A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Molecular survey of infectious bronchitis virus on poultry farms in Gifu Prefecture, Japan from 2021 to 2022 by RT-PCR with an enhanced level of detection sensitivity for the S1 gene.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB) in chickens. There is a correlation between cross-protection and percentage of similarity between nucleotide sequences encoding the S1 subunit, which is responsible for generating neutralizing and serotype-specific antibodies. Therefore, RT-PCR is commonly used to amplify the IBV-S1 gene following DNA sequencing in order to predict the efficacy of vaccines against IBV strains. We successfully enhanced the sensitivity for detection of the IBV-S1 gene by second PCR after purification of the 1st RT-PCR product. Using that method, we obtained detailed information on the prevalence of IBV on poultry farms in Gifu Prefecture, Japan. The IBV-S1 gene detection method used in the current study will enable accurate information on the prevalence of IBV in Japan to be obtained.

Diagnostic Accuracy of Ultrasonography to Detect False Layers in a Commercial Laying Flock Infected by an Infectious Bronchitis Virus Delmarva Genotype Causing Cystic Oviducts.

Infection of the oviduct by an infectious bronchitis virus (IBV) in laying hens has been associated with the false layer syndrome. Because the diagnostic procedure for the detection of cystic oviducts by postmortem examinations in IBV-positive replacement pullet flocks could involve the unnecessary sacrifice of numerous healthy pullets without reproductive tract anomalies, the development of a noninvasive and nonlethal diagnostic procedure would be desirable. The first objective of the study was to evaluate the diagnostic accuracy of a transcutaneous ultrasonography method to predict the presence of cystic oviducts compared to postmortem examinations in a commercial pullet flock positive for an IBV genotype Delmarva (DMV) variant. The second objective was to evaluate the performance of the same ultrasonography method to later detect false layers in the same flock in sexually mature hens by identifying the presence of an egg in the oviduct due to the presence of atretic oviducts undetectable by ultrasonography and the absence of cystic oviducts at that age. In replacement pullets, the sensitivity (Se) and specificity (Sp) of the ultrasonography (index test) compared to the postmortem examination (reference standard test) were 73% and 91%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 67% and 93%. The ultrasonography technique showed a positive likelihood ratio (LR+) of 7.82 and a negative likelihood ratio (LR-) of 0.30. In sexually mature hens, the Se, Sp, PPV, and NPV of the ultrasonography compared to the laying status were 98%. The LR+ was 49.00 and the LR- was 0.02 when compared to the laying status. In conclusion, the ultrasonography could replace postmortem examinations to detect cystic oviducts in commercial flocks of replacement pullets previously infected with an IBV-DMV 1639 variant. Although the test accuracy of ultrasonography was excellent for the hens at production peak to identify laying and nonlaying hens based on the presence of an egg in the reproductive tract, its practicality was limited due to atretic oviducts being not detectable.

Precisión diagnóstica de la ultrasonografía para detectar gallinas falsas ponedoras en una parvada comercial infectada por un virus de la bronquitis infecciosa genotipo Delmarva que causa oviductos quísticos. La infección del oviducto por el virus de bronquitis infecciosa (IBV) en gallinas de postura se ha asociado con el síndrome de la falsa ponedora. Debido a que el procedimiento de diagnóstico para la detección de oviductos quísticos mediante exámenes post mortem en parvadas de pollitas de reemplazo positivas para bronquitis infecciosa podría involucrar el sacrificio innecesario de numerosas pollitas sanas sin anomalías del tracto reproductivo, por lo tanto es deseable el desarrollo de un procedimiento de diagnóstico no invasivo y no letal. El primer objetivo del estudio fue evaluar la precisión diagnóstica de un método de ultrasonografía transcutánea para predecir la presencia de oviductos quísticos en comparación con los exámenes post mortem en un lote comercial de pollitas que resultó positivo para una variante del genotipo Delmarva (DMV) del virus de la bronquitos infecciosa. El segundo objetivo fue evaluar el desempeño del mismo método de ultrasonografía para detectar posteriormente gallinas falsas en la misma parvada en las gallinas sexualmente maduras mediante la identificación de la presencia de un huevo en el oviducto debido a la presencia de oviductos atrésicos indetectables por ultrasonografía y la ausencia de oviductos quísticos a esa edad. En las pollitas de reemplazo, la sensibilidad (Se) y la especificidad (Sp) de la ultrasonografía (prueba de índice) en comparación con el examen post mortem (prueba estándar de referencia) fueron de 73% y 91%, respectivamente. El valor predictivo positivo (VPP) y el valor predictivo negativo (VPN) fueron 67% y 93%. La técnica de ultrasonografía mostró una razón de probabilidad positiva (LR+) de 7.82 y una razón de probabilidad negativa (LR­) de 0.30. En las gallinas sexualmente maduras, la Se, Sp, PPV y NPV de la ultrasonografía en comparación con el estado de postura fueron del 98%. El LR + fue 49.00 y el LR­fue 0.02 en comparación con el estado de la postura. En conclusión, la ultrasonografía podría reemplazar los exámenes post mortem para detectar oviductos quísticos en parvadas comerciales de pollitas de reemplazo previamente infectadas con una variante DMV-1639 del virus de la bronquitis infecciosa. Aunque la precisión de la prueba de la ecografía fue excelente para las gallinas en el pico de producción para identificar gallinas ponedoras y no ponedoras en función de la presencia de un huevo en el tracto reproductivo, su funcionalidad fue limitada debido a que los oviductos atrésicos no fueron detectables.

Research Note: Rapid detection of avian infectious laryngotracheitis virus with real-time fluorescence-based recombinase-aided amplification.

In this study, specific primers and fluorescent probes were designed to target the thymidine kinase (TK) gene sequence of avian infectious laryngotracheitis virus (ILTV). Through specificity and sensitivity tests, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method for detecting ILTV was established. The results showed that the method was specific and could be used to accurately detect ILTV, and there was no cross-reaction with Newcastle disease virus (NDV), avian influenza virus (AIV), or infectious bronchitis virus (IBV). Real-time fluorescence-based recombinase-aided amplification had high sensitivity, and the lowest detectable limit (LDL) for ILTV could reach 10 copies/µL, 1,000 times more sensitive than conventional PCR (104 copies/µL), to rival that of real-time fluorescence-based quantitative PCR (RFQ-PCR) (10 copies/µL). This method and RFQ-PCR were used to detect 96 samples of chicken throat swabs with ILT initially diagnosed in clinic from the north of China, and the coincidence rate of the 2 methods was 100%. The RF-RAA reaction required only 20-30 minutes to completing, and its sensitivity was much higher than that of conventional PCR. Real-time fluorescence-based recombinase-aided amplification is similar to RFQ-PCR and has the advantages of specificity, sensitivity, and high efficiency, so it is suitable for early clinical detection and epidemiological investigation of ILTV.