Morphological and molecular characterization of Prosthogonimus falconis n. sp. (Trematoda; Prosthogonimidae), found in a peregrine falcon (Falco peregrinus) (Aves: Falconidae) in the United Arab Emirates

At a routine health check of a female peregrine falcon, 23 trematodes preliminary identified as Prosthogonimus sp. were removed from the bursa of Fabricius. Based on morphological and molecular examination, a new species, Prosthogonimus falconis, was described. The pear-shaped flukes were 4.3–6.9 mm long, with greatest width posterior to testes. Tegumental spines measuring between 17 and 21 μm long covered the whole body. Length and width ratio of oral to ventral suckers were 1:1.3. Extracaecal, multifollicular vitelline glands commenced prior to acetabulum and terminated posterior to testes. Eggs in the distal uterus measured 21 × 12 μm. Molecular analysis of internal transcribed spacer 2, cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1 gene regions revealed that the new species described here is phylogenetically closest to Prosthogonimus cuneatus and Prosthogonimus pellucidus clusters.

Molecular Survey of Viral Poultry Diseases with an Indirect Public Health Significance in Central Ethiopia

The importance of poultry production is globally increasing, in Ethiopia as well, where high-quality protein and contained costs make poultry a valuable food resource. However, this entails some problems linked to rural, backyard and intensively reared flock proximity and pathogen circulation. This study is aimed at monitoring the presence of important viral pathogens in poultry (infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV)) in Ethiopia. Respiratory and cloacal swabs and bursa of Fabricius and kidney imprints on FTA cards were collected in 2021 from 16 farms and tested for IBV, aMPV, NDV and IBDV. One farm was positive for IBDV, resulting in strains similar to those present in vaccines, belonging to genogroup A1a; two farms were positive for IBV but, due to sensitivity limits, only one sample was sequenced, resulting in a 4/91-like strain (GI-13); a layer farm tested positive for NDV with a Lasota-like vaccine strain. These findings suggest a low presence of these pathogens, probably due to the implementation of vaccination strategies, which is also testified by the detection of vaccine strains. A close diagnostic activity should be implemented on a routine basis in order to monitor pathogen circulation, ameliorate biosecurity measures and protect animal health and production levels.

Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand

Background and Aim: A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand. Materials and Methods: Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test. Results: The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR. Conclusion: These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

From nasal to basal: single-cell sequencing of the bursa of Fabricius highlights the IBDV infection mechanism in chickens

Background Chickens, important food animals and model organisms, are susceptible to many RNA viruses that invade via the nasal cavity. To determine the nasal entry site of the virus and clarify why avians are susceptible to RNA viruses, infectious bursal disease virus (IBDV) was selected because it is a typical avian RNA virus that infects chickens mainly via the nasal route. Results First, we found that IBDV infected the posterior part of the nasal cavity in chickens, which is rich in lymphoid tissue and allows the virus to be easily transferred to the blood. Via the blood circulation, IBDV infected peripheral blood mononuclear cells (PBMCs) and was transferred to the bursa of Fabricius to damage the IgM + B lymphocyte population. Subsequently, the single-cell RNA sequencing (scRNA-seq) results suggested the more detailed response of different bursal cell populations (B cells, epithelial cells, dendritic cells, and fibroblasts) to IBDV. Regarding B cells, IBDV infection greatly decreased the IgM + B cell population but increased the IgA + B cell population in the bursal follicles. In contrast to B cells, bursal epithelial cells, especially basal cells, accumulated a large number of IBDV particles. Furthermore, we found that both innate RNA sensors and interferon-stimulated genes (ISGs) were highly expressed in the IBDV-infected groups, while dicer and ago2 expression was largely blocked by IBDV infection. This result suggests that dicer-related RNA interference (RNAi) might be an effective antiviral strategy for IBDV infection in avian. Conclusion Our study not only comprehensively elaborates on the transmission of airborne IBDV via the intranasal route and establishes the main target cell types for productive IBDV infection but also provides sufficient evidence to explain the cellular antiviral mechanism against IBDV infection. Graphical Abstract

Influence on Mycotox® NG effects on relative weights of some internal organs in Pekin ducks with experimentally reproduced aflatoxicosis В1

Contamination of poultry feeds with mycotoxins is a global problem faced by poultry industry due to increased demands and availability of poor-quality cereals. The aim of the present study was to evaluate the beneficial effects of a mycotoxin binder (Mycotox NG) on relative weights of internal organs in Pekin ducks with experimental aflatoxicosis. The birds were divided into one control and six experimental groups (n=10) as follows: group І (0 mg/kg AFB1 without Mycotox NG); group ІI (0.5 g/kg Mycotox NG); group ІІI (1.0 g/kg Mycotox NG); group IV (0.2 mg/kg AFB1); group V (0.4 mg/kg AFB1); group VI (0.2 mg/kg AFB1 + 0.5 g/kg Mycotox NG) and group VII (0.4 mg/kg AFB1 + 1.0 g/kg Mycotox NG). Trial duration was 42 days. It was established that ducks fed AFB1-contaminated feed had increased relative weights of liver, kidneys, pancreas, heart, gizzard and proventriculus compared to the control group. At the same time, the relative weights of immunocompetent organs (thymus, spleen and bursa of Fabricius) were reduced. The addition of Mycotox NG to the feed contaminated with AFB1 compensated partly the changes in relative weights of visceral organs. The results from the present study demonstrated that the tested toxin binder could be effective for reduction of toxic effects of aflatoxins in domestic ducks.