Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry.

In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.

Unexpected pattern of antibody response of hens following infectious bursal disease virus vaccine.

The antibody titers to infectious bursal disease virus (IBDV) of a group of hens were determined every 2 weeks during the laying period using a kinetic-based enzyme-linked immunosorbent assay (ELISA). When the titers of the flock were regressed against time, the flock titer decayed with statistically significant linearity. However, when the antibody titers of individual hens were measured, their titers regressed on time in a significant quartic curvilinear fashion. Since these hens were not reimmunized, this suggests that a anamnestic response was stimulated from an unknown external source.

The relationship between the hemagglutination-inhibition test and the enzyme-linked immunosorbent assay for the detection of antibody to Newcastle disease.

An assay of 364 chicken serum samples for Newcastle disease virus antibodies determined that a commercial NDV enzyme-linked immunosorbent assay (ELISA) had a 98.2% sensitivity and a 91.7% specificity relative to the NDV HI test. The ELISA values regressed significantly (F = 930, df = 1/362, P less than 0.001) on the hemagglutination-inhibition (HI) titers. The correlational coefficient was 0.85. For individuals, two tests can have the same result based upon chance alone. Kappa is a measure of agreement between two tests that corrects for this chance agreement. The kappa between the ELISA and HI test was calculated to be 0.84 (Z = 7.74, P = 0.00001), which indicates a highly significant agreement between the two tests.

Respiratory coccidiosis (Cryptosporidium baileyi) among northern Georgia broilers in one company.

Cryptosporidium baileyi causes respiratory disease in chickens. The purposes of this prospective study were to determine the incidence of C. baileyi tracheitis among broilers in a commercial setting, and the relationship between C. baileyi tracheitis and production performance parameters. All samples came from 56 farms that grow broilers for one company in northern Georgia. Tracheas were collected and examined with a light microscope and cultured for viruses and bacteria. Overall, 23 of 56 (41%) broiler flocks had C. baileyi tracheitis. Parasitism rates among C. baileyi-infected flocks ranged from a low of 10% to a high of 60%. Cryptosporidium baileyi tracheitis was very highly correlated (rho = 0.81, n = 56, P < 0.00001) to severity of tracheitis, negatively correlated (rho = -0.27, n = 56, P < 0.04) with average body weight, and correlated with airsacculitis (rho = 0.30, n = 56, P < 0.03) and condemnations (rho = 0.27, n = 56, P < 0.05). The present study indicates that C. baileyi infection rates are high, and the role that this parasite plays in the pathogenesis of respiratory disease and production losses could be unexpectedly large.

The relationship of egg yolk and serum antibody. I. Infectious bursal disease virus.

Eggs and serum samples were collected from hens every 2 weeks for 50 weeks from hens caged individually so that the yolk and serum samples could be paired. It was found that the antibody titer of the yolk sample regressed significantly (F = 403.3, df = 1/135, P less than 0.001) on the titer of the serum sample. This permitted the use of the yolk titer to predict the serum titer.

Comparison of histopathology to the direct immunofluorescent antibody test for the diagnosis of infectious laryngotracheitis in chickens.

Histopathology and direct immunofluorescent antibody (DIFA) tests were compared for diagnoses of infectious laryngotracheitis (ILT) in 144 cases of spontaneous respiratory disease in chickens presented to the Georgia Poultry Laboratory during 1988. For the 48 cases in which ILT was diagnosed, correct histologic diagnoses were made 100% (48/48) of the time and correct DIFA diagnoses were made 96% (46/48) of the time. For the 96 cases in which ILT was not diagnosed, correct histologic and DIFA diagnoses were made in every case. A Kappa test showed that DIFA was as effective as histopathology for the diagnosis of ILT in chickens (P = 0.0). It was concluded that increased use of DIFA could be instrumental in the abatement of ILT in chickens.

Infectious laryngotracheitis virus in commercial hens: a serological study based on enzyme-linked immunosorbent assay.

Serum samples collected from 23 flocks of commercial hens from three different companies were tested by enzyme-linked immunosorbent assay (ELISA) for antibodies against infectious laryngotracheitis (ILT) virus, and data were analyzed statistically. Geometric mean titers (GMTs) were compared from hens that were unvaccinated, once-vaccinated, or twice-vaccinated, from single-age farms or multiple-age farms, from molted or unmolted flocks, and from different companies. There were significant differences among the groups compared by vaccination, between the single-age and multiple-age groups, and between the molted and unmolted groups. The GMT of unvaccinated flocks and the GMT of molted flocks that had been vaccinated once as pullets with a chick-tissue-culture-origin (CTCO) live vaccine could not be differentiated. The ELISA is useful to detect and quantitate ILT vaccine-induced antibody, but it does not reliably identify older flocks that were vaccinated at a young age with CTCO vaccine.

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction.

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.

Pasteurella challenge and ELISA serology evaluation of broiler breeders vaccinated with live cholera vaccine.

Broiler breeder pullets were vaccinated against fowl cholera at 10 and 20 wk of age using a live PM-1 Pasteurella multocida vaccine administered by wing web stick. Antibody production for P. multocida was measured at vaccination and at 1-4-wk intervals following vaccination by enzyme-linked immunosorbent assay. Groups of vaccinated birds were challenged at 23 and 32 wk of age. Two doses of a live PM-1 P. multocida vaccine protected broiler breeder hens against virulent challenge up to 32 wk of age when measured antibody levels had a range of 1951-4346 and a geometric titer of 3000.

DNA in situ hybridization for the rapid diagnosis of massive necrotizing avian adenovirus hepatitis and pancreatitis in chicks.

The present study describes the use of DNA in situ hybridization for the rapid diagnosis of massive necrotizing adenovirus hepatitis and pancreatitis in broiler chicks. A light microscope and DNA probes were used to identify avian adenovirus in replicate sections of formalin-fixed paraffin-embedded liver and pancreas from field and experimental chicks. Avian adenovirus infection was confirmed by transmission electron microscopy and virus isolation.

Studies of infectious laryngotracheitis vaccines: immunity in broilers.

Broiler chickens were vaccinated at 18 days of age against infectious laryngotracheitis (ILT) using chicken-embryo-origin (CEO) and tissue-culture-origin (TCO) vaccines, each vaccine given either by drinking water, spray, or eyedrop. Controls were not vaccinated. The broilers were challenged 3 weeks later with virulent ILT virus (USDA challenge strain). Serum samples taken before challenge were analyzed by a virus neutralization (VN) test to determine titers due to vaccination. Both vaccines, regardless of route of administration, produced low VN titers, geometric mean titer (GMT) being less than 4.0 in all vaccinated groups. When administered by the same route, the CEO vaccine produced higher titers than the TCO vaccine. Titers following drinking-water or eyedrop administration of vaccines were higher than titers following spray vaccination. There was an inverse relationship between pre-challenge VN titers of groups of birds and the percentage of birds in the groups dying from ILT virus challenge. The drinking-water route of vaccination provided the most protection, while the spray provided the least.

Studies of infectious laryngotracheitis vaccines: immunity in layers.

Ten-week-old layer chickens obtained from a commercial source were eye-drop vaccinated with chicken-embryo-origin (CEO) or tissue-culture-origin (TCO) vaccines for infectious laryngotracheitis (ILT). Controls were not vaccinated. Approximately one-third of the layers were challenged with virulent ILT virus at 21, 40, or 60 weeks of age. Serum samples taken from the layers before challenge were used in a virus neutralization (VN) test to determine vaccination titers at those three ages. Both vaccines induced low VN titers (geometric mean titer [GMT] less than 6). At 21 weeks of age, the titers produced by the two vaccines were not significantly different, but at 40 and 60 weeks of age the VN GMT of the CEO-vaccinated group was significantly greater than that of the TCO-vaccinated group. The VN GMTs did not drop over time in either group and actually rose between 21 and 60 weeks of age in the CEO group. Both vaccines protected layers against severe challenge with virulent ILT virus, neither being significantly better than the other under these experimental conditions. Unvaccinated sentinel chickens were maintained in contact with the vaccinated layers during three intervals between 1 day and 6 weeks post-vaccination. Diagnostic tests performed on the sentinels to detect lateral spread of vaccine virus from vaccinated to unvaccinated chickens showed scattered positive results.

Respiratory cryptosporidiosis in chickens.

In order to better characterize spontaneous respiratory cryptosporidiosis in chickens, a retrospective examination of histopathology reports from the Georgia Poultry Laboratories for an 18-mo period (4/1/86 to 9/30/87) was made; 12 cases were found. Collected data were analyzed and certain epidemiologic and histologic features were identified. Eleven of the 12 cases involved broiler type chickens. The ages of chickens with respiratory cryptosporidiosis were evenly distributed between 17 and 52 days of age. The infected birds were always clinically ill. Viruses or bacteria or both often accompanied respiratory Cryptosporidium sp. infections. Histologic lesions (including those of ciliary-adherent bacteria) are described. As the inflammatory response in infected organs became progressively nonpurulent (lymphocytes and plasma cells predominate), numbers of Cryptosporidium diminished. Cytologic preparations were useful for making diagnoses of respiratory cryptosporidiosis in chickens. Identification of epidemiologic features of respiratory cryptosporidiosis, and improved ability to make accurate and prompt diagnoses of Cryptosporidium sp. infection, are vital for a more complete understanding of the impact of this disease on poultry health.