Prevalence of Middle East Respiratory Syndrome Coronavirus in Dromedary Camels, Tunisia.

Free-roaming camels, especially those crossing national borders, pose a high risk for spreading Middle East respiratory syndrome coronavirus (MERS-CoV). To prevent outbreaks, active surveillance is necessary. We found that a high percentage of dromedaries in Tunisia are MERS-CoV seropositive (80.4%) or actively infected (19.8%), indicating extensive MERS-CoV circulation in Northern Africa.

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.

Viral and Bacterial Zoonotic Agents in Dromedary Camels from Southern Tunisia: A Seroprevalence Study.

The rapid spread of SARS-CoV-2 clearly demonstrated the potential of zoonotic diseases to cause severe harm to public health. Having limited access to medical care combined with severe underreporting and a lack of active surveillance, Africa carries a high burden of neglected zoonotic diseases. Therefore, the epidemiological monitoring of pathogen circulation is essential. Recently, we found extensive Middle East respiratory syndrome coronavirus (MERS-CoV) prevalence in free-roaming dromedary camels from southern Tunisia. In this study, we aimed to investigate the seroprevalence, and thus the risk posed to public health, of two additional viral and two bacterial pathogens in Tunisian dromedaries: Rift Valley fever virus (RVFV), foot-and-mouth disease virus (FMDV), Coxiella burnetii and Brucella spp. via ELISA. With 73.6% seropositivity, most animals had previously been exposed to the causative agent of Q fever, C. burnetii. Additionally, 7.4% and 1.0% of the dromedaries had antibodies against Brucella and RVFV, respectively, while no evidence was found for the occurrence of FMDV. Our studies revealed considerable immunological evidence of various pathogens within Tunisian dromedary camels. Since these animals have intense contact with humans, they pose a high risk of transmitting serious zoonotic diseases during active infection. The identification of appropriate countermeasures is therefore highly desirable.

Molecular epidemiology and phylogeny of spotted fever group Rickettsia in camels (Camelus dromedarius) and their infesting ticks from Tunisia.

Rickettsia species are adapted to a wide range of specific animal hosts. Camels (Camelus dromedarius) have been identified as a carrier of various zoonotic pathogens and became a focus of growing public health interest. This study reported the occurrence of rickettsial infection in camels and infesting ticks from five Tunisian governorates. Based on ompB PCR, eight out of 293 camels (2.7%) were found to be infected with Rickettsia spp. Furthermore, 13 tick specimens of Hyalomma impeltatum (10.4%) and 9 of H. dromedarii (8.0%) harboured DNA of Rickettsia bacteria with an overall prevalence rate of 9.2% (22/237). Molecular prevalence of Rickettsia infection varied significantly according to tick infestation for camels and among tick genders. Five rickettsial species, showing a potential public health interest, were revealed by sequencing. Based on ompB partial sequences, five species were identified corresponding to R. aeschlimannii, R. monacensis, R. helvetica and R. massiliae in camels and to R. africae, R. aeschlimannii, R. monacensis and R. helvetica in ticks. Based on ompA typing, three species were revealed corresponding to R. africae and R. monacensis in camels and to R. africae, R. aeschlimannii and R. monacensis in ticks. This is the first report consolidating the hypothesis that camels may serve as potential hosts for Rickettsia spp. and Hyalomma spp. ticks as possible vectors in arid and Saharan areas of Tunisia. The present data highlight the importance of preventive measures and survey that must be implemented in camel herds in order to limit the spread of these vector-borne bacteria to animals and humans.

Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia.

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

First report on Bartonella henselae in dromedary camels (Camelus dromedarius).

Bartonellosis is one of the clinically underdiagnosed emerging bacterial diseases among domestic livestock, particularly in camels. Until now, the natural infection of camels with Bartonella species was not investigated in Tunisia. In the attempt of filling this gap in knowledge, a total of 412 dromedary camels (Camelus dromedarius) as well as 300 associated ticks (Hyalomma dromedarii (160; 53.4%), H. impeltatum (131; 43.6%) and H. excavatum (9; 3%) were screened for the presence of Bartonella spp. by PCR followed by a sequencing step through the amplification of the rpoB gene. Positive samples were then tested and further characterized by the combined use of the ftsZ and gltA genes. Fifteen camels (3.6%) were found to be positive to Bartonella spp. However, there was no evidence of Bartonella DNA in any of the analyzed ticks. Risk factors' analysis shows that camels derived from arid and semi-arid bioclimatic areas were more infected than those originated from desert area. Molecular characterization and phylogenetic analysis revealed the occurrence of novel B. henselae genotypes closely related to those isolated from humans, cats, and lions. By combining the characteristics of each single gene with those of concatenated sequences, we report here the first molecular detection of B. henselae in the dromedary camel suggesting a possible involvement of camelids as hosts or reservoirs in the transmission cycle of this emerging bacterium in arid and saharan areas.

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.

Improving specific detection and updating phylogenetic data related to Anaplasma platys-like strains infecting camels (Camelus dromedarius) and their ticks.

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.

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.