Comparative Evaluation of the Efficacy of Two Ectoparasiticides in Preventing the Acquisition of Borrelia burgdorferi by Ixodes scapularis and Ixodes ricinus: A Canine Ex Vivo Model.

In dogs, tick infestation can cause damage ranging from a simple skin irritation to severe diseases and/or paralysis leading to animal death. For example, Ixodes ricinus and I. scapularis are among the tick species incriminated the most in the transmission of Borrelia burgdorferi, the agent of human and canine Lyme borreliosis (LB). In this study, we aimed to compare the efficacy of two products designed for dogs-an oral systemic ectoparasiticide and a topical repellent ectoparasiticide-against the acquisition of B. burgdorferi by adult I. scapularis and I. ricinus using an ex vivo model. Thirty-two beagle dogs were included in a parallel-group-designed, randomized, single-center, negative-controlled efficacy study. The dogs were allocated to three groups based on gender and body weight: a fluralaner (F, Bravecto®) treatment group (n = 8), administered a single oral treatment on day 0 at the recommended dose; a dinotefuran-permethrin-pyriproxyfen (DPP, Vectra® 3D) treatment group (n = 8), topically treated on day 56 at the recommended dose; and an untreated control group (n = 16). Blood and hair were collected from each dog on days 58, 63, 70, 77, and 84. Hair was added to the silicone-based membrane separating two glass chambers forming the feeding unit (FU). Chamber 1 was filled with blood spiked with B. burgdorferi sensu stricto, strain B31 (105 cells/mL). Chamber 2, glued below chamber 1, was seeded with 20 adult I. scapularis or I. ricinus. The FUs (n = 240) were incubated at 37 °C with a humidity >90%. Tick survival, attachment, and feces presence were observed from 1 h up to 72 h after tick seeding. The uptake of B. burgdorferi was determined in ticks using nested polymerase chain reaction (nPCR). The acaricidal efficacy of DPP-treated hair was 100% within 1 h of tick release on every study day for both I. ricinus and I. scapularis. The speed of kill associated with DPP was sufficiently fast to prevent tick attachment and engorgement, and, consequently, to prevent the acquisition of B. burgdorferi. In the F-treated group, the acaricidal efficacy observed at 12 h, throughout the study, was <20% and <28% for I. scapularis and I. ricinus, respectively. Furthermore, tick feces were observed in the FUs, and several female ticks (I. scapularis (n = 55) and I. ricinus (n = 94)) tested positive for B. burgdorferi. The results provide proof of concept for the use of an ex vivo model based on an artificial feeding system to compare two ectoparasiticides against the acquisition of B. burgdorferi by I. ricinus and I. scapularis. In addition, our results demonstrate the superiority of DPP compared to F in the speed of acaricidal activity against ticks, as well as in preventing the acquisition of B. burgdorferi.

Vectra 3D (dinotefuran, pyriproxyfen and permethrin) prevents acquisition of Borrelia burgdorferi sensu stricto by Ixodes ricinus and Ixodes scapularis ticks in an ex vivo feeding model.

BACKGROUND: We evaluated the efficiency of an ex vivo feeding technique using a silicone membrane-based feeding chamber to (i) assess the anti-feeding and acaricidal efficacy of a spot-on combination of dinotefuran, pyriproxyfen and permethrin (DPP, Vectra® 3D) against adult Ixodes scapularis and Ixodes ricinus ticks, and to (ii) explore its effect on blocking the acquisition of Borrelia burgdorferi sensu stricto. METHODS: Eight purpose-bred dogs were randomly allocated to two equal-size groups based on body weight assessed on day 2. DPP was administered topically, as spot-on, to four dogs on day 0. Hair from the eight dogs was collected individually by brushing the whole body on days 2, 7, 14, 21, 28 and 35. On each day of hair collection, 0.05 g of sampled hair was applied on the membrane corresponding to each feeding unit (FU). Seventy-two FU were each seeded with 30 adults of I. scapularis (n = 24 FU) or I. ricinus ticks (n = 48 FU). Bovine blood spiked with B. burgdorferi sensu stricto (strain B31) was added into each unit and changed every 12 h for 4 days. Tick mortality was assessed 1 h after seeding. One additional hour of incubation was added for live/moribund specimens and reassessed for viability. All remaining live/moribund ticks were left in the feeders and tick engorgement status was recorded at 96 h after seeding, and the uptake of B. burgdorferi s.s. was examined in the collected ticks by applying quantitative real-time PCR. RESULTS: Exposure to DPP-treated hair was 100% effective in blocking B. burgdorferi s.s. acquisition. The anti-feeding efficacy remained stable (100%) against both Ixodes species throughout the study. The acaricidal efficacy of DPP evaluated at 1 and 2 h after exposure was 100% throughout the study for I. ricinus, except the 1-h assessment on day 28 (95.9%) and day 35 (95.3%). The 1-h assessment of acaricidal efficacy was 100% at all time points for I. scapularis. CONCLUSIONS: The ex vivo feeding system developed here demonstrated a protective effect of DPP against the acquisition of B. burgdorferi without exposing the animals to the vectors or to the pathogen.

Broad diversity of Mycobacterium tuberculosis complex strains isolated from humans and cattle in Northern Algeria suggests a zoonotic transmission cycle.

Mycobacterium tuberculosis complex (MTBC) comprises closely related species responsible for human and animal tuberculosis (TB). Efficient species determination is useful for epidemiological purposes, especially for the elucidation of the zoonotic contribution. In Algeria, data on MTBC genotypes are largely unknown. In this study, we aimed to investigate the occurrence and diversity of MTBC genotypes causing human and bovine TB in Northern Algeria. During a two-year sampling period (2017-2019) in two regions of Northern Algeria, we observed an overall prevalence of 6.5% of tuberculosis (TB) among slaughtered cattle, which is higher than previous Algerian data yet comparable to neighboring countries. A total of 296 Mycobacterium tuberculosis complex (MTBC) isolates were genotyped by spoligotyping: 181 from tissues with TB-like lesions collected from 181 cattle carcasses and 115 from TB patients. In human isolates, we identified 107 M. tuberculosis, seven M. bovis and one "M. pinnipedii-like", while for bovine samples, 174 isolates were identified as M. bovis, three as M. caprae, three as "M. pinnipedii-like" and one as "M. microti-like". The majority of isolates (89.2%) belonged to 72 different known Shared International Types (SIT) or M. bovis spoligotypes (SB), while we also identified seven new SB profiles (SB2695 to SB2701). Twenty-eight of the SB profiles were new to Algeria. Our data suggest zoonotic transmission in Sétif, where significantly more TB was observed among cattle (20%) compared to the slaughterhouses from the three other regions (5.4%-7.3%) (p < 0.0001), with the isolation of the same M. bovis genotypes from TB patients. The present study showed a high genetic diversity of MTBC isolated from human and cattle in Northern Algeria. Even though relatively small in terms of numbers, our data suggest the zoonotic transmission of TB from cattle to humans, suggesting the need for stronger eradication strategies for bovine TB.

Aedes (Stegomyia) aegypti mosquito bite hypersensitivity in a dog: a case report.

BACKGROUND: Mosquitoes are vectors of several pathogens of considerable importance to humans and companion animals, including nematode helminths such as Dirofilaria immitis and Dirofilaria repens that cause heartworm disease and subcutaneous dirofilariosis, respectively. In addition to mosquito-borne pathogen transmission, mosquito bites can cause discomfort and irritation in pets, and even lead to severe hypersensitivity reactions. In the present study, we report an acute local hypersensitivity reaction in a dog following experimental exposure to Aedes (Stegomyia) aegypti. CASE PRESENTATION: A healthy six-year-old male beagle was included in an efficacy study in which dogs (n = 28) were exposed to Ae. aegypti mosquitoes. On Day - 6, the dog was allocated to one of the study groups, consisting of seven dogs to be treated on Day 0 with an imidacloprid/flumethrin collar. After sedation, animals were exposed to approximately 50 females of Ae. aegypti for 60 (± 5) minutes on Days - 6, 1, 7, 14, 21, 28, 55, and 83. On Day - 6, no allergic reaction to the mosquito bites was observed. However, on Day 1, corresponding to the second challenge, the dog demonstrated an acute allergic reaction characterized by swelling of the face (especially in the base of the muzzle and around the eyes), redness of the eyes, and conjunctival edema of the right eye was also observed. The dog was immediately treated with an intramuscular injection of a commercially available antihistamine treatment, Pen-Hista-Strep® containing a suspension of benzylpenicillin, chlorphenamine, dexamethasone, dihydrostreptomycin, and procaine at a dosage of 1 mL per 10 kg. A few hours after treatment, the dog showed noticeable improvement. CONCLUSIONS: This case provides the first evidence of canine acute local hypersensitivity reaction to mosquito bites under laboratory conditions. This observation suggests that invasive mosquito species such as Aedes spp. may affect the health and comfort of our companion animals, especially for pets with outdoor access without individual protective measures against insect bites.

Effect of Dinotefuran, Permethrin, and Pyriproxyfen (Vectra® 3D) on the Foraging and Blood-Feeding Behaviors of Aedes albopictus Using Laboratory Rodent Model.

Dinotefuran-Permethrin-Pyriproxyfen (DPP) is used to kill and repel mosquitoes from dogs. However, the influence of the product on the host-seeking behavior of mosquitoes remains unknown. The interference of DPP with the host selection of unfed female Aedes albopictus was investigated. A total of 18 animals (9 mice and 9 rats) were divided into three groups of six animals each. DU: DPP treated rats (n = 3) with untreated mice (n = 3), UD: DPP treated mice (n = 3) with untreated rats (n = 3) and control UU: untreated mice (n = 3) and untreated rats (n = 3). In each group, the rats and mice were placed 30 cm apart. After sedation, the animals in each group were exposed twice (Day 1 and Day 7 post-treatment) for one hour to 71 ± 3 female mosquitoes. Mosquitoes were categorized after the 2-h post-exposure period as dead or alive. Blood-meal origin was determined from mosquitoes using a newly customized duplex qPCR. The highest values of forage ratio (1.36 ≥ wi ≤ 1.88) and selection index (0.63 ≥ Bi ≤ 0.94) for rat hosts indicates a preference of mosquitoes for this species as compared to mice when co-housed during the exposure. The mosquitoes only seldom fed on mice, even in the untreated group. The anti-feeding effect of DPP was therefore only assessed on rat's hosts. The results showed that DPP, when directly applied on rats, provided a direct protection of 82% and 61% on Day 1 and Day 7, respectively, while when applied on mice hosts (UD), the DPP provided an indirect protection of 21% and 10% on Day 1 and Day 7, respectively. The results showed also that DPP, when applied on rats, provided a direct protection against Ae. albopictus bites. This effect did not result in increased exposure of the untreated host placed in the same cage at a distance of 30 cm.

Interrupted Blood Feeding in Ticks: Causes and Consequences.

Ticks are obligate hematophagous arthropods and act as vectors for a great variety of pathogens, including viruses, bacteria, protozoa, and helminths. Some tick-borne viruses, such as Powassan virus and tick-borne encephalitis virus, are transmissible within 15-60 min after tick attachment. However, a minimum of 3-24 h of tick attachment is necessary to effectively transmit bacterial agents such as Ehrlichia spp., Anaplasma spp., and Rickettsia spp. to a new host. Longer transmission periods were reported for Borrelia spp. and protozoans such as Babesia spp., which require a minimum duration of 24-48 h of tick attachment for maturation and migration of the pathogen. Laboratory observations indicate that the probability of transmission of tick-borne pathogens increases with the duration an infected tick is allowed to remain attached to the host. However, the transmission time may be shortened when partially fed infected ticks detach from their initial host and reattach to a new host, on which they complete their engorgement. For example, early transmission of tick-borne pathogens (e.g., Rickettsia rickettsii, Borrelia burgdorferi, and Brucella canis) and a significantly shorter transmission time were demonstrated in laboratory experiments by interrupted blood feeding. The relevance of such situations under field conditions remains poorly documented. In this review, we explore parameters of, and causes leading to, spontaneous interrupted feeding in nature, as well as the effects of this behavior on the minimum time required for transmission of tick-borne pathogens.

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.

Vector-borne nematode diseases in pets and humans in the Mediterranean Basin: An update.

Vector-borne diseases (VBDs) are among the leading causes of morbidity and mortality in humans and animals. The scale of VBDs is increasing worldwide, including in the Mediterranean Basin, a region exposed to climate changes. Indeed, weather conditions may influence the abundance and distribution of vectors. The vector-borne nematode diseases of dogs and cats, such as dirofilariosis, onchocercosis, thelaziosis, Cercopithifilaria, and Acanthocheilonema infections, are some of these vectorized diseases, several of which are zoonoses. They are all caused by parasitic nematodes transmitted by arthropods, including mosquitoes (Dirofilaria spp.), black flies (Onchocerca lupi), drosophilids (Thelazia callipaeda), ticks (Acanthocheilonema dracunculoides and Cercopithifilaria bainae), and fleas and lice (Acanthocheilonema reconditum). The control and prevention of these infections and diseases require a multidisciplinary approach based on strengthening collaboration between the different actors in the fields of health, research, sociology, economics, governments and citizens, to improve human, animal, and ecosystem health. This is the concept of "one health." The review aimed to provide a general update on the spatial and temporal distribution of vector-borne nematodes diseases affecting companion animals and humans, as well as the vectors involved in the Mediterranean area. Simultaneously, certain epidemiological parameters, diagnosis, treatment, and control of these diseases based on the "one health" concept will also be discussed.

Molecular detection of Leishmania infantum DNA and host blood meal identification in Phlebotomus in a hypoendemic focus of human leishmaniasis in northern Algeria.

BACKGROUND: Leishmania parasites are transmitted by female phlebotomine sand flies that maintain the enzootic cycle by circulating between sylvatic and domestic mammals. Humans are part of this cycle as accidental hosts due to the vector's search for a source of blood. In Algeria, Human Leishmaniases (HL) are endemic and represent a serious public health problem because of their high annual incidence and their spread across the country. The aim of this study is to identify sand fly species fauna (vectors of Leishmania), determine their infection rate and identify their feeding preferences using molecular tools in a hypoendemic focus of HL located in the province of Tipaza, northern Algeria. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey using CDC light traps was conducted between July and October of 2015 in four HL affected peri-urban locations in the province of Tipaza, northern Algeria. Sand flies were identified using the morphological criteria of the genitalia for the males and spermathecae for the females. Leishmania DNA was detected in pooled female sand flies (N = 81 pools with 8-10 specimens per pool) using quantitative real-time polymerase chain reaction (qPCR) targeting two different genes: kDNA-PCR and 18S rRNA. To identify their blood meal sources, blood-fed female sand flies were analyzed by PCR-sequencing targeting the vertebrate cytochrome c oxidase I (COI) gene. A total of 4,045 sand flies were caught, of which 3,727 specimens were morphologically identified. Seven species were recorded: P. (L.) perniciosus (50.28%), P. (L.) perfiliewi (26.13%), P. (L.) longicuspis (21.92%), Sergentomyia (S.) minuta (0.85%), P. (P.) papatasi (0.42%), P. (L.) langeroni (0.32%) and P. (L.) ariasi (0.05%). Afterwards, 740 female specimens were randomly selected and divided into 81 pools and were then screened to investigate the presence of Leishmania spp. L. infantum DNA was detected in three pools, corresponding to three sand fly specimens (one each). The infection rate was 0.33% (2/600) for P. (L.) perniciosus and 2.56% (1/39) for P. (L.) perfiliewi. Analysis of the blood feeding sources (N = 88 specimens) revealed that sand flies belonging to Larroussius subgenera, mainly (71.5%) feed on small ruminants. Human blood is the second feeding source (17%), eight specimens (9%) were found to feed on equines and no domestic reservoir (dog) blood was found. CONCLUSIONS/SIGNIFICANCE: The presence of human leishmaniasis cases, the high abundance of Phlebotomus (Larroussius) species which are proven or suspected vectors of L. infantum, and the detection of L. infantum DNA from its natural vectors (P. (L.) perniciosus, P. (L.) perfiliewi), in addition to the blood-feeding of positive females for L. infantum on humans blood, prove that the major elements of the epidemiological transmission cycle of L. infantum are present and indicate risk factors for an outbreak of the disease in the province of Tipaza.

Prevalence of Anaplasmataceae and Filariidae species in unowned and military dogs in New Caledonia.

Dogs are competent reservoir hosts of several zoonotic agents, including Filariidae nematodes and Anaplasmataceae family bacteria. The latter family unites human and veterinary pathogens (Anaplasma, Ehrlichia and Neorickettsia bacteria) with Wolbachia, some of which are obligatory endosymbionts of pathogenic filarial nematodes. The epidemiology of Anaplasmataceae and Filariidae species infecting dogs living in kennels in New Caledonia was studied. 64 EDTA blood samples were screened for the presence of Anaplasmataceae and filarial nematodes. Molecular study was conducted using primers and probe targeting the of 23S rRNA long fragment of Anaplasmataceae species. Next, all blood sample was screened for the presence of Filariidae species targeting the primers and probe targeting the COI gene, as well as primers targeting the COI and 5S rRNA genes of all filarial worms. Anaplasma platys was identified in 8/64 (12.5, 95% confidence interval [CI]: 4.4-20.6%) and Wolbachia endosymbiont of Dirofilaria immitis in 8/64 (12.5%, CI: 4.4-20.6%). Filariidae species investigation was performed and showed that 11/64 (17.2%, CI: 7.9-26.4%) dogs were infected with D. immitis, whereas, 2/64 (3.1%, CI: 0.0-7.3%) were infected with Acanthocheilonema reconditum. Finally, we checked the occurrence of co-infection between Anaplasmataceae and Filariidae species. Co-occurrence with Wolbachia endosymbiont of D. immitis was observed in seven dogs, one dog was co-infected with A. platys and A. reconditum and another was co-infected with Wolbachia endosymbiont of D. immitis and A. reconditum. These results are the first report of Anaplasmataceae and Filariidae occurring in dogs in New Caledonia.

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.

Assessment of MALDI-TOF mass spectrometry for filariae detection in Aedes aegypti mosquitoes.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% of D. immitis, B. malayi and B. pahangi respectively. This study presents the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes. Considering that the results might vary in other mosquito species, further studies are needed to consolidate the obtained preliminary results before applying this tool in entomological surveillance as a fast mass screening method of filariosis vectors in endemic areas.

Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France.

BACKGROUNDS: Corsica is a French island situated in the Mediterranean Sea. The island provides suitable natural conditions to study disease ecology, especially tick-borne diseases and emerging diseases in animals and ticks. The family Anaplasmataceae is a member of the order Rickettsiales; it includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia. Anaplasmosis and ehrlichiosis traditionally refer to diseases caused by obligate intracellular bacteria of the genera Anaplasma and Ehrlichia. The aim of this study was to identify and estimate the prevalence of Anaplasmataceae species infecting domestic animals and ticks in Corsica. METHODS: In this study, 458 blood samples from sheep, cattle, horses, goats, dogs, and 123 ticks removed from cattle, were collected in Corsica. Quantitative real-time PCR screening and genetic characterisation of Anaplasmataceae bacteria were based on the 23S rRNA, rpoB and groEl genes. RESULTS: Two tick species were collected in the present study: Rhipicephalus bursa (118) and Hyalomma marginatum marginatum (5). Molecular investigation showed that 32.1% (147/458) of blood samples were positive for Anaplasmataceae infection. Anaplasma ovis was identified in 42.3% (93/220) of sheep. Anaplasma marginale was amplified from 100% (12/12) of cattle and two R. bursa (2/123). Several potentially new species were also identified: Anaplasma cf. ovis, "Candidatus Anaplasma corsicanum", "Candidatus Anaplasma mediterraneum" were amplified from 17.3% (38/220) of sheep, and Anaplasma sp. marginale-like was amplified from 80% (4/5) of goats. Finally, one R. bursa tick was found to harbour the DNA of E. canis. All samples from horses and dogs were negative for Anaplasmataceae infection. CONCLUSIONS: To our knowledge, this study is the first epidemiological survey on Anaplasmataceae species infecting animals and ticks in Corsica and contributes toward the identification of current Anaplasmataceae species circulating in Corsica.

First molecular detection of Dirofilaria immitis (Spirurida: Onchocercidae) infection in dogs from Northern Algeria.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioid nematodes that affect dogs and other domestic and wild carnivores, causing heartworm disease and subcutaneous dirofilariosis, respectively. In Algeria, the data about the epidemiology of these infections is largely unknown. The present study was designed to establish the occurrence of D. immitis and D. repens in dogs in Algeria using molecular tools. In 2014 and 2015, a total of 209 dogs over one year of age of different breed and sex, living in Northern Algeria, were examined and blood samples were collected from each dog. The presence of D. immitis and D. repens in these samples was detected by real-time PCR followed by standard PCR and sequencing. Overall, the blood of 209 dogs from two departments was collected and only 3 (1.4%) of the blood samples were found positive for D. immitis DNA. Sequencing of the corresponding amplicon displayed a 99.8% identity to D. immitis, confirming the presence of this mosquito-borne nematode in Algeria. Furthermore, all tested samples were negative for D. repens.

Molecular survey of Dirofilaria immitis and Dirofilaria repens by new real-time TaqMan® PCR assay in dogs and mosquitoes (Diptera: Culicidae) in Corsica (France).

Dirofilaria immitis and D. repens are filarioid nematodes of animals and humans, transmitted by the bite of infected mosquitoes. Domestic and wild canids are a major natural host and reservoir for these parasites. In this study, we designed a duplex real-time PCR protocol targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, detecting both D. immitis and D. repens using two primer pairs and two Dirofilaria-specific hydrolysable probes. The sensitivity and specificity of the primers and probes were tested in both experimental and naturally infected samples. The detection limits of this assay were evaluated using plasmid DNA from D. immitis and D. repens. No cross-reaction was observed when testing this system against DNA from other filarial nematodes. The detection limit of the real-time PCR system was one copy per reaction mixture containing 5µl of template DNA. Field application of the new duplex real-time assay was conducted in Corsica. The prevalence rate of D. immitis was 21.3% (20/94) in dogs. In a locality where most dogs with Dirofilaria spp. infection were found, D. immitis and D. repens were detected in 5% (20/389) and 1.5% (6/389) of the Aedes albopictus population, respectively. These results suggest that this sensitive assay is a powerful tool for monitoring dirofilariosis in endemic or high risk areas.

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.