Pathogenicity and molecular characterization of Mycoplasma bovis isolate from calves in Morocco.

Bovine respiratory disease caused by Mycoplasma bovis (M. bovis) represents a major health problem for cattle worldwide that causes considerable financial losses. This study reports for the first time the molecular and pathogenic characterization of a strain of M. bovis isolated from a dead local calf with respiratory symptoms in Morocco. M. bovis was isolated from lung tissue, purified by cloning, and subtyped using MLST analysis. Experimental infection was conducted in naïve calves to evaluate pathogenicity. The isolate was identified as a new subtype ST-204 that shares similarities with the 2019-2021 Spanish strains (ST-169, ST-170, ST-171) and the 2018 Algeria isolate (ST-4). Experimental infection resulted in fever and respiratory symptoms with serous nasal discharge. At postmortem, lung lesions of congestion and hepatization were observed with lymph node enlargement and foci of necrosis. The study confirms the high pathogenicity of the isolate and the important role of M. bovis in bovine respiratory disease.

Neutralising antibodies against SARS-CoV-2 give important information on Covid-19 epidemic evolution in Rabat, Morocco, March 2020-February 2021.

Background: The SARS-CoV-2 is an extremely contagious and acute viral disease mainly affecting humans. Objective: To estimate seroprevalence of SARS-CoV-2 neutralizing antibodies (NAbs) for illegible armed force individuals living in Rabat, Morocco. Method: A convenience sample (N = 2662) was conducted from May 2020 to February 2021. We used the standard neutralization assay to quantify the NAbs titers. A serum was positive when the titer was 1:4. High positive NAbs titers were defined when ≥ 1:32. Results: Demographic and socioeconomic status did not affect seroprevalence data. An overall seroprevalence of 24,9% was found. Sera from blood donors, young recruits and auto-immune population had lower NAbs titers. However, titers were above 1:16 in 9% of the population with high risk of SARS-CoV-2 exposure. Seropositivity increased over time with values reaching peaks after the epidemic waves (2.4% in May 2020; 16.2% in August 2020; 22.7% in December 2020 and 37% in February 2021). Conclusion: And increase of NAbs was observed over time and correlated with the post-epidemic waves of COVID-19 in Morocco.

Avian Influenza a H9N2 Viruses in Morocco, 2018-2019.

Low pathogenic H9N2 avian influenza (LPAI H9N2) is considered one of the most important diseases found in poultry (broiler, laying hens, breeding chickens, and turkeys). This infection causes considerable economic losses. The objective of this work was to monitor and assess the presence of avian influenza virus (AIV) H9N2 in eight different regions of Morocco using real-time RT-PCR, and to assess the phylogenetic and molecular evolution of the H9N2 viruses between 2016 and 2019. Field samples were collected from 108 farms suspected of being infected with LPAI H9N2 virus. Samples were analyzed using H9N2-specific real-time RT-PCR. Highly positive samples were subjected to virus isolation and seven isolates were fully sequenced. Low pathogenic H9N2 avian influenza virus was introduced in Morocco in 2016. We show that in 2018-2019, the virus was still present irrespective of vaccination status. Phylogenetic and molecular analyses showed mutations related to virulence, although our viruses were related to 2016 Moroccan viruses and grouped in the G1 lineage. Specific amino acid substitutions were identified in Moroccan H9N2 viruses that are believed to lead to increased resistance to antiviral drugs.

Assessing the Efficacy of Recombinant Newcastle Disease Virus Vaccines (Double-Insert vHVT-IBD-ND and Single-Insert vHVT-ND) Followed by a Vaccination with a Live Newcastle Disease Vaccine Against a Moroccan Velogenic Newcastle Disease Challenge in Commercial Broilers.

The advent of turkey herpesvirus (HVT) vector vaccine technology (vHVT) has made a huge improvement in the prevention and control of several poultry diseases. The objective of this study was to compare, under experimental conditions, the protection conferred by different vaccination programs based on an HVT double-insert (infectious bursal disease {IBD] and Newcastle disease [ND]) vector vaccine (vHVT-IBD-ND) and an HVT single-insert (vHVT-ND) vector vaccine followed by a vaccination with a live ND vaccine at Day 1 only or at Days 1 and 14. Commercial broilers were vaccinated by the recombinant ND virus vaccines subcutaneously at 1 day old, in the hatchery, and challenged at 30 days of age using the Moroccan ND virus velogenic viscerotropic JEL strain. The results showed that the tested vaccine induced 95% to 100% clinical protection against mortality and clinical signs. The humoral immune response to vaccination was detected from 3 wk of age using enzyme-linked immunosorbent assay and hemagglutination inhibition tests. ND challenge virus shedding was significantly reduced in the vaccinated birds as compared to controls. Significant reduction of the cloacal shedding suggests that the vHVT-IBD-ND vaccine stimulates actively the immunity against the tested ND challenge virus. No significant differences were found between the vaccination programs based on vHVT-IBD-ND or on vHVT-ND.

Evaluación de la eficacia de las vacunas recombinantes contra el virus de la enfermedad de Newcastle (vHVT-IBD-ND de doble inserto y vHVT-ND de inserto único) seguidas de una vacunación con una vacuna viva para la enfermedad de Newcastle contra un desafío de la enfermedad de Newcastle velogénico marroquí en pollos de engorde comerciales. El advenimiento de la tecnología de vacunas recombinantes (vHVT) del virus herpes del pavo (HVT) ha provocado una mejora en la prevención y el control de varias enfermedades avícolas. El objetivo de este estudio fue comparar, en condiciones experimentales, la protección conferida por diferentes programas vacunales basados en una vacuna recombinante HVT con doble inserto (bursitis infecciosa [EII] y enfermedad de Newcastle [ND]) (vHVT-IBD-ND) y una vacuna recombinante HVT con inserto única (vHVT-ND) seguida de una vacunación con una vacuna para Newcastle viva aplicada en el día 1 o en los días 1 y 14. Pollos de engorde comerciales se vacunaron con las vacunas recombinantes del virus de la enfermedad de Newcastle por vía subcutánea al día de edad, en la incubadora y se expusieron a los 30 días de edad utilizando la cepa JEL viscerotrópica velogénica del virus de la enfermedad de Newcastle de Marruecos. Los resultados mostraron que la vacuna evaluada indujo una protección clínica del 95% al 100% contra la mortalidad y los signos clínicos. La respuesta inmune humoral a la vacunación se detectó a partir de las 3 semanas de edad mediante ensayo inmunoabsorbente ligado a enzimas y pruebas de inhibición de la hemaglutinación. La excreción del virus de Newcastle de desafío se redujo significativamente en las aves vacunadas en comparación con los controles. La reducción significativa de la eliminación cloacal sugiere que la vacuna vHVT-IBD-ND estimula activamente la inmunidad contra el virus de Newcastle de desafío analizado. No se encontraron diferencias significativas entre los programas de vacunación basados en vHVT-IBD-ND o en vHVT-ND.

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.

Pathogenesis of Avian Influenza Virus Subtype H9N2 in Turkeys and Evaluation of Inactivated Vaccine Efficacy.

Avian influenza H9N2 viruses circulate in all types of poultry species, including turkeys, and cause significant losses for the poultry industry in many parts of the word. The aim of this study was to assess the pathogenesis of the Moroccan avian influenza virus (AIV) H9N2 under experimental conditions in turkeys and the protection efficacy of an inactivated commercial vaccine against AIV H9N2. Unvaccinated turkeys showed marked depression sinusitis, respiratory distress characterized by bronchiolar and tracheal rales of moderate severity, and a mortality rate of 50%. Postmortem examinations of dead and euthanatized birds revealed the presence of fibrinous tracheitis and airsacculitis lesions. Vaccination reduced the mortality rate to 20%. Vaccinated birds recovered at day 10 postchallenge, and only 12.5% (1/8) and 37.5% of birds still displayed fibrinous and nonfibrinous airsacculitis lesions, respectively, at day 15 postinoculation. Viral shedding in cloacal and tracheal swabs was lower in vaccinated than in control birds. Although viral RNA was detected in the cloacal swabs of all unvaccinated turkeys at day 3 postinoculation, only 50% of the vaccinated turkeys were positive for virus detection. At day 11 postinoculation, no viral RNA was detected in oropharyngeal swabs of vaccinated turkeys, whereas 40% of the unvaccinated turkeys were still shedding virus.

Artículo regular­Patogenia del subtipo H9N2 del virus de la influenza aviar en pavos y evaluación de la eficacia de una vacuna inactivada. Los virus de la influenza aviar H9N2 circulan en todo tipo de especies de aves comerciales, incluidos los pavos, y causan pérdidas significativas para la industria avícola en muchas partes del mundo. El objetivo de este estudio fue evaluar la patogenia del virus de la influenza aviar de Marruecos (AIV) H9N2 bajo condiciones experimentales en pavos y la eficacia de protección de una vacuna comercial inactivada contra el virus de la influenza aviar H9N2. Los pavos no vacunados mostraron una marcada sinusitis, depresión, dificultad respiratoria caracterizada por estertores bronquiolares y traqueales de severidad moderada y una tasa de mortalidad del 50%. Los exámenes post mortem de aves muertas y sacrificadas revelaron la presencia de traqueítis fibrinosa y aerosaculitis. La vacunación redujo la tasa de mortalidad al 30%. Las aves vacunadas se recuperaron en el día 10 después del desafío, y solo el 12.5% (1/8) y el 37.5% de las aves todavía mostraban aerosaculitis fibrinosa y no fibrinosa, respectivamente, el día 15 después de la inoculación. La diseminación viral en los hisopos cloacales y traqueales fue menor en las aves vacunadas que en las aves control. Aunque se detectó ARN viral en los hisopados cloacales de todos los pavos no vacunados en el día tres después de la inoculación, solo el 50% de los pavos vacunados dieron positivo para la detección del virus. En el día 11 después de la inoculación, no se detectó ARN viral en hisopados orofaríngeos de pavos vacunados, mientras que el 40% de los pavos no vacunados todavía estaban diseminando virus.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

Co-infections of chickens with avian influenza virus H9N2 and Moroccan Italy 02 infectious bronchitis virus: effect on pathogenesis and protection conferred by different vaccination programmes.

Since the emergence of low pathogenic avian influenza (LPAI) H9N2 viruses in Morocco in 2016, severe respiratory problems have been encountered in the field. Infectious bronchitis virus (IBV) is often detected together with H9N2, suggesting disease exacerbation in cases of co-infections. This hypothesis was therefore tested and confirmed in laboratory conditions using specific-pathogen-free chickens. Most common field vaccine programmes were then tested to compare their efficacies against these two co-infecting agents. IBV γCoV/chicken/Morocco/I38/2014 (Mor-IT02) and LPAI virus A/chicken/Morocco/SF1/2016 (Mor-H9N2) were thus inoculated to commercial chickens. We showed that vaccination with two heterologous IBV vaccines (H120 at day one and 4/91 at day 14 of age) reduced the severity of clinical signs as well as macroscopic lesions after simultaneous experimental challenge. In addition, LPAI H9N2 vaccination was more efficient at day 7 than at day 1 in limiting disease post simultaneous challenge.RESEARCH HIGHLIGHTS Simultaneous challenge with IBV and AIV H9N2 induced higher pathogenicity in SPF birds than inoculation with IBV or AIV H9N2 alone.Recommended vaccination programme in commercial broilers to counter Mor-IT02 IBV and LPAIV H9N2 simultaneous infections: IB live vaccine H120 (d1), AIV H9N2 inactivated vaccine (d7), IB live vaccine 4-91 (d14).

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco.

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin. In a phylogenetic tree, nucleotide sequences of segment A and B formed a common branch with those vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of deduced amino acid sequences segment B, confirmed the presence of the conserved TDN tripeptide found in all of the vvIBDV genotype and revealed the presence of 2 substitutions I472L and E688D specific for the vvIBDV Moroccan isolates. The deduced amino acid sequences of segment A genes showed the presence of the "signature" typical of the vvIBDV genotype and revealed the presence of 7 aa substitutions specific for the vvIBDV Moroccan strains. The evolution rate for IBDV VP2 gene was estimated at 5.875 × 10-4 substitutions/site/year. The estimation of the time to most common recent ancestor of Moroccan vvIBDV based on the VP2 sequences available was 31 years, corresponding to 3 years earlier than the first vvIBDV case detection in layers in the country.

First Report of Isolation of Gallibacterium anatis from Layer Chickens in Morocco with Decrease in Laying Performance.

Gallibacterium is a genus of the family of Pasteurellaceae. It is well known as a commensal inhabitant of the respiratory and reproductive tract of healthy chickens. But in the last years, Gallibacterium anatis is increasingly reported in field cases with a decrease in laying performance due to infections of the reproductive tract. The aim of the present study was to investigate the implication of G. anatis infection in layer flocks facing a decrease in laying performance in Morocco. Birds were received from five different laying hen farms in two regions in Morocco showing a drop of egg production. Necropsy revealed 46.1 % (24/52) of sampled birds showed variable lesions in ovaries, salpinx, and trachea. In fact, 24 birds were affected by salpingitis, 18 by oophoritis, and 11 birds by atrophy of ovaries. Furthermore, tracheitis was observed in 24 birds. Bacteriological investigation was done from different organs, and G. anatis was found in ovaries (n = 20), trachea (n = 17), and cloaca (n = 3). Identification was based on growth morphology, Gram staining, and biochemical properties. Additionally, polymerase chain reaction test using specific primers for the genus identification was carried out. All isolates showed bands of 925 bp specific for G. anatis expressing the virulent toxin GtxA. Antibiotic resistance testing was performed and revealed that isolates were sensitive to enrofloxacin, florfenicol, and gentamycin but resistant to ampicillin, erythromycin, oxytetracycline, and sulfamethoxazole-trimethoprim. The present study is the first report of G. anatis in Morocco, demonstrating the need for further epidemiologic investigations as well as in regard to antibiotic resistance development.

Primer informe de aislamiento de Gallibacterium anatis de gallinas de postura en Marruecos con disminución en la eficiencia de la postura. Gallibacterium es un género de la familia Pasteurellaceae. Es bien conocido como un habitante comensal del tracto respiratorio y reproductivo de pollos sanos. Pero en los últimos años, han aumentado los reportes de Gallibacterium anatis en casos de campo con una disminución en la eficiencia de la postura debido a infecciones del tracto reproductivo. El objetivo del presente estudio fue investigar la implicación de la infección por G. anatis en parvadas de gallinas de postura que presentaban una disminución en la eficiencia de la postura en Marruecos. Se recibieron aves de cinco granjas diferentes de gallinas postura en dos regiones de Marruecos, mostrando una caída en la producción de huevos. La necropsia reveló que 46.1% (24/52) de las aves muestreadas mostraron lesiones variables en ovarios, oviducto y tráquea. De hecho, 24 aves mostraron salpingitis, 18 ooforitis y 11 aves atrofia de ovarios. Además, se observó traqueítis en 24 aves. Se realizó la investigación bacteriológica de diferentes órganos, y se encontró G. anatis en ovarios (n = 20), tráquea (n = 17) y cloaca (n = 3). La identificación se basó en la morfología del crecimiento, en la tinción de Gram y en las propiedades bioquímicas. Además, se realizó una prueba de reacción en cadena de la polimerasa utilizando iniciadores específicos para la identificación del género. Todos los aislamientos mostraron bandas de 925 pares de bases específicas para G. anatis que expresaban la toxina virulenta GtxA. Se realizaron pruebas de resistencia a los antibióticos y revelaron que los aislamientos eran sensibles a enrofloxacina, florfenicol y gentamicina, pero resistentes a ampicilina, eritromicina, oxitetraciclina y sulfametoxazol-trimetoprima. El presente estudio es el primer informe de G. anatis en Marruecos, que demuestra la necesidad de más investigaciones epidemiológicas, así como en relación con el desarrollo de resistencia a los antibióticos.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

Molecular characterization and phylogenetic analysis of very virulent infectious bursal disease virus circulating in Morocco during 2016-2017.

Very virulent infectious bursal disease virus (vvIBDV), the cause of significant economic losses in many poultry-producing areas, has been present in Morocco since 1991. In spite of the introduction of vaccination, disease outbreaks are frequently observed. To ascertain if vaccines failure may be due to the emergence of new strains, the aim of this study was to perform for the first time the molecular characterization of vvIBDV strains circulating in Morocco by focusing on the hypervariable region (HVR) of the VP2 protein, which is frequently used for molecular epidemiology and phylogenetic studies. Field samples of haemorrhagic bursae of Fabricius were collected for molecular characterization in different parts of the country during 2016-2017 from 48 chicken flocks showing symptoms of disease. In a phylogenetic tree, nucleotide sequences containing the VP2 HVR of 13 samples that were positive for vvIBDV formed a common branch with those of vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of the deduced amino acid sequences, in addition to confirming the presence of the "signature" typical of the vvIBDV HVR, also revealed the presence of substitutions in hydrophilic loops that are known to be involved in the elicitation of neutralizing antibodies. One of these substitutions is unique to the Moroccan isolates. These results represent the first molecular characterization of vvIBDV isolates in Morocco and may indicate that one of the causes of vaccine ineffectiveness is antigenic drift.