Circulation of a multi-resistance clone of bacteria associated with genetic elements in diseased animals constitutes a global public health problem. Our study focused on the characterization of the support of ESBL in cefotaxime resistant E. coli (CTX^R) isolates recovered from poultry with diarrhea, analysis of their clonal lineage, and virulence-associated genes. The study was carried out on 130 samples of chickens with diarrhea, collected in 2015 from poultry farms in Tunisia. Isolates of 20 CTX^R E. coli strains were identified as ESBL and AmpC β- lactamase producers. The following β-lactamase genes (number of isolates) were detected: bla_CTX-M-15+ bla_OXA1 (4), bla_CTX-M-15 + bla_OXA1 + bla_TEM-1b (2), bla_CTX-M-1 + bla_TEM-1b (9), bla_CTX-M-1 (2), bla_CMY2 + bla_TEM-1b (3). Six E. coli harboring bla_CTXM-15 were allocated to ST131-B2-O25b-; six and three bla_CTX-M-1 were grouped in ST155, ST10, and ST58, respectively, related to the phylogroup D and A. The qnrB gene, the variant aac(6′)-Ib-cr, and the class 1 integrons with different gene cassettes, were detected amongst our 20 isolated strains, which were classified as ExPEC and aEPEC. Our findings highlighted the emergence of the human pandemic ST131-CTX-M-15-O25-B2 clone and the high risk of such clonal lineage strains in diarrheic poultry, in Tunisia, which could constitute a risk of their transfer to healthy animals and humans.

H9N2 avian influenza virus (AIV) has been isolated from various species of wild birds and domestic poultry worldwide. It has been reported since the late 1990s, that H9N2 AIV has infected humans as reported in some Asian and North African countries. This subtype has already been circulating and constituting a serious threat to the poultry industry in Tunisia back in 2009. To investigate zoonotic potential and pathogenicity of H9N2 AIV in chickens and mice in Tunisia, five strains have been isolated during the period from 2014 to 2018. Samples were withdrawn from several wild bird species and environment (Lagoon water) of Maamoura and Korba Lagoons as well as Kuriat Island. Phylogenetic analyzes demonstrated that the isolated H9N2 strains belonged to the G1-like sublineage and were close to AIV H9N2 poultry viruses from North Africa, West Africa and the Middle East. All strains carried in their hemagglutinin the residue 226 L, which is an important marker for avian-to-human viral transmission. The hemagglutinin cleavage site has several motifs: PSKSSR/G, PARSSR/G and HARSSR/G. The neuraminidase showed S372A and R403W substitutions that have been previously detected in H3N2 and H2N2 viruses that were reported in human pandemics. Many mutations associated with mammalian infections have been detected in internal proteins. Pathogenicity evaluation in chickens showed that GF/14 replicates effectively in the lungs, tracheas, spleens, kidneys and brains and that it was transmitted among contact chickens. However, GHG/18 replicates poorly in chickens and has not an efficient transmission in contact chickens. GF/14 and GHG/18 could not kill mice though they replicated in their respiratory tract and caused a significant body weight loss (p < 0.05). This study highlights the importance of H9N2 AIV monitoring in both migratory birds and the environment to prevent virus transmission to humans.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

West Nile virus (WNV) is an arthropod borne virus, the most widely distributed of the encephalitic Orthoflaviviruses. It can cause severe neurological symptoms in both humans and horses. It poses an emerging threat to both public and animal health. In this study, we retrospectively screened 25 suspected WNV samples collected from 2018 to 2023. A total number of 25 samples (brain tissue and blood) were collected from clinically affected horses and those already deceased across several locations in Tunisia. All samples were tested for the presence of WNV NS2A gene using qRT-PCR. Eleven positive samples underwent virus isolation in Vero cells and partial sequencing of their envelope (E) glycoprotein gene. The resulting sequences were analyzed to gain molecular and phylogenetic insights. Eleven of the 25 (44%) samples were WNV positive and 11 partial E protein sequences were taken. The phylogenetic analysis revealed that all Tunisian isolates belonged to lineage 1a and were closely related to each other and formed a distinct group within the Mediterranean subtype of clade 1a. Tunisian WNV were characterized by the presence of a potential N-linked glycosylation site at residue 154–156 (Asn-Tyr-Ser) (NYS) and did not have the mutation E-I159V or E-I159A. Genomic monitoring of horses has revealed the circulation of WNV in several regions of Tunisia. Our findings highlight the critical need for prompt investigation of WNV infections in horses, as these cases may precede outbreaks of human cases in the country.

H9N2 avian influenza viruses (AIV) have been isolated from various species of wild birds and domestic poultry worldwide. They have been described to not only infect mammals directly, but also provide gene segments to generate novel but lethal human reassortants. H9N2 AIV has circulated in Tunisian commercial poultry since 2009 and poses a serious threat to the poultry industry. In the present study, we investigate five H9N2 viruses isolated from greater flamingoes (GF) (Phoenicopterus roseus), gray-headed gull (GHG) (Chroicocephalus crirrocephalus), black-winged stilt (BWS) (Himantopus himantopus), and lagoon water (EN) from Maamoura Lagoon, Korba Lagoon, and Kuriat Island in Tunisia from 2014–2018. Full-length genome sequences of AIV H9N2.Viral sequence analyses showed several amino acid changes related to viral adaptation to mammals. Phylogenetic analyses demonstrated that the five isolates belonged to the same sublineage G1and were close to AIV H9N2 poultry strains isolated in North Africa, West Africa, and the Middle East. At the hemagglutinin (HA) cleavage site, viruses (GF/120/15), (BSW/119/18), (GHG/216/18), and (EN/117/18) had the 335RSSR/GLF341 motif. However, (GF/121/14) undergo punctual substitutions at the HA1/HA2genesand acquires the dibasic pattern 335KSSR/GLF341.All Tunisian viruses carried in their hemagglutinins the human-like marker 226L, which is important for avian-to-human viral transmission. The neuraminidase (NA) of the five viruses showed S372A and R403W substitutions previously detected in H9N2 from Asia and the Middle East and in H3N2 and H2N2 strains reported in human pandemics. Many mutations associated with mammalian infections have been detected in internal proteins. Chicken experiments (IPIV) show that the five Tunisian viruses displayed low pathogenicity, causing mild symptoms in chickens. The virus could not kill mice, and merely replicated when administrated to mice. To our knowledge, this is the first study to address the evolution, pathogenicity, and transmission of the H9N2 viruses isolated from wild birds, waterfowl, and lagoon water in North Africa. Our results indicate that we should pay greater attention to H9N2 AIV surveillance in wilds birds. These results provide new insights into the measures that need to be undertaken regarding the role of wild birds in AIV introduction and persistence in Tunisia.