Four Kinds Of Pathogens Investigation Of Chicken Flocks With Oviduct Cyst And S1 Gene Variation Analysis Of IBV Isolates

Gallibacterium is a new genue of Pasteurellacease, and Gallibacterium anatis is typical strains. Many foreign scholars have confirmed that Gallibacterium have a certain relationship with salpingitis and tubal cyst; Chuan-Qing Wang reported there were higher infection rate of Gallibacterium in partial flocks infected with tubal cyst in Henan Province for the first time. Foreign information reported that flocks infected with Gallibacterium simultiously often mixed infected egg drop syndrome virus(EDS76V), avian infectious bronchitis virus (IBV) and Mycoplasma gallisepticum (MG) and other, while Gallibacterium may induce and enhance other infections. In order to find out the Gallibacterium infection situation of domestic flocks suffering from oviduct cyst, while clearly make sure the role of EDS76V, MG and IBV play in above flocks, samples collected from oviduct cyst and clinical healthy flocks from Henan and Shanxi were investigated correlative pathogens: heart, liver, spleen, lung, trachea, oviduct, ovary and duodenum were collected from oviduct oyst flocks, while the cloaca, cleft palate, throat and tracheal swabs were collected, all samples were cultured on blood agar plate, suspicious colonies of Gallibacterium were further identificated by specific PCR amplification located on the internal transcribed 16S to 23S rRNA gene sequence, the results showed that: the positive rate of Gallibacterium infection is 41.67% in oviduct cyst flocks bacteria (40/96), and clinical healthy flocks is 27.3% bacteria; PCR test results of cotton swab samples showed that there were individual hens mixed infected with IBV, EDS76V and MG, this result initially excluded the major roles of IBV, MG and EDS76V in these flocks of tubal cysts, and indicated Gallibacterium play an important role in the chicken oviduct cystic disease, while Gallibacterium existed in clinically healthy flocks, this suggested that we should not overlook the threat of Gallibacterium in the prevention and treatment of chicken oviduct cystic disease.IBV has many serotypes, and there are only partial or not cross-protective actions among serotypes and variations strains, immune response induced by a Massachusetts-type vaccine is often not sufficiently effective against other IBV of new serotype, this problem has brought great difficulties to prevention and treatment of IB, and seriously endanger the development of poultry industry. It is important to type for the IBV isolates, which contribute to carry out effective control IB by choosing vaccine of apt serotype. S1 gene is the main immunogen gene of IBV, sequence analysis based on S1 gene contributed to determine the serotype of IBV isolates. However, amplification and sequencing of the entire S1 gene is not efficient and not suitable for diagnostic methods because it is time consuming and costly. There is a hypervariable region (HVR) in 159 ~ 444nt after the start codon of S1 gene 5 'end, someone reported that genetic typing based on sequence analysis or RFLP analysis of HVR I for those isolates which have close genetic origin but different serotype can be used to typing for most of the IBV isolates of different genotype. In this study, some 4 strains of the virus were isolated from floks suspected infected Nephropathogentic-type IB in Henan province, and carried out the following research:1,Four IBV strains were isolated from different flocks suspected Nephropathogentic-type IBV infection in Henan province through chicken embryo culture; when obtained the allantoic fluid, white floc attached to the embryos were observed, chorioallantoic membrane thickening, embryos developed slowly and there were bleeding spots on the head of embyros; allantoic fluid had no direct hemagglutination; isolates injuried the tracheal cilia obviously; with the passage number increased, chicken embryo hatched to 18-day-old presented the typical dwarf embryo change; RT-PCR amplification of nucleocapsid gene of isolates showed single and specific aim band ; restriction analysis showed that PCR amplified products of HN / HL contained a single restriction sites of HaeⅢ; sequence analysis of HN / HL further confirmed that four isolates were the infectious bronchitis virus (IBV).2,Spike protein gene of 7 isolates and H120 strain were amplified by RT-PCR and sequenced. These sequences were analyzed by using DNA Star and Mega 4.0. Four lengths of S1 gene of 8 IBV strains were 1632bp,1620bp,1617bp and 1611bp, respectively. There were great mutations in many loci sites among 8 sequences, insertion and deletion of base were existed simultaneously; Analysis for deduced amino acid sequences showed that sequential patterns of S protein cleavage sites of 8 IBV strains were H-R-R-R-R, R-R-F-R-R and R-R-S-R-R, respectively; Homolog analysis showed that Jin-13 had 97.3% identity to H120. Simultaneously, YI had 99.8% identity to ArkDPI11, and the homologous rate between HN/SG and other strains only were 79.5%～88.4%, between Henan isolates (HN/HL,XP/1/09,XP/3,WZL ) and Shandong isolates were 94.8%～99.1%. Phylogenetic analysis showed that YI and Jin-13 respectively had close relationship with ArkDPI11 and H120. HN/HL, XP/1/09, XP/3, WZL isolates had a near relationship to Shandong Nephropathogenic-type isolates and had a distant relationship to HN99. HN/SG had a distant relationship to domestic Nephropathogenic-type isolates. All results showed that there were different IBV epidemic isolates in Henan province and they had happened obvious variation at the molecular level, and the current vaccine can't provide effective protection to the immunized flocks. The variation law of Henan isolates revealed at molecular level could provide scientific reference to prevention flocks from IB.3,By analyzing the complete sequences of the IBV S1 gene published on GenBank, the relatively conservative differential sequences of respiratory-type vaccine strain and local Nephropathogenic epidemic strains were found out. Two pairs of primers were designed and synthesized and the reverse transcription -multiple PCR methods were established to type for the two serotypes of IBV. Using this method, the partial sequences of S1 gene were amplified from the clinical positive IBV strains and the vaccine strains conserved in the laboratory. The results showed that: the methods could distinguish respiratory-type strains from the local Nephropathogenic strains isolated in recent years better; in recent years, the separation of strains that could infect chickens and cause high mortality mainly were Nephropathogenic IBV. The method has characteristics of strong specificity,high sensitivity and good repetition. And the method can distinguish completely the local Nephropathogenic IBV from the respiratory-type IBV vaccine strains and supply reference for detection and vaccine options of IB.