BotCl, the First Chlorotoxin-like Peptide Inhibiting Newcastle Disease Virus: The Emergence of a New Scorpion Venom AMPs Family.

Newcastle disease virus (NDV) is one of the most serious contagions affecting domestic poultry and other avian species. It causes high morbidity and mortality, resulting in huge economic losses to the poultry industry worldwide. Despite vaccination, NDV outbreaks increase the need for alternative prevention and control means. In this study, we have screened fractions of Buthus occitanus tunetanus (Bot) scorpion venom and isolated the first scorpion peptide inhibiting the NDV multiplication. It showed a dose dependent effect on NDV growth in vitro, with an IC50 of 0.69 µM, and a low cytotoxicity on cultured Vero cells (CC50 > 55 µM). Furthermore, tests carried out in specific pathogen-free embryonated chicken eggs demonstrated that the isolated peptide has a protective effect on chicken embryos against NDV, and reduced by 73% the virus titer in allantoic fluid. The N-terminal sequence, as well as the number of cysteine residues of the isolated peptide, showed that it belongs to the scorpion venom Chlorotoxin-like peptides family, which led us to designate it "BotCl". Interestingly, at 10 µg/mL, BotCl showed an inhibiting effect three times higher than its analogue AaCtx, from Androctonus australis (Aa) scorpion venom, on NDV development. Altogether, our results highlight the chlorotoxin-like peptides as a new scorpion venom AMPs family.

Full-length genome sequences of the first H9N2 avian influenza viruses isolated in the Northeast of Algeria.

BACKGROUND: H9N2 avian influenza viruses (AIV) has a worldwide geographic distribution and affects poultry of different types of production. H9N2 AIV was first reported in the Northeast of Algeria in April 2017, following an outbreak associated with high mortality, in broiler flocks. In the present study, we report full-length genome sequences of AIV H9N2, and the detailed phylogeny and molecular genetic analyses. METHODS: Ten AIV H9N2 strains, collected in broiler flocks, were amplified in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. Their full-length genomes were successfully sequenced and phylogenetic and molecular characterizations were conducted. RESULTS: Phylogenetic analysis showed that the isolates were monophyletic, grouped within the G-1 lineage and were very close to Moroccan and Algerian strains identified in 2016 and 2017, respectively. The low pathogenicity of the strains was confirmed by the sequence motif (335RSSR/GLF341) at the hemagglutinin (HA) cleavage site. An exclusive substitution (T197A) that had not been previously reported for H9N2 viruses; but, conserved in some pandemic H1N1 viruses, was observed. When compared to the G1-like H9N2 prototype, the studied strains showed one less glycosylation site in HA, but 2-3 additional ones in the stalk of the neuraminidase (NA). The HA protein harbored the substitution 234 L, suggesting binding preference to human-like receptors. The NA protein harbored S372A and R403W substitutions, previously detected in H9N2 from Asia and the Middle East, and especially in H2N2 and H3N2 strains that caused human pandemics. Different molecular markers associated with virulence and mammalian infections have been detected in the viral internal proteins. The matrix M2 protein possessed the S31N substitution associated with drug resistance. The non-structural 1 (NS1) protein showed the "GSEV" PDZ ligand (PL) C-terminal motif and no 80-84 deletion. CONCLUSION: Characterized Algerian AIV isolates showed mutations that suggest increased zoonotic potential. Additional studies in animal models are required to investigate the pathogenicity of these H9N2 AIV strains. Monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. Implementation of adequate biosecurity measures that limit the introduction and the propagation of AIV H9N2 in Algerian poultry farm is crucial.

Newly detected mutations in the Meq oncogene and molecular pathotyping of very virulent Marek's disease herpesvirus in Tunisia.

Marek's disease (MD) is a contagious avian viral disease that is responsible for large economic losses to farmers. The disease is caused by Marek's disease virus (species Gallid alphaherpesvirus 2), which causes neurological lesions, immune suppression, and tumor proliferation of lymphoid cells that invade a large number of organs and tissues. Despite widespread vaccination, Marek's disease virus (MDV), has shown a continuous increase in its virulence and has acquired the ability to overcome immune responses induced by vaccines. In the present study, the oncogenic serotype MDV-1 was detected by real-time PCR in DNA samples extracted from organs developing tumor infiltrations. Identification of the pathotype based on a 132-bp tandem repeat and sequencing and phylogenetic analysis of the Meq gene and its encoded protein allowed classification of the isolated viruses as "very virulent", with two new and unique mutations in the Meq gene resulting in amino acid substitutions. Sequencing of pp38, vIl-8, UL1 and UL44 genes did not reveal any new mutations that were characteristic of the Tunisian isolates or correlated with virulence. These results raised concerns about the ability of HVT and CVI988 vaccines, which are currently used in Tunisia and other countries, to protect chickens against highly virulent virus strains.

Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization.

During 2009-2012, several outbreaks of avian influenza virus H9N2 were reported in Tunisian poultry. The circulating strains carried in their hemagglutinins the human-like marker 226L, which is known to be important for avian-to-human viral transmission. To investigate the origins and zoonotic potential of the Tunisian H9N2 viruses, five new isolates were identified during 2012-2016 and their whole genomes were sequenced. Bayesian-based phylogeny showed that the HA, NA, M and NP segments belong to the G1-like lineage. The PB1, PB2, PA and NS segments appeared to have undergone multiple intersubtype reassortments and to be only distantly related to all of the Eurasian lineages (G1-like, Y280-like and Korean-like). The spatiotemporal dynamic of virus spread revealed that the H9N2 virus was transferred to Tunisia from the UAE through Asian and European pathways. As indicated by Bayesian analysis of host traits, ducks and terrestrial birds played an important role in virus transmission to Tunisia. The subtype phylodynamics showed that the history of the PB1 and PB2 segments was marked by intersubtype reassortments with H4N6, H10N4 and H2N2 subtypes. Most of these transitions between locations, hosts and subtypes were statistically supported (BF > 3) and not influenced by sampling bias. Evidence of genetic evolution was observed in the predicted amino acid sequences of the viral proteins of recent Tunisian H9N2 viruses, which were characterized by the acquisition of new mutations involved in virus adaptation to avian and mammalian hosts and amantadine resistance. This study is the first comprehensive analysis of the evolutionary history of Tunisian H9N2 viruses and highlights the zoonotic risk associated with their circulation in poultry, indicating the need for continuous surveillance of their molecular evolution.

Inhibition of HSV-2 infection by pure compounds from Thymus capitatus extract in vitro.

Thymus capitatus represents 1 of the 5 Tunisian species of the genus Thymus, which has long­standing use for flavouring and preserving several food products. Its constituents have been reported to endow antimicrobial properties, but little is known about their antiviral activities. The aim of this study was to examine the antiviral activity of pure compounds from the most bioactive inhibitory T. capitatus extract in vitro against herpes simplex virus Type 2 (HSV­2) infection and to identify their mechanism of action. Either the extracts or the essential oil exert inhibitory activity against HSV­2 infection, with the ethanolic extract showing the lowest EC50 value (2.3 µg/ml). Three pure compounds were then isolated from the ethanolic extract and investigated for their antiviral activity. ß­sitosterol showed the most favourable selectivity index and both cinnamaldehyde and carvacrol exerted moderate antiviral effect. Investigation of the mechanism of action revealed that all three compounds directly inactivated the infectivity of the virus particles. These findings suggest the use of T. capitatus ethanolic extract as source of anti­HSV­2 pure compounds and warrant further studies to evaluate their therapeutic potential.

Accurate Detection of Avian Respiratory Viruses by Use of Multiplex PCR-Based Luminex Suspension Microarray Assay.

A novel oligonucleotide suspension microarray (Luminex microsphere system) was developed for the rapid detection of avian respiratory viruses of major clinical importance. This test was optimized and validated with 70 clinical samples. The developed tool was accurate for high-throughput detection and differentiation of the most important avian respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) in single- and mixed-virus infections. A multiplex reverse transcriptase PCR (RT-PCR), followed by a monoplex or a multiplex Luminex assays, were realized using a Luminex 200 analyzer instrument. The sensitivity, specificity, and reproducibility of the multiplex DNA suspension microarray system were evaluated. The results showed no significant differences in the median fluorescence intensity (MFI) value in monoplex and multiplex Luminex assays. The sensitivity and specificity proved to be completely concordant with monoplex real-time RT-PCR. We demonstrated that the multiplex DNA suspension microarray system is an accurate, high-throughput, and relatively simple method for the rapid detection of the main respiratory viruses of poultry.

Variability of tropism and replicative capacity of two naturally occurring influenza A H9N2 viruses in cell cultures from different tissues.

Studies carried out on cell permissivity are of great interest to understand virus replication and pathogenicity. We described the results of a comparative analysis of replication efficiency of two naturally occurring influenza A H9N2 variants isolated from poultry and wild birds, differing by only two substitutions Q226L and T384N, in the receptor-binding site of haemagglutinin and the 380 loop region of NA proteins, respectively. Considering the overall growth of both viruses, lung cultures ensured the most efficient growth of TUN12L226N384 strain with titres up to 10(9) TCID50/ml whereas small intestine culture was highly susceptible to the TUN51Q226T384 virus reaching a titre of 10(6) TCID50/ml. The lowest replication was shown in liver cells. The addition of trypsin was essential for the replication of either virus in primary fibroblasts, but it had a marginal positive effect on virus replication in the four other culture types with maximum titres of 10(8) TCID50/ml. This means that in chicken, the proteolytic activation of the H9N2 viruses with the cleavage motif RSSR may be mediated by other endoproteases than trypsin. Further investigations should concentrate on the production of the appropriate set of viruses by a reverse genetics approach and the examination of cellular protease expression in chicken tissues. This would lead to a more complete understanding of the tropism of low-pathogenic Influenza A viruses.

Production of a truncated recombinant HA1 for influenza A H9 subtype screening.

Hemagglutinin is the major component of membrane protein and plays a major role in virus entry into host cells through their receptors and it is predicted to elicit the production neutralizing antibodies. Our aim is to assess the potential of a truncated rHA1 domain, encoding residues 157-260 to detect influenza A H9 specific antibodies. The predicted characteristics of this protein revealed that it is a hydrophobic protein possessing predominant antigenicity and composed of random coils (48%) and extended strand (28%) but few α-helix (6.33%) and ß-sheet (7%). A 312 pb HA1 gene was amplified and cloned in pET23b(+) vector including an C-terminal polyHis as a fusion partner, transformed and expressed in Escherichia coli cells as inclusion bodies. The truncated protein was solubilized with 8 M urea, purified by immobilized metal affinity chromatography and then detected by western blot with anti-His and H9-specific polyclonal antibodies. The test demonstrated high specificity (100%) and sensibility (98%). The immunoreactivity of the truncated rHA1 assessed revealed that only antisera against H9 yielded a specific and strong reactivity, with no cross-reactivity against negative sera. This study demonstrates that the truncated rHA1 may serve as a useful tool for rapid and easy surveillance of H9 infection.

Simultaneous alteration of residues 279 and 284 of the VP2 major capsid protein of a very virulent Infectious Bursal Disease Virus (vvIBDV) strain did not lead to attenuation in chickens.

BACKGROUND: Cell culture adaptation of very virulent infectious bursal disease virus (vvIBDV) was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284. The single mutation A284T proved critical for cell culture tropism, but did not confer efficient virus replication, which at least required one additional mutation, Q253H or D279N. While the double mutation Q253H/A284T was unambiguously shown to confer both efficient replication in cell culture and attenuation in chickens, conflicting results have been reported regarding the replication efficiency of vvIBDV mutants bearing the D279N/A284T double mutation, and no data are hitherto available on their virulence in chickens. FINDINGS: Here we used an in vivo reverse genetics system to assess the impact of the D279N/A284T double mutation on the replication and attenuation of a chimeric IBDV virus, whose polyprotein derived from a non-culturable vvIBDV clinical isolate. We found that the D279N/A284T double mutation did indeed confer efficient replication in chicken embryo fibroblast (CEF) cell culture, but the mutant virus remained highly pathogenic to chickens. CONCLUSIONS: The double mutation D279N/A284T of the VP2 major capsid protein of vvIBDV is sufficient to confer cell culture tropism and replication efficiency, but does not necessarily lead to virus attenuation.

Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review.

In recent years there has been enormous interest in researching oxidative stress. Reactive oxygen species (ROS) are derived from the metabolism of oxygen as by-products of cell respiration, and are continuously produced in all aerobic organisms. Oxidative stress occurs as a consequence of an imbalance between ROS production and the available antioxidant defence against them. Nowadays, a variety of diseases and degenerative processes such as cancer, Alzheimer's and autoimmune diseases are mediated by oxidative stress. Heat stress was suggested to be an environmental factor responsible for stimulating ROS production because of similarities in responses observed following heat stress compared with that occurring following exposure to oxidative stress. This manuscript describes the main mitochondrial sources of ROS and the antioxidant defences involved to prevent oxidative damage in all the mitochondrial compartments. It also deals with discussions concerning the cytotoxic effect of heat stress, mitochondrial heat-induced alterations, as well as heat shock protein (HSP) expression as a defence mechanism.

Non-cytotoxic Thymus capitata extracts prevent Bovine herpesvirus-1 infection in cell cultures.

BACKGROUND: Bovine herpes virus type 1 (BHV-1) still causes great economic loss to the livestock industry and trade because there aren't any available drugs that proved to be fully effective against it. In this study, the cytotoxicity and the antiviral activities of the Thymus capitata extracts were evaluated for the development of new, non toxic and specific anti-herpesvirus drug. Aqueous extracts (AE), ethanolic extracts (EE) and essential oil (EO) of the aerial parts of Thymus capitata were analyzed to determine their chemical compositions by gas chromatography, and high performance liquid chromatography combined with mass spectrometry. Their cytotoxicity and antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by quantifying the reduction of the viral cytopathic effect using Madin-Darby Bovine Kidney cell line with colorimetric assay. T. capitata extracts were added at different stages of the viral infection to investigate and better quantify their potential inhibitory effects. RESULTS: Polyphenols and flavonoids were the major compounds found in T. capitata EO, EE and AE. The cytotoxic concentrations at 50% were 48.70, 189 and 289 µg ml(-1) for EO, EE and AE, respectively. The inhibitor concentrations at 50% for the EO, EE and AE, were 3.36, 47.80 and 164 µg ml(-1), respectively. The selectivity index anti-BHV-1 values were 14.49, 3.95 and 1.81 for EO, EE and AE, respectively. Thus, the EO extracts were the most efficient antiviral compounds. T. capitata extracts affect mainly the adsorption of BHV-1 virus to host cells. CONCLUSION: T. capitata extracts inhibit the viral replication by interfering with the early stages of viral adsorption and replication. Thus, T. capitata is a potential candidate for anti-herpesvirus treatment.

Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

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.

Infection dynamics of subtype H9N2 low pathogenic avian influenza a virus in turkeys.

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems.

Molecular characterization of avian reovirus isolates in Tunisia.

BACKGROUND: Genotype analyses of avian reoviruses isolated from organ samples collected from chickens with suspicious clinical symptoms, between 1997-2008, was based on sequences for both σC and σB genes and aligned with those published in the Genbank, making it possible to carry out studies of molecular classification and relationships. METHODS: The full length of the known variable protein σC and part of the σB encoding genes, were amplified with RT-PCR, using conserved primers. PCR products were sequenced and the sequences were analyzed and aligned with avian reovirus sequences from the Genbank database. RESULTS: The sequences of σC-encoding genes of all the isolated strains indicated their close relationship with the American, Chinese and Indian strains. Taking the American strain S1133 as a reference, the two Tunisian isolates 97.1 and 97.2 showed some nucleotide substitutions. For isolate 97.1, the substitution was silent whereas for strain 97.2 the mutation was at the first position of the corresponding codon and induced the substitution of the amino acid encoded. For the σB-encoding gene, the sequences of the Tunisian strains showed mutations at positions two or three of the corresponding codons, inducing substitutions of amino acids at these positions. The phylogenic trees based on σC and σB encoding genes indicated closer relationship between Tunisian, American and Taiwanese isolates of genotype I. CONCLUSION: Our study describes the genotype of avian reoviruses that are not yet well characterized genetically. The characterization and classification of these viruses might be significant for understanding the epidemiology of malabsorption syndrome and viral arthritis, and improving our knowledge of the genotype of strains circulating in Tunisian flocks. Furthermore, the study of their variable pathogenicity could be extremely important in the choice of the appropriate vaccine strain to control disease.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus.

Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

Phylogenetic and phylodynamic analyses of subtype-B metapneumovirus from chickens in Tunisia.

Swollen Head Syndrome (SHS) is an economically important viral disease of chickens caused by avian metapneumovirus (aMPV). The virus comprises 6 different subtypes (A,B,C,D, New-1 and New-2). To date, no information was available on the presence of the virus in Tunisian poultry. The present work aims to detect the presence of (aMPV) in broiler chicken in Tunisia, then to characterise the isolates in order to determine their subtype and to estimate their geographic origin of introduction. A total of 289 samples were collected, aMPV detection was detected by real time RT-PCR and molecular characterization was warried out by Sanger sequencing on the glycoprotein (G) gene. Phylogenetic analysis was carried out using Beast 2 software. Out of the 289 samples, 21 were revealed positive to aMPV. Only 2 isolates have been confirmed by sequencing analysis ; one isolate sampled in 2015 and another in 2019. Based on the partial G gene sequence, analysis of these 2 Tunisian isolates showed that they belong to subtype B. The isolate sampled in 2015, appeared to be phylogenetically related to derived vaccine strain. However, the one sampled in 2019 appeared to be a field strain. Phylodynamic analysis provided evidence that this field strain derived from a Spanish strain and probably the virus has been introduced from Spain to North Africa back in 2016. This study is the first that highlighted the circulation of (aMPV) in Tunisia. It is possible that aMPV has been circulating in Tunisia and neighboring countries without being detected. Also, multiple strains could be present and therefore multiple introductions have happened. Through this study, we shed the light on the importance of reinforcing farms biosecurity as well as virological surveillance.

MBT/Pas mouse: a relevant model for the evaluation of Rift Valley fever vaccines.

Currently, there are no worldwide licensed vaccines for Rift Valley fever (RVF) that are both safe and effective. Development and evaluation of vaccines, diagnostics and treatments depend on the availability of appropriate animal models. Animal models are also necessary to understand the basic pathobiology of infection. Here, we report the use of an inbred MBT/Pas mouse model that consistently reproduces RVF disease and serves our purpose for testing the efficacy of vaccine candidates; an attenuated Rift Valley fever virus (RVFV) and a recombinant RVFV-capripoxvirus. We show that this model is relevant for vaccine testing.

Thymus capitatus flavonoids inhibit infection of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpes virus 8 (HHV-8), causes primary effusion lymphoma, multicentric Castleman's disease, and Kaposi's sarcoma. Few antiviral drugs are available to efficiently control KSHV infection, and therefore, the development of novel, effective anti-KSHV treatments is needed. The aim of this study was to determine the antiviral activity of ethanolic and aqueous extracts, essential oils, and certain flavonoids (hesperidin, eupafolin, and vicenin) derived from Thymus capitatus (commonly known as thyme). We assessed the toxicity of these different extracts and components in RPE-1 cell cultures using the MTS test and evaluated their antiviral effect using the TCID50 method. The mechanism of action was determined through time-of-addition tests as well as viral entry, attachment, and virucidal assays. Additionally, western blot analysis was also used to assess their modes of action. Total treatment assay showed that the aqueous extract of T. capitatus has the highest inhibitory effect against KSHVLYT with an EC50 value of 0.2388 µg·mL-1 . Both hesperidin and eupafolin showed the ability to inactivate viral infection in a dose-response manner (EC50 values of 0.2399 and 1.396 µm, respectively). Moreover, they were able to inactivate KSHVLyt postinfection by reducing viral protein expression. In summary, the effective antiviral property of the aqueous extract is likely a result of the inhibition of viral growth within the host cells by both hesperidin and eupafolin.

Distribution of virulence and antibiotic resistance genes in Campylobacter jejuni and Campylobacter coli isolated from broiler chickens in Tunisia.

BACKGROUND: Thermo-tolerant Campylobacter species are the major cause of foodborne diseases worldwide. This study aimed to evaluate the prevalence of virulence genes and antibiotic resistance determinants in Campylobacter jejuni and Campylobacter coli isolates, and to investigate the relationship between these two traits. METHODS: A total of 132 Campylobacter isolates from poultry were tested for the presence of 13 virulence genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB and ceuE. The mechanisms underlying antibiotic resistance phenotypes were also studied by PCR and MAMA-PCR. RESULTS: PCR results revealed the presence of antimicrobial resistance genes in C. jejuni and C. coli as follows: cmeB (80% and 100%), tet(O) (100% and 80%), and the blaOXA-61 (81% and 93%), respectively. None of these strains harbored the aphA-3 gene. The Thr-86-Ile mutation associated with resistance to quinolones was found in 90% of C. jejuni and 80% of C. coli isolates. While the A2075G and A2074C mutations linked to the erythromycin resistance were detected in 100% of both species. Virulence genes were prevalent and ranged from 40 to 100%. A positive relationship was revealed between cadF, racR, and ciaB genes and resistance to ampicillin, amoxicillin/clavulanic acid, chloramphenicol, and nalidixic acid, in C. jejuni. However, no association was observed for C. coli isolated strains. CONCLUSION: This study provides for the first time an overview of antibiotic resistance mechanisms and pathogenic profiles of Campylobacter isolates, which emphasizes the potential risk for consumer health.

Lineages, Virulence Gene Associated and Integrons among Extended Spectrum ß-Lactamase (ESBL) and CMY-2 Producing Enterobacteriaceae from Bovine Mastitis, in Tunisia.

Extended Spectrum Beta-Lactamase (ESBL) Enterobacteriaceae are becoming widespread enzymes in food-producing animals worldwide. Escherichia coli and Klebseilla pneumoniae are two of the most significant pathogens causing mastitis. Our study focused on the characterization of the genetic support of ESBL/pAmpC and antibiotic resistance mechanisms in cefotaxime-resistant (CTXR) and susceptible (CTXS) Enterobacteriaceae isolates, recovered from bovine mastitis in Tunisia, as well as the analyses of their clonal lineage and virulence-associated genes. The study was carried out on 17 ESBL/pAmpC E. coli and K. pneumoniae and 50 CTXS E. coli. Detection of resistance genes and clonal diversity was performed by PCR amplification and sequencing. The following ß-lactamase genes were detected: blaCTX-M-15 (n = 6), blaCTX-M-15 + blaOXA-1 (2), bla CTX-M-15 + blaOXA-1 + blaTEM-1b (2), blaCTX-M-15 + blaTEM-1b (4), blaCMY-2 (3). The MLST showed the following STs: ST405 (n = 4 strains); ST58 (n = 3); ST155 (n = 3); ST471 (n = 2); and ST101 (n = 2). ST399 (n = 1) and ST617 (n = 1) were identified in p(AmpC) E. coli producer strains. The phylogroups A and B1 were the most detected ones, followed by the pathogenic phylogroup B2 that harbored the shigatoxin genes stx1/stx2, associated with the cnf, fimA, and aer virulence factors. The qnrA/qnrB, aac(6')-Ib-cr genes and integrons class 1 with different gene cassettes were detected amongst these CTXR/S isolated strains. The presence of different genetic lineages, associated with resistance and virulence genes in pathogenic bacteria in dairy farms, may complicate antibiotic therapies and pose a potential risk to public health.