Colonization Pattern, Genetyping And Synergistic Pathogenicity With REV Of Bordetella Avium

Bordetella avium (B. avium) was first isolated from young turkeys in1967. It mainlycauses respiratory diseases, including ophthalmia and rhinitis, in poultries. However, in recentyears our research group constantly isolated B. avium strains from dead embryos in somebreeder hatcheries with reduced hatchability rate and increased hatched-weak chicken rate. Toanalyze the pathogenicity of B. avium isolates from chick embryos, the study conducted ananalysis of the causatives in scale hatcheries of2001-2011. B. avium strains isolated fromembryos were gene typed by RAPD and screened for virulen strain whose colonizationpattern would be detected and pathogenicity changes under immune inhibition would bestudied.This study is divided into five parts:1Pathogen analysis of embryonal diseases in scale hatcheriesIn this experiment, pathogen detection and analysis were conducted on dead chickembryos or newly hatched chicks from72chicken hatcheries in8provinces of China from2001to2011. Multiplex PCR and RT-PCR amplification, as well as bacterial andmycoplasma isolation were done about the samples. Single infection and multiple infectionsof CIAV, REV and ALV were detected from33hatcheries, accounting for45.8%.Mycoplasma infection lied in6hatcheries, with a rate of8.33%. Single infection and multipleinfections of bacteria were detected from52hatcheries, accounting for72.2%. Five kinds ofbacteria were isolated with a total number of105, including Staphylococcus aureus,Escherichia coli, Bordetella avium, Salmonella and Pseudomonas aeruginosa. Number oftolerated drugs of5kinds of bacteria was15-32from47tested drugs. General antibioticresistance is on the rise, with the most serious drug-resistance of Staphylococcus. The declinein hatchability and increase of weak chick rate are primarily caused by multiple bacterialinfection (47.22%) and viral and bacterial co-infection (19.44%). Single virus, bacteria andmycoplasma infections accounted for only a very small proportion, with infection rates of6.94%,9.72%and4.71%respectively.2Biological characteristic and pathogenicity of B.avium isolates from chickembryos Culture characteristics and biochemical characteristics of22B. avium isolates in1991-2010are basically the same, and all of them have resistance to drugs. Tolerated drugnumbers of different isolates vary from8to30in47detected drugs. ELD50to11-day-oldchick embryo and LD50to1-day-old chicks were detected about12isolates from chickembryo in recent years. Results showed that their ELD50were between6.75-27.95CFU andLD50between9.2×105-3.4×108CFU. Accordingly three virulent strains Ba15, Ba17, andBa21were screened from them. Ba15had strongest inhibition of growth and immuneresponse to NDV among the3isolates after nasal infection in1-day-old chicks, with a rate of100%infection in the lungs and trachea, and30%-40%infection in various internal organs. Itwas discovered by challenging chicks with Ba15in different ways that intra-abdominalinfection with highest proportion of visceral infections (100%), close to fetal infections, hadstrongest growth inhibition and immune suppression. Nasal infection with a infection rate of30-40%in viscera organs was more serious than ocular infection (30%).3Colonization pattern of B. avium in nasal challenged SPF chickensBacterial counting of the homogenate, inoculating loop line method, smear stainingmethod, indirect immunoenzyme histochemistry, as well as indirect immunofluorescencehistochemistry were used to detect B. avium infection in different tissues after intranasalinfection of Ba15. Results showed that the bacteria were isolated from tracheas and lungs at1h post-infection. As time prolonged, B. avium continually increased in lungs within12hpost-infection. Afterwards, they colonized livers, hearts and spleens at5d and then infectedkidneys at7d. The peak of bacteria count appeared between14-28days of age. After35daysof age, the proportion of infections in the viscera organs gradually decreased. It was directlyrelated with antibody levels of B. avium and enhanced disease resistance. Visceral organswere colonized extensively by B. avium which were distributed densely in intercellular spaceand cytoplasm by detecting with indirect immunoenzyme histochemistry and indirectimmunofluorescence histochemistry.4Gene typing of B. avium isolates by RAPD analysisBacterial genomic DNAs of high purity and high concentration were obtained by using acommercially available kit. RAPD amplification procedure which had good stability andreproducibility were established. Accordingly, a set of20commercially available primers wasscreened out to identify suitable primers for RAPD analysis of B. avium isolates. At last6primers R1, R2, R4, R6, R8and R10resulted in clear fingerprints of well polymorphism andwere used to evaluate the B. avium isolates. Based on their RAPD patterns, a dendrogram allowed the separation of the B. avium isolates into3genetic similarity clusters. The firstcluster could be divided into3subsets, and the third cluster could be divided into2subsets.The first cluster included10isolates before2000and isolate Ba12, Ba13, Ba14and Ba20after2001. The isolates in the second cluster and the third cluster were all after2001. Ba15,Ba17, Ba18and Ba22were in the second cluster and Ba11, Ba16, Ba19and Ba21were in thethird cluster.5Synergistic effects of co-infection of REV and B. aviumB. avium of single infection in chick embryo had weak virulence with late death time ofchick embryos. The main harm is to generate a large number of weak chicks, and the chickswould be carriers for a long time, grow slowly and egg laying days delay. The death of chickembryos in co-infection of REV and B. avium were ahead of time with a hatching rate of only30%. Dead embryo rate and weak young chick rate increased, and the ultimate survival rate ofweak young chicks was0. Results of LD50detection of single B. avium infection andco-infection of REV and B. avium at different ages showed that infection age is very essential.Whether in single infection or co-infection, LD50value of high age was significantly higherthan that of low age. REV infection both at the embryonic period and1day old significantlyreduced the LD50values of B. avium, especially REV infection at the embryonic period. REV,as an immunosuppressive virus, earlier infects in chicks, greater effect to pathogenicity of B.avium.Effects of B. avium single nasal infection on growth inhibition and immune inhibition areweak. REV infection to embryos then nasal infection of B. avium to chicks had synergy ongrowth inhibition and immunosuppressive. Immunosuppression, although mainly caused byREV, B. avium infection accelerated the immune organ cell apoptosis, necrosis, and shortenedthe duration of antibody levels. In addition, co-infection improved the proportion of stiffchicken number. The detection time of B. avium in the visceral organs were ahead of time, theinfection rate increased, infection time extended in the viscera, and secondary infectionnumber also increased.It was discovered that infection age is crucial in single infection of B. avium at1,7,14,and21ages. At the age of1and7, chicks are more sensitive. Symptoms showed onlytransient infection when challenged at21ages. Compared between infections at different ages,the lower infection age was, the more serious effect on growth inhibition and immunesuppression, as well as the more numbers of cases of B. avium invaded into visceral organs.When B. avium was challenged at1,7,14, and21ages after REV infection at1age, it was showed that a certain degree of synergy appeared on growth inhibition and immunesuppression. Compared to single infection of B. avium, the proportion of viscera invaded by B.avium in co-infection increased. The chicks of low ages were still more susceptible than thoseof high ages, however, the difference between the infections at different ages reduced.In short, the study results show that multi-pathogen infection was widespread in chickenhatcheries of our country. B. avium isolates in nearly ten years were divided into3clusters.One virulent strain was screened and its colonization pattern in chickens and synergisticpathogenicity with REV were studied. Under state of immune suppression B. avium invadedthe visceral ahead of time, the infection ratio increased and pathological changes were moreserious. At the same time co-infection of REV and B. avium had synergistic effect on growthsuppression and immune suppression. Immunosuppressive virus infection significantlyincreased the hazard of B. avium in the chickens.