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Introduction: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in humans and is considered an important cause of food-borne diseases. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n = 160) and from five farms (n = 100). Methodology: E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey medium to detect non-sorbitol fermenting colonies. These bacteria were examined for the presence of O157:H7 antigen by latex agglutination. The isolation of E. coli O157:H7 has been confirmed with PCR amplification of rfbEO157 and fliCH7 specific genes for serogroup O157 and with multiplex PCR of stx1, stx2, eaeA, and ehxA. All isolates were examined for their susceptibility to 21 antibiotics by the disc diffusion method. Results: Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) and ehxA were present in 70% of isolates, stx1 and eae were confirmed in 60% of the isolates. Other virulence factors screened (fimH, sfa/focDE, cdt3, traT, iutA, and hlyA) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E. Conclusions: This is the first study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our finding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of food-borne disease.

Background Abortion is a serious problem for sheep flocks and it is responsible for considerable economic losses. The epidemiological situation of abortion causing agents in sheep is poorly documented in Tunisia. This study aims to investigate the status of three abortion causing agents (Brucella spp, Toxoplasma gondii, and Coxiella burnetii) among organized flocks in Tunisia. Results A total of 793 sample blood collected from twenty-six flocks in seven governorates in Tunisia, were tested by indirect enzyme-linked immunosorbent assay (i-ELISA) for antibodies against three abortion causing agents (Brucella spp, Toxoplasma gondii, and Coxiella burnetii). Risk factors for individual-level seroprevalence were analyzed using a logistic regression model. Results revealed that 19.7%, 17.2%, and 16.1% of the tested sera were positive for toxoplasmosis, Q fever, and brucellosis, respectively. Mixed infection was found in all the flocks with 3 to 5 responsible abortive agents simultaneously. Logistic regression showed that the management practices (control of new introduction, common grazing and watering point, workers exchange, presence of lambing box on the farm) and the history of infertility and the presence of abortion in neighboring flocks were likely to increase the probability of being infected by the three abortive agents. Conclusions Evidence of the positive relationship between seroprevalence of abortion causing agents and several risk factors, suggests further investigations to better understand the etiology of infectious abortions in flocks to develop an applicable preventive and control program.

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This review presents updated knowledge on the main tick-borne bacteria infecting one-humped camels (Camelus dromedarius) around the world. Camels are increasingly the subject of several scientific investigations, showing that they are receptive and carriers of several zoonotic bacteria. An appraisal is also given of the relative public health importance of these bacterial infections according to One Health concept. Microscopic, serologic and molecular findings are appropriately generated in order to exploit epidemiological data, and phylogeographic specificities associated to each vector-borne bacterium. Indeed, camels and their ticks harbour similar species and genotypes of pathogenic bacteria commonly identified in other animals, e.g., Anaplasma spp.,Ehrlichia spp., Borrelia spp., Rickettsia spp., Coxiella burnetii, Bartonella spp. and hemotrophic mycoplasmas. This evidence suggests an epidemiological role of camels in the spread of these pathogens in their natural habitats. However, these infections are commonly asymptomatic in camels resulting in underestimation of the impact of these infections. Furthermore, camels have recently been proven to have their own specific unclassified strains, such as Candidatus Anaplasma camelii and Candidatus Bartonella camelii, implying that possible interactions may lead to the emergence of pathogenic and zoonotic bacteria. In camel-rearing areas of the world, spatial and temporal spread of these infections, due to climatic and ecological changes and human activities such as development projects and urbanization, is expected. Hence the data presented herein provides a basis for strategic frameworks for the research and the development of novel diagnosis and control strategies worldwide, which are needed to protect camels, other livestock, and people in contact with dromedaries from threats that arthropod-borne pathogens can pose.

Multidrug resistance in Enterobacteriaceae has become a major concern worldwide that is increasingly observed in human, animals, and also cockroaches. In our study, we found that cockroaches may play an important role as a potential vector of multidrug-resistant Enterobacteriaceae in the hospital environment and collective catering.

Objectives Resistance to extended-spectrum cephalosporins, fluoroquinolones and colistin is under constant scrutiny in food-producing animals worldwide. However, little is known about camels, which provide milk and meat for human consumption, and are attractions for tourists to ride in arid regions. This study assessed the role of camels as potential reservoirs of these resistance determinants. Methods Faecal swabs were collected from 232 camels in Tunisia between April 2016 and July 2018. Enterobacteriaceae were detected on MacConkey agar and extended-spectrum β-lactamase (ESBL)-producers on the same medium supplemented with cefotaxime. Antimicrobial resistance was assessed by disc diffusion, and ESBL-producing isolates were further characterised by phylogrouping (for Escherichia coli, E. coli) and multilocus sequence typing. Genetic support of the blaESBL and mcr-1 genes was identified by plasmid-typing and Southern blot. Results E. coli were identified in 163 of 232 (70.3%) and Klebsiella pneumoniae (K. pneumoniae) in 16 of 232 (6.9%) of the dominant flora. Three E. coli and one K. pneumoniae (1.3% and 0.4%, respectively) were found on cefotaxime-enriched media. One K. pneumoniae and one E. coli from a tourist farm harboured the blaCTX-M-15 gene on an IncY plasmid, while the two E. coli from the butchery sector displayed the blaCTX-M-15 gene on an IncI1 plasmid and colocalisation of the blaCTX-M-1 and mcr-1 genes on an IncHI2 plasmid. Conclusions This study reported ESBL-producing Enterobacteriaceae in Tunisian camels from both tourist and meat-producing sectors. This was the first description of the mcr-1 gene in a meat-producing camel. Although not alarming, this context needs specific attention to avoid camels becoming a bigger reservoir for multidrug-resistant Enterobacteriaceae.

Enterobacteriaceae resistant to extended-spectrum cephalosporins (ESC-R) are listed as "priority pathogens" by the World Health Organization, and the Agri-food sector has regularly been pointed out as a potential source of ESC-R for humans through food consumption and animal handling. Chicken industry and chicken meat have recurrently been under specific scrutiny due to the high proportions of ESC-R reported worldwide in this sector. In Tunisia, recent studies suggested that the plasmidic AmpC blaCMY-2 gene may have emerged in chicken. We thus collected 258 cloacal swabs from five different farms and selected ESC-R isolates to determine the current ESC-R prevalence and epidemiology. All five farms were ESC-R positive with proportions ranging from 4% to 67.3%. blaCTX-M-1/IncI1/ST3 was the dominant gene/plasmid association in chicken, but several other CTX-M genes and plasmid backgrounds were shown to spread ESC-R. Surprisingly, the CMY-2 enzyme was only identified in one isolate. In addition, we also reported the sporadic presence of the mcr-1 gene carried by an IncHI2 plasmid. Our data suggest that the high diversity of Enterobacteriaceae clones and plasmids circulating in healthy chicken in Tunisia maintains a high ESC-R proportion in flocks and constitutes a major source of ESC-R determinants further disseminating in the food chain.

Antimicrobial resistance (AMR) is recognized as an emerging and growing public health problem worldwide. In Tunisia, knowledge is still limited to domestic animals and humans, and only few data are available regarding the role of wildlife. This research determined the antibiotic susceptibility profiles of Beta-lactamase producing Gram-negative bacteria isolated from the faeces of 110 wild boars (Sus scrofa) in northern Tunisia. Fecal samples, obtained post mortem from boar carcasses, were cultured on MacConkey agar and MacConkey agar containing 2 mg/L of cefotaxime. A total of 102 Enterobacterales isolates were identified from 94(85%) fecal samples. Escherichia coli (56, 54%), Citrobacter freundii (14, 13%), Klebsiella oxytoca (11, 10%), and Klebsiella pneumoniae (7, 6%) were the most predominantly identified Enterobacterales. However, Pantoea spp. (4, 4%), Enterobacter spp. (3,3%), Enterobacter cloacae (1, 1%), Enterobacter gergoviae (2, 2%), Proteus mirabilis (2, 2%), Yersinia sp. (1, 1%), and Citrobacter diversus (1, 1%) were rarely identified. Antimicrobial susceptibility tests revealed that 55% (57/102) of the identified strains were multidrug resistant (MDR). A total of 30% (31/102) of the tested isolates were recognized as Extended Spectrum β-Lactamase (ESBL)-producing strains and blaCTX-M-G1, blaTEM, blaSHV β-lactamases were the main encoding genes revealed. Furthermore, identified isolates showed a high level of AMR, especially for amoxicillin-clavulanic acid (77.67%), ticarcillin-clavulanic acid (71.85%), streptomycin (76.69%), amoxicillin (75.73%), and cephalotin (74.76%). Alarming levels of resistance to colistin (2.9%) and ertapenem (9.7%) were revealed and confirmed by the detection of mcr-1, and bla

Bovine anaplasmosis caused byAnaplasma marginaleis a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification ofA. marginaleisolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range ofA. marginalestrains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence ofA. marginaleDNA throughmsp4gene fragment amplification. Phylogeographic analysis was performed by usinglipAandsucBgene analyses, and the genetic relationship with previously characterizedA. marginaleisolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost completeompAnucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells.A. marginaleDNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis oflipAphylogeographic marker indicated a higher diversity of TunisianA. marginaleisolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multipleA. marginaleworldwide strains.

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin, lukE–lukD, encoding bicomponent leukotoxin LukE–LukD, the clfB gene, encoding clumping factor B, the laminin gene, encoding laminin-binding protein, and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Rift Valley fever (RVF) has been reported in the sub-Saharan region of Africa, Egypt and Arabian Peninsula - Yemen and Saudi Arabia, over the past 20 years and is a threat to both the animal and human populations in Tunisia. Tunisia is considered as a high-risk country for the introduction of RVF due to the informal movements of diseased animals already reported in the neighboring countries. The objective of this study was to assess the status of RVF in small ruminants and camels in Tunisia. A risk-based serological survey was conducted to evaluate the presence of RVF based on spatial qualitative risk analysis (SQRA). Samples were collected from small ruminants (sheep and goats) (n = 1,114), and camels (n = 173) samples, belonging to 18 breeders in 14 governorates between November 2017 and January 2018. Samples were tested using an RVF specific multispecies competitive ELISA. Out of the 1,287 samples tested for the presence of RVF IgG antibodies by ELISA, only one positive sample 0.07% (1/1 287) was detected but not confirmed with the virus neutralization test (VNT) used for confirmation. So far, no RVF outbreaks have been reported in Tunisia and our study confirmed the absence of RVF in livestock up to January 2018. Further investigations are needed to confirm the RVF-free status of Tunisia today., QRA methodology, Risk mapping, Survey, Tunisia, Rift valley fever, Small ruminants, Camels

Rabbit hemorrhagic disease (RHD) causes high mortality and morbidity in European rabbits (Oryctolagus cuniculus). In Africa, the presence of the causative agent, the rabbit hemorrhagic disease virus (RHDV), was first confirmed in 1992 (genotype Lagovirus europaeus/GI.1). In 2015, the new genotype Lagovirus europaeus/GI.2 (RHDV2/b) was detected in Tunisia. Currently, GI.2 strains are present in several North and Sub-Saharan African countries. Considerable economic losses have been observed in industrial and traditional African rabbitries due to RHDV. Like other RNA viruses, this virus presents high recombination rates, with the emergence of GI.2 being associated with a recombinant strain. Recombination events have been detected with both pathogenic (GI.1b and GII.1) and benign (GI.3 and GI.4) strains. We obtained complete genome sequences of Tunisian GI.2 strains collected between 2018 and 2020 and carried out phylogenetic analyses. The results revealed that Tunisian strains are GI.3P-GI.2 strains that were most likely introduced from Europe. In addition, the results support the occurrence of multiple introductions of GI.2 into Africa, stressing the need for characterizing complete genome sequences of the circulating lagoviruses to uncover their origin. Continued monitoring and control of rabbit trade will grant a better containment of the disease and reduce the disease-associated economic losses.

Background: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in human and considered a main cause of food-borne diseases. It was isolated from animals, human and food. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n=160) and from farms (n=100).Methods: E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey to detect non-sorbitol fermenting colonies that were examined for the presence of O157 antigen by latex agglutination, and positive bacteria were screened for the existence of stx1, stx2, eaeA and ehxA by PCR as well as rfbEO157 and fliCH7 genes specific for serotype O157. All isolates were examined for the susceptibility against 21antibiotics discs.Results: Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) was present in 70% of isolates, stx1 and ehxA were confirmed in 60% of the isolates, whereas eae was identified in two isolates. Other virulence factors screened (fimH, sfa/focDE, cdt3, traT, iutA and hly) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E.Conclusion: This is the first study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our finding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of human contamination.

Species belonging to the genus Anaplasma (Rickettsiales) include bacteria of veterinary and public health importance. Beside the zoonotic Anaplasma phagocytophilum, A. platys, the etiological agent of canine cyclic thrombocytopenia, has been sporadically reported in clinically ill human patients. The ongoing emergence of novel strains related to this species in vertebrate hosts emphasises the need for genetic comparisons among strains identified in different regions of the world. In this paper we developed a PCR test suitable for amplification of the still undescribed gltA gene of Anaplasma strains related to A. platys from Mediterranean ruminants and applied on a panel of 248 samples. gltA sequencing allowed phylogenetic comparison with strains related to A. platys recently identified in China, and strains representative of the Anaplasmataceae family. Results suggest the designation of Candidatus A. turritanum, including Mediterranean A. platys - like strains, and Candidatus A. cinensis, including strains isolated in China. Data generated in this study are a solid reference for future epidemiological studies of novel unclassified strains related to A. platys and for their diagnosis and raise concern on their potential veterinary and public health implications encouraging investigating the suspected unexplored diversity within the genus Anaplasma in animals and human.

Bovine mastitis is a major disease in dairy cattle that causes high economic losses annually. Staphylococci, streptococci, and coliforms are among the major pathogens responsible for such infections. While data on bovine mastitis are numerous in Europe where the efficacy of farm management was monitored, those are scarce in African countries. In this study, we reported the occurrence of Escherichia coli (118/372, 31.7%) and Klebsiella pneumoniae (77/372, 20.7%), two environmental pathogens known to cause bovine mastitis. Resistance phenotypes were frequently identified for tetracycline (E. coli, 46.6%/K. pneumoniae, 20.8%), sulfonamides-trimethoprim (17.8%/11.7%), gentamicin (19.5%/14.3%), and enrofloxacin (11.0%/6.5%). No carbapenem-resistant isolate was detected. Extended-spectrum beta-lactamases (ESBLs) were detected on selective medium in three E. coli and six K. pneumoniae, all carrying the bla

Anaplasma marginale, which is responsible for bovine anaplasmosis in tropical and subtropical regions, is a tick-borne obligatory intraerythrocytic bacterium of cattle and wild ruminants. In Tunisia, information about the genetic diversity and the phylogeny of A. marginale strains are limited to the msp4 gene analysis. The purpose of this study is to investigate A. marginale isolates infecting 16 cattle located in different bioclimatic areas of northern Tunisia with single gene analysis and multilocus sequence typing methods on the basis of seven partial genes (dnaA, ftsZ, groEL, lipA, secY, recA and sucB). The single gene analysis confirmed the presence of different and novel heterogenic A. marginale strains infecting cattle from the north of Tunisia. The concatenated sequence analysis showed a phylogeographical resolution at the global level and that most of the Tunisian sequence types (STs) formed a separate cluster from a South African isolate and from all New World isolates and strains. By combining the characteristics of each single locus with those of the multi-loci scheme, these results provide a more detailed understanding on the diversity and the evolution of Tunisian A. marginale strains.

Introduction: Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. Methodology: This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). Results: About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson’s diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. Conclusions: Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Wild rodents are considered as potential carriers of several zoonotic vector-borne bacteria but their epidemiology is poorly understood in Tunisia. A total of 305 biological samples (100 spleens, 100 livers, 100 kidneys, and 5 pooled ectoparasites (Xenopsylla cheopis, Laelaps echidninus, Ornithonyssus sp., Hoplopleura sp. and eggs of the rat fleas)) were collected from 100 wild rodents from three Tunisian governorates. Molecular screening was performed to reveal infections with main vector-borne bacteria. Captured rodents belonged to three rodent genera and species including Rattus rattus (n = 51, 51%), Meriones shawi (n = 24, 24%) and Mus musculus (n = 25, 25%). Examined rodents were found to be heavily infested by the rat flea X. cheopis (n = 32, 47%) and the rat mite L. echidninus (n = 22, 32.3%). However, the rat mite Ornithonyssus sp. (n = 13, 19.1%) and the rat lice Hoplopleura sp. (n = 1, 1.5%) were rarely identified. Based on 16S rRNA and msp4 genes, infection with Anaplasmataceae bacteria was detected in six specimens of R. rattus and one M. shawi. Pathogenic A. phagocytophilum (n = 1), A. phagocytophilum-like 1 (Anaplasma sp. Japan) (n = 1), and A. ovis (n = 5) were identified. On the basis of ompB, ompA and gltA genes, infection with Rickettsia spp. was identified in three specimens of R. rattus and one of M. shawi. Five Rickettsia species of the spotted fever group, corresponding to R. monacensis, R. helvetica, R. massiliae, R. africae, and R. aeschlimannii, were detected in mixed infections. Bartonella henselae DNA was also found in two R. rattus, based on rpoB partial sequences. All revealed Anaplasma, Rickettsia and Bartonella bacteria were detected in spleen samples. Ehrlichia, Coxiella and Borrelia spp. were not identified in any of the tested samples. In Tunisia, this is the first report indicating infections with Anaplasma, Rickettsia and Bartonella spp. in wild rodents, particularly present alongside domestic livestock and human. This represents a serious risk of potential bacterial transmission. Thus, controlling rodent population in animal herds, residential areas and sensitizing local people to this risk seem absolutely necessary.

Anaplasma ovis, the causative agent of ovine anaplasmosis in tropical and subtropical countries, is a tick-borne obligatory intraerythrocytic bacterium of sheep, goats and wild ruminants. In Tunisia, data about the molecular phylogeny and the genetic diversity of A. ovis isolates are limited to the analysis of msp4 and groEL genes. The aim of this study was to genetic characterize 40 A. ovis isolates infecting 28 goats, 10 sheep, one camel and one Rhipicephalus turanicus tick located in different geographic regions of Tunisia on the basis of 3 partial genes (gltA, groEL and msp1a). Sequence analysis revealed 6 and 17 different genotypes in the partial gltA and groEL genes, respectively. Phylogenetic analysis revealed, as expected for the groEL gene, that sequences from small ruminants and their infesting ticks clustered separately from those isolated from camels. The analysis of amino-acid Msp1a sequences identified 18 novel genotypes of Msp1a repeats from 20 A. ovis isolates. These Msp1a repeats were highly variable with 33-47 amino-acids, and the number of repeats is one for 19 isolates infecting 18 goats and one R. turanicus tick, and 4 for a single isolate found in one sheep. Phylogenetic trees based on Msp1a partial sequences revealed that the N-terminal region of Msp1a protein appear to be relatively more informative phylogeographically compared to other markers especially according to countries. The presented data give a more detailed knowledge regarding the molecular phylogeny and the genetic diversity of A. ovis isolates occurring in different animal species and their associated ticks in Tunisia.

Anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys. Understanding the phylogenetic relations among these species is fundamental to perform an accurate identification and an informative intra-specific analysis. Heat shock groESL operon is frequently employed in phylogenetic analysis of Anaplasma species and, for the most cases, the use of partial sequences of this operon is randomly done without knowing the most appropriate regions to be used either in species attribution or in intra-specific diversity analysis. In this study, on the basis of all fully and nearly complete groESL sequences available in the GenBank, we firstly selected a minimum partial length sequence which allows species delineation and gives a similar topology to that found by analyzing the complete sequence. By using other in silico analyses, we obtained two minimal partial sequences that are the most interesting to describe intra-specific diversity within A. ovis and A. centrale. Our results raise concern on the use of randomly selected partial sequences of groESL operon employed for the detection and the characterization of Anaplasma species and provide additional background about minimum length groESL operon required for Anaplasma species attribution and strains diversity analysis.

Tick-borne bacteria are considered to be emerging in camels, but data about their occurrence in Tunisian dromedaries and their infesting ticks remain scarce. In this study, 412 camel blood samples and 327 partially engorged ticks were collected and tested for the presence of Coxiella burnetii and/or related strains, and Rickettsiales bacteria. Coxiella burnetii was detected in 9 Hyalomma impeltatum and 3 H. dromedarii with an overall prevalence rate of 3.6% (12/327). Candidatus Midichloria mitochondrii DNA was identified in 16 H. impeltatum and 10 H. dromedarii with an overall prevalence rate of 8% (26/327). Six ticks (2%) were found to be co-infected with these two bacteria. No positive DNA camel blood sample was observed for both bacteria. Genotyping and phylogenetic analysis of obtained C. burnetii partial sequences based on the IS1111 and htpB genes revealed 99-100% similarity to the pathogenic C. burnetii strains isolated from humans, ruminants and ticks, and that were genetically distant to those isolated from all endosymbiotic related strains (Coxiella-like bacteria). The analysis of the rickettsial partial sequences of the 16S rRNA gene showed 100% similarity to Ca. M. mitochondrii strains infecting Ixodid ticks and dogs. This is the first time that C. burnetii and Ca. M. mitochondrii have been detected in ticks from Tunisia, which raises the possibility of the involvement of Hyalomma tick species in the active diffusion of these bacteria among camels, other domestic animals and humans.

Resistance to extended-spectrum cephalosporins, fluoroquinolones and colistin is under constant scrutiny in food-producing animals worldwide. However, little is known about camels, which provide milk and meat for human consumption, and are attractions for tourists to ride in arid regions. This study assessed the role of camels as potential reservoirs of these resistance determinants.Faecal swabs were collected from 232 camels in Tunisia between April 2016 and July 2018. Enterobacteriaceae were detected on MacConkey agar and extended-spectrum β-lactamase (ESBL)-producers on the same medium supplemented with cefotaxime. Antimicrobial resistance was assessed by disc diffusion, and ESBL-producing isolates were further characterised by phylogrouping (for Escherichia coli, E. coli) and multilocus sequence typing. Genetic support of the blaE. coli were identified in 163 of 232 (70.3%) and Klebsiella pneumoniae (K. pneumoniae) in 16 of 232 (6.9%) of the dominant flora. Three E. coli and one K. pneumoniae (1.3% and 0.4%, respectively) were found on cefotaxime-enriched media. One K. pneumoniae and one E. coli from a tourist farm harboured the blaThis study reported ESBL-producing Enterobacteriaceae in Tunisian camels from both tourist and meat-producing sectors. This was the first description of the mcr-1 gene in a meat-producing camel. Although not alarming, this context needs specific attention to avoid camels becoming a bigger reservoir for multidrug-resistant Enterobacteriaceae.

Salmonella enterica subsp. enterica serovar Eppendorf, with antigenic formula 1,4,12,[27]:d:1,5, is an infrequent serovar. However, 14% (20 of 142) of the isolates recovered during June-July 2012 in chicken farms in Tunisia belonged to S. Eppendorf. These isolates were analysed for resistance and virulence profiles. None of them were susceptible to all antimicrobials tested, while 70%, 60%, 50%, 50%, 20% and 5% were resistant to sulphonamides (sul1, sul2 and sul3), streptomycin (aadA1-like), trimethoprim (dfrA1-like), nalidixic acid (GyrA Asp87 →Asn and not identified), gentamicin (not identified) and ampicillin (blaTEM -1-like). About 30% of the isolates showed decreased susceptibility to ciprofloxacin and carried the qnrB gene; 65% of the isolates were multidrug resistant and contained class 1 integrons with sul1 or sul3 in the 3' conserved segment. The orgA, ssaQ, mgtC, siiD and sopB virulence genes located on SPI1 to SPI5 and the fimbrial bcfC gene were present in all isolates; the sopE1 and sodC1 carried by prophages were variably detected; however, the prophage gipA gene and the spvC gene of serovar-specific virulence plasmids were absent. Altogether, ten resistance and three virulence profiles were identified. Typing of the isolates with XbaI- and BlnI-PFGE supports a close relationship, although they appear to be evolving under selective pressure probably caused by antimicrobial use in chicken husbandry. As far as we know, this is the first study investigating the molecular bases of antimicrobial drug resistance, the virulence gene content and the PFGE profiles of S. Eppendorf. The epidemiological surveillance of this serovar would be necessary to evaluate its possible impact on human health, particularly in Tunisia and other African countries where it was already reported.

Enzootic abortion of ewes (EAE) caused by Chlamydia abortus is a disease of ruminants that results in serious economic losses in livestock industry. The zoonotic potential of the pathogen adds a public health concern on the efforts to control the disease. We report herein a cross-sectional study that was conducted during the lambing season (June and July) in Tunisia to estimate the seroprevalence of C. abortus infection in large sheep herds with abortion history. A total of 803 ewes were sampled and tested using indirect enzyme linked immunosorbent assay (iELISA). The overall apparent seroprevalence at herd and individual levels were 58 % (95 %CI = 39–74.5 %) and 6.6 % (95 %CI = 4.9–8.3 %), respectively. Significant risk factors investigated using univariate and multivariate analyses were history of infertility (OR = 5.7; 95 %CI = 3.05–10.66), the number of reproductive ewes (OR = 2.1; 95 %CI = 1.12–3.94), the control of new animals at introduction (OR = 4.35; 95 %CI = 2.46–7.68), the sharing of drinking water (OR = 2.18; 95 %CI = 1.22–3.9), the exchange of breeding males (OR = 2.56; 95 %CI = 1.003–6.54), the disposal of abortion materials without precaution (OR = 4.36; 95 %CI = 2.42–7.87), the lack of lambing barn (OR = 2.39; 95 %CI = 1.13–5.04), the non-application of hygienic post-abortion measures (OR = 10.35; 95 %CI = 5.28–20.26) and the manure management (OR = 11.35; 95 %CI = 3.26–39.48). To the best of our knowledge, this is the first sero-epidemiological survey conducted on an abortive disease in Tunisian ewes that investigated the risk factors of C. abortus infection.

Rift Valley fever (RVF) is a mosquito-borne zoonosis that severely impacts livelihoods, national and international economies, and human health. Few studies have investigated the prevalence of this infection in Tunisian livestock. The present report aimed to update the epidemiological status and identify the risk factors associated with this RVF virus infection in the one-humped dromedary camel from arid areas. A total of 470 sera of apparently healthy camels (Camelus dromedarius) were collected from six governorates from southern and central Tunisia. Samples were tested by a competitive Enzyme Linked Immunosorbent Assay (ELISA). An overall, 162 camels (34%, 95%CI: 0.1-0.4) were seropositive to RVF virus antigen. Logistic regression model revealed three potential risk factors associated with the infection. A meaningful high seropositivity was observed among aged camels (>10 years-old) (40%) (P=0.001; OR=3.367). Besides, camels raised in small flocks particularly intended for meat production showed a high level of seropositivity (37%) (P=0.013; OR=13.173). Animals having close contact with other ruminants showed high seroprevalence (37%) (P=0.022; OR=10.919). This report indicated that Tunisian one-humped dromedaries were exposed to this virus and may contribute to its dissemination among farmers and other livestock. Furthers studies are urgently required to isolate and characterize this virus, evaluate the potential risk of human infection particularly in farmers, veterinarians and slaughterhouse workers and finally to program a serious strategy for RVF control.

The genus Anaplasma currently comprises 6 bacterial species mostly pathogenic to animals and/or human, including the zoonotic species Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) of ruminants, and of granulocytic anaplasmosis of horses, dogs and human. Recently, novel potentially non-pathogenic strains related to A. phagocytophilum have been identified in Japan, China, and Tunisia. This paper reports the identification, molecular typing, and evolutionary history of novel Anaplasma strains (A. phagocytophilum-like 1 and 2), related to but distinct from A. phagocytophilum in Mediterranean area of Europe and Africa. PCR-RFLP and phylogenetic analyses based on 16S rRNA provided evidence for the circulation of A. phagocytophilum-like 1 strains in Europe. Phylogeny based on groEL gene showed the inclusion of Sardinian and Tunisian A. phagocytophilum-like 1 strains in a unique clade distinct from, but related to that of Japanese strains. Results suggest that genetic diversity within the genus Anaplasma is much greater than expected and provide information useful for the development of specific and effective diagnostic and prophylactic tools.

To date, no information is available regarding the infection of camels (Camelus dromedarius) by Anaplasma ovis in North African region. Several animal species can be infected by A. ovis which further complicates its natural infection cycle. In this paper, we investigated the occurrence and the genetic diversity of A. ovis in camels and ticks collected from them in Tunisia and the risk factor analysis. Camel blood samples (n = 412) and tick (n = 300) samples, identified as Hyalomma dromedarii (n = 149, 49.6%), H. impeltatum (n = 142, 47.3%) and H. excavatum (n = 9, 3%), were analyzed by conventional PCR followed by the sequencing of msp4 and groEL genes. A. ovis DNA was identified in five camels (1.2%), but not in infesting ticks (0%). The microscopic examination revealed the specific infection of camel erythrocytes by Anaplasma inclusions. The msp4 and groEL typing confirmed the natural infection of camels by A. ovis and revealed two different msp4 genotypes earlier detected in Tunisian small ruminants and their infested ticks, and five different and novel groEL genetic variants forming a separately sub-cluster within A. ovis cluster. The occurrence of different A. ovis strains specific to camels associated with a low prevalence of this Anaplasma species in camels may enrich knowledge regarding the distribution and the transmission cycle of this bacterium in arid and Saharan areas of Tunisia.

In Tunisia, most of Anaplasma species and unclassified strains have been detected in several animals, but data on the occurrence of Anaplasma spp. in ticks are still lacking. In this study, we report the molecular evidence, genetic characterization and phylogeny of Anaplasma spp. in ticks collected from small ruminants. A total of 395 ticks (178 males and 179 females) were collected from sheep (n = 215) and goats (n = 180). Tick species were identified as 232 Rhipicephalus turanicus, 99 Rhipicephalus sanguineus sensu lato, 34 Rhipicephalus bursa and 30 Rhipicephalus annulatus. Overall infection rate of Anaplasma spp. was 5.6% (20/357 analyzed ticks). All positive ticks were collected from goats and found to be infected by A. ovis. R. turanicus is the most infected tick species by A. ovis (7.9%) followed by R. sanguineus s.l. (2.5%) with an absence of infection in R. bursa and R. annulatus. A. ovis prevalence rate varied significantly according to bioclimatic areas and geographic regions. GroEL typing and phylogenetic analysis revealed that these analyzed ticks are infected with various and novel strains of A. ovis. The use of PCR-RFLP method complemented with sequencing and phylogenetic analysis based on 16S rRNA gene confirm that one R. turanicus tick, positive to A. ovis, is co-infected with A. phagocytophilum-like 2 (0.3%). Specific A. phagocytophilum, A. phagocytophilum-like 1, A. marginale, A. centrale, A. bovis, and A. platys and related strains were not detected in any of the tested ticks. Present data expand knowledge about tick-borne bacteria present in ticks and further clarify the transmission cycles of these bacteria and their different elements in Tunisia.

Q fever, caused by Coxiella burnetii, is a zoonotic disease responsible of abortion in ruminants. Few studies have investigated the prevalence of this infection in camels (Camelus dromedarius). The present report aimed to highlight the epidemiological status and identify the risk factors associated with C. burnetii infection in one-humped dromedary that is the most productive livestock species in arid areas. A total of 534 sera of healthy camels were collected in eight governorates from southern and central Tunisia. Samples were tested by an indirect Enzyme linked Immunosorbent Assay (ELISA). Results were analyzed using the Chi-square test and logistic regression. Overall, 237 camels (44%, 95%CI: 0.40-0.49) were seropositive to C. burnetii. Statistical analysis pointed out four potential risk factors associated with infection. A meaningful high seropositivity was observed in female camels with a previous history of abortion (70%) (OR = 4.186, 95%CI: 2.05–8.51). Seroprevalence was higher in aged camels (>10 years-old) (48%) (OR = 2.91, 95%CI: 1.37–6.17). Besides, camels, intended for meat production from small herds showed a high level of infection (52%) (OR = 2.43, 95%CI: 1.3–4.5). Coxiellosis evolved in dromedary herds throughout the year, however infection was significantly important in autumn (60%) (OR = 4.13, 95%CI: 1.86–9.17) and winter (56%) (OR = 5.52, 95%CI: 2.50–12.16). Bioclimatic stage, gender, tick infestation and contact with other ruminants were not risk factors in camel’s infection by C. burnetii. Our reports confirm that Tunisian one-humped dromedaries had been exposed to this bacterium and could contribute to its dissemination among farmers and other livestock animals. Furthers studies are required to evaluate the prevalence of Q fever among people professionally exposed like farmers, veterinarians and slaughterhouse workers.

Introduction: Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. Methodology: A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. Results: Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale , A. centrale , A. bovis , and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma ( H. dromedarii , H. impeltatum , and H. excavatum ). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys . Conclusions: This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys .

A survey of Anaplasma species in small ruminants is still lacking in North African countries. In this study, the presence of A. phagocytophilum, A. phagocytophilum–related species, and A. ovis was investigated in a total of 563 healthy small ruminants (303 goats and 260 sheep), from 25 randomly selected flocks sampled in Tunisia. Anaplasma spp. and A. ovis overall infection rates were 95.0% and 93.8% in sheep and 69.6% and 65.3% in goats, respectively. A. phagocytophilum was not detected in any of tested animals. A total of 20 sheep (7.7%) and 144 goats (47.5%) were infected by Anaplasma strains genetically related to A. phagocytophilum. Both in sheep and goats A. ovis prevalence was higher in adults (≥2 years) than in young (