Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites.

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89-111%. Cross-reaction was not detected with 33 non-A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

Cross-protection study of the nine serovars of Haemophilus paragallinarum in the Kume haemagglutinin scheme.

The cross-protection and haemagglutination-inhibition antibodies present in chickens vaccinated with one of the nine currently recognized Kume haemagglutinin serovars of Haemophilus paragallinarum were investigated. The results confirmed the widely accepted dogma that serogroups A, B, and C represent three distinct immunovars. Within Kume serogroup A, there was generally good cross-protection among all four serovars. However, within Kume serogroup C, there was evidence of a reduced level of cross-protection between some of the four serovars. The haemagglutination-inhibition antibody levels generally showed the same trend as with the cross-protection results. This study suggests that some apparent field failures of infectious coryza vaccines may be due to a lack of cross-protection between the vaccine strains and the field strains. Our results will help guide the selection of strains for inclusion in infectious coryza vaccines.

Evaluación de dos protocolos de bacterinización de gallinas de postura, mediante detección de anticuerpos inhibidores de la hemoaglutinación y protección ante un desafío controlado con Haemophilus paragallinarum / Evaluation of two bacterinization programs of field-immunized layer-hens by detection of hemagglutination-inhibition antibodies and protection against a controlled-challenge with Haemophilus paragallinarum

Se evaluaron dos protocolos de bacterinización de gallinas de postura inmunizadas en campo, de forma bivalente o trivalente, contra la coriza infecciosa, mediante detección de anticuerpos circulantes inhibidores de la hemoaglutinación y protección ante desafío controlado con los serovares A, B y C de H. paragallinarum. Gallinas inmunizadas de forma bivalente mostraron la más baja protección contra signos clínicos de coriza infecciosa cuando fueron desafiadas con el serovar A. No obstante, gallinas inmunizadas de forma bivalente mostraron protección al desafío y anticuerpos circulantes inhibidores de la hemoaglutinación para el serovar B. Gallinas inmunizadas de forma trivalente mostraron protección contra signos de coriza infecciosa para los tres serovares. No se observaron diferencias estadísticas entre los títulos de anticuerpos circulantes inhibidores de la hemoaglutinación contra un mismo serovar conferidos por bacterinas bivalentes o trivalentes. Los resultados indican que títulos elevados de anticuerpos inhibidores de la hemoaglutinación conferidos por bacterinas bivalentes o trivalentes pueden predecir protección frente a un desafío con los serovares A, B y C de H. paragallinarum.

Influence of inoculation site of combined oil-adjuvanted vaccine on the antibody response in chickens.

Inactivated oil-adjuvanted vaccines for ND, IB, and IC serotypes A and C (OILVAX NB2AC) have been marketed from 1993. In the outdoors, various inoculation sites have been used in chickens because to make the inoculation procedure easier. We examined whether differences would be obtained in the antibody response according to the inoculation sites; the subcutaneous inoculation into the back of the neck for OILVAX use, and the thigh, lower thigh, breast and shoulder muscle for the possible application of the inoculation outdoors. The clear order was not found in the NDV-HI titer, IBV-SN titer against Nerima or TM-86 among the inoculation sites used during the examination period. The IC serotypes A and C HI titers did vary among the inoculation sites; the subcutaneous inoculation produced the highest antibody titer, and high antibody titers were observed in the order of lower thigh muscle > or = thigh muscle > breast muscle > or = shoulder muscle inoculation.

Chronic social stress, social status, and susceptibility to upper respiratory infections in nonhuman primates.

OBJECTIVE: The objective of the study was to assess the roles of social stress and social status in susceptibility to upper respiratory infection. METHOD: Sixty male cynomolgus monkeys were randomly assigned to stable or unstable social conditions for 15 months. Two markers of social status, social rank and percent of behaviors that were submissive, were assessed at independent observation periods. Endocrine, immune, and behavioral responses were each assessed (at 3-month intervals) during the 9th through 14th months of the study. At the beginning of the 15th month, all animals were exposed to a virus (adenovirus) that causes a common-cold-like illness. The primary outcome was whether or not an animal developed an infection (shed virus) after viral exposure. RESULTS: Although the social instability manipulation was associated with increased agonistic behavior as indicated by minor injuries and elevated norepinephrine responses to social reorganizations, the manipulation did not influence the probability of being infected by the virus. However, low social status (as assessed by either marker) was associated with a substantially greater probability of being infected. It was also associated with less body weight, greater elevated cortisol responses to social reorganizations, and less aggressive behavior. However, none of these characteristics could account for the relation between social status and infection. CONCLUSIONS: Social stress was not associated with susceptibility to infection. However, animals with lower social status were at higher risk than high social status animals.

Turkey coryza: lack of correlation between plasmids and pathogenicity of Bordetella avium.

Plasmids were removed from pathogenic Bordetella avium using a variety of treatments. The plasmid-cure rates depended on the treatment and isolate. Pathogenicity of B. avium in turkey poults was not altered by removal of plasmids.

Efficacy of a commercial turkey coryza vaccine (Art-Vax) in turkey poults.

Four laboratory experiments were designed to study the efficacy of the only available commercial vaccine for turkey coryza, Art-Vax. Poults were vaccinated either once or twice at different ages and challenged with pathogenic Alcaligenes faecalis. In another study, commercial turkeys vaccinated at 1 and 12 days of age on a commercial farm were brought to the laboratory for challenge with pathogenic A. faecalis. Both the laboratory- and field-vaccinated poults were given the manufacturer's recommended dosage of the vaccine strain. Regardless of the vaccine schedule or source of poults, the vaccine was not effective in protecting challenged turkeys from infection. Furthermore, the vaccine was not effective in protecting poults less than 3 weeks of age from disease, but it was effective in protecting poults more than 3 weeks of age from disease. These results indicate that although vaccinated turkeys older than 3 weeks of age were not susceptible to disease, they were susceptible to infection and could act as carriers of field strains of A. faecalis, thus perpetuating the risk of infection to flocks subsequently raised in the same buildings.

Genetic and phenotypic comparison of three new avian Haemophilus-like taxa and of Haemophilus paragallinarum Biberstein and White 1969 with other members of the family Pasteurellaceae Pohl 1981.

In the course of post-mortem bacteriological examinations, several previously unreported bacterial strains were isolated from budgerigars, pigeons, kestrels, and a goose. They have been separated into three distinct collectives according to their cultural, morphological, and biochemical characteristics. Since they require V factors, they were tentatively assigned to the genus Haemophilus Winslow et al. 1917. This preliminary classification was checked by determination of guanine + cytosine contents and genome sizes and by DNA:DNA hybridization tests among reference strains of the three new avian taxa and recognized species of the family Pasteurellaceae Pohl 1981. With the same methods, the genetic relationships of Haemophilus paragallinarum Biberstein and White 1969 within the family were determined. It could be shown that the three avian Haemophilus-like taxa have to be regarded as new species within the family Pasteurellaceae not affiliated with the recognized genera Actinobacillus, Haemophilus and Pasteurella. H. paragallinarum must be excluded from the genus Haemophilus because of its closer relationship to the actinobacilli. All strains investigated can be differentiated from each other and from recognized species of Pasteurellaceae using an appropriate set of biochemical tests.

Further characterization of the agent causing coryza in turkeys.

A total of 128 isolates of Alcaligenes faecalis, from the respiratory tract of turkeys and chickens, were identified and divided into two types designated type I and type II. Type I isolates were pathogenic in poults, hemagglutinated guinea pig red blood cells (RBCs), and did not grow on minimal essential medium (MEM) agar, and most did not grow in 6.5% NaCl broth. Type II isolates were nonpathogenic and nonhemagglutinating and grew on MEM agar, and most grew in 6.5% NaCl broth. Hemagglutination of guinea pig RBCs was a reliable characteristic for distinguishing type I from type II isolates, and it correlated with pathogenicity. In serological studies using 62 type I and 21 type II isolates, cross-reactions were observed when type I but not type II antigens were used to test antisera in the microagglutination test. Eleven bacterial isolates, different from type I and type II isolates, were urease-positive. Although frequently isolated from turkeys with coryza, these isolates were nonpathogenic and were always found in association with type I A. faecalis. Urease-positive isolates and type I and type II A. faecalis isolates were stable following 50 in vitro passages. Bordetella avium sp. nov. (the nomenclature suggested in Europe for A. faecalis) was pathogenic in poults. The colonial morphology, biochemical characteristics, and hemagglutinating activity of B. avium sp. nov. were the same as those of type I A. faecalis isolates. Based on the results of these studies, it was concluded that type I A. faecalis is the etiologic agent of turkey coryza.

Immune response of poults following intranasal inoculation with Artvax vaccine and a formalin-inactivated Bordetella avium bacterin.

Poults 3 weeks and older developed temporary tracheal resistance to intranasal challenge following inoculation of either Artvax vaccine or formalin-inactivated Bordetella avium bacterin by the intranasal and eyedrop routes. Resistance usually persisted for 3-4 weeks after B. avium challenge. However, with constant exposure to infected controls, the vaccinated birds eventually developed tracheal infection. Day-old poults did not respond to either the Artvax or the bacterin and were completely susceptible to challenge. Two-week-old poults responded to some degree, but poults 3 weeks old and older responded best. Poults inoculated with bacterin by aerosol or by drinking water did not respond as well as those inoculated by the intranasal and eyedrop routes. When poults were given a single subcutaneous injection at 3 weeks of age and challenged 2 weeks later, three of five resisted infection for 18 days.

Pathogenicity of Australian isolates of Haemophilus paragallinarum and Haemophilus avium in chickens.

Twenty-seven Australian avian Haemophilus isolates were tested for their ability to cause infectious coryza in specific pathogen-free chickens. All 15 isolates, identified as H. paragallinarum, produced infectious coryza, whereas all 12 H. avium isolates were nonpathogenic, but spread to in-contact chickens.