LAT assay. The result demonstrated that the coincident rate was 83.1% between 16S rRNA-based PCR and LAT test. In summery, Latex agglutination test is a very simple, rapid and effective method and it could be used in field investigations. 2. Development and application of ERIC-PCR for epidemiological survey of Haemophilus parasuis isolates from ChinaIn recent time, the co-infection of H parasuis and viral diseases which relate to immune suppression has taken a very high percentage within herds and resulted in high morbidity and mortality. Therefore, it is essential to know the prevalent serovars which caused widespread infections in a given area to effectively control this disease. The majority of the epidemiological studies on H.parasuis infections are based on serotyping information. 15 serovars have been identified, but considerable H.parasuis strains are not typeable both by gel diffusion (GD) or Indirect haemagglution (IHA). Serovar diversity and the substantial proportion of non-typeable isolates have affected the development of effective cross-protective vaccines. As an advanced technique for epidemiology studies, genotyping is more discriminative than serotyping. Genotyping of Haemophilus parasuis by ERIC-PCR allows detections of the prevalent strains causing nursery mortality, and this technique is very suitable for the selection of strains to be used in autogenous vaccines.From September 2007 to September 2008, 201 strains of Haemophilus parasuis were isolated from 13 provinces of China. All these isolates were identified and genotyped with the enterobacterial repetitive intergenic consensus based-polymerase chain reaction (ERIC-PCR) technique and their serovar groups were predicted by comparing 15 unique ERIC-PCR fingerprints that represented 15 serovar reference strains of Haemophilus parasuis. The results demonstrated serovar 5 (50; 25%) was the most prevalent, followed by serovars 9 (15; 7%) and 13 (11; 5%). Nontypable isolates were also frequently identified, accounting for 31% of the evaluated isolates. Furthermore, a total of 59 isolates from 20 farms in 8 provinces of China were genotyped, and the remarkable diversity of the prevalent H parasuis serovars and strains in different areas even among the farms in the same area was observed. In three specific farms, strains which caused several outbreaks of Gl(a|¨)sser's disease were isolated and DNA fingerprints presented some differences each outbreak. In short, isolates of Hparasuis were found to be very heterogeneous at the genetic level in China. High diversity of the prevalent H parasuis strains exacerbated the difficulties of disease control and commercial vaccines had already not enabled to provide good protective immunity for herds suffered from H parasuis infection. Thus, ERIC-PCR fingerprinting was used for a guideline for selection of vaccine strains to provide more effective protection in our study.