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In August 2023, ehrlichiosis was confirmed in a patient in Italy with a Haemaphysalis punctata tick attached to his neck. Gene sequences of Ehrlichia canis from the tick and the patient were identical, indicating a potential risk for this uncommon infection for persons participating in outdoor activities.

Abstract Background Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). Methods We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. Results The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. Conclusions Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease. Graphical Abstract

Abstract Canine leishmaniasis is a widespread disease on the American continent, with cases reported from Uruguay to the USA and Canada. While numerous Leishmania spp. have been reported in dogs in this region, Leishmania infantum and Leishmania braziliensis are the most common etiological agents of canine leishmaniasis from a continental perspective. Nonetheless, other species may predominate locally in some countries. The participation of dogs in the transmission cycle of various Leishmania spp. has long been speculated, but evidence indicates that their role as reservoirs of species other than L. infantum is negligible. Various native wildlife (e.g., small rodents, marsupials, sloths, and monkeys) are, in fact, the primary hosts of Leishmania spp. in the Americas. In this review, an updated list of Leishmania spp. infecting dogs in the Americas is presented, along with their distribution and clinical and zoonotic importance Graphical Abstract

Abstract Background Despite the increasing focus on strengthening One Health capacity building on global level, challenges remain in devising and implementing real-world interventions particularly in the Asia-Pacific region. Recognizing these gaps, the One Health Action Commission (OHAC) was established as an academic community for One Health action with an emphasis on research agenda setting to identify actions for highest impact. Main text This viewpoint describes the agenda of, and motivation for, the recently formed OHAC. Recognizing the urgent need for evidence to support the formulation of necessary action plans, OHAC advocates the adoption of both bottom-up and top-down approaches to identify the current gaps in combating zoonoses, antimicrobial resistance, addressing food safety, and to enhance capacity building for context-sensitive One Health implementation. Conclusions By promoting broader engagement and connection of multidisciplinary stakeholders, OHAC envisions a collaborative global platform for the generation of innovative One Health knowledge, distilled practical experience and actionable policy advice, guided by strong ethical principles of One Health. Graphical Abstract

Abstract ParSCo (Parasitology Summer Course) is an intense, 1-week-long summer course organized by the Parasitology Unit of the Department of Veterinary Medicine, University of Bari, Italy, with the support of the World Association for the Advancement of Veterinary Parasitology (WAAVP), the European Veterinary Parasitology College (EVPC) and Parasites and Vectors. The course, which is conducted in southern Italy, is planned for parasitologists and post-graduate students working in the field of parasitology. The course consists of theoretical and practical lessons, which include the collection, identification and diagnosis of parasites of pets, livestock and wildlife. The participants in ParSCo are afforded the opportunity to be involved in clinical examination and sample collection for the diagnosis of parasitic diseases (e.g. leishmaniosis, thelaziosis and many tick-borne diseases) present in the Mediterranean Basin. The course is conducted at Casa di Caccia, a hunting lodge situated in the Gallipoli Cognato Forest near the Basento River in the Basilicata region in southern Italy. In addition to the training purpose, ParSCo is a great opportunity for sharing knowledge and expertise while becoming part of the parasitology community in a pleasant environment. In this editorial, we share some information and celebrate 10 years of ParSCo, looking forward to forthcoming sessions of this unique parasitology summer course. Graphical Abstract

Among the parasitic and infectious diseases affecting cats, those caused by vector-borne pathogens deserve attention due to their ability to cause nonspecific clinical signs and clinicopathological abnormalities. We studied the presence of Cytauxzoon spp., Ehrlichia spp., and Mycoplasma spp. in blood samples from 135 cats referred to the veterinary teaching hospital of the Federal University of Goiás in midwestern Brazil. We also investigated co-infections with Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) as well as the correlation between Mycoplasma spp. infection and cat variables, including age, sex, breed, and complete blood count abnormalities. Upon PCR testing, 20.7% (28/135) of samples were positive for Mycoplasma spp., 1.5% (2/135) for Cytauxzoon spp., and none for Ehrlichia spp. Co-infections with Mycoplasma spp. and Cytauxzoon spp. were detected in the two cats with the latter infection. Mycoplasma spp. infection was statistically associated with the simultaneous presence of thrombocytopenia and leukocytosis. This study confirms a high frequence of Mycoplasma spp. infection, with both M. haemofelis and ‘Candidatus Mycoplasma haemominutum’ circulating in this cat population. The clinical significance of Mycoplasma spp. infection in cats should be further explored and this infection should eventually be included in the differential diagnosis of thrombocytopenia and leukocytosis in otherwise apparently healthy cats.

ABSTRACT Background: Pressatia choti is a common sand fly found in the Atlantic Forest of Brazil, which is suspected to be involved in the transmission of Leishmania braziliensis. Herein, we aimed to establish a Pr. choti laboratory colony. Methods: Wild-caught female sand flies were blood fed on hamsters and maintained under controlled conditions (temperature: 26 °C; relative humidity: 70%). Results: Of the 301 collected female sandflies, 288 were identified as Pr. choti. The life cycle duration ranged from 31 to 56 days. Conclusions: We successfully established a Pr. choti colony, whose biological parameters were similar to those of other neotropical sand flies.

Abstract Toxoplasma gondii is an intracellular parasite with a worldwide distribution. Toxoplasma gondii infections are of great concern for public health, and their impact is usually most severe in pregnant women and their foetuses, and in immunocompromised individuals. Displaying considerable genetic diversity, T. gondii strains differ widely according to geographical location, with archetypal strains predominantly found in the Northern Hemisphere and non-archetypal (atypical) strains, with highly diverse genotypes, found mainly in South America. In this review, we present an overview of the identification and distribution of non-archetypal strains of T. gondii. Special attention is paid to the strains that have been isolated in Brazil, their interaction with the host immunological response, and their impact on disease outcomes. The genetic differences among the strains are pivotal to the distinct immunological responses that they elicit. These differences arise from polymorphisms of key proteins released by the parasite, which represent important virulence factors. Infection with divergent non-archetypal strains can lead to unusual manifestations of the disease, even in immunocompetent individuals. Graphical Abstract

Abstract Filarioids of the genus Cercopithifilaria are little studied, yet widespread parasites, that are relatively unique in being one of the very few nematodes transmitted by hard ticks. These filarioids live in the subcutis while microfilariae are found in the dermis. Definitive hosts include domestic dogs as well as a wide range of vertebrates, such as ruminants, non-human primates, murids, marsupials, porcupines, viverrids, bears and lagomorphs. The genus Cercopithifilaria contains three taxa (i.e. C. bainae, C. grassii and a yet undescribed species, namely Cercopithifilaria sp. II) that are known to infect dogs worldwide, with their occurrence overlapping the distribution of the main tick vector, Rhipicephalus sanguineus sensu lato. In recent decades, more attention has focused on these filarioids since they have been associated with clinical signs of infection, such as dermatitis, chronic polyarthritis and cutaneous cysts, and possibly with facilitating infections caused by other tick-borne pathogens. Nevertheless, these parasites remain largely underdiagnosed in clinical practice due to the lack of awareness of veterinary practitioners and to major obstacles to their diagnosis. In this review, we have assessed currently available data on Cercopithifilaria spp. infecting dogs worldwide and discussed the biological, clinical and epidemiological aspects of these filarioids, with the overall aim to gain a better understanding of their potential role in skin diseases. Graphical abstract

Abstract Background The tick Amblyomma cajennense sensu stricto (A. cajennense s.s.) frequently parasitizes animals and humans in the Amazon biome, in addition to being a vector of Rickettsia amblyommatis. In the present study, we evaluated both the population dynamics of A. cajennense s.s. in a degraded area of the Amazon biome and the presence of rickettsial organisms in this tick population. Methods The study was carried out in a rural area of the Santa Inês municipality (altitude: 24 m a.s.l.), Maranhão state, Brazil. Ticks were collected from the environment for 24 consecutive months, from June 2021 to May 2023. The region is characterized by two warm seasons: a rainy season (November–May) and a dry season (June–October). We characterized the temporal activity of A. cajennense s.s. on the vegetation by examining questing activity for each life stage (larvae, nymphs, adults [males and females]) in relation to the dry and rainy season. Ticks collected in this study were randomly selected and individually tested by a TaqMan real-time PCR assay that targeted a 147-bp fragment of the rickettsial gltA gene. Results Overall, 1843 (62.4%) adults (52.6% females, 47.4% males), 1110 (37.6%) nymphs and 398 larval clusters were collected. All adult females and nymphs were morphologically identified as A. cajennense s.s. Larval activity was observed from April to December, with a peak from June to September (dry season); nymph abundance peaked from September to November (transition period between dry and rainy seasons); and adult ticks were abundant from October to May (spring/summer/early autumn). The infection rate by R. amblyommatis in A. cajennense s.s. ticks was at least 7% (7/99). Conclusion Our data suggest a 1-year generation pattern for A. cajennense s.s., with a well-defined seasonality of larvae, nymphs and adults in the Amazon biome. Larvae predominate during the dry season, nymphs are most abundant in the dry-rainy season transition and adults are most abundant in the rainy season. The presence of R. amblyommatis in adult ticks suggests that animals and humans in the study region are at risk of infection by this species belonging to the spotted fever group of Rickettsia. Graphical Abstract

Abstract Dirofilaria immitis (the canine heartworm) is widespread in the tropics, with prevalence surpassing 30% in high-risk areas. In addition to the suitable climatic conditions that favour mosquito abundance and filarial larva development, there is low compliance with the recommended year-round use of preventives in these transmission hotspots. This represents a major concern, considering that melarsomine (first-line heartworm adulticide) is unavailable in several tropical countries, resulting in the so-called slow-kill protocol being the only available adulticide treatment option. In this article, the members of TroCCAP (Tropical Council for Companion Animal Parasites) review the current distribution of heartworm in the tropics and the availability of melarsomine, and discuss alternatives for the management of heartworm infections in dogs. Graphical Abstract

Abstract Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat’s lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats. Graphical Abstract

Abstract Background Chagas disease (CD) is caused by Trypanosoma cruzi, which is transmitted mainly through the feces/urine of infected triatomine bugs. The acute phase lasts 2–3 months and is characterized by high parasitemia and nonspecific symptoms, whereas the lifelong chronic phase features symptoms affecting the heart and/or digestive tract occurring in 30–40% of infected individuals. As in humans, cardiac abnormalities are observed in T. cruzi-infected dogs and cats. We reviewed the technological advances in the serological diagnosis of CD in dogs and cats. Methods A review of the published literature during the last 54 years (1968–2022) on the epidemiology, clinical features, diagnosis, treatment and prevention of CD in dogs and cats was conducted. Results Using predefined eligibility criteria for a search of the published literature, we retrieved and screened 436 publications. Of these, 84 original studies were considered for inclusion in this review. Dogs and cats are considered as sentinels, potentially indicating an active T. cruzi transmission and thus the risk for human infection. Although dogs and cats are reputed to be important for maintaining the T. cruzi domestic transmission cycle, there are no commercial tests to detect past or active infections in these animals. Most published research on CD in dogs and cats have used in-house serological tests prepared with native and/or full-length recombinant antigens, resulting in variable diagnostic performance. In recent years, chimeric antigens have been used to improve the diagnosis of chronic CD in humans with encouraging results. Some of them have high performance values (> 95%) and extremely low cross-reactivity rates for Leishmania spp., especially the antigens IBMP-8.1 to IBMP-8.4. The diagnostic performance of IBMP antigens was also investigated in dogs, showing high diagnostic performance with negligible cross-reactivity with anti-Leishmania infantum antibodies. Conclusions The development of a commercial immunodiagnostic tool to identify past or active T. cruzi infections in dogs and cats is urgently needed. The use of chimeric recombinant T. cruzi antigens may help to fill this gap and is discussed in this review. Graphical Abstract

Abstract Several zoonotic vector-borne helminths (VBHs) infesting canids cause serious veterinary and medical diseases worldwide. Increasing the knowledge about their genetic structures is pivotal to identify them and therefore to settle effective surveillance and control measures. To overcome the limitation due to the heterogeneity of large DNA sequence-datasets used for their genetic characterization, available cytochrome c oxidase subunit 1 (cox1) (n = 546) and the 12S rRNA (n = 280) sequences were examined using combined bioinformatic approach (i.e., distance-clustering, maximum likelihood phylogeny and phylogenetic evolutionary placement). Out of the 826 DNA available sequences from GenBank, 94.7% were characterized at the haplotype level regardless sequence size, completeness and/or their position. A total of 89 different haplotypes were delineated either by cox1 (n = 35), 12S rRNA (n = 21) or by both genes (n = 33), for 14 VBHs (e.g., Acanthocheilonema reconditum, Brugia spp., Dirofilaria immitis, Dirofilaria repens, Onchocerca lupi and Thelazia spp.). Overall, the present approach could be useful for studying global genetic diversity and phylogeography of VBHs. However, as barcoding sequences were restricted to two mitochondrial loci (cox1 and 12S rRNA), the haplotype delineation proposed herein should be confirmed by the characterization of other nuclear loci also to overcome potential limitations caused by the heteroplasmy phenomenon within the mitogenome of VBHs.

This study was carried out from February 2020 to September 2021 in Parque Nacional das Emas (PNE), a national park located in the Cerrado biome, midwestern Brazil, as well as in surrounding rural properties. Serum and tick samples were collected from dogs, terrestrial small mammals, and humans. Ticks were also collected from the environment. Dogs were infested with Rhipicephalus linnaei adults, whereas small mammals were infested by immature stages of Amblyomma spp., Amblyomma triste, Amblyomma dubitatum, and Amblyomma coelebs. Ticks collected from vegetation belonged to several species of the genus Amblyomma, including A. coelebs, A. dubitatum, Amblyomma naponense, Amblyomma sculptum, and A. triste. Two Rickettsia species were molecularly detected in ticks: Rickettsia parkeri in A. triste from the vegetation and a Rickettsia sp. (designated Rickettsia sp. strain PNE) in A. sculptum and A. triste collected from lowland tapirs (Tapirus terrestris). Based on short gltA gene fragments, this rickettsial organism showed 99.7–100% to Rickettsia tillamookensis. Seroreactivity to Rickettsia antigens was detected in 21.9% of dogs, 15.4% of small mammals, and 23.5% of humans. The present study reveals the richness of ticks and demonstrates the circulation of rickettsial agents in one of the largest conservation units in the Cerrado biome in Brazil. To our knowledge, this is the first report of a rickettsial phylogenetically related to R. tillamookensis in Brazil.

Amblyomma sculptum is a relevant tick species from a One Health perspective, playing an important role as a vector of Rickettsia rickettsii, the main agent of spotted fever rickettsiosis in Brazil. In this study, we evaluated the susceptibility of two A. sculptum populations from Goiás state (midwestern Brazil) to different acaricides. The first tick population (GYN strain) originated from an experimental farm, where the ticks are annually exposed to acaricides. The second (PNE strain) was collected in a national park (Emas National Park), where the ticks had not been exposed to acaricides. Immersion tests were conducted with 21-day-old laboratory-reared larvae and nymphs originating from adult ticks collected in the areas mentioned above. The chosen acaricides were two synthetic pyrethroids (cypermethrin and deltamethrin), one organophosphate (chlorfenvinphos), one formamidine (amitraz), and two combinations of pyrethroids and organophosphates (cypermethrin, chlorpyrifos and citronellal; cypermethrin, fenthion and chlorpyrifos). Mortality data were used to determine the lethal concentration (LC) values at which 50%, 90%, and 99% of the ticks died (LC50, LC90, and LC99, respectively), and resistance ratios (RR) were calculated based on the LC values. The RR revealed differences between the acaricide-exposed (GYN) and unexposed (PNE) tick strains. The PNE strain larvae and nymphs were susceptible to all the tested acaricides. The GYN strain larvae were tolerant to cypermethrin, whereas the nymphs were tolerant to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal (2 < RR ≤ 10). The GYN strain nymphs were resistant to amitraz (RR > 10). This is the first report of A. sculptum nymphs with resistance to amitraz and tolerance to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal.

ABSTRACT Background: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. Methods: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. Results: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. Conclusions: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.

Piroplasmids (order Piroplasmida) are a diverse group of tick-borne protozoa that may cause disease in animals and occasionally in humans. Novel Piroplasmida clades and species have been found in wild animals from Brazil based on the phylogenetic assessment of near-complete 18S rRNA, mitochondrial and heat-shock protein genes. For instance, a putative novel Babesia species has been detected in capybaras and Amblyomma ticks in three Brazilian states. The present work aimed to describe, using phylogenetic assessments based on distinct molecular markers, this novel Babesia species in capybaras and associated ticks (Amblyomma sculptum and Amblyomma dubitatum) sampled in Goiânia city, Goiás state, midwestern Brazil. While the phylogenetic analysis based on both near-complete 18S rRNA and hsp-70 genes positioned the sequences obtained from capybara blood samples into a new clade sister to the Babesia sensu stricto clade, the phylogenetic inference based on the COX-3 amino acid positioned the obtained sequences from capybara blood samples and A. sculptum ticks also into a clade sister to the Theileria sensu stricto clade, highlighting the inappropriateness of this marker inferring evolutionary relationships among piroplasmids. Pairwise distance analysis demonstrated that the divergence rates between the 18S rRNA sequences detected in capybaras and other Piroplasmida already described were very high and ranged from 9.4 to 12.9%. Genotype analysis based on the near-full 18S rRNA sequences of the Piroplasmida detected in capybaras and associated ticks demonstrated the occurrence of high genotype diversity at an intra-species level. In conclusion, phylogenetic analyses based on distinct molecular markers supported the description of Babesia goianiaensis nov. sp. in capybaras and associated Amblyomma ticks. Additionally, a novel phylogenetic clade, apart from the previously described ones, was described in the present study and contributed to untangling the complex evolutionary history of the Piroplasmida.

Brazilian spotted fever (BSF) is the most important tick-borne diseases affecting humans in Brazil. Cases of BSF have recently been reported in the Goiás state, midwestern Brazil. All cases have been confirmed by reference laboratories by seroconversion to Rickettsia rickettsii antigens. Because serological cross-reactions among different rickettsial species that belong to the spotted fever group (SFG) are common, the agent responsible for BSF cases in Goiás remains unknown. From March 2020 to April 2022, ticks and plasma were collected from dogs, horses and capybaras (Hydrochoerus hydrochaeris), and from the vegetation in an area where BSF cases have been reported and two areas under epidemiological surveillance in Goiás. Horses were infested by Amblyomma sculptum, Dermacentor nitens and Rhipicephalus microplus; dogs by Rhipicephalus sanguineus sensu lato (s.l.), Amblyomma ovale and A. sculptum, and capybaras by A. sculptum and Amblyomma dubitatum. Adults of A. sculptum, A. dubitatum, Amblyomma rotundatum and immature stages of A. sculptum and A. dubitatum, and Amblyomma spp. were collected from the vegetation. DNA of Rickettsia that did not belong to the SFG was detected in A. dubitatum, which was identified by DNA sequencing as Rickettsia bellii. Seroreactivity to SFG and Rickettsia bellii antigens was detected in 25.4% (42/165) of dogs, 22.7% (10/44) of horses and 41.2% (7/17) of capybaras, with higher titers for R. bellii in dogs and capybaras. The seropositivity of animals to SFG Rickettsia spp. antigens demonstrates the circulation of SFG rickettsiae in the region. Further research is needed to fully determine the agent responsible for rickettsiosis cases in this area.

Abstract The Companion Vector-Borne Diseases (CVBD) World Forum is a working group of leading international experts who meet annually to evaluate current scientific findings and future trends concerning the distribution, pathogenesis, clinical presentation, diagnosis and prevention of vector-borne infections of dogs and cats. At the 14th Symposium of the CVBD World Forum in Trieste, Italy (March 25–28, 2019), we identified the need to (i) bring attention to the potential spread of parasites and vectors with relocated dogs, and (ii) provide advice to the veterinary profession regarding the importance of surveillance and treatment for parasites and vector-borne infections when rehoming dogs. This letter shares a consensus statement from the CVBD World Forum as well as a summary of the problem faced, including the role of veterinary professionals in parasite surveillance, causal issues, and the importance of interdisciplinary cooperation in addressing the problem. To limit opportunities for dissemination of parasites and vectors, whenever possible, underlying problems creating the need for dog rehoming should be addressed. However, when it is necessary to rehome dogs, this should ideally take place in the country and national region of origin. When geographically distant relocation occurs, veterinary professionals have a vital role to play in public education, vigilance for detection of exotic vectors and infections, and alerting the medical community to the risk(s) for pathogen spread. With appropriate veterinary intervention, dog welfare needs can be met without inadvertently allowing global spread of parasites and their vectors.

Abstract Background Different methods have been used to preserve phlebotomine sand flies for research purposes, including for taxonomic studies and detection of Leishmania spp. Here, we evaluated the effect of various preservation methods at different storage times on phlebotomine sand fly DNA concentration and purity. Methods Field-collected phlebotomine sand flies were individually stored in 70% ethanol (G1) and 95% ethanol (G2) at room temperature, 70% ethanol (G3) and 95% ethanol (G4) at 8 °C or frozen dry (i.e. no preservation solution) at − 20 °C (G5). DNA concentration and purity were assessed at various storage times (T1, ≤ 12 h; T2, 3 months; T3, 6 months; T4, 9 months; and T5, 12 months). Fragments of the cytochrome c oxidase subunit 1 (cox1) and cacophony (CAC) genes of phlebotomine sand flies were also amplified. Results Mean DNA concentration (P = 0.178) and 260/280 purity ratios (P = 0.584) did not vary significantly among various preservation methods and storage times. Within each group, DNA concentration varied in G1 (Kruskal-Wallis H-test, P = 0.009) for T3 vs T4 (Dunn’s post-hoc, P

Abstract Background Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks and fleas from companion dogs and cats in East and Southeast Asia. Methods A total of 392 ticks and 248 fleas were collected from 401 infested animals (i.e. 271 dogs and 130 cats) from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, the Philippines and Vietnam, and molecularly screened for the presence of pathogens. Ticks were tested for Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. while fleas were screened for the presence of Rickettsia spp. and Bartonella spp. Result Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%) and Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and “Candidatus Rickettsia senegalensis” (0.4%). Furthermore, 35 Rhipicephalus sanguineus ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus (sensu stricto)). Conclusion Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health.

We detected Leishmania infantum infection in 45% of tigers and 5.3% of sand flies tested at a zoo in southern Italy in 2019. These infections in tigers and the abundance of Phlebotomus perniciosus sand flies represent a potential risk to other animals and humans living in or visiting the zoo.

Abstract Background The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies. Methods Primers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA. Results The multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA). Conclusions This multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.

Abstract Background Various vector-borne pathogens (VBPs) affect dogs worldwide, with their diversity and force of infection being usually higher in the tropics. Cross-sectional studies have been conducted to investigate the prevalence of VBPs in dogs, but data from longitudinal studies are scarce. Herein, we assessed the prevalence and the year-crude incidence (YCI) of Leishmania spp. and other VBPs in privately-owned dogs from two geographical regions of Brazil. Methods A total of 823 dogs were initially screened for Leishmania spp. by both serology and polymerase chain reaction (PCR). From the negatives, 307 (103 from São Joaquim de Bicas, Minas Gerais, and 204 from Goiana, Pernambuco) were randomly selected for the longitudinal study. These dogs were tested for various VBPs at baseline, after 8 and 12 months. Results Out of 823 dogs initially screened, 131 (15.9%) were positive for Leishmania spp. Out of the 307 dogs enrolled in the longitudinal study, 120 (39.1%) were lost for different reasons (e.g. animal death, owner decision, and lost to follow-up). In São Joaquim de Bicas, the baseline prevalence and YCI were as follows: 16.5% and 7.1% for Anaplasma spp.; 81.6% and 100% for Babesia spp.; 0% and 1.3% (only one faint positive) for Dirofilaria immitis; 37.9% and 22.9% for Ehrlichia spp.; 19.5% and 43.8% for Leishmania spp. In Goiana, the baseline prevalence and YCI were as follows: 45.1% and 38.3% for Anaplasma spp.; 79.9% and 96.0% for Babesia spp.; 36.3% and 39.8% for D. immitis; 64.7% and 58.5% for Ehrlichia spp.; 14.7% and 19.6% for Leishmania spp. Anti-Borrelia burgdorferi antibodies were not detected in any of the samples tested herein. The prevalence and YCI of Anaplasma spp., D. immitis and Ehrlichia spp. were significantly higher in Goiana. In contrast, the YCI of Leishmania spp. infection was significantly higher in São Joaquim de Bicas. Conclusions We confirmed a high prevalence and YCI of various VBPs among privately-owned dogs in two geographical regions of Brazil. Our data also indicate that the risk of infection varies significantly for individual VBPs and between the regions, which may be related to several factors that are still poorly understood.

Abstract Background Feline vector-borne pathogens (FeVBPs) have been increasingly investigated for their impact on cat health and their zoonotic potential. The aim of the present study was to assess the prevalence of FeVBPs and haemoplasmas in cats across Italy and to identify potential risk factors linked to their occurrence. Methods Blood samples from 958 owned cats living in the North (n = 556), Centre (n = 173) and South (n = 229) of Italy were tested for Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids by conventional PCR (cPCR) and for haemoplasmas and Bartonella spp. by SYBR green real-time PCR. Cats included in the study represent a sub-sample from a larger number of animals enrolled in a previous study, which were selected based on the geographical origin. Data on cats’ positivity for Leishmania infantum, feline leukaemia virus (FeLV) and for feline immunodeficiency virus (FIV), available from the previous study, were included and examined. Potential risk factors for pathogen infection were assessed in relationship to categorical variables including sex, geographical origin, breed, neutering status and age of cats. Results Out of the 958 cats, 194 (20.2%) were positive for at least one of the tested pathogens, 89 (16%) from the North, 32 (18.5%) from the Centre and 73 (31.9%) from the South of Italy. A high prevalence of FeVBPs was detected in male cats (n = 125, 27.8%), living in the southern part of the country (n = 73, 31.9%), younger than 18 months of age (n = 24, 22.4%) and not neutered (n = 39; 27.5%). In particular, 24 cats (2.5%) tested PCR-positive for Bartonella spp., of which 1.6% for B. henselae and 0.9% for B. clarridgeiae. A total of 111 cats scored PCR-positive for haemoplasmas (11.6%), specifically “Candidatus Mycoplasma haemominutum” (n = 95, 9.9%), M. haemofelis (n = 14, 1.5%) and “Candidatus Mycoplasma turicensis” (n = 2, 0.2%). Moreover, 39, 31 and 8 cats were positive for FeLV (4.1%), L. infantum (3.2%) and FIV (0.8%), respectively. Co-infections were registered for 19 (9.8%) cats. Conclusions These results confirm the occurrence of haemoplasmas and FeVBPs throughout Italy. Preventive measures to protect both animal and human health should be carried out also for owned cats, even if no health status of animals has been assessed in this study.

Abstract Background Camel production in Saudi Arabia is severely affected by various diseases and by inadequate veterinary services. Ticks and tick-borne pathogens (TBPs) affect the health and wellbeing of camels consequently diminishing their productivity and performances. In addition, camels may act as hosts for TBPs (e.g. Anaplasma phagocytophilum) causing diseases in humans. The current study aimed to determine the prevalence of ixodid ticks and molecularly investigate the associated pathogens in camels from Saudi Arabia. Methods Blood and tick samples were collected from camels (n = 170) in Riyad Province of Saudi Arabia. Ticks were morphologically identified, and blood of camels were molecularly screened for apicomplexan (i.e. Babesia spp., Theileria spp., Hepatozoon spp.) and rickettsial parasites (i.e. Ehrlichia spp. and Anaplasma spp.). Results Of the 170 camels examined, 116 (68.2%; 95% CI: 60.9–75.1%) were infested by ticks with a mean intensity of 2.53 (95% CI: 2.4–2.6). In total of 296 ticks collected, Hyalomma dromedarii was the most prevalent (76.4%), followed by Hyalomma impeltatum (23.3%) and Hyalomma excavatum (0.3%). Of the tested animals, 13 (7.6%; 95% CI: 4.3–12.8%) scored positive to at least one TBP, with Anaplasma platys (5.3%; 95% CI: 2.7–9.9%) being the most prevalent species, followed by Anaplasma phagocytophilum, Anaplasma sp., Ehrlichia canis and Hepatozoon canis (0.6% each; 95% CI: 0.04–3.4%). None of the camels were found to be co-infected with more than one pathogen. All samples tested negative for Babesia spp. and Theileria spp. Conclusions The present study reveals the occurrence of different tick species and TBPs in camels from Saudi Arabia. Importantly, these camels may carry A. phagocytophilum and A. platys, representing a potential risk to humans.

Capybaras (Hydrochoerus hydrochaeris) are the largest rodents on Earth. While capybaras are hosts for various tick species, there is limited information regarding the tick-borne pathogens they can carry. We investigated the presence of piroplasmids and Ehrlichia spp. in capybaras and their associated ticks in two peri-urban areas in Goiás state, central-western Brazil. Blood samples collected from 23 capybaras were used to investigate the presence of piroplasmids and Ehrlichia spp. in stained-blood smears and by PCR. Ticks collected from the capybaras were identified morphologically and also tested using PCR for the same pathogens. A total of 955 ticks were collected, including 822 (86.1%) Amblyomma sculptum, 132 (13.8%) Amblyomma dubitatum, and one (0.1%) unidentified larva of Amblyomma sp. Neither the capybaras nor ticks were positive for Ehrlichia spp. However, a stained-blood smear examination revealed the presence of ring-stage and pyriform-shaped merozoites in the erythrocytes of one (4.4%) capybara. In the same way, 47.8% (11/23) and 19.9% (36/181) of blood samples and ticks, respectively, were positive for piroplasmids in the PCR. We successfully sequenced a partial 18S rRNA gene fragment of four samples (two capybaras, one A. sculptum, and one A. dubitatum), and the phylogenetic reconstruction disclosed that the organism reported in the present study clusters within the genus Babesia. Further research is required for a formal delineation of this species (designated as Babesia sp. strain Capybara) and to investigate the hypothesis of A. dubitatum and A. sculptum ticks being vectors.

Abstract The aim of this study was to investigate the level of exposure to Leishmania infection in stray dogs in an area of intense visceral leishmaniasis transmission in the state of Pernambuco, Brazil. Blood samples from 178 dogs were analyzed using serological and molecular assays: rapid immunochromatographic test (ICT), enzyme-linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT), and conventional and quantitative polymerase chain reaction (cPCR and qPCR). Positivity values obtained with serological tests were 71.4% (127/178), 70.2% (125/178), and 50.6% (90/178) using ICT, ELISA, and IFAT, respectively, with 38.8% (69/178) of the dogs were simultaneously positive for all three tests. The positivity values obtained with cPCR and qPCR were 20.2% (36/178) and 38.8% (69/178), respectively, with 11.8% (21/178) testing positive in both molecular assays. Overall, 87.1% (155/178) were positive for anti-Leishmania spp. antibodies and/or Leishmania spp. DNA. Positivity to one or more tests was statistically associated with lymphadenomegaly, skin lesions, lymphocytosis, anemia and hyperproteinemia. The results of this study revealed a high level of exposure to Leishmania in stray dogs in an area of intense human visceral leishmaniasis transmission, suggesting that dogs play a role as reservoirs in the transmission cycle of this zoonosis.

ABSTRACT: Ticks are significant parasites of dogs in the tropics, where tick-borne pathogens are highly prevalent, especially in areas where tick control measures are frequently neglected. This study investigated the seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in Central-western Brazil. Out of 264 dogs tested for anti-Ehrlichia canis antibodies by an indirect immunofluorescence assay (IFA), 59.1% (156/264) were positive. Seropositivity was significantly associated to anemia and thrombocytopenia, alone or in combination, and to leukopenia. Conversely, there were no differences in terms of seroprevalence according to sex, breed and age. This study demonstrated that dogs referred to a veterinary teaching hospital in Central-western Brazil are highly exposed to E. canis and that seropositive dogs are more likely to present hematological abnormalities, particularly anemia, thrombocytopenia and leukopenia. To our knowledge, this is the first study on detection of anti-E. canis antibodies by means of IFA among dogs in the state of Goiás. These findings highlighted the need for increasing awareness among dog owners regarding tick control measures in Central-western Brazil, ultimately to reduce the risk of exposure to E. canis and other tick-borne pathogens.

Abstract The fast development of molecular taxonomy is impacting our knowledge of the world parasite diversity at an unprecedented level. A number of operational taxonomic units have been uncovered and new species described. However, it is not always that new parasite species are being described in compliance with the International Code of Zoological Nomenclature. This is the case of “Candidatus Dirofilaria hongkongensis”, a nematode found in dogs, jackals and humans in Hong Kong and parts of India. This name has been proposed without a formal description and without the designation of a holotype, and therefore is an unavailable name. Finally, we argue that using the provisional status Candidatus in zoological nomenclature is inappropriate, considering this term is not considered in the International Code of Zoological Nomenclature.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19–22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

Abstract Background From 2012 to 2013, an outbreak of cutaneous leishmaniasis by Leishmania braziliensis was detected in indigenous villages located in a remote rural area of Pernambuco state, north-eastern Brazil. Considering that the principal activities of this indigenous community are farming and crop plantation, and also that the outbreak involved many children, we investigated the presence of sand fly vectors inside human houses and also the exposure of dogs to leishmanial parasites. Our general objective was to gather epidemiological data that could indicate the occurrence of a peri-domestic/domestic transmission cycle of L. braziliensis in these indigenous villages. Methods From March 2015 to March 2016, sand flies were collected using light traps in the indoor and immediate outdoor environments in the three indigenous villages that reported the most cutaneous leishmaniasis cases during the 2012–2013 outbreak. Moreover, samples obtained from 300 dogs living in the outbreak villages and two nearby villages were tested by a rapid immunochromatographic test and by a real-time PCR for detecting anti-Leishmania antibodies and Leishmania DNA, respectively. Results In total, 5640 sand flies belonging to 11 species were identified. Males (n = 3540) predominated over females (n = 2100). Migonemyia migonei (84.3%) was the most abundant species, followed by Evandromyia lenti (5.5%), Lutzomyia longipalpis (4.1%), Nyssomyia intermedia (1.6%) and Micropygomyia capixaba (1.4%), representing together ~97% of the sand flies collected. Nine out of the 11 species identified in this study were found indoors, including M. migonei, L. longipalpis and N. intermedia, which are proven vectors of Leishmania spp. Out of 300 dogs tested, 26 (8.7%) presented anti-Leishmania antibodies and six (2%) were Leishmania DNA-positive. The level of exposure in dogs living in the indigenous villages where the 2012–2013 outbreak of human CL was detected was almost 2-fold higher than in the two nearby villages (11.0 vs 6.2% for serology and 2.6 vs 1.4% for real-time PCR). Conclusions The results suggest that different sand fly vectors may be adapted to human dwellings, thus increasing the risk of transmission in the indoor and immediate outdoor environments. The adaptation of sand flies to the indoor environment in the studied indigenous villages may be partly explained by the poor housing conditions and the proximity of the houses to crop plantations and forest fragments.

Abstract Background The aim of this study was to detect and molecularly identify Rickettsia spp. in Rhipicephalus sanguineus (sensu lato) collected from free-roaming dogs in 30 communities from five municipalities in the south of Coahuila State, northern Mexico, where Rocky Mountain spotted fever is endemic. Methods In total, 60 dogs from each municipality were examined for engorged ticks. DNA was isolated from tick pools and conventional PCR assays targeting the 23S-5S ribosomal RNA intergenic spacer and outer membrane protein (ompA) gene of Rickettsia spp. were performed. Results All ticks (n = 1238) were morphologically identified as R. sanguineus (s.l.). Six pools (each with six engorged females) from four municipalities were positive to Rickettsia spp. DNA sequencing and phylogenetic analyses confirmed the presence of R. rickettsii and R. rhipicephali in R. sanguineus (s.l.) in these ticks. Conclusions This study confirms the presence of R. rickettsii and R. rhipicephali in R. sanguineus (s.l.) from stray dogs in the south of Coahuila. This suggests that stray dogs may play a role in the inter-municipal dissemination of infected ticks in this region. Further research is required to assess whether ticks from stray dogs could serve as good indicators for the molecular xenomonitoring of R. rickettsii in this region. Considering that R. sanguineus (s.l.) is a proven vector of R. rickettsii in Mexico, increased awareness regarding permanent tick control in dogs is warranted.

Abstract Background Bartonella henselae, Bartonella clarridgeiae and the rare Bartonella koehlerae are zoonotic pathogens, with cats being regarded as the main reservoir hosts. The spread of the infection among cats occurs mainly via fleas and specific preventive measures need to be implemented. The effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar (Seresto®, Bayer Animal Health), registered to prevent flea and tick infestations, in reducing the risk of Bartonella spp. infection in privately owned cats, was assessed in a prospective longitudinal study. Methods In March-May 2015 [Day 0 (D0)], 204 privately-owned cats from the Aeolian Islands (Sicily) were collared (G1, n = 104) or left as controls (G2, n = 100). The bacteraemia of Bartonella spp. was assessed at enrolment (D0) and study closure (D360) by PCR and DNA sequencing both prior to and after an enrichment step, using Bartonella alpha proteobacteria growth medium (BAPGM). Results A total of 152 cats completed the study with 3 in G1 and 10 in G2 being positive for Bartonella spp. Bartonella henselae genotype I ZF1 (1.35%) and genotype II Fizz/Cal-1 (6.76%) as well as B. clarridgeiae (5.41%) were detected in cats of G2. Bartonella clarridgeiae was the only species detected in G1. Based on the yearly crude incidence of Bartonella spp. infection (i.e. 3.85% in G1 and 13.51% in G2; P = 0.03) the Seresto® collar achieved a preventative efficacy of 71.54%. The incidence of Bartonella spp. infection was more frequent in flea-infested cats (6/33, 18.18%) than in uninfested ones (7/112, 5.88%) (P = 0.036). Conclusions Cats living in the Aeolian Islands are exposed to B. henselae and B. clarridgeiae. The Seresto® collar provided significant risk reduction against Bartonella spp. infection in outdoor cats under field conditions. Such a preventative tool could be a key contribution for decreasing the risk of Bartonella spp. infection in cats and thus ultimately to humans.

Abstract Background Borrelia burgdorferi (sensu lato) is a complex containing pathogenic bacteria of which some species, such as Borrelia lusitaniae, use birds, small mammals and reptiles as reservoirs. In Italy, the bacteria have been detected in reptilian and avian reservoirs in the northern and central regions. Results Here, 211 reptiles from three orders [Squamata (Sauria with seven species in five families and Ophidia with 11 species in three families), Crocodylia (one family and two species), and Testudines (two families and two species)] were examined for ectoparasites and molecular detection of B. burgdorferi (s.l.) in three different sites of southern Italy, an area for which no information was previously available on the occurrence of borreliosis in animals and humans. Borrelia lusitaniae was molecularly detected in larvae and nymphs (11.6%) of Ixodes ricinus infesting lizards (i.e. Podarcis muralis, Podarcis siculus and Lacerta bilineata) and in 12.3% blood samples of P. siculus. Finally, B. lusitaniae and Borrelia garinii were detected in 5.1% (32/630) of questing I. ricinus. Conclusions These results show the circulation of B. lusitaniae in southern Italy and suggest that P. siculus could play a role as a reservoir, representing a potential medical threat to humans living in or visiting these localities.

We assessed the presence of Rickettsia spp., Coxiella burnetii and Anaplasma phagocytophilum in reptiles, their ectoparasites and in questing ticks collected in a nature preserve park in southern Italy, as well as in a peri-urban area in another region. We also investigated the exposure to these pathogens in forestry workers, farmers and livestock breeders living or working in the nature preserve park given the report of anecdotal cases of spotted fever rickettsioses. Rickettsia spp. were molecularly detected in Podarcis muralis and Podarcis siculus lizards (i.e., 3.1%), in Ixodes ricinus (up to 87.5%) and in Neotrombicula autumnalis (up to 8.3%) collected from them as well as in I. ricinus collected from the environment (up to 28.4%). Rickettsia monacensis was the most prevalent species followed by Rickettsia helvetica. An undescribed member of the family Anaplasmataceae was detected in 2.4% and 0.8% of the reptiles and ectoparasites, respectively. Sera from human subjects (n = 50) were serologically screened and antibodies to Rickettsia spp. (n = 4; 8%), C. burnetti (n = 8; 16%) and A. phagocytophilum (n = 11; 22%) were detected. Two ticks collected from two forestry workers were positive for spotted fever group (SFG) rickettsiae. Ixodes ricinus is involved in the transmission of SFG rickettsiae (R. monacensis and R. helvetica) in southern Europe and lizards could play a role in the sylvatic cycle of R. monacensis, as amplifying hosts. Meanwhile, N. autumnalis could be involved in the enzootic cycle of some SFG rickettsiae among these animals. People living or working in the southern Italian nature preserve park investigated are exposed to SFG rickettsiae, C. burnetii and A. phagocytophilum.

Dogs and cats are important hosts and reservoirs of many viral, bacterial, protozoal, and helminthic pathogens transmitted by arthropods, including some of zoonotic concern. By sharing the same environment, these companion animals play an important role in the transmission of zoonotic pathogens to humans in various regions and socioeconomic contexts. While canine and feline vector-borne diseases (VBD) are of major concern in wealthy regions (e.g. Europe and North America), less attention has been received in developing countries such as those in Southeast Asia (SEA). This review provides summarized and updated information on canine and feline VBD with emphasis on those of zoonotic concern in SEA. Of these, zoonotic bacteria (i.e. Bartonella henselae, Bartonella clarridgeiae, and Rickettsia felis) and filarial nematodes (i.e. Brugia malayi, Dirofilaria repens, and Dirofilaria immitis) stand out as the most important in veterinary and human medicine. Additionally, the recent finding of Leishmania infantum in dogs in SEA raised more concerns about the spreading of this zoonotic agent in this region. Further epidemiological surveys, especially in countries with extremely scant information such as Cambodia, Laos, Myanmar, and Timor-Leste are advocated. Additionally, effective control measures of canine and feline VBD as well as their arthropod vectors should be simultaneously performed for the management of zoonotic infections.

ABSTRACT This study has estimated the risk of Leishmania transmission via blood transfusion in one of the largest blood banks in Northeastern Brazil, where visceral leishmaniasis is endemic. Five hundred blood samples from donors were tested for circulating Leishmania spp. DNA by real-time PCR. Positive samples were tested by a species-specific conventional PCR targeting Leishmania infantum . Overall, 6.2% (95% CI: 4.1–8.3%) of the samples carried Leishmania DNA and in one sample the species was confirmed as L. infantum . No statistically significant differences were found in relation to gender, sex, education level, incomeas well as the place of residence between positive and negative blood donors. Our results confirm the presence of asymptomatic Leishmania carriers among blood donors in a large blood bank in Northeastern Brazil. Considering the studied population, we estimate that for every 1,000 blood donors screened, 41 to 83 will be positive for Leishmania DNA. This finding reinforces the urgent need for elaborating specific Blood bank guidelines to allow the early detection of asymptomatic Leishmania carriers among blood donors before their blood products are transfused to uninfected individuals.

Cutaneous leishmaniasis is a disease caused by various Leishmania spp., which are transmitted by phlebotomine sand flies. Algeria is one of the most affected countries, with thousands of cutaneous leishmaniasis cases registered every year. From March to November of 2016 and 2017, sand flies were collected in 12 municipalities in Setif province, North-Eastern Algeria. Sand flies were identified and females were tested by PCR for detecting Leishmania DNA. Additionally, cutaneous leishmaniasis cases notified during the study period were analysed. Out of 1804 sand flies collected, 1737 were identified as belonging to seven species, with Phlebotomus perniciosus (76.2%), Ph. papatasi (16.7%) and Ph. sergenti (5.0%) being the most common species, representing together 97.9% of the collected specimens. The remaining specimens were identified as Sergentomyia minuta, Se. fallax, Ph. longicuspis and Ph. perfiliewi. The number of sand flies collected monthly was positively correlated with temperature. Out of 804 females tested, nine Ph. perniciosus (1.1%) scored positive for Leishmania infantum (n = 5), L. major (n = 3) and L. tropica (n = 1), respectively. During the study period, 34 cutaneous leishmaniasis cases were notified in Setif, of which 58.8% were patients residing in two urban and peri-urban municipalities and 41.2% in rural areas. The finding of Ph. perniciosus as the most abundant species in Setif suggests that this sand fly may be adapted to different biotopes in the North-East region of Algeria. The detection of different Leishmania spp. in Ph. perniciosus suggests a complex epidemiological picture of cutaneous leishmaniasis in Setif, with the involvement of different etiological agents and possibly with different reservoir hosts and vectors.

Abstract Background The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called “temperate” and “tropical” lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal). Methods Ticks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted. Results Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically. Conclusions The temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks.

Abstract Background Ehrlichia canis is a tick-borne bacterium that causes severe, life-threatening disease in dogs, being more prevalent in tropical and subtropical countries. Randomized studies conducted in Brazil indicate that the prevalence of E. canis infection in dogs ranges from 0.7% to over 50.0%. In a study conducted in northern Brazil, the prevalence was higher in dogs from urban areas, as compared to dogs from rural areas. In the present study, we investigated the exposure to Ehrlichia spp. infection in dogs from remote indigenous villages located in a rural area in north-eastern Brazil. Methods From March to June 2015, 300 privately owned dogs were blood sampled and tested by a rapid ELISA and by a conventional PCR in order to detect anti-Ehrlichia spp. antibodies and E. canis DNA, respectively. Additionally, dogs were also tested for anti-Anaplasma spp. antibodies and Anaplasma platys DNA, using the same diagnostic approaches. Positivity was correlated with tick infestation and dogs’ data (gender, age and level of restriction). Results Overall, 212 (70.7%) dogs were positive for at least one test targeting Ehrlichia spp. In particular, 173 (57.7%) dogs were positive only by rapid ELISA, 5 (1.7%) only by PCR and 34 (11.4%) were simultaneously positive by both tests. In the same way, 39 (13.0%) dogs presented detectable E. canis DNA in their blood, whereas 18 (6.0%) dogs were A. platys DNA-positive. Coupling serological and PCR data, 63 (21.0%) dogs were simultaneously positive to Ehrlichia spp. and Anaplasma spp. Positivity rates for both Ehrlichia spp. and Anaplasma spp. were higher among dogs more than 1 year of age. Sick dogs were more positive to Ehrlichia spp. as compared to healthy dogs. Conclusions Dogs from rural areas in north-eastern Brazil are highly exposed to Ehrlichia spp. infection and positivity rates do not necessarily correlate with current tick infestation load, since only one infected tick bite is needed to get the infection. This reinforces the importance of keeping dogs free of ticks, in order to reduce as much as possible the risk of infection by E. canis and other tick-borne pathogens such as Babesia vogeli, which are usually co-endemic.

Cats naturally exposed to Ehrlichia canis have been described in different regions of the world, but little is known about the genotypes associated with infection in these animals. To detect E. canis-specific antibodies and investigate the E. canis TRP genotypes in cats, serum samples from 76 domestic cats reactive to crude E. canis antigens by the indirect fluorescence antibody test (IFAT) were analyzed by ELISA, using E. canis-specific peptides (i.e., TRP19 and TRP36 /BR/US/CR). Of these, 25 (32.9%) cats reacted to at least one TRP peptide, confirming their specific exposure to E. canis. Eighteen (23.7%) cats reacted to TRP19, 15 (19.8%) to BRTRP36, and 11 (14.5%) to USTRP36, but none of them reacted to CRTRP36. Eight (10.5%) cats reacted to TRP19 but not to any TRP36 genotype, demonstrating the possible existence of a new E. canis genotype infecting felines. Nevertheless, this study provides the first report of anti-E. canis-specific antibodies in domestic cats.

Abstract Background Leishmaniosis caused by Leishmania infantum is one of the most important vector-borne diseases affecting animals and humans worldwide. Dogs are considered main reservoirs of the zoonotic forms, though in the last years the role of cats as reservoirs has been increasingly investigated. Feline leishmaniosis (FeL) occurs in endemic areas and no specific preventive measures have been investigated so far. In this study the efficacy of a 10% imidacloprid/4.5% flumethrin polymer matrix collar, licensed for tick and flea prevention, has been assessed against FeL in a longitudinal study on 204 privately owned cats from the Aeolian islands (Sicily), an area highly endemic for the disease. From March to May 2015 [Study Day 0 (SD 0)], cats negative for FeL were collared (G1, n = 104) or left untreated (G2, n = 100). Diagnosis consisted of serology and qPCR on blood and conjunctival swabs, which were collected at baseline (SD 0) and at the end of the study (SD 360). Interim clinical examinations were performed on SD 210 (when collars were replaced in G1) and SD 270. Results Of the 159 cats which completed the study, 5 in G1 and 20 in G2 were positive for L. infantum infection, in at least one of the diagnostic tests leading to a yearly crude incidence of 6.3% and 25.0% in G1 and G2, respectively (P = 0.0026). This translates into an efficacy of the collar of 75.0% in preventing feline Leishmania infection. The collar was generally well tolerated with no systemic adverse reactions and few local skin reactions were observed in the application area in four out of 104 treated cats (3.8%). Conclusions The 10% imidacloprid/4.5% flumethrin collar significantly reduced the risk of L. infantum infection in cats. To our knowledge, this is the first study in which a preventative strategy against feline Leishmania infection is assessed under natural conditions. These findings close a gap in veterinary medicine, in that they confirm this collar as a tool in reducing the risk of Leishmania infection in cats. Such a preventative tool could contribute to the reduction of the risk of the disease in animal and in human populations when included in integrated leishmaniosis control programmes.