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.

Salmonella Broiler Meat's Contamination in Tunisia: Prevalence, Serotypes, Antimicrobial Resistance and Molecular Characterization of Isolated Strains.

This study was conducted in north-eastern Tunisia to estimate the contamination prevalence of Salmonella in broilers' meat, to rank serotypes and to characterize the isolated multidrug-resistant (MDR) strains. A total number of 1288 meat samples were collected from 322 broiler batches; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disc diffusion method on Mueller-Hinton agar. Some antimicrobial resistance genes were identified using Polymerase Chain Reaction (PCR). The prevalence rates of Salmonella in the neck skin and the breast muscle contamination were estimated at 11.8% (38/322) and 0.9% (3/322), respectively. The prevalence rate of Salmonella in meat cutting parts contamination was estimated at 5.1% (33/644). Eight serotypes of Salmonella were identified, namely S. Enteritidis, S. Kentucky, S. Anatum, S. Infantis, S. Mbandaka, S. Zanzibar, S. Hadar and S. Agona. High rate of resistance was identified against amoxicillin (91.9%), nalidixic acid (83.8%), tetracycline (75.7%), streptomycin (73%), ciprofloxacin (70%), sulfamides (68.9%), cefalotin (68.9%), cefotaxim (67.6%) and cefoxitin (60.8%). The majority (90.5%; 67/74) of isolated strains was recognized as MDR. Nine MDR strains were identified as Extended-Spectrum ß-Lactamase (ESBL) producers. The blaCTX-M gene was identified by PCR in all the nine ESBL strains. TetA, tetB and dfrA1 genes were amplified in 3.6% (2/56), 1.8% (1/56) and 19.3% (5/26) of tetracycline and trimethoprim-resistant strains, respectively. The integrase gene (class 2) was identified in only 8.1% (6/74) of the Salmonella-isolated strains. Our findings highlight the emergence of MDR Salmonella isolates in Tunisia.

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.

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.

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.

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.

Prevalence, Risk Factors, Antimicrobial Resistance and Molecular Characterization of Salmonella in Northeast Tunisia Broiler Flocks.

This study was conducted in northeastern Tunisia to estimate both the prevalence and the risk factors of Salmonella in broiler flocks as well as to characterize the isolated multidrug-resistant (MDR) Salmonella strains. In the present study, a total number of 124 farms were sampled; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disk diffusion method on Mueller-Hinton agar using antimicrobial discs. Some antimicrobial resistance genes were identified using PCR. The prevalence rate of Salmonella infection, in the sampled farms, was estimated at 19.9% (64/322). Moreover, a total number of 13 different serotypes were identified. High rate of resistance was identified against nalidixic acid (82.85%), amoxicillin (81.25%), streptomycin (75%), and ciprofloxacin (75%). Alarming level of resistance to ertapenem (12.5%) was noticed. A total of 87.5% (56/64) of isolated strains were recognized as MDR. Three MDR strains were extended-spectrum ß-lactamases (ESBL)-producers and three MDR strains were cephalosporinase-producers. The blaCTX-M gene was amplified in all the three ESBL strains. The qnrB gene was not amplified in fluoroquinolones-resistant strains. The tetA and tetB genes were amplified in 5% (2/40) and 2.5% (1/40) of tetracycline-resistant strains, respectively. The dfrA1 gene was amplified in five of the 20 trimethoprim-resistant strains. The mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were not amplified in any of the phenotypically colistin-resistant strains. In terms of integrase genes int1 and int2, only gene class 2 was amplified in 11% (7/64) of analyzed strains. Risk factors, such as the poor level of cleaning and disinfection, the lack of antimicrobial treatment at the start of the breeding, and a crawl space duration lower than 15 days, were associated with high Salmonella infection in birds. These data should be considered when preparing salmonellosis control programs in Tunisian broiler flocks.

First Serological Evidence of Crimean-Congo Hemorrhagic Fever Virus and Rift Valley Fever Virus in Ruminants in Tunisia.

Crimean-Congo hemorrhagic fever virus (CCHFV, Nairoviridae family) and Rift Valley fever virus (RVFV, Phenuiviridae family) are zoonotic vector-borne pathogens with clinical relevance worldwide. Our study aimed to determine seroprevalences of these viruses and potential risk factors among livestock (cattle, sheep, and goats) in Tunisia. Sera were tested for antibodies against CCHFV (n = 879) and RVFV (n = 699) using various enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence assays (IIFA). The overall seroprevalence of IgG antibodies was 8.6% (76/879) and 2.3% (16/699) against CCHFV and RVFV, respectively. For CCHF seropositivity bioclimatic zones and breed were potential risk factors for the three tested animal species; while the season was associated with cattle and sheep seropositivity, tick infestation was associated with cattle and goats seropositivity and age as a risk factor was only associated with cattle seropositivity. Age and season were significantly associated with RVFV seropositivity in sheep. Our results confirm the circulation of CCHFV and RVFV in Tunisia and identified the principal risk factors in ruminants. This knowledge could help to mitigate the risk of ruminant infections and subsequently also human infections.

Analysis of Fluoride Contamination in Sheep Serum and in Water in the North East of Tunisia Ore Deposits.

The north east of Tunisia ore deposits (Hammam Zriba) contain a large amount of fluorides constituting a risk of environmental pollution as well as risks to human and animal health. The aim of our work was to assess the levels of fluorides in the water and blood of sheep in the east of Tunisia region. This analysis study included 78 water samples and 60 blood samples taken during 3 days in the year 2019, from a sheep herd at Hammam Zriba delegation. The determination of fluorine concentration in the samples was carried out by potentiometry with a selective electrode type ISE combined Fluoride perfectION™. High Fluoride concentrations were found at a mean of 1.62 ± 1.18, 1.45 ± 0.98, and 2.65 ± 1.61 mg/l, respectively, in running, deep, and stagnant waters. In addition, 83.3% of the animals reared within 400 m of the study area had elevated fluoride levels in the blood exceeding the usual values (fluoremia> 0.15 mg/l). The concentration of fluoride in the blood of the animals decreases with increasing distance from the mine.

Risk based serological survey of Rift Valley fever in Tunisia (2017-2018).

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.

First serological evidence of the Rift Valley fever Phlebovirus in Tunisian camels.

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.

Seroprevalence and associated risk factors of Chlamydia abortus infection in ewes in Tunisia.

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.

High Genetic Diversity of Enterobacteriaceae Clones and Plasmids Disseminating Resistance to Extended-Spectrum Cephalosporins and Colistin in Healthy Chicken in Tunisia.

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.

Various Inc-type plasmids and lineages of Escherichia coli and Klebsiella pneumoniae spreading blaCTX-M-15,blaCTX-M-1 and mcr-1 genes in camels in Tunisia.

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 ß-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.

Coxiella burnetii in Tunisian dromedary camels (Camelus dromedarius): Seroprevalence, associated risk factors and seasonal dynamics.

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.

Epidemiology, Antimicrobial Resistance, and Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Clinical Bovine Mastitis in Tunisia.

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 blaCTX-M-15 gene. The K. pneumoniae belonged to two highly uncommon sequence types (ST471 and ST1083), while E. coli clustered in the ST167/617 clones, which have been widely reported in humans, animals, and the environment. These data point out the necessity to improve farm management in Tunisia to reduce the occurrence of coliform-induced mastitis and to avoid the dissemination in this sector of ESBL-producing E. coli and K. pneumoniae, which are of public health concern.

Molecular Survey of Anaplasma Species in Small Ruminants Reveals the Presence of Novel Strains Closely Related to A. phagocytophilum in Tunisia.

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 (<2 years) subjects (p = 0.001 and 0.002 for goats and sheep, respectively). In sheep, A. ovis prevalence was higher in ewes with respect to rams (p = 0.010). The A. ovis infection rate was significantly lower in goats of the local breed (p = 0.049) and it was higher in goats infested by ticks than in not infested animals (p = 0.005). Genetic analysis of the msp4 gene of A. ovis indicated the presence of strains shared by Tunisian sheep and goats. Sequence analysis and phylogenetic studies on the basis of the 16S rRNA gene provided evidence for the circulation of at least two different potentially novel species genetically related to A. phagocytophilum in Tunisian small ruminants. These findings cause concern about specificity of serological tests used for detection of A. phagocytophilum in ruminants and provide additional information for elucidating pathogenesis and molecular epidemiology of A. phagocytophilum and related species.