Seroprevalence of zoonotic abortive diseases and their associated risk factors in Tunisian sheep.

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.

Prevalence, risk factors and emergence of extended-spectrum ß-lactamase producing-, carbapenem- and colistin-resistant Enterobacterales isolated from wild boar (Sus scrofa) in Tunisia.

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 ß-Lactamase (ESBL)-producing strains and blaCTX-M-G1, blaTEM, blaSHV ß-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 blaIMP and blaVIM genes, respectively. Various phenotypes of AMR were obtained in this study highlighting the important role of wild boars as hosts and even carriers for several resistant Enterobacterales isolates. This may represents a focal risk factor allowing the transmission of these strains between domestic, wild animals, environment and humans.

Molecular detection and genetic characterization of the potentially pathogenic Coxiella burnetii and the endosymbiotic Candidatus Midichloria mitochondrii in ticks infesting camels (Camelus dromedarius) from Tunisia.

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.

Computational selection of minimum length groESL operon required for Anaplasma species attribution and strain diversity analysis.

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.

Geographic distribution of MERS coronavirus among dromedary camels, Africa.

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.

First detection of Rickettsia felis and Ehrlichia canis in the common bed bug Cimex lectularius.

Bed bugs, common blood-feeding pests, have received limited attention regarding their potential involvement in emerging pathogen transmission. This study aimed to investigate the main vector-borne bacteria within bed bugs collected from Tunisian governorates and to genetically characterize the identified species. Molecular screening was conducted on field-collected bed bug samples, targeting zoonotic vector-borne bacteria from the Anaplasmataceae family, as well as the genera Rickettsia, Ehrlichia, Bartonella, and Borrelia. A total of 119 Cimex lectularius specimens were collected and grouped into 14 pools based on sampling Tunisian sites. Using genus-specific PCR assays, DNA of Rickettsia and Ehrlichia spp. was detected in a single pool. Sequencing and BLAST analysis of the obtained partial ompB and dsb sequences from positive samples revealed 100% similarity with those of Ehrlichia canis and Rickettsia felis available in GenBank. Obtained partial sequences showed phylogenetic similarity to R. felis and E. canis isolates found in dogs and ticks from American and European countries. To the best of our knowledge, this study is the first to investigate bed bugs in Tunisia and to report the worldwide identification of R. felis and E. canis DNA in the common bed bug, C. lectularius. These findings highlight the need for further research to explore the potential role of bed bugs in the epidemiology of these vector-borne bacteria.

Serological and molecular survey of brucellosis and chlamydiosis in dromedary camels from Tunisia.

The present sero-epidemiological survey was designed and conducted to scrutinize the current status of camel-related brucellosis and chlamydiosis in Tunisia. Whole blood and serum samples were collected from 470 dromedaries (Camelus dromedarius) from eight different Tunisian governorates. Serum samples were subjected to indirect enzyme-linked immunosorbent assay (iELISA). The detection of Brucella and Chlamydia DNA was performed using conventional PCR targeting the bcsp-31 and 16 S rRNA gene, respectively. Overall, 10/470(2.12%) and 27/470 (5.75%) camels were revealed seropositive to Brucella and Chlamydia, respectively. Multivariate logistic regression analysis showed different risk factors associated with these infections. Meaningful high rates of seropositivity of brucellosis (9.5%; p = 0.000; OR=64.193) and chlamydiosis (22.6%; p = 0.000; OR=42.860) were noted among camels showing previous abortions in particular for aged females. Besides, Chlamydia seropositivity is significantly important during winter (12.5%; p = 0.009; OR= 27.533), and in camels raised in small farms (11.4%, p = 0.000, OR=86.052). Molecular analysis revealed no positivity from all analyzed blood samples. These findings indicate the involvement of camels in the epidemiology of these abortive infectious diseases. This raises awareness and serious public health concern for infectious camel diseases in order to develop further diagnostic improvements and effective control strategies.

Detection and genetic identification of Borrelia lusitaniae in questing Ixodes inopinatus tick from Tunisia.

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.

Antimicrobial resistance, virulence genes, and genetic lineages of Staphylococcus pseudintermedius in healthy dogs in tunisia.

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.

Occurrence and characteristics of extended-spectrum-ß-lactamase producing Escherichia coli (blaTEM-128) isolated from Mus musculus captured from a veterinary clinic and houses in Tunis, Tunisia.

Antimicrobial resistance in Enterobacteriaceae is a public health problem. Rodents, can be a potential vector for transmission of multidrug resistant bacteria between animals, humans, and environment. The aim of our study was to assess the level of Enterobacteriaceae present in the intestines of rats collected from different locations in Tunisia, then to determine their antimicrobial susceptibility profiles, to screen extended spectrum ß-lactamases-producing strains and determine the molecular mechanism of ß-lactams resistance. Between July 2017 and June 2018, 55 strains of Enterobacteriaceae were isolated from 71 rats captured in various locations in Tunisia. Antibiotic susceptibility testing was performed using the disc diffusion method. Genes encoding ESBL and mcr genes were investigated by RT-PCR, standard PCR and sequencing when these genes were found. Fifty-five strains of Enterobacteriaceae were identified. The overall prevalence of ESBL production found in our study was 12.7 % (7/55) of which two E. coli strains were DDST positive, one isolated from a house-caught rat and one from the veterinary clinic and harbored the blaTEM-128 gene. In addition, the other five strains were DDST negative and harbored the blaTEM gene, including three strains isolated from collective restaurant (n = 2: blaTEM-163; n = 1: blaTEM-1), one strain isolated from the veterinary clinic (blaTEM-82), and one strain isolated from a house (blaTEM-128). The results of our study suggest that rodents may play a role in the spread of antimicrobial resistant E. coli, highlighting the need to protect the environment and monitor antimicrobial resistant bacteria in rodents to prevent their spread to other wildlife and humans.

First Report of OXA-48 and IMP Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Diarrheic Calves in Tunisia.

Antimicrobial resistance is one of the most serious threats to human and animal health. Evidence suggests that the overuse of antimicrobial agents in animal production has led to the emergence and dissemination of multidrug-resistant isolates. The objective of this study was to assess the rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in calf feces and to characterize their resistance genes for antibiotics like beta-lactams and colistin, but also to determine their virulence genes. Fecal samples were collected from 100 diarrheic calves in the region of Bizerte, Tunisia. After isolation, E. coli isolates were screened for antimicrobial resistance against 21 antibiotics by the disc diffusion method. Characterization of ß-lactamase genes and determination of associated resistance genes were performed by polymerase chain reaction. Among 71 E. coli isolates, 26 (36.6%) strains were ESBL-producing. Most of these isolates were multidrug-resistant (92.3%) and the most prevalent beta-lactamase genes detected were blaCTX-M (n = 26), blaSHV (n = 11), and blaTEM (n = 8), whereas only 1 isolate carried the blaCMY gene. In addition, resistance to carbapenems was detected in two isolates; one of them harbored both blaOXA-48 and blaIMP genes and the other isolate carried only the blaIMP gene. Several resistance genes were identified for the first time in Tunisia from cases of diarrheic calves. Furthermore, to the best of our knowledge, this is the first report of detection and identification of carbapenem resistance genes and virulence genes from calves in North Africa. A high occurrence of antimicrobial resistance of E. coli recovered from fecal samples of calves with diarrhea was observed, highlighting the need for prudent use of antimicrobial agents in veterinary medicine to decrease the incidence of multidrug-resistant bacteria for both animals and humans.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia.

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.

Epidemiology and genetic characteristics of tick-borne bacteria in dromedary camels of the world.

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.

First Report of Extended-Spectrum ß-Lactamase (blaCTX-M1) and Colistin Resistance Gene mcr-1 in E. coli of Lineage ST648 from Cockroaches in Tunisia.

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 ß-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 ß-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.

Prevalence of Escherichia coli O157:H7 isolated from fecal samples of diarrheic camels in Tunisia.

Shiga­toxin­producing E. coli (STEC) is a foodborne pathogen associated with outbreaks worldwide that can be identified in the feces and in the meat of food­producing 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. Non­sorbitol­fermenting 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 sulfamethoxazole­trimethoprim. 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 food­producing animals and meat products, to perform early and rapid identification of food­borne pathogens.

gltA typing of Anaplasma strains related to A. platys: Taxonomical and one health implications.

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.

Prevalence, virulence genes, and antimicrobial profiles of Escherichia coli O157:H7 isolated from healthy cattle in Tunisia.

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.

Anaplasma ovis Prevalence Assessment and Cross Validation Using Multiparametric Screening Approach in Sheep from Central 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.

Zoonotic Rickettsia Species in Small Ruminant Ticks From Tunisia.

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.

Zoonotic vector-borne bacteria in wild rodents and associated ectoparasites from Tunisia.

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.