Search

The first Tunisian national molecular survey of coagulase-positive staphylococci (CoPS) isolated from dogs with pyoderma and otitis externa was conducted to evaluate the prevalence of CoPS and identify its phenotypic and genotypic diversities. A total of 99 out of the 195 samples collected from 39 sick dogs were identified across multiple sites as methicillin-susceptible CoPS belonging to the species S. pseudintermedius (64.4%), S. aureus (20.2%), S. coagulans (10.1%), and S. hyicus (5%). Fifteen sampled dogs carried more than one Staphylococcus species. Their antibiotic resistance and virulence factors were determined using conventional and molecular methods. Of the S. pseudintermedius isolates found, 17.4% were multidrug-resistant, whereas high rates of virulence genes were observed among the S. aureus isolates. On polystyrene surfaces, 75% of S. aureus isolates were biofilm producers, of which 15% were classified as strong producers. The capsular polysaccharide cap8 genotype was predominant among them. A MultiLocus Sequence Typing (MLST) analysis clustered the S.aureus isolates into five distinct sequence types (STs), with four assigned for the first time. Our findings highlight the spread of CoPS among diseased dogs and, especially, the emergence of S. hyicus, S. coagulans, multidrug-resistant S. pseudintermedius and S. aureus isolates with high genetic variability. The precise characterization of these strains, as well as their continuous monitoring, is necessary for the implementation of preventive strategies given the significant public health risk.

Escherichia coli is an important opportunistic pathogen, causing several infections in dogs. The antimicrobial resistance of E. coli occurring in companion animals becomes an emerging problem. This study aimed to estimate the prevalence of ESBL-producing E. coli in diarrheic dogs, investigate the occurrence and molecular characterization of carbapenem-resistant isolates, and determine their virulence genes. Fecal samples were collected from 150 diarrheic dogs in Tunisia. E. coli isolates were screened for antimicrobial resistance against 21 antibiotics by the disk diffusion method. The characterization of β-lactamase genes, associated resistance genes, and virulence genes was studied using PCR. Among 95 E. coli strains, 25 were ESBL-producing, and most of them were multidrug-resistant. The most prevalent β-lactamase genes were blaCTX-M1 (n = 14), blaTEM (n = 3), and blaCMY (n = 2). The blaIMP carbapenemase gene was found in two carbapenem-resistant isolates, which showed that carbapenemase-producing E. coli spread to companion animals in Tunisia. Different virulence genes associated with extraintestinal pathogenic E. coli were detected. This is the first report of the characterization of carbapenem resistance and virulence genes in dogs in North Africa. Our study showed that diarrheic dogs in Tunisia can be a potential reservoir of ESBL- or carbapenemase-producing E. coli with a possible risk of transmission to humans.

Background: Until now, there has been limited information on the prevalence and the phylogeny of Borrelia burgdorferi sensu lato in Ixodes ticks in Tunisia, particularly in Ixodes inopinatus. Methods: The present study aimed to determine the prevalence and the phylogeny of B. burgdorferi s.l., in coexisted I. ricinus and I. inopinatus ticks collected from Northern Tunisia. One hundred questig ticks were collected during winter 2020 by tick-dragging method in Beja gouvernorate located in the north of Tunisia. Real-time PCR panel targeting B. burgdorferi s.l. 23S rRNA gene were performed. Positive DNA samples were subjected to conventional PCRs targeting 457 bp fragment of the Borrelia sp. flagellin (fla) gene using primers FlaF/FlaR. The identified Borrelia sp. isolate underwent partial sequence analysis to determine genospecies and evaluate their phylogenetic position. Results: The study revealed a prevalence rate of 28% (28/100) for B. burgdorferi sensu lato in the Ixodes ticks. The prevalence rates across tick species and genders did not show significant variations (p > 0.05). Interestingly, the study underlines the coexistence of I. inopinatus and I. ricinus sharing the same geographic areas in Northern Tunisia. Furthermore, DNA of B. lusitaniae was detected in I. inopinatus ticks for the first time in Tunisia. Revealed B. lusitaniae bacterium is similar to previously identified strains in Mediterranean region, but distinct from those isolated exclusively from countries of Eastern and Central Europe, such as Serbia, Romania, and Poland. This study highlights the prevalence of B. burgdorferi s.l. in I. ricinus/I. inopinatus ticks, and reveals B. lusitaniae in I. inopinatus ticks for the first time in Tunisia. Conclusion: These findings suggest the involvement of I. inopinatus as a potential vector of this pathogenic genospeciess in Tunisia. This may help understanding the ecology of Ixodes ticks, the natural infection and the transmission dynamics of Borrelia species in this country.

Abstract 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.

Shiga‑toxin‑producing E. coli (STEC) is a foodborne pathogen associated with outbreaks worldwide that can be identified in the feces and in the meat of food‑producing animals. Our study aimed to evaluate the incidence of E. coli O157:H7 in the feces of diarrheic camels (Camelus dromedarius) in Tunisia. From January 2018 to April 2019, 120 unduplicated fecal samples were obtained from diarrheic camels located in southern Tunisia. Non‑sorbitol‑fermenting colonies were confirmed as E. coli O157 via latex agglutination test and were screened for the presence of rfbEO157, fliCH7, stx1, stx2, eaeA, and ehxA genes by PCR. All isolates were examined for their susceptibility to 21 antibiotics. Of the 70 E. coli isolates that were recovered from 120 diarrheic camels, 4 (5.7%) were identified as STEC O157:H7. All isolates harbored ehxA and eae genes. Shiga toxin genes stx2 and stx1 were present in 50% and 25% of isolates, respectively. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole‑trimethoprim. All isolates belonged to the phylogroup E. This is the first report of E. coli O157:H7 isolates from diarrheic camels in Tunisia with a prevalence of 4 isolates (3.3%) amongst 120 fecal samples. This study supports the necessity for a platform purposed for regular screening and surveillance programs in food‑producing animals and meat products, to perform early and rapid identification of food‑borne pathogens.

Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.

ABSTRACT The emergence of multidrug-resistant bacteria has become a major problem. Cockroaches may play an important role in the spread of those bacteria between the environment and humans. This study was designed to screen extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant strains and to investigate the molecular support of multidrug-resistant Enterobacteriaceae in the external surface and gut homogenates of cockroaches collected from different locations in Tunisia. Between July 2017 and June 2018, 144 Enterobacteriaceae samples were isolated from 115 trapped cockroaches (collective catering, houses, and a hospital). Antibiotic susceptibility testing was performed using the disk diffusion method. Extended-spectrum β-lactamase-encoding genes and the mcr-1 gene were investigated by real-time PCR (RT-PCR) and standard PCR. The genetic relationship among isolates was studied with the help of multilocus sequence type (MLST) analysis. Of the 144 Enterobacteriaceae isolates, 22 strains exhibited a positive ESBL-screening test (73.3%), including 17 Escherichia coli isolates and 5 Klebsiella pneumoniae isolates. Among them, 9 Escherichia coli isolates were resistant to colistin, with an MIC ranging from 8 to16 μg/L, all of which harbored the mcr-1 gene. Eight blaCTX-M-15 genes were detected; two among them were associated with blaTEM-117 and blaTEM-128, and seven blaCTX-M-1 genes were detected that also harbored the mcr-1 gene. Genotyping analysis revealed 7 different sequence types already described in humans and animals. We report the first survey of mcr-1 in ESBL-producing E. coli isolates from cockroaches. Our findings highlight cockroaches as a source of nosocomial infections, and they are a reservoir of colistin-resistant E. coli, which is a carrier of other additional risk genes such as blaESBL, especially in hospitals. IMPORTANCE 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. Our study describes the first survey of mcr-1 in ESBL-producing E. coli isolates from hospital cockroaches. Our results further highlight the possibility that mcr-1 may enter humans via cockroach contamination and thereby threaten public health. Our results show that these cockroaches are an important reservoir of colistin-resistant E. coli and carriers of other additional risk genes such as blaESBL, hence the importance of strengthening prevention strategies and of strictly respecting hygiene measures in order to control their distribution and spread in Tunisia.

We conducted a 5-month-long screening of Anaplasma spp. and Anaplasma ovis infection in sheep from central Tunisia. During this longitudinal study, we investigated the infection dynamics using both direct and indirect assessments validated with a polymerase chain reaction (PCR) as the gold standard method. The experimental design included 84 male lambs aged from 6 to 8 months, and 32 ewes, both chosen randomly from June to November with a periodicity of 2 weeks approximately between June and September, and 1 month between September and November. A total of 9 field visits were carried out in this period during which animals were clinically examined and biological samples were extracted. Thus, a total of 716 blood smears, 698 sera from the nine sampling dates, as well as 220 blood samples from the first and the ninth sampling dates were collected from apparently healthy lambs and ewes, respectively, and analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and polymerase chain reaction (PCR) assay, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. Sera were analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and PCR, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. The Anaplasma spp. initial seroprevalence rate was 33.3% in lambs and 100% in ewes, and it then flowed in an upward trend to reach a maximum of 52.6% in lambs, whereas in ewes, the Anaplasma spp. seroprevalence rate remained unchanged and equal to 100%. Meanwhile, the A. ovis initial molecular prevalence was 22.6% at the first visit and 26.3% at the last visit in lambs, whereas in ewes, the molecular prevalence rates of A. ovis were higher in both the first and the last visit estimated at 100% and 85.7%, respectively. The Kappa coefficient between cELISA and PCR indicated a moderate level of agreement on the first sampling date (0.67) and a low agreement level on the last (0.43). Furthermore, an exploratory data analysis using a multimodal machine learning approach highlighted the underlying pattern of each analytical technique used in this study. In this prospect, we were able to establish the performance of each technique at detecting Anaplasma spp. in sheep. The combination of these approaches should improve the field assessment while promoting a data-based decision in precision epidemiology. The genetic follow-up test relevant to A. ovis msp4 sequences revealed three different genotypes, two of which were previously described in Italy.

Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates 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 of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide 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. marginale DNA 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 of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates 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 multiple A. marginale worldwide strains.

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.

Tick-borne rickettsioses present a significant public health threat among emerging tick-borne diseases. In Tunisia, little is known about tick-borne Rickettsia pathogens. Therefore, the aim of this study was to investigate the presence of Rickettsia species in small ruminant ticks from Tunisia. Adult ticks (n = 694) were collected from goats and sheep in northern Tunisia. Obtained ticks were identified as Rhipicephalus turanicus (n = 434) and Rhipicephalus sanguineus sensu lato (n = 260). Selected ticks (n = 666) were screened for the presence of Rickettsia spp. by PCR targeting a partial sequence of the ompB gene followed by sequence analysis. Rickettsial DNA was detected in 122 (18.3%) tested tick samples. The infection rates in Rh. turanicus and Rh. sanguineus s.l. ticks were 23.4 and 9.5%, respectively. The overall prevalence of rickettsial DNA was markedly higher in ticks collected from goats (23.2%) compared to those infesting sheep (7.9%). The detection of rickettsial DNA was significantly higher in ticks from the governorate of Beja (39.0%) than those from the governorate of Bizerte (13.9%). Two additional genes, the outer membrane protein A gene (ompA) and the citrate synthase gene (gltA), were also targeted for further characterization of the detected Rickettsia species. Genotyping and phylogenetic analysis based on partial sequences (n = 106) of the three different genes revealed that positive ticks are infected with different isolates of two Spotted Fever Group (SFG) Rickettsia, namely, Rickettsia massiliae and Rickettsia monacensis, closely related to those infecting camels and associated ticks from Tunisia, and humans and small ruminant ticks from neighboring countries like Italy, France, and Spain.

This study focused on 77 isolates of Salmonella enterica serotype Enteritidis collected during 2009 to 2013 from healthy and sick chickens and environmental farm samples in Tunisia. Resistance to 14 antimicrobials and the encoding genes were analyzed. 66, 26, 6.5, 3.9 and 1.3% were pan-susceptible or showed resistance to nalidixic acid (Asp87 to Tyr and Asp87 to Asn substitutions in GyrA), ampicillin (blaTEM-1-like and blaSHV), sulfonamides (sul1and sul3) and streptomycin (strB), respectively. A single isolate with intermediate susceptibility to ciprofloxacin was positive for qnrB, whereas qnrA, qnrS or aac(6′)-Ib-cr were not detected. The virulotype of the isolates was established by testing ten virulence genes. The orgA, ssaQ, mgtC, siiD, sopB genes, located on Salmonella pathogenicity islands, and spvC of the serotype-specific virulence plasmid, were common to all isolates. In contrast, the prophage-associated sopE-1, sodC1 and gipA genes and the fimbrial bcfC gene were variably represented. All isolates except one contained the virulence plasmid, which appeared either alone or together with one or more additional plasmids. One isolate carried a single plasmid of ca. 90 Kb which may be derived from the virulence plasmid (60 Kb). Overall, seven resistotypes, six virulotypes and six plasmid profiles were identified. XbaI-PFGE revealed four related pulsotypes (X1–X4), with 80% of the isolates sharing the X1 pattern. The latter isolates exhibited different resistance, virulence and plasmid profiles, suggesting that mobile genetic elements, particularly prophages and plasmids, are of central importance for the evolution and adaptation of S. Enteritidis circulating in chicken farms in Tunisia. Keywords: Poultry, Antimicrobial drug resistance, qnrB, Virulence genes, PFGE

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.

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.

Borrelia burgdorferi sensu lato (s.l.) are tick-transmitted spirochaetes of veterinary and human importance. Molecular epidemiology data on ruminants are still lacking in most countries of the world. Therefore, the aim of this study was to estimate the rate of B. burgdorferi s.l. infection in ruminants from Tunisia. A total of 1,021 ruminants (303 goats, 260 sheep, 232 cattle and 226 camels) from different bioclimatic areas in Tunisia were investigated for the presence of B. burgdorferi s.l. DNA in blood by real time PCR. Prevalence rates were 30.4% (92/303) in goats, 6.2% (16/260) in sheep, 1.3% (3/232) in cattle, and 1.8% (4/226) in camels. Only tick species belonging to Rhipicephalus and Hyalomma genera were found on the investigated animals. In small ruminants, the prevalence of B. burgdorferi s.l. varied significantly according to localities and farms. Goats located in humid areas were statistically more infected than those located in sub-humid areas. Prevalence rates varied significantly according to age and breed in sheep, and age and tick infestation in goats. This study provides the first insight into the presence of B. burgdorferi s.l. DNA in ruminants in Tunisia, and demonstrates that host species such as goats and sheep may play an important role in natural Lyme disease cycles in this country.

We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula.