Phenotypic and genotypic characterization of acaricide resistance in Rhipicephalus microplus field isolates from South Africa and Brazil

AVAILABILITY OF DATA AND MATERIALS : The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.Rhipicephalus (Boophilus) microplus is one of the most successful ticks infesting cattle around the world. This highly-invasive species transmits cattle parasites that cause cattle fever leading to a high socio-economic burden. Tick eradication programs have often failed, due to the development of acaricide resistance. Here we characterize acaricide resistance in a large number of tick isolates from regions in South Africa (KwaZulu Natal, Mpumalanga, Western & Eastern Cape provinces) and two Brazilian regions. By means of Larval Packet Tests (LPT's) acaricide resistance was evaluated against five commonly used acaricides (chlorfenvinphos, fipronil, deltamethrin, amitraz, and ivermectin). Furthermore, the coding region containing the knock down resistance (kdr) mutation, known to result in pyrethroid resistance, was sequenced. Resistance to at least one acaricide class was reported in each of the five regions, and a high proportion of tick isolates exhibited multi-resistance to at least two acaricide classes (range: 22.2–80.0%). Furthermore, resistance ratios (RR) showed high spatial variation (intercontinental, as well as regional) but low regional spatial autocorrelation. Previous and current acaricide use correlated with current RR, and several combinations of acaricide RR were positively correlated. Moreover, fipronil resistance tended to be higher in farms with more intense acaricide use. The kdr-mutations provided the ticks a fitness advantage under the selection pressure of synthetic pyrethroids based on population (kdr-allele frequency) and individual level data (genotypes). The data show the threat of acaricide (multi-)resistance is high in Brazil and South Africa, but acaricide specific levels need to be assessed locally. For this purpose, gathering complementary molecular information on mutations that underlie resistance can reduce costs and expedite necessary actions. In an era of human-caused habitat alterations, implementing molecular data-driven programs becomes essential in overcoming tick-induced socio-economic losses.The Bill & Melinda Gates Foundation (BMGF).https://www.elsevier.com/locate/ijpddrhj2024Veterinary Tropical DiseasesSDG-03:Good heatlh and well-bein

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks.

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria

Occurrence of Aelurostrongylus abstrusus and Troglostrongylus brevior in domestic cats in Greece

Despite the evidence that Mediterranean Europe offers suitable conditions for the biology of felid respiratory metastrongyloids, no updated data on the presence of felid lungworms are available for Greece. Although the cat lungworm Aelurostrongylus abstrusus is considered as enzootic in domestic cats (Felis silvestris catus) living in some areas of continental Greece, conversely, Troglostrongylus brevior, has only been reported in the island of Crete. The present study aimed to evaluate the occurrence of Aelurostrongylus abstrusus and Troglostrongylus brevior in domestic cats from four different Greek locations including islands where European wildcats (Felis silvestris silvestris), believed to be the natural reservoir of T. brevior, are considered absent

Six-month field efficacy and safety of the combined treatment of dogs with Frontline Tri-Act® and NexGard Spectra®

Abstract Background Safety and efficacy of the combined monthly use of spot-on fipronil 6.76% w/v / permethrin 50.48% w/v (Frontline Tri-Act®) and chewable tablets of afoxolaner 1.9% w/w / milbemycin oxime 0.4% w/w (NexGard Spectra®) in dogs was evaluated in a field study over a period of 6 months. Methods Forty-one healthy dogs living in highly endemic area for canine leishmaniosis and other canine vector borne diseases (VBD) were included in the study at the beginning of the Leishmania transmission season. Sixteen dogs were pet dogs living each in a single household; twenty-five dogs were hunting dogs living in three kennels. At inclusion, the dogs were ELISA (rapid test) negative for antibodies to Anaplasma, Borrelia, Ehrlichia, and for antigens of Dirofilaria. The dogs were also negative for blood microfilariae at the Knott’s test, and no clinical or haematological abnormalities were observed. Of the included dogs, six hunting, apparently healthy, dogs were ELISA (rapid test) positive to Leishmania, and some were naturally infected by gastrointestinal nematodes (58.5%) and/or infested by fleas (58.5%) and ticks (9.8%). All the included dogs were treated at Days 0, 28, 56, 84, 112 and 140, and followed-up for efficacy until the study end (Day 168). Results No adverse events related to the two products, nor skin reactions, general signs, or changes in the haematological profile, were observed during the study. At Day 14, anthelminthic efficacy was 100% for Toxocara canis, Toxascaris leonina and Capillaria aerophila, while few hunting dogs were still shedding eggs of Trichuris vulpis (1/25 hunting dog) and Ancylostomatidae (9/25 hunting dogs). All pet dogs were nematode free at the end of the study. Hunting dogs were free of roundworms and whipworms. Twenty-four hours after the first treatment, 95.8% of the ectoparasite infested dogs were free from fleas and ticks. Ectoparasites were significantly controlled during the 6-month study period, with 100% efficacy on both fleas and ticks from Day 56 to Day 168. Blood and serum samples collected on Day 168 were tested for vector-borne pathogens using same methods of the inclusion and no new seroconversions or circulating blood microfilariae were observed. Conclusions Concomitant use of Frontline Tri-Act® and NexGard Spectra® in dogs for six months was well tolerated. The combination was effective in controlling fleas, ticks, gastro-intestinal nematodes, and neither new seroconversion to the tested vector-borne pathogens nor blood microfilariae were detected in treated dogs at the end of the study

Parasites of domestic owned cats in Europe: co-infestations and risk factors.

BACKGROUND: Domestic cats can be infested by a large range of parasite species. Parasitic infestations may cause very different clinical signs. Endoparasites and ectoparasites are rarely explored in the same study and therefore multiparasitism is poorly documented. The present survey aimed to improve knowledge of the prevalence and risk factors associated with ecto- and endoparasite infestations in owned cats in Europe. METHODS: From March 2012 to May 2013, 1519 owned cats were included in a multicenter study conducted in 9 veterinary faculties throughout Europe (Austria, Belgium, France, Hungary, Italy, Romania and Spain). For each cat, ectoparasites were checked by combing of the coat surface associated with otoscopic evaluation and microscopy on cerumen samples. Endoparasites were identified by standard coproscopical examinations performed on fresh faecal samples. Risk factors and their influence on parasitism were evaluated by univariate analysis followed by a multivariate statistical analysis (including center of examination, age, outdoor access, multipet status, and frequency of treatments as main criteria) with logistic regression models. RESULTS: Overall, 50.7% of cats resulted positive for at least one internal or one external parasite species. Ectoparasites were found in 29.6% of cats (CI95 27.3-32.0%). Otodectes cynotis was the most frequently identified species (17.4%), followed by fleas (15.5%). Endoparasites were identified in 35.1% of the cats (CI95 32.7-35.7%), including gastro-intestinal helminths in 25.7% (CI95 23.5-28.0), respiratory nematodes in 5.5% (CI95 4.2-7.0%) and protozoans in 13.5% (CI95 11.8-15.3%). Toxocara cati was the most commonly diagnosed endoparasite (19.7%, CI95 17.8-21.8%). Co-infestation with endoparasites and ectoparasites was found in 14.0% of the cats, and 11.9% harbored both ectoparasites and gastro-intestinal helminths.Age, outdoor access, living with other pets, and anthelmintic or insecticide treatments were significantly associated with the prevalence of various parasites. CONCLUSIONS: This survey demonstrates that parasitism is not a rare event in European owned cat populations. The prevalence of multi-parasitism is significantly greater than expected by chance and hence there is tendency for some individual cats to be more prone to infestation by both endo- and ectoparasites due to common risk factors

Questionnaire-based survey on the distribution and incidence of canine babesiosis in countries of Western Europe.

The incidence of canine babesiosis may vary considerably from one country to another depending on the distribution of the causative parasite species and their specific vectors. The aim of the present study was to evaluate the clinical occurrence of canine babesiosis diagnosed in European veterinary clinics and propose an updated map of the disease distribution in Western Europe. Questionnaires were sent to companion animal veterinary clinics in Spain, France, Benelux, Germany and Austria. The annual number of babesiosis cases in 2010, the number of practitioners in the clinic and the location of the clinic were recorded. The total numbers of dogs and practitioners in each country were used for definition of the reference populations and the annual incidence of canine babesiosis was calculated by dividing the total number of reported babesiosis cases by the total number of dogs in the veterinary practices involved in the study. Data were georeferenced for distribution map construction. The overall annual incidence of clinical babesiosis amongst the investigated dog population was 0.7%, with significant variations amongst countries and regions. Three epidemiological situations were described: (i) Spain, with co-existence of several species of piroplasms and patchy distribution of babesiosis, (ii) France, with overall presence of babesiosis due to Babesia canis and local variations and (iii) Benelux, Germany and Austria, with overall low prevalence of the disease associated with localised description related either to imported cases or to small autochthonous foci of B. canis infection

Ticks and associated pathogens in dogs from Greece

Background: With the aim to assess the occurrence of hard ticks and the pathogens they may carry in dogs from Greece, ixodid specimens (n = 757) were collected from 310 animals living in six provinces across the Greek peninsula. All ticks were morphologically identified, and genomic DNA was extracted from 344 (45.5%) representative specimens, according to their species, engorgement status and sampling area. The occurrence of Anaplasma spp., Ehrlichia spp., Hepatozoon spp., Rickettsia spp., Babesia spp., Theileria spp. and Cercopithifilaria spp. was assessed by conventional and quantitative real-time PCR. Results: Overall, 150 dogs (48.4%) were infested by ticks, with Rhipicephalus sanguineus (sensu lato) being the most prevalent (70.1%), followed by Haemaphysalis parva (14.7%), Rhipicephalus turanicus (11.4%), and Haemaphysalis concinna (2.4%). Out of 344 specimens molecularly examined, 41 (11.1%) were positive for at least one microorganism (i.e. 5.5% for Cercopithifilaria bainae, 2.9% for Hepatozoon canis, 1.7% for Rickettsia hoogstraalii, 1.2% for Hepatozoon felis, 0.6% for Rickettsia massiliae, 0.6% for Theileria ovis, 0.3% for Anaplasma platys and 0.3% for Coxiella like-endosymbiont). Conclusions: The results of this study show that different tick species parasitize dogs in Greece, carrying a range of microorganisms potentially pathogenic for dogs and humans. Consequently, control strategies against ticks are of great importance to prevent the risk of tick-borne diseases. The relationship between ticks infesting dogs and associated microorganisms is described according to collection site and dog lifestyle

Occurrence and bacterial loads of Bartonella and haemotropic Mycoplasma species in privately owned cats and dogs and their fleas from East and Southeast Asia

Bartonella spp. and haemoplasmas are pathogens of veterinary and medical interest with ectoparasites mainly involved in their transmission. This study aimed at molecular detection of Bartonella spp. and haemoplasmas in cats (n = 93) and dogs (n = 96), and their related fleas (n = 189) from countries in East and Southeast Asia. Ctenocephalides felis was the dominant flea species infesting both cats (97.85%) and dogs (75%) followed by Ctenocephalides orientis in dogs (18.75%) and rarely in cats (5.2%). Bartonella spp. DNA was only detected in blood samples of flea-infested cats (21.51%) (p < .0001, OR = 27.70) with Bartonella henselae more frequently detected than Bartonella clarridgeiae in cat hosts (15.05%, 6.45%) and their associated fleas (17.24%, 13.79%). Out of three Bartonella-positive fleas from dogs, two Ct. orientis fleas carried Bartonella vinsonii subsp. berkhoffii and Bartonella clarridgeiae, while the 3rd flea (Ct. felis) carried Candidatus Bartonella merieuxii. Felines represented a risk factor for Bartonella spp. infections, where fleas collected from cats (32.25%) presented an increased likelihood for Bartonella spp. occurrence (p < .0001, OR = 14.76) than those from dogs (3.13%). Moreover, when analysing infectious status, higher Bartonella spp. DNA loads were detected in fleas from bacteraemic cats compared to those from non-bacteraemic ones (p < .05). The haemoplasma occurrence was 16.13% (15/93) and 4.17% (4/96) in cat and dog blood samples from different countries (i.e. Indonesia, Malaysia, the Philippines, Taiwan and Thailand), with cats more at risk of infection (p < .01, OR = 5.96) than dogs. Unlike Bartonella spp., there was no evidence for flea involvement in the hemoplasmas' transmission cycle, thus supporting the hypothesis of non-vectorial transmission for these pathogens. In conclusion, client-owned cats and dogs living in East and Southeast Asia countries are exposed to vector-borne pathogens with fleas from cats playing a key role in Bartonella spp. transmission, thus posing a high risk of infection for humans sharing the same environment