Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

First Report of Low Pathogenic Avian Influenza Subtype H9N2 in African Houbara Bustards (Chlamydotis undulata undulata) and Gamebirds in Morocco: Clinico-Pathological Findings, Molecular Characterization, and Associated Coinfections.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

First Seroprevalence Survey of Avian Reovirus in Broiler Breeders Chicken Flocks in Morocco.

Avian reovirus (ARV) is a prevalent infectious agent that has the potential to cause respiratory and gastrointestinal illnesses in poultry, leading to substantial financial losses in the poultry sector. Until now, there have been no investigations conducted to examine the epidemiological status of ARV infections in Morocco. The aim of this study was to investigate the seroprevalence of ARV infections with respect to area, types of chickens (broiler breeder, and broiler), vaccination status, and age of chickens. A total of 826 serum samples were collected from 36 broiler and broiler breeder flocks, with 14 of them being unvaccinated, fromsix different regions of Morocco, namely Casablanca-Settat, Rabat-Salé-Kénitra, Tanger-Tétouan-Al Hoceïma, Oriental, Marrakech-Safi, and Fez-Meknès between 2021 and 2022.These serum samples were screened using a commercial indirect ELISA ARV antibody test kit (IDEXX REO). The study found that all tested flocks were positive for ARV-specific antibodies, indicating that the virus was present in these flocks. Out of the 826 serum samples tested, 782 were positive for ARV-specific antibodies. The overall prevalence of ARV infections in breeder and broiler flocks was calculated to be 94.6% ± 0.78. To summarize, the current study provides evidence of the widespread distribution of ARV infections in Morocco, suggesting that the poultry industry in the country is highly infected with ARV.

Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum in Poultry: Review of Pathogenesis, Antibiotic Resistance, Diagnosis and Control in the Genomic Era.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) has two distinct biovars, Pullorum and Gallinarum. They are bacterial pathogens that exhibit host specificity for poultry and aquatic birds, causing severe systemic diseases known as fowl typhoid (FT) and Pullorum disease (PD), respectively. The virulence mechanisms of biovars Gallinarum and Pullorum are multifactorial, involving a variety of genes and pathways that contribute to their pathogenicity. In addition, these serovars have developed resistance to various antimicrobial agents, leading to the emergence of multidrug-resistant strains. Due to their economic and public health significance, rapid and accurate diagnosis is crucial for effective control and prevention of these diseases. Conventional methods, such as bacterial culture and serological tests, have been used for screening and diagnosis. However, molecular-based methods are becoming increasingly important due to their rapidity, high sensitivity, and specificity, opening new horizons for the development of innovative approaches to control FT and PD. The aim of this review is to highlight the current state of knowledge on biovars Gallinarum and Pullorum, emphasizing the importance of continued research into their pathogenesis, drug resistance and diagnosis to better understand and control these pathogens in poultry farms.

Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021.

The H9N2 virus continues to spread in wild birds and poultry worldwide. At the beginning of 2016, the H9N2 Avian influenza virus (AIV) was detected in Morocco for the first time; despite the implementation of vaccination strategies to control the disease, the virus has become endemic in poultry in the country. The present study was carried out to investigate the origins, zoonotic potential, as well as the impact of vaccination on the molecular evolution of Moroccan H9N2 viruses. Twenty-eight (28) H9N2 viruses collected from 2016 to 2021 in Moroccan poultry flocks were isolated and their whole genomes sequenced. Phylogenetic and evolutionary analyses showed that Moroccan H9N2 viruses belong to the G1-like lineage and are closely related to viruses isolated in Africa and the Middle East. A high similarity among all the 2016-2017 hemagglutinin sequences was observed, while the viruses identified in 2018-2019 and 2020-2021 were separated from their 2016-2017 ancestors by long branches. Mutations in the HA protein associated with antigenic drift and increased zoonotic potential were also found. The Bayesian phylogeographic analyses revealed the Middle East as being the region where the Moroccan H9N2 virus may have originated, before spreading to the other African countries. Our study is the first comprehensive analysis of the evolutionary history of the H9N2 viruses in the country, highlighting their zoonotic potential and pointing out the importance of implementing effective monitoring systems.

Pathogenicity of field strain of fowl aviadenovirus serotype 11 isolated from chickens with inclusion body hepatitis in Morocco.

Outbreaks of inclusion body hepatitis have emerged in Morocco since 2013 and has resulted in significant economic losses to poultry farms. Three isolates of the causative virus, Fowl adenonovirus (FAdV)were characterized from chickens with IBH, but their pathogenicity has never been investigated. In this work, the pathogenicity of an isolate FAdV 11 (MOR300315 strain) was evaluated by inoculating a group of 40 SPF chickens at 3 days of age by oral route. A group of 40 chicks injected with phosphate-buffered saline solution was used as a control group. The infected chickens showed decreased weight gain from 3dpi. Necropsy displayed pallor and enlargement in liver, swelling and slight hemorrhage in kidney and spleen at 6 dpi. Histopathological changes were mainly characterized by severe and extensive hepatic necrosis associated with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The FAdV was reisolated in chicken embryo fibroblast cell culture from liver tissue homogenate of infected chicken from 3 to 6 dpi. Viral DNA was detected by PCR in liver, kidney, spleen and cloacal swabs from 3 to 13 dpi. Antibody response against inoculated FAdV was appeared from 9 dpi. These results confirmed that the FAdV 11 strain is pathogenic in chicken. This study is the first experimental infection of FAdV 11 in chicken in Morocco, which increase our understanding of its pathogenicity in chickens and indicate that preventive measures against FAdV infection in poultry farms should be implemented in Morocco.