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.

Prevalence of major infectious diseases in backyard chickens from rural markets in Morocco.

Background and Aim: Raising backyard chickens is a common practice in Morocco, mainly in rural or periurban areas. Constraints due to devastating avian diseases have been recognized as a major limiting factor in backyard poultry production. Consequently, these flocks could potentially be implicated as reservoirs for poultry diseases. However, there is a considerable lack of information on disease prevalence in this production system, and the risk represented by these small flocks remains under debate. This study aimed to estimate the seroprevalence and identify related risk factors of a range of bacterial and viral pathogens of outstanding importance for the economy and public health in backyard poultry in Morocco. Materials and Methods: A total of 712 sera samples and 258 cloacal swabs were collected from 712 backyard chickens from 15 rural markets in the Khemisset and Skhirat-Temara provinces. None of the sampled chickens received any vaccination. Sera samples were screened for antibodies against Newcastle disease virus (NDV) and low pathogenic avian influenza H9N2 subtype (LPAI H9N2) using a hemagglutination-inhibition test, against bursal infectious disease virus (IBDV) and infectious bronchitis virus (IBV) using enzyme-linked immunosorbent assay, and against Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using a rapid serum agglutination test. Swab samples were compiled into 86 pools and submitted for molecular detection using real-time reverse-transcription-polymerase chain reaction (RT-PCR). Results: The seroprevalences in backyard chickens for NDV, LPAI H9N2, IBDV, IBV, MG, and MS were 52.1% (371/712), 63.5% (452/712), 84.7% (603/712), 82.2% (585/712), 58% (413/712), and 74.8% (533/712), respectively. Based on the RT-PCR results, 2.3% (2/86), 62.8% (54/86), 2.3% (2/86), 63.9% (55/86), 40.7% (35/86), and 29.1% (25/86) of the pools were positive for NDV, H9N2 LPAI, IBDV, IBV, MG, and MS, respectively. Multiple coinfections (H9N2-IBV-MG), (H9N2-IBV-MS), or (IBV-MG-MS) were observed in 15.1%, 8.5%, and 8.5% of the tested samples, respectively. Conclusion: The results show that backyard chicken flocks and rural markets have the potential to serve as reservoirs or amplifiers for poultry pathogens and could pose a risk to the commercial poultry sector. This highlights the need for a comprehensive and adapted vaccination plan for backyard chickens, and extension of efforts to increase flock owners' awareness of avian diseases and incite the implementation of biosecurity measures at the farm level.

Backyard Poultry Flocks in Morocco: Demographic Characteristics, Husbandry Practices, and Disease and Biosecurity Management.

Backyard poultry farming is an important tool for poverty alleviation and food security in rural areas of Morocco. A descriptive epidemiologic survey was conducted in 286 backyard poultry flocks from the provinces of Khemisset and Skhirat-Temara to gain baseline data on the current status of backyard poultry flocks in Morocco as well as its potential implications on the transmission and spread of avian diseases. The findings indicated that 88.8% of flocks were raised in a mixed confinement system, with an average flock size of 30 birds (range 1-352). Chickens accounted for 83% of the overall reported birds. More than two-thirds of respondents (69%) kept chickens only, while the remaining flocks raising multiple bird species in total promiscuity. Diseases were the highest cause of mortality (84.7%), followed by predation (15.3%). According to 56.1% of the owners, respiratory symptoms were among the major disease signs reported, besides ectoparasite infestation. Flock health management revealed a lack of preventive vaccination, lack of veterinary consulting, lack of biosecurity practices, and irrational self-medication of diseased birds using antibiotics, pesticides, and hazardous chemicals that could be a significant health risk for consumers. The need for an outreach program about disease prevention and biosecurity practices, along with prophylactic campaigns, should be emphasized to further mitigate the risks of backyard poultry flocks on the commercial sector and public health.

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.

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.

Canine Leishmaniasis in Morocco: A Descriptive Prospective Clinical Study.

Canine leishmaniasis (CanL) is a zoonotic vector-borne disease that is endemic in the Mediterranean Basin including Morocco. Dogs play a major epidemiological role in this zoonosis as reservoir hosts. This study investigated the clinical manifestations of CanL in dogs naturally infected with Leishmania infantum. A total of 96 dogs presented to the Small Animal Clinic of the Hassan II Agronomy and Veterinary Institute (IAV Hassan II) of Rabat, Morocco, and were tested by RT-PCR and/or serology. Among them, 32 (33.3%) were positive to Leishmania infantum infection. The majority of the positive dogs (93.7%) came from urban areas. Most of them were male (62.5%) and purebreds (65.6%), were aged between 3 and 7 years (71.8%), and had outside activities (guarding, hunting, livestock guarding, and service activities) (71.8%) and all of them were living exclusively outdoor or had free access to the outdoor environment. Lymphadenomegaly (81.2%), dermatological disorders (65.6%) (mostly exfoliative dermatitis), weight loss (59.3%), exercise intolerance (56.2%), anorexia (28.1%), hyporexia (15.6%), and ocular lesions (28.1%) were the most frequent clinical signs and complaints recorded. Anemia and hyperproteinemia due to hyperglobulinemia were observed in 68.7% and 72.7% of the cases, respectively. These results suggest that CanL leads to various nonspecific clinical signs as described previously, making the diagnosis challenging. Since CanL is endemic in Morocco, it should be recommended to systematically test dogs displaying clinical signs compatible with this disease and to regularly screen asymptomatic at-risk dogs. It is also crucial to educate dog owners about the zoonotic aspect of the disease and to encourage intersectorial collaboration following the "One Health" concept, in order to contribute to a more effective control/prevention of human and canine leishmaniasis.

Using geostatistics to better understand the epidemiology of animal rabies in Morocco: what is the contribution of the predictive value?

This study aims to characterize the spatial distribution of animal rabies in Morocco in order to provide appropriate control approaches. Descriptive analyses of the epidemiological data show that the number of reported canine rabies cases greatly underestimates the true incidence of the disease. Underreporting subsequently affects the coherence of its spatial distribution. To perform accurate geographic distribution mapping of the disease based on interpolation methods, a data set was created using data between 2000 and 2018 to compare the derived disease cases with known true values in order to identify disease clusters. The subsequent interpolation was conducted using Ordinary Kriging regression methods and the semi variogram to focus on short distances and reduce uncertainty. The estimated clusters of rabies were evaluated using a cross validation step which revealed predicted cases close to the true values. To improve the precision of analysis, the authors displayed georeferenced dog and human rabies cases reported during the last three years, demonstrating reliable results that correspond to the estimated cluster areas similar to the true disease incidence on the field. This work highlights a strong correlation between infrastructure projects (i.e. railways, roads, facilities) and rabies epizootics for several specific locations. This study is the first attempt to use geostatistics to build upon the understanding of animal rabies in Morocco and shed light on the most appropriate strategies to sustainably reduce and mitigate the risk of rabies. There has been little literature on the use of kriging methods in animal health research. Thus, this study also aimed to explore a novel method in the veterinary sciences to establish kriging as a valid and coherent analysis tool to identify the extent to which the geostatistic area can objectively support understanding on animal rabies and saw it as being highly instrumental in coping with gaps in the data.

Evaluation of antidiabetic effect of Cistus salviifolius L. (Cistaceae) in streptozotocin-nicotinamide induced diabetic mice.

OBJECTIVES: Cistus salviifoluis L. is a shrub from Cistaceae family used in many traditional medicines for the treatment of various diseases including diabetes mellitus. The aim of this study was to evaluate the in vivo antidiabetic potential of the aerial parts aqueous extract of Cistus salviifolius L. (CSA). METHODS: Experimental diabetes was induced in adult male mice by intra-peritoneal injection of Streptozotocin-nicotinamide (STZ-NC). CSA at a dose of 500 mg/kg was administered daily to the diabetic mice for four weeks. The effect of the extract on hyperglycemia, body weight, serum total cholesterol, triglycerides, hepatic and renal functional markers were determined. Histopathological examination of the mice pancreas was also performed. The diabetic animals treated with CSA were compared with animals treated by the standard drug metformin. RESULTS: Treatment with CSA showed a significant reduction in blood glucose, total triglycerides and creatinine levels and prevented the reduction of body weight caused by diabetes. Furthermore, histopathological analysis of the mice pancreas showed that the group treated with CSA reduced damage induced by STZ-NC on islets of Langerhans cells when compared to the diabetic control. CONCLUSIONS: The results suggest that the aqueous extract of Moroccan C. salviifolius L. possesses beneficial effect on treatment of diabetes.

Isolation and characterization of fowl aviadenovirus serotype 11 from chickens with inclusion body hepatitis in Morocco.

The present study was conducted in order to isolate, identify and characterize fowl aviadenovirus associated with inclusion body hepatitis (IBH) in three poultry farms (two of broiler chickens and one of breeder broiler chickens) in Morocco during 2015. Liver samples collected from affected three poultry farms were examined by histopathological examination. Tissue samples showing necrosis of hepatocytes associated with basophilic intranuclear inclusion bodies were homogenized and submitted to FAdV isolation in chicken embryo fibroblast (CEF) cell cultures and in SPF embryonated eggs. The cytopathic effect (CPE) was observed in the second passage with swelling and rounding of infected cells. The inoculated embryos were hemorrhagic and showed hepatitis with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The presence of the virus was confirmed by conventional polymerase chain reaction based on hexon gene from all investigated samples. Moreover, phylogenetic analysis of the hexon gene revealed that FAdVs isolated from different affected poultry belonged to FAdV 11 serotype of the D genotype group. This work is the first isolation in cell culture and SPF embryonated eggs of FAdV from Moroccan broilers and breeder broiler chickens with IBH.

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform.

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.

Efficacy of Massachusetts and 793B Vaccines Against Infectious Bronchitis Moroccan-Italy 02 Virus in Specific-Pathogen-Free Chickens and Commercial Broilers.

The ability of commercial vaccines H120 and 4/91 to protect against Moroccan-Italy 02 infectious bronchitis virus (Mor-It02) was investigated in specific-pathogen-free (SPF) chickens and commercial broiler chickens. Commercial broiler chicks (Experiment 1) were vaccinated at the hatchery with H120 vaccine at Day 1, and challenged at Day 21 with 104 50% egg-infective dose (EID50) of Mor-It02. All chicks were observed daily for clinical signs attributable to Mor-It02 infection during the 10 days postchallenge (pc). At 5 and 10 days pc, chicks were humanely sacrificed for necropsy examination, and tissues were collected for histopathology evaluation. To better understand the findings on commercial broilers, day-old SPF chicks were divided into five groups in a second experiment: Group Mass/4-91, vaccinated with H120 and 4/91 respectively at Days 1 and 15 of age; Group Mass/Mass, vaccinated by H120 at Days 1 and 15; Group Mass, vaccinated with H120 at Day 1; Group NV, kept unvaccinated; and Group NC, kept as a negative control (unchallenged). At Day 24 of age, Groups Mass/4-91, Mass/Mass, Mass, and NV were challenged with 104 EID50 of Mor-It02. In both experiments, blood samples were collected at different periods for serologic analyses. Oropharyngeal swabs were collected for virus detection by reverse-transcription PCR. In Experiments 1 and 2, respiratory signs started as early as 24 hr pc and maximum severity was observed on Days 3 and 4 pc. The viral shedding rate was significantly lower in Group Mass/4-91 compared to other challenged groups. Serologic analysis in both experiments showed that the sera of challenged group exhibited significantly higher antibody titers than sera collected before challenge. Histopathologic investigations in SPF birds showed deciliation and hyperplasia in Group NV and less-pronounced lesions in Groups Mass/Mass and Mass. In commercial broilers vaccinated with H120 alone, hyperplasia and deciliation were observed in 90% of the tracheas. These experiments illustrated that Mor-It02 is pathogenic for chickens and a combination of live H120 and 4/91 vaccines given respectively at Day 1 and Day 15 of age confer a good protection against Mor-It02.

Assessment of pathogenicity and tissue distribution of infectious bronchitis virus strains (Italy 02 genotype) isolated from moroccan broiler chickens.

BACKGROUND: Avian infectious bronchitis (IB) is one of the most important viral diseases of poultry, affecting chickens of all ages and causing major economic losses in poultry flocks. Mass vaccination is conducted in Morocco using a vaccine against Massachusetts, which is the most dominant serotype; however no information about the pathogenesis and tissue distribution of the Moroccan Italy 02 genotype was reported. 40 one-day-old specific pathogen free chickens were divided randomly into four groups. Group1, 2 and 3 were inoculated intra oculo-nasally with 103.5 EID50 of Italy02 viruses, and group 4 was kept as control. Chickens in each group were monitored for 14 days post-infection (pi). RESULTS: Chickens in all infected groups showed severe respiratory signs, which most of them have been reproduced on 2dpi, with varying times of appearance and disappearance. The infected birds appeared lethargic, reluctant to move, with specific respiratory clinical signs and macroscopic lesions. However no nephritis lesions or mortality were recorded in all groups. The specific histological lesions finding in all infected birds, exhibited tracheal lesions with mucosal thickening, hyperplasia of the surface epithelium, mononuclear inflammatory cell infiltrate of lamina propria. Primary and secondary bronchi, epithelial hyperplasia and mononuclear inflammatory cell infiltrate of the lamina propria were also observed. Tracheal lesions developed in all infected birds, confirm the ability of the three tested strains to induce respiratory disease. The results at 14 dpi also revealed that all strains were able to induce serological response. Virus re-isolation from infected organs and amplification of the viral RNA by real-time PCR proved the presence of the virus in lung and trachea of infected chicks. Neither re-isolation nor significant viral RNA detection were detected in the kidney. CONCLUSION: The results demonstrated that the three strains Italy02 genotype emerging in Moroccan poultry farms have a wide distribution for respiratory system, without kidney damage and without causing mortality.