Colistin Resistance Mechanism in Enterobacter hormaechei subsp. steigerwaltii Isolated from Wild Boar (Sus scrofa) in France.

Wild animals may act as efficient antimicrobial-resistance reservoirs and epidemiological links between humans, livestock, and natural environments. By using phenotypic and genotypic characterization, the present study highlighted the occurrence of an antimicrobial-resistant (i.e., amoxicillin, amoxicillin-clavulanic acid, cephalothin, and colistin) Enterobacter hormaechei subsp. steigerwaltii strain in wild boar (Sus scrofa) from France. The molecular analysis conducted showed non-synonymous mutations in the pmrA/pmrB and phoQ/phoP operons and the phoP/Q regulator mgrB gene, leading to colistin resistance. The present data highlight the need for continuous monitoring of multidrug-resistant bacteria in wild animals to limit the spread of these threatening pathogens.

Serological and molecular survey of Leptospira spp. infections in wild boars and red foxes from Southeastern France.

BACKGROUND AND AIM: Leptospirosis is a zoonotic disease. Information on the recent prevalence of Leptospira in hunted wild animals is limited, particularly in southeastern France. A cross-sectional survey was conducted to assess the prevalence and diversity of Leptospira spp. among wild boars (Sus scrofa) and red foxes (Vulpes vulpes) from two military camps in Southeastern France. MATERIALS AND METHODS: Serological analyses were performed using microscopic agglutination tests and polymerase chain reaction (PCR) assays were used to demonstrate Leptospira spp. infection from boar kidney DNA extracts. RESULTS: According to the species, the positive sera were obtained from 18% of 358 boars and 6 % of 64 foxes tested. The prevalence rate is significantly higher (p≤0.02) in boars than in foxes. In wild boar, Australis represents the most recorded serogroup (15.9%), followed by Sejroe (2.8%) and icterohaemorhagiae (2.8%). In red fox, icterohaemorhagiae represents the most recorded serogroup (6.25%), followed by Sejroe (1.57%) and Hebdomadis (1.57%). PCR-based detection of Leptospira DNA was positive in 6/62 (9.6%) of the wild boars tested. CONCLUSION: The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Southeastern France. Due to their predatory behavior and their varied diet, mainly composed of small mammals, red foxes could be considered sentinel animals of environmental contamination with leptospires.

SARS-CoV-2 antibodies seroprevalence in dogs from France using ELISA and an automated western blotting assay.

Dogs are occasionally susceptible to SARS-CoV-2, developing few or no clinical signs. Epidemiological surveillance of SARS-CoV-2 in dogs requires testing to distinguish it from other canine coronaviruses. In the last year, significant advances have been made in the diagnosis of SARS-CoV-2, allowing its surveillance in both human and animal populations. Here, using ELISA and automated western blotting (AWB) assays, we performed a longitudinal study on 809 apparently healthy dogs from different regions of France to investigate anti-SARS-CoV-2 antibodies. There were three main groups: (i) 356 dogs sampled once before the pandemic, (ii) 235 dogs sampled once during the pandemic, and (iii) 218 dogs, including 82 dogs sampled twice (before and during the pandemic), 125 dogs sampled twice during the pandemic and 11 dogs sampled three times (once before and twice during the pandemic). Using ELISA, seroprevalence was significantly higher during the pandemic [5.5% (25/453)] than during the pre-pandemic period [1.1% (5/449)]. Among the 218 dogs sampled twice, at least 8 ELISA-seroconversions were observed. ELISA positive pre-pandemic sera were not confirmed in serial tests by AWB, indicating possible ELISA cross-reactivity, probably with other canine coronaviruses. A significant difference was observed between these two serological tests (Q = 88, p = 0.008). A clear correlation was observed between SARS-CoV-2 seroprevalence in dogs and the incidence of SARS-CoV-2 infection in human population from the same area. AWB could be used as a second line assay to confirm the doubtful and discrepant ELISA results in dogs. Our results confirm the previous experimental models regarding the susceptibility of dogs to SARS-CoV-2, suggesting that viral transmission from and between dogs is weak or absent. However, the new variants with multiple mutations could adapt to dogs; this hypothesis cannot be ruled out in the absence of genomic data on SARS-CoV-2 from dogs.

Enzootic Hepatic Capillariasis (Calodium hepaticum) in Street Rats (Rattus norvegicus) from Marseille City, France.

Hepatic capillariasis is a rare and neglected zoonosis affecting wild and synanthropic small rodents. It is caused by infection with Calodium hepaticum in liver. Despite the worldwide distribution of the host Rattus norvegicus (brown or street rats) in the urban area, the epidemiological status of this parasitosis remains unknown. In the present study, we examined a total of 27 brown rats from the city centre and a garden (four km from the city centre) of Marseille, France. All rats were autopsied and 52% showed the presence of C. hepaticum eggs in the liver. This result draws general attention to public health risks, since street rats are living near the human population.

MOLECULAR INVESTIGATION OF VECTOR-BORNE PATHOGENS IN RED FOXES (VULPES VULPES) FROM SOUTHERN FRANCE.

Because of their free-ranging nature, the probability of wild animals being exposed to vector-borne pathogens is likely higher than that of humans and pets. Recently, the red fox (Vulpes vulpes) has been suspected as being a reservoir or host of several pathogens of veterinary and public health importance. We conducted a molecular survey on 93 red foxes hunted in 2008-18, in the departments of Bouches-du-Rhône and Var, in southeastern France, for pathogens including Leishmania infantum, Piroplasmida, Hepatozoon spp., nematodes, Coxiella burnetii, Borrelia spp., Rickettsia spp., and Anaplasmataceae. Spleen samples were screened for the presence of vector-borne pathogens by PCR followed by sequencing. Pathogens were detected in 94% (87/93) of red foxes, and coinfections were identified in 24% (22/93) of foxes. We identified DNA from Hepatozoon canis, L. infantum, and Babesia vogeli in 92% (86/93), 15% (14/93), and 3% (3/93) of red foxes, respectively. We also found DNA of nematodes in 3% (3/93) of foxes; Spirocerca vulpis was identified in one fox and Dirofilaria immitis in the two others. Interestingly, C. burnetii genotype 3, previously described in humans from the same region, was identified in 3% (3/93) of foxes and Anaplasma platys in 2% (2/93) of foxes. We did not detect DNA of Borrelia spp., Bartonella spp., or Rickettsia spp. In our study, the prevalence of pathogens did not vary by fox origin, sex, or tick carriage. Molecular evidence of B. vogeli, H. canis, S. vulpis, D. immitis, C. burnetii, and A. platys in red foxes has not previously, to our knowledge, been reported from southern France. We propose that red foxes are potential reservoirs for several pathogens, including major zoonotic agents such as L. infantum. They could be incidental hosts for pathogens, such C. burnetii. The high prevalence for H. canis suggests an important role of foxes in domestic dog (Canis lupus familiaris) infection. These animals may pose a threat to human and animal health.

Detection of Canine Vector-Borne Filariasis and Their Wolbachia Endosymbionts in French Guiana.

In French Guiana, canine heartworm disease is well known, but the diversity of filarial parasites of dogs remains largely unknown. A total of 98 canine blood samples from Cayenne and Kourou were assessed by a blood wet mount preparation, heartworm antigen test and molecular exploration of filarioid and Wolbachia DNAs, followed by a multiplex species-specific qPCR's identification and a subsequent sequencing analysis. Thereafter, a phylogeny based on maximum likelihood was carried out to facilitate specific identification. Five dogs were microfilaremic. Heartworm antigens were detected in 15 (15.3%) dogs. Of these, six (6.1%) were considered as occult infections as neither microfilariae nor Dirofilaria immitis DNA were detected. The 11 (11.2%) D. immitis isolates corresponded to a low virulent strain. Six of the D. immitis isolates were positive for Wolbachia endosymbionts of D. immitis belonging to the clade C DNA. Acanthocheilonema reconditum DNA was detected in 3 (3.1%) samples. Of these latter, one was found co-infected with the Brugia sp. genotype and the DNA of the clade D of the Wolbachia endosymbiont of Brugia species. This latter was also detected in two filarioid DNA-free samples. Finally, two samples were positive for Cercopithifilaria bainae genotype, which is distinct from those identified in Europe. The present study highlights the urgent need to implement chemoprophylaxis associated with anti-Wolbachia drugs to control these potential zoonoses.

Frequent transmission of hepatitis E virus among piglets in farms in Southern France.

The present study aimed to assess whether hepatitis E virus (HEV) is present in domestic pigs in Southern France, and to determine the relationship between HEV sequences detected from pigs and from humans. Two hundred fifteen sera, 207 stools, and 107 bile samples were collected from 3- or 6-month-old pigs from different regions of Southern France. Pig IgG anti-HEV antibodies testing was performed using a commercial ELISA kit with minor modifications. Pig HEV RNA was tested by real-time PCR and sequencing assays using "in-house" protocols. Forty percent of pigs were HEV-seropositive. Sixty-five percent of 3-month-old pigs and none of 6-month-old pigs were HEV RNA-positive. HEV RNA was significantly more frequently detected from stools than from sera (65% vs. 22%; P < 0.001). Phylogenetic analysis showed that pig HEV sequences belonged to genotype 3 and formed two clusters of genotype 3f and 3e. Nucleotide homology between pig HEV sequences of each cluster was high (>97%), and clusters were correlated with the geographical origin of pigs and with their repartition into pens and buildings in the pig farm. Based on analysis of 331 nucleotides, pig HEV sequences were close genetically to HEV sequences found from humans or pigs in Europe, and one showed complete nucleotide identity with an HEV sequence obtained in France from a human. The present data indicate that 3-month-old pigs from Southern France might represent a potential source of HEV transmission to humans, and stress the potential of HEV to cause epizootic infections in population of farm pigs.

Potential animal reservoirs (dogs and bats) of human visceral leishmaniasis due to Leishmania infantum in French Guiana.

In French Guiana, cutaneous leishmaniasis is highly endemic, whereas no autochthonous case of visceral leishmaniasis have been reported so far. However, due to its proximity to Brazil which is highly endemic for visceral leishmaniasis, and the high transboundary population flow, an epidemiological challenge could arise at any time. As an overseas department and region and the largest outermost region of the European Union, epidemiological surveillance of visceral leishmaniasis is of great importance. Our study aimed to investigate the presence of Leishmania spp. in domestic (dogs) and sylvatic (bats) animals from French Guiana. Over the 2008-2018 period, samples from 349 animals were collected. They included blood from 179 autochthonous dogs and 59 bats, spleen samples from 33 bats and, blood from 78 military working dogs (MWD) collected before their departure from continental France and at the end of their four-month stay in French Guiana. Samples were screened using real-time polymerase chain reaction (qPCR) assays targeting Leishmania DNA followed by sequencing of 18S rRNA, kDNA and ITS2 genes. L. infantum was detected in 2.3% (8/349) of animals with 1.7% (3/179) of autochthonous dogs, 5.1% (4/78) of MWD returning from French Guiana, whereas they were negative before their departure. One of them dates back to 2012. All these dogs were positive for serological tests. In addition, L. infantum DNA was detectable in one bat spleen sample, belonging to Carollia perspicillata species. We report here for the first time an infection with L. infantum in dogs and bat from French Guiana. Our results suggest the existence of potential reservoir and transmission cycle for visceral leishmaniasis, at least since 2012, which was unknown in this territory until now. Further studies are needed to determine how these animals were infected and which vectors are involved in the transmission in this area.

[Prevention of zoonoses: creation of a unit for early detection of animal infections]. / Pour une approche préventive des zoonoses: la création d'une cellule de détection précoce des infections animales.

In a context in which known and potential zoonoses are emerging (simian retrovirus infections, SRAS, West Nile, H5N1 avian influenza, etc.), French army veterinarians have been tasked with assessing epidemiological risks related to animals in proximity with troops, particularly during foreign operations. They have already completed surveys of more than 70 infections (toxoplasmosis, leishmaniasis, rickettiosis, leptospirosis, Q fever, hepatitis E, Rift valley fever, etc.). The strategy consists of detecting pathogenic agents ("the right sample, at the right time, and kept in the right conditions") in reservoir animals and vectors, upstream of epidemics. The authors propose to set up a flexible and mobile animal infection detection unit, working closely with hospital physicians, veterinarians and specialized microbiology laboratories. This would be an efficient tool for anticipating, preventing and combating zoonoses.

Draft Genome Sequence of "Nocardia suismassiliense" Strain S-137 (CSUR P4007).

"Nocardia suismassiliense" strain S-137 isolated from Sus scrofa feces exhibits a 9.4-Mb (67.1% GC content) draft genome sequence containing 8,658 protein-coding genes, 66 tRNAs, and 9 rRNAs. In silico DNA-DNA hybridization confirmed strain S-137 as representative of a new species, "Nocardia suismassiliense," closely related to N. tenerifensis and N. brasiliensis.

Molecular and serological investigation of Trypanosoma cruzi infection in dogs in French Guiana.

Clinical cases of Chagas disease, an infection caused by the parasite Trypanosoma cruzi, have been recently described in humans and dogs in French Guiana, a French overseas department located in South America. Elsewhere in endemic countries for this disease, cases of asymptomatic infections have been described. We performed a prevalence survey of the infection in dogs in Cayenne and Kourou, the main cities of French Guiana. In 2014 and 2016, blood samples were taken from 153 dogs from Cayenne and Kourou. All dogs were apparently healthy at the time of sampling. Sex and age of the dogs were recorded as well as the location where they lived. Serum samples from dogs were screened using a rapid immunochromatographic test (Chagas Stat-Pak®Assay, Chembio, USA) detecting anti-T. cruzi antibodies. Simultaneously, a real-time PCR targeting T. cruzi kDNA was performed on the blood samples of the dog. Six dogs (3.9%) were positive only in serology and one (0.6%) only in qPCR. Two dogs were positive for both tests. The prevalence of infection (positivity for one of the two tests) was 5.8% (9/153). There was no significant difference (χ2 test) between Cayenne (5/100) and Kourou (4/53), between males (3/60) and females (6/93), or between 2014 (2/55) and 2016 (7/98). Canine surveillance is a useful tool for the public health risk assessment of Chagas disease. Positive dogs, even when asymptomatic, should be treated as they can serve as a reservoir for T. cruzi.

Q fever epidemic in Cayenne, French Guiana, epidemiologically linked to three-toed sloth.

A Q fever epidemic occurred in 2013 in a small military residential area in Cayenne, French Guiana. A retrospective cohort study was conducted to identify Q fever risk factors. Confirmed acute Q fever case was defined as positive serology (IgM ≥ 50 and phase II IgG ≥ 200) and/or positive qPCR on serum or blood. In addition, wild mammals were captured at the study site and tested by serology and real-time PCR performed on blood, vaginal swabs and ticks. The attack rate was 20 percent (11/54). All the cases were symptomatic with fever >38.5 °C and community-acquired pneumonia for four cases. Log binomial multivariate models identified two independent risk factors associated with Q fever: to clean the house (RRa = 7.5 CI95% [1.03-55.3]) and to carry a three-toed sloth in arms (RRa = 2.6 CI95% [1.1-5.8]). Eighteen marsupial individuals were captured, all PCRs were negative but 17% (3/18) had a positive serology. Another study conducted after the epidemic found only one (1/4) three-tooth sloth (Bradypus tridactylus) with feces highly infectious for C. burnetii MST17. The same strain C. burnetii genotype 17 has been laboratory- confirmed in this mammal and in human cases. These results support the implication of three-toed-sloth in this epidemic. Human contamination mainly occurs through inhalation of infectious aerosols as suggested by high relative risk associated with house cleaning activities and pulmonary forms of the disease, and through direct contact with three- toed-sloth. Positive serological results among marsupials confirm wildlife exposure and suggest a more complex sylvatic transmission cycle among wild mammals.

First Serological Evidence of West Nile Virus in Horses and Dogs from Corsica Island, France.

West Nile virus (WNV) is widely distributed over the world, including Europe, Africa, and Asia and spread over the past two decades to North and South America. In the south of France, sporadic cases are frequently described and the virus is endemic in Italy with frequent cases and outbreaks. The aim of this study was to identify a possible WNV circulation in Corsica (French island in the Mediterranean Sea) in sheep, horses, and dogs as sentinel animals for the virus surveillance. In 2014, 386 blood samples were collected from 219 sheep, 96 horses, and 71 dogs, in 12 localities in Corsica, in the oriental coast of Corsica. Each sample was systematically tested for WNV immunoglobulin G using an in-house enzyme-linked immunosorbent assay (ELISA) with inactivated WNV as antigen. The result of the ELISA for the WNV antibody test on the sheep sera was all negative, whereas 9 of 96 horses (9.4%) and 6 of 71 dogs (8.4%) presented WNV antibodies. All the positive samples from horses and dogs were confirmed by serum neutralization test. Although no clinical case in humans and horses was reported to date, this report highlights the necessity to improve WNV surveillance in animals and humans, as well as in blood donors in Corsica.

Molecular detection of Bartonella quintana, B. Elizabethae, B. Koehlerae, B. Doshiae, B. Taylorii, and Rickettsia felis in rodent fleas collected in Kabul, Afghanistan.

The prevalences of Bartonella spp. and Rickettsia spp. were investigated using molecular methods in 77 rodent fleas collected in November 2002 by the French forces detachment in Kabul, Afghanistan. Overall, Bartonella DNA was detected in 15.5% of gerbil fleas and 40.5% of rat fleas, whereas Rickettsia felis was found in 9% of gerbil fleas. We described for the first time in this country Bartonella quintana, B. koehlerae, B. taylorii, and Rickettsia felis in fleas from the gerbil species Meriones lybicus, and B. elizabethae and B. doshiae in rat fleas. Of these, B. quintana, B. elizabethae, B. koehlerae, and R. felis are recognized human pathogens. These results emphasize the potential risk of flea-borne infections transmitted by rodents in this area, and suggest that preventive measures should be taken in the general framework of zoonoses management.

Seroprevalence of Toscana virus in dogs from Corsica, France.

BACKGROUND: Toscana virus (TOSV) is an arbovirus belonging to the Bunyaviridae, a family of negative-stranded, enveloped RNA viruses. The virus can be transmitted to humans through the bite of an infected female sand fly of the genus Phlebotomus. Infections are usually asymptomatic but the virus is known to cause aseptic meningitis and/or meningo-encephalitis in the Mediterranean countries. Dogs are good sentinels for detection of viral circulation and are more easily accessible than wild animals. FINDINGS: In 2013 and 2014, we collected sera from 231 adult dogs living in 26 counties in two departments in Corsica, a French island in the Mediterranean. The virus microneutralization-based seroprevalence assay revealed a seropositivity of 3.9 % dogs on the eastern coast of Corsica. CONCLUSIONS: Our study confirms the circulation of TOSV in Corsica. Accordingly, in geographical areas where dogs possess TOSV neutralizing antibodies, direct and indirect TOSV diagnosis should be implemented in patients presenting with febrile illnesses and central nervous system infections such as meningitis and encephalitis.

Evidence of Bartonella spp. in Blood and Ticks (Ornithodoros hasei) of Bats, in French Guiana.

We screened blood from 59 bats from French Guiana for Bartonella spp. PCRs were positive for 13.6% and culture was positive in one Noctilio albiventris and one Pteronotus parnellii, as well as in Ornithodoros hasei ticks collected from bats. Two isolated strains represent possible two new species.

New Rickettsia species in soft ticks Ornithodoros hasei collected from bats in French Guiana.

In French Guiana, located on the northeastern coast of South America, bats of different species are very numerous. The infection of bats and their ticks with zoonotic bacteria, especially Rickettsia species, is so far unknown. In order to improve knowledge of these zoonotic pathogens in this French overseas department, the presence and diversity of tick-borne bacteria was investigated with molecular tools in bat ticks. In the beginning of 2013, 32 bats were caught in Saint-Jean-du-Maroni, an area close to the coast of French Guiana, and the ticks of these animals were collected. A total of 354 larvae of Argasidae soft ticks (Ornithodoros hasei) from 12 bats (Noctilio albiventris) were collected and 107 of them were analysed. DNA was extracted from the samples and quantitative real-time PCR was carried out to detect Rickettsia spp., Bartonella spp., Borrelia spp. and Coxiella burnetii. All tested samples were negative for Bartonella spp., Borrelia spp. and Coxiella burnetii. Rickettsia DNA was detected in 31 (28.9%) ticks. An almost entire (1118 base pairs long) sequence of the gltA gene was obtained after the amplification of some positive samples on conventional PCR and sequencing. A Bayesian tree was constructed using concatenated rrs, gltA, ompA, ompB, and gene D sequences. The study of characteristic sequences shows that this Rickettsia species is very close (98.3-99.8%) genetically to R. peacockii. Nevertheless, the comparative analysis of sequences obtained from gltA, ompA, ompB, rrs and gene D fragments demonstrated that this Rickettsia is different from the other members of the spotted fever group. The sequences of this new species were deposited in GenBank as Candidatus Rickettsia wissemanii. This is the first report showing the presence of nucleic acid of Rickettsia in Ornithodoros hasei ticks from South American bats.

Undiagnosed leptospirosis cases in naïve and vaccinated dogs: properties of a serological test based on a synthetic peptide derived from Hap1/LipL32 (residues 154-178).

Leptospirosis is a common disease in dogs, despite having current vaccinations. However, leptospirosis diagnosis based on the routine Microscopic Agglutination Test (MAT) leads to confusing conclusions, especially for infected vaccinated dogs. Indeed, both bacterin and natural infection stimulate the production of agglutinating antibodies. In experimentally infected dogs, antibodies against the peptide PP derived from Hap1/Lipl32 were raised earlier than agglutinating antibodies. The background level of these antibodies was determined in a group of 109 healthy dogs, either vaccinated or not against leptospirosis, with a specificity for IgM of 96.4% and for IgG of 95.5%. PP ELISA was subsequently performed with 118 sera from dogs with suspected leptospirosis that was not confirmed by MAT. New leptospirosis cases based on the PP ELISA results were suspected in 14 out of 102 vaccinated dogs and in two out of 16 non-vaccinated dogs. These results highlight the importance of serological diagnosis corresponding to an interesting window when it is too late for PCR detection and too early to be confirmed by MAT.