Real-time polymerase chain reaction for the qualitative and quantitative detection of Mycoplasma gallisepticum.

The aim of this study was to evaluate the sensitivity and the detection limit of a real-time polymerase chain reaction (Q-PCR) developed for the qualitative and quantitative detection of Mycoplasma gallisepticum. No cross-reactivity was observed with DNA from other important avian mycoplasmas, including Mycoplasma synoviae and Mycoplasma meleagridis. However, the Q-PCR could not distinguish between M. gallisepticum and Mycoplasma imitans. The Q-PCR had detection limits 10 to 1000 times lower than a conventional commercial PCR method and than culture. The Q-PCR was used quantitatively by incorporating a set of external M. gallisepticum DNA standards, derived from a M. gallisepticum log-phase culture of a known concentration. The number of colony-forming unit equivalents per millilitre in tracheal swabs from experimentally infected birds could be determined from a single sample. The method had good reproducibility and correlated well with standard counting techniques using culture. It can be concluded that the Q-PCR described is suitable for qualitative and quantitative detection of M. gallisepticum in clinical samples.

An experimental model to quantify horizontal transmission of Mycoplasma gallisepticum.

Before interventions to control horizontal transmission of Mycoplasma gallisepticum can be tested, a suitable experimental model should be available. Transmission dynamics in a flock can be quantified by two parameters: the average number of secondary cases infected by one typical infectious case (R0) and the number of new infections that occur due to one infectious animal per unit of time (beta). The transmission dynamics of M. gallisepticum have not been studied experimentally, so the aim of this study was to examine the horizontal transmission of M. gallisepticum. The study was carried out using a pairwise design with three different inoculation doses. Every pair consisted of an inoculated chicken and a susceptible in-contact chicken. Five susceptible individually housed chickens were placed in between pairs in order to measure airborne transmission. Infection was detected by serology, quantitative polymerase chain reaction and culture. The inoculated and in-contact chickens were equally infectious and the pairs could be regarded as independent. The R0 was estimated to be greater than 1 (infinity; 95% confidence interval, 4.5 to infinity), the estimated beta was 0.22 per day and there was no significant difference between the different inoculation doses. It was concluded that the animal model as described in this study meets the conditions for the establishment of transmission dynamics of M. gallisepticum and therefore can be used to establish the quantitative effect of intervention measures on horizontal transmission.

Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections.

In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.

Validation of five commercially available ELISAs for the detection of antibodies against infectious bursal disease virus (serotype 1).

In this study, the results are reported from a validation study of five commercially available enzymelinked immunosorbent assays (ELISAs) for the detection of antibodies against infectious bursal disease virus (IBDV), serotype 1. The specificity of the ELISAs varied from 63.8 to 100%. All ELISAs reached a sensitivity of 100% on sera between 14 and 21 days post-vaccination (d.p.v.) with two classical vaccines and a Delaware variant-E virus. Overall, most birds became positive between 8 and 11 d.p.v. As expected, the ELISA with the lowest specificity showed the highest sensitivity at 5 d.p.v. When the decrease in maternally derived antibodies against IBDV was measured, a highly significant correlation (P < 0.001) was found for all ELISAs and the virus neutralization test (VNT).

Arthropathic and amyloidogenic Enterococcus faecalis infections in brown layers: A study on infection routes.

Intravenous, intra-articular and intraperitoneal inoculation of 6-week-old brown-layer pullets with an arthropathic and amyloidogenic strain of Enterococcus faecalis resulted in amyloid arthropathy, while intramuscular, oral and intratracheal inoculation did not. Oral inoculation of 1-day-old chickens did not cause any pathology. However, intramuscular inoculation with 106 colony forming units resulted in severe growth retardation and arthritis in 60% of the birds, and amyloid arthropathy in approximately 40%. In egg transmission studies, neither egg dipping nor inoculation of the air chamber with E. faecalis reproduced the condition, although a few chicks became septicaemic. Yolk sac inoculation of 6-day-old embryos caused embryonic death within 2 days. In contrast, egg albumen inoculation with E. faecalis led to arthritis in one of six of the progeny, indicating the possibility that vertical transmission of E. faecalis by the oviductal route could lead to arthritis. The presence of antibodies to E. faecalis was confirmed by enzyme-linked immunosorbent assay in 14/15 of experimental birds that had developed arthritis.

A study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis.

Ten brown layer parent hens were injected intravenously with arthropathic and amyloidogenic Enterococcus faecalis at 27 weeks of age to assess its vertical transmission during the subsequent 6-week production period. All inoculated hens developed chronic bacteraemia and arthritis, four died due to septicaemia and two of the remaining six showed amyloid arthropathy. The egg production was maintained at a lower level than the controls. Of eggs collected during the first 2 weeks after inoculation, E. faecalis was re-isolated from the yolk sac of 76% (13/17) of infertile eggs and dead embryos detected at the 18-day candling, and 100% (6/6) of non-hatching eggs, and from arthritic joints of 3% (2/66) offspring chicks of the same batch, although the latter did not develop joint amyloidosis by 8 weeks of age. E. faecalis was also re-isolated from ovary and oviduct of parent birds that died due to septicaemia. The E. faecalis organisms re-isolated from blood, ovaries and joints of diseased parent stock, yolk sac of infertile eggs and dead embryos detected at the 18-day candling, and non-hatching eggs, as well as organs and joints of offspring, had the same pulsed-field gel electrophoresis patterns as the E. faecalis isolate used to infect the parent birds. These findings indicate that vertical transmission of arthropathic and amyloidogenic E. faecalis may occur on a small scale.

Detection of specific IgM antibodies to infectious bronchitis virus by an antibody-capture ELISA.

The results of a new antibody-capture ELISA (alpha-IgM-IBV ELISA), specific for IgM directed against Infectious Bronchitis Virus (IBV) show that this assay is a useful tool for diagnosing IBV infections. The data include individual results of the alpha-IgM-IBV ELISA in sequential SPF chicken sera after vaccination with H120 and challenge with M41, the specificity is based on results of 499 SPF sera, and the sensitivity on sera from experimentally vaccinated and challenged birds. Also reported are ELISA results on 168 field sera originating from seven broiler flocks (24 samples per flock) collected during the acute phase of an IBV infection. The alpha-IBV-IgM ELISA results obtained with field sera from broiler flocks infected by Massachusetts, D207, D212 or an unidentified serotype of IBV indicated that the ELISA, in which only M41-strain was used as antigen, was able to detect IgM responses to IBV serotypes other than Massachusetts. The specificity of the alpha-IBV-IgM ELISA was 99%, the sensitivity based on the experimental vaccination was 83 to 100%, depending on the day post vaccination. The sensitivity, based on the experimental challenge, was 83%, while the sensitivity, based on 168 field sera, was 93.5%. The IgM responses were rapid and transient and therefore indicative for acute IBV infections.

Comparison of three commercial ELISA kits for the detection of turkey rhinotracheitis virus antibodies.

Three commercial ELISAs (Pathasure, Svanovir and Flockscreen) were compared in their ability to detect turkey rhinotracheitis virus (TRTV) vaccine-induced antibodies. Sequential sera taken from specified pathogen-free chickens, vaccinated with two combinations of live and inactivated TRTV vaccines were used. The vaccines were based on TRTV strains from the United Kingdom (Intervet) and from France (Rhone Merieux). One ELISA failed to detect antibodies after live vaccination with both French and UK vaccines, but detected antibodies induced by both inactivated vaccines by 8 days after vaccination. The two other ELISAs responded to both live vaccinations equally well and both detected a rise in antibody level 8 days after the inactivated vaccination. The specificity of the three ELISAs was 100%. When tested on field samples, all three ELISAs indicated a high prevalence of TRTV antibodies in turkey flocks in the Netherlands.

Sensitivity and specificity of serological tests for infectious bronchitis virus antibodies in broilers.

Broilers with maternally-derived immunity (MDI) to infectious bronchitis (IB) were either spray-vaccinated with H120 at 1 day old, or not vaccinated, then challenged at 28 days with one of four different IBV serotypes. Birds were bled frequently and the sera tested by agar gel precipitation (AGP), haemagglutination inhibition (HI), 2 commercial ELISAs, and virus neutralization (VN) to compare the sensitivity and specificity of the assays. The AGP detected a transient response to challenge with a specificity of 100% and a sensitivity of approximately 40%. The ELISAs showed moderate sensitivity and high specificity with sera from non-vaccinated broilers, and high sensitivity and variable specificity with vaccinated birds. Depending on the cut-off value used, the specificity of HI tests was 55 to 100%, while the sensitivity varied widely, making identification of the serotype of an IB challenge unreliable. In vaccinated broilers the sensitivity of the VN tests (used at 21 days post-challenge only) varied from 20 to 100%, while the specificity was dependant on the cut-off value selected. Increases in HI, VN and ELISA titres in vaccinates were generally about half those in non-vaccinates. It is concluded that AGP and ELISA are adequate to detect antibody responses to IBV challenge in both vaccinated and non-vaccinated broilers. In the ELISA, a cut-off value higher than that suggested by the manufacturers is preferred in vaccinated broilers. Similarly, a cut-off value of at least log(2)7 is desirable when attempting to use HI for IB serotyping.

[Viral hemorrhagic disease: an overview and personal data]. / Viral haemorrhagic disease: een overzicht en eigen bevindingen.

A review is given of current knowledge of taxonomy, clinical symptoms, pathogenesis and pathology, diagnosis, epizootiology and prevention of Viral Haemorrhagic Disease (VHD). We also report our own experiences with the (histo)pathology, laboratory diagnosis, and epizootiology of this disease. The frequency of other diagnoses in an eighteen months period.