Q3 lab-on-chip real-time PCR for the diagnosis of Leishmania infantum infection in dogs.

Leishmaniasis is a vector-borne disease caused by many Leishmania spp. which infect humans and other mammalian hosts. Leishmania infantum is the main agent of canine leishmaniasis (CanL) whose diagnosis is usually confirmed by serological and molecular tests. This study aimed to evaluate the clinical and analytical sensitivities of a lab-on-chip (LOC) real-time PCR applied on the portable Q3-Plus V2 platform (Q3 qPCR) in the detection of L. infantum. The Q3 qPCR performance was assessed by comparing the quantification cycle (Cq) values with those obtained using the qPCR run on a CFX96 Real-Time System (CFX96 qPCR). A total of 173 DNA samples (extracted from bone marrow, lymph node, blood, buffy coat, conjunctival swab, and skin) as well as 93 non-extracted samples (NES) (bone marrow, lymph node, blood, and buffy coat) collected from dogs were tested with both systems. Serial dilutions of each representative DNA and NES sample were used to assess the analytical sensitivity of the Q3 qPCR system. Overlapping Cq values were obtained with the Q3 qPCR and CFX96 qPCR, both using DNA extracted from L. infantum promastigotes (limit of detection, <1 promastigote per milliliter) and from biological samples as well as with NES. However, the Q3 qPCR system showed a higher sensitivity in detecting L. infantum in NES as compared with the CFX96 qPCR. Our data indicate that the Q3 qPCR system could be a reliable tool for detecting L. infantum DNA in biological samples, bypassing the DNA extraction step, which represents an advance in the point-of-care diagnostic of CanL.

Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial.

The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adult antigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.

Anaplasmosis outbreaks in taurine and zebu cattle in Brazil: causes, treatments, and Anaplasma marginale genetic diversity.

This study aimed to investigate seven outbreaks of A. marginale infection in two regions of Brazil, affecting taurine, zebu, and crossbred cattle. We assessed the possible causes, treatment measures, and genetic diversity of A. marginale. These outbreaks occurred in two states (Goiás: outbreaks 1-7; Mato Grosso do Sul: outbreak 3), breeds (Holstein, Nellore, and crossbreed), age groups (beef cattle: 18-25 days old and 7-8 months; dairy cattle: 18-25 days old, 13-14 months, and cow after the first birth) and rearing systems (feedlot, pasture, pen in a wood shaving bedding system and compost bedded-pack barns). Metaphylactic or prophylactic treatments varied according to outbreak (imidocarb dipropionate: outbreaks 1-4 and 6; enrofloxacin: outbreaks 5 and 7; diminazene diaceturate: outbreak 5). In outbreaks 6 and 7, the packed cell volume was monitored. In all outbreaks, the practice of needle/syringe sharing was discontinued. For outbreaks 1-3, clinical signs and mortality (range, 4.8-13.3%) occurred 36-45 days after entry into the feedlot. In outbreak 4, A. marginale was diagnosed in 66.2% of the calves (bacteremia, 0-4.5%), with a mortality of 8.6%. Among nursing calves aged 60 days during outbreak 5, 53.8% were infected with A. marginale, with average bacteremia of 2.7% (range, 0-21.3%), and a mortality of 13.8%. In dairy heifers aged 14 months, raised in paddocks lacking vegetation cover and infested with R. microplus, then transitioned to a rotational grazing system also infested with R. microplus, the A. marginale bacteremia ranged from 3.2 to 6.7%, with a mortality of 20%. Before monitoring during outbreak 7, the mortality was 17.9%, but no further deaths were observed after monitoring initiation. In conclusion, possible causes triggering the outbreaks included primary tick infestation, needle/syringe sharing, and stress factors which may have affected the immunological statues of animals in the feedlots. Control measures performed in all outbreaks were effective. The partial msp4 gene sequences of A. marginale generated herein belonged to two haplotypes, but further research would be needed to investigate if this finding has any clinical significance.

Evidence against Zika virus infection of pets and peri-domestic animals in Latin America and Africa.

Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013-2018 in Brazil and 2006-2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire (n=259) and Brazil (n=1416). In contrast, ZIKV seroprevalence was 22.2% (2/9, 95% CI, 2.8-60.1) in West African chimpanzees (Pan troglodytes verus) and 11.1% (1/9, 95% CI, 0.3-48.3) in king colobus (Colobus polycomos). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.

Vector-borne pathogens of zoonotic concern in dogs from a Quilombola community in northeastern Brazil.

Canine vector-borne pathogens (CVBPs) comprise a group of disease agents mainly transmitted by ticks, fleas, mosquitoes and sand flies. In this study, we assessed the presence of CVBPs in an Afro-descendent community (Quilombola) of northeastern, Brazil. Dog blood samples (n = 201) were collected and analyzed by rapid test for the detection of antibodies against Leishmania spp., Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi sensu lato (s.l.), and antigens of Dirofilaria immitis. In addition, polymerase chain reactions were performed for Anaplasmataceae, Babesia spp., Hepatozoon spp., Rickettsia spp. and B. burgdorferi s.l. Overall, 66.7% of the dogs scored positive to at least one pathogen at serological and/or molecular methods. Antibodies against Ehrlichia spp. were the most frequently detected (57.2%; n = 115/201), followed by Anaplasma spp. (8.5%; n = 17/201), Leishmania spp. (8.5%; n = 17/201) and B. burgdorferi s.l. (0.5%; n = 1/201). For D. immitis, 11 out of 201 (5.5%) animals scored positive. At the molecular analysis, 10.4% (n = 21/201) of the samples scored positive for Babesia spp./Hepatozoon spp., followed by Anaplasmataceae (5.0%; n = 10/201) and Rickettsia spp. (3.0%; n = 6/201). All samples were negative for B. burgdorferi s.l. Our data demonstrated the presence of CVBPs in the studied population, with a high seropositivity for Ehrlichia spp. In addition, considering the detection of zoonotic pathogens in dogs and their relationship with people from Quilombola communities, effective control strategies are advocated for minimizing the risk of infection in this socially vulnerable human population and their pets.

Ticks on reptiles and amphibians in Central Amazonia, with notes on rickettsial infections.

Reptiles and amphibians are exceptional hosts for different ectoparasites, including mites and ticks. In this study, we investigated tick infestations on reptiles and amphibians trapped in Central Amazonia, and also assessed the presence of rickettsial infections in the collected ticks. From September 2016 to September 2019, 385 reptiles (350 lizards, 20 snakes, 12 tortoises, and three caimans) and 120 amphibians (119 anurans and one caecilian) were captured and examined for ectoparasites. Overall, 35 (10%) lizards, three (25%) tortoises and one (0.8%) toad were parasitized by ticks (124 larvae, 32 nymphs, and 22 adults). In lizards, tick infestation varied significantly according to landscape category and age group. Based on combined morphological and molecular analyses, these ticks were identified as Amblyomma humerale (14 larvae, 12 nymphs, 19 males, and one female), Amblyomma nodosum (three larvae, one nymph, and one female), and Amblyomma rotundatum (four larvae, three nymphs, and one female), and Amblyomma spp. (103 larvae and 16 nymphs). Our study presents the first records of A. nodosum in the Amazonas state and suggests that teiid lizards are important hosts for larvae and nymphs of A. humerale in Central Amazonia. Moreover, a nymph of A. humerale collected from a common tegu (Tupinambis teguixin) was found positive for Rickettsia amblyommatis, which agrees with previous reports, suggesting that the A. humerale-R. amblyommatis relationship may be more common than currently recognized.

Didelphis spp. opossums and their parasites in the Americas: A One Health perspective.

Medium sized opossums (Didelphis spp.) are among the most fascinating mammals of the Americas, playing important ecological roles (e.g., dispersal of seeds and control of insect populations) in the environment they inhabit. Nevertheless, as synanthropic animals, they are well adapted to human dwellings, occupying shelters within the cities, peripheral areas, and rural settings. These marsupials can harbor numerous pathogens, which may affect people, pets, and livestock. Among those, some protozoa (e.g., Leishmania infantum, Trypanosoma cruzi, Toxoplasma gondii), helminths (e.g., Ancylostoma caninum, Trichinella spiralis, Alaria marcianae, Paragonimus spp.) and arthropods (e.g., ticks, fleas) present substantial public health and veterinary importance, due to their capacity to cause disease in humans, domestic animals, and wildlife. Here, we reviewed the role played by opossums on the spreading of zoonotic parasites, vectors, and vector-borne pathogens, highlighting the risks of pathogens transmission due to the direct and indirect interaction of humans and domestic animals with Didelphis spp. in the Americas.

Vector-borne pathogens in dogs of different regions of Iran and Pakistan.

Canine vector-borne diseases (CVBDs) are highly prevalent in tropical and subtropical countries, mainly due to favorable climate conditions and reduced adoption of preventive measures. This study aimed to provide a comprehensive overview on the prevalence of CVBDs in Iran and Pakistan where limited data are available. Blood samples were collected from 403 dogs from six provinces in Iran and Pakistan to assess the presence of pathogen DNA (i.e., Anaplasma spp., Coxiella burnetii, Ehrlichia spp., Rickettsia spp., Babesia spp., Hepatozoon spp., filarioids, and Leishmania spp.). Sera were also screened by an immunofluorescence antibody test for the detection of antibodies against Leishmania infantum. In total, 46.9% of dogs scored positive to Hepatozoon canis being the most frequently detected (41.4%), followed by Anaplasma platys (6.4%), Ehrlichia canis (3.4%), Rickettsia spp. (2.2%), Babesia vogeli (1.0%), and L. infantum (0.3%). A seroprevalence of 9.6% to anti-L. infantum IgG was also recorded. Data reported herein demonstrate that dogs from Iran and Pakistan are at a high risk of CVBDs, particularly of canine hepatozoonosis. Effective control strategies are advocated for minimizing the risk of infection in animals and humans, also in consideration of the zoonotic potential of some pathogens detected.

Molecular epidemiology and prevalence of babesial infections in dogs in two hyperendemic foci in Brazil.

Babesial parasites are some of the most ubiquitous blood pathogens and consequently have considerable worldwide veterinary impact. Dogs living in the tropics are highly exposed to babesial parasites, particularly to Babesia vogeli. Limited data on the seroprevalence and molecular prevalence of Babesia spp. in dogs are available in Latin America. We conducted a cross-sectional study combining serological and molecular tests to estimate the seroprevalence and molecular epidemiology of Babesia spp. infections in dogs in two hyperendemic foci in Brazil. A total of 630 privately owned dogs (417 from Goiana municipality, Pernambuco state, north-eastern Brazil, and 213 from São Joaquim de Bicas municipality, Minas Gerais state, south-eastern Brazil) were sampled and molecularly and serologically tested for Babesia spp. Overall, 519 dogs (82.4%) presented detectable IgG antibodies against Babesia spp., and seropositivity was significantly higher in dogs older than 1 year. Molecularly, 34 dogs (5.4%) were positive for a ~ 200 bp fragment of the 18S rRNA gene of Babesia spp. and 88 (14.0%) for a longer fragment (~ 450 bp) of the same gene of Babesia spp. and other protozoa. The 18S rRNA gene sequences generated herein corresponded to B. vogeli (n = 52) or Hepatozoon canis (n = 20). This study confirms a high level of exposure to B. vogeli in two areas of Brazil and highlights that most of the dogs living in these areas are infected during the course of their life, reflected by increased seroprevalence in older dogs. Increased awareness and prevention of tick-borne protozoa infections in dogs from Brazil and Latin America are urgently needed.

Tick infestation on birds in an urban Atlantic Forest fragment in north-eastern Brazil.

Birds are important hosts for various tick species, playing a significant role in their biological life cycle and dispersion. In this study, we investigated tick infestations on birds trapped in an urban remnant of Atlantic Forest in Pernambuco state, Brazil. From February 2015 to March 2017, 541 birds belonging to 52 species were trapped with mist nets and examined for ectoparasites. Birds trapped in the late successional forest were significantly more infested than birds trapped in the early successional forest. In the same way, ectoparasite infestation varied significantly according to bird weight and collection plot. Overall, 198 birds (36.6%) belonging to 27 species were parasitized by ectoparasites (i.e., ticks, lice and/or mites). Ectoparasites were effectively collected from 111 birds, of which 99 belonging to 20 species were infested by ticks (n = 261), namely, Amblyomma longirostre (13 nymphs), Amblyomma nodosum (21 nymphs), Amblyomma varium (one nymph), and Amblyomma spp. (five nymphs and 221 larvae). Most of the ticks (> 90%) were collected from Passeriformes. This study provides the second record of A. varium in Pernambuco state and confirms that birds, especially Passeriformes, are important hosts for larvae and nymphs of Amblyomma spp. in the Atlantic Forest biome of Pernambuco.

Seasonal dynamics of Amblyomma sculptum in two areas of the Cerrado biome midwestern Brazil, where human cases of rickettsiosis have been reported.

Amblyomma sculptum is a common human-biting tick in Brazil, where it plays an important role as a vector of Rickettsia rickettsii, the agent of the Brazilian spotted fever. Herein, we studied the seasonal dynamics of A. sculptum in an urban area of the Cerrado biome in midwestern Brazil, where human rickettsiosis is endemic. Ticks were collected in two sites located within the campus of Federal University of Goiás. The collections were done by dragging, flagging and visual search. In total, 117,685 ticks were collected, including 100,627 Amblyomma spp. larvae, 10,055 nymphs and 6977 adults of A. sculptum, and one nymph and 25 adults of Amblyomma dubitatum. The highest peak of larvae occurred in June 2018 and in July 2019, whereas nymphs peaked in July 2018 and September 2019. Adults reached their highest numbers in March 2018 and November 2019. These data suggest that A. sculptum develops one generation per year in this urban area of the Cerrado biome in midwestern Brazil. Interestingly, the peak of nymphs occurred during the same period of all confirmed cases of rickettsiosis in Goiás, suggesting a possible relationship between the seasonal dynamics of this tick stage and rickettsiosis transmission in this state.

Leishmania infantum in Tigers and Sand Flies from a Leishmaniasis-Endemic Area, Southern Italy.

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.

Ecology of Antricola ticks in a bat cave in north-eastern Brazil.

Argasid ticks are a diverse group of acarines that parasitize numerous vertebrate hosts. Along with birds, bats serve as hosts for several argasid ticks, which are commonly found in bat caves. Argasid ticks have regained attention from tick taxonomists in recent decades, with a number of new species described in various zoogeographical regions. Nonetheless, studies on their ecology are still scarce. We conducted a 1-year longitudinal study to assess the presence of argasid ticks in a bat cave in the drylands of north-eastern Brazil and evaluate their possible response to abiotic factors. From July 2014 to June 2015, 490 ticks were collected (272 nymphs, 169 males and 49 females) in a cave chamber hosting a large colony of Pteronotus spp. bats, being relatively more frequent from July to December 2014. Adults were identified as Antricola guglielmonei, whereas nymphs were assigned to the genus Antricola. Almost all ticks (98%) were collected on the cave walls. Only 2% were on the ceiling and, surprisingly, no specimens were found on the floor and/or guano. Adults were usually clustered in the crevices and little mobile, whereas nymphs were dispersed and more active, moving over the walls or ceiling of the cave. Although present in most of the studied period, there was a significantly negative correlation between tick abundance and relatively humidity, and A. guglielmonei was more frequent during the dry season. Moreover, there was no evident correlation between the abundance of ticks and bats. Further long-term studies will be able to verify whether this pattern is repeated over time, and even whether other variables can influence the population dynamics of A. guglielmonei.

Canine Leishmaniasis Control in the Context of One Health.

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.

Ticks and associated pathogens from dogs in northern Vietnam.

The medical and veterinary significance of ticks and tick-borne pathogens (TBPs) in tropical and subtropical zones is well recognized. Although ticks and TBPs are known to occur in Southeast Asia, limited data is available in the international literature for some countries, such as Vietnam. The aim of this study was to investigate the species of ticks and TBPs associated with dogs in northern Vietnam. Out of 359 dogs enrolled in this study, 26.2% (n = 94) were infested by 466 ticks (i.e., 287 males, 139 females, 30 nymphs, and 10 larvae). All ticks were morphologically identified as Rhipicephalus sanguineus sensu lato, and some of them genetically characterized as belonging to the tropical lineage. A total of 302 ticks were molecularly screened for the detection of selected TBPs. Three ticks were positive for Hepatozoon canis, one for Ehrlichia canis, and one for Babesia vogeli, representing the first molecular characterization of these pathogens in Vietnam. In conclusion, the tropical lineage of R. sanguineus s.l. is the dominant tick taxon infesting dogs from northern Vietnam, where different TBPs are circulating.

Tick infestation on caimans: a casual tick-host association in the Atlantic rainforest biome?

Ticks are parasites of birds, mammals, amphibians, and reptiles, but information about tick communities that parasitize reptiles in the Neotropical region is still fragmentary. In the present study, we assessed the presence of ticks on broad-snouted caimans (Caiman latirostris) and Cuvier's dwarf caimans (Paleosuchus palpebrosus) trapped in the Atlantic rainforest biome in Pernambuco state, north-eastern Brazil, to determine which tick species feed on these animals and how frequent or rare this parasite-wildlife association is. We also report an occasional finding of Amblyomma rotundatum on a smooth-fronted caiman (Paleosuchus trigonatus) in the Amazon biome in Pará state, northern Brazil. Out of 490 animals trapped in the Atlantic rainforest biome, four (0.82%) broad-snouted caimans were infested by ticks. Ticks belonged to two Amblyomma species: A. rotundatum (three females) and A. fuscum (one male). Our findings indicate that ticks are infrequent parasites of caimans in the Atlantic rainforest biome. Tick infestation on broad-snouted caimans is probably of minor clinical significance and probably a casual finding due to habitat sharing with the common tick hosts.

Leishmania-FAST15: A rapid, sensitive and low-cost real-time PCR assay for the detection of Leishmania infantum and Leishmania braziliensis kinetoplast DNA in canine blood samples.

We describe an improved real-time PCR assay (designated as "Leishmania-FAST15") for the detection and quantification of Leishmania infantum and Leishmania braziliensis kinetoplast DNA minicircles in canine blood samples. The analytical sensitivity of this technique is 0.1 fg of DNA, which is equivalent to 0.002 parasite per reaction. This assay uses a small reaction volume (15 µl) and is rapid to perform, with the results being available in less than 34 min. This improved assay might also be suitable for detecting and quantifying L. infantum and L. braziliensis DNA in other tissues, such as bone marrow and lymph nodes.

Transcriptome of larvae representing the Rhipicephalus sanguineus complex.

Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) is possibly the most widespread tick species worldwide, responsible for transmitting several vector-borne pathogens of medical and veterinary importance. Here, we explore the transcriptome of R. sanguineus s.l. larvae (Putignano strain). We sequenced total RNA from R. sanguineus s.l. larvae. A total of 15,566,986 short paired-end reads were de novo-assembled into 33,396 transcripts and then annotated and analyzed. Particular attention was paid to transcripts putatively encoding ATP-binding proteins, due to their importance as mechanisms of detoxification and acaricide resistance. Additionally, microsatellite loci were investigated, as these are useful markers for population genetic studies. The present data and analyses provide a comprehensive transcriptomic resource for R. sanguineus. The results presented here will aid further genetic and genomic studies of this important tick species.

Rhipicephalus turanicus, a new vector of Hepatozoon canis.

The distribution of Hepatozoon canis mainly encompasses areas where its main tick vector, Rhipicephalus sanguineus sensu lato, is present. However, the detection of this pathogen in dogs, foxes and golden jackals well outside the areas inhabited by this tick species reinforced the hypothesis that additional ixodids are involved in the life cycle and transmission of this protozoon. The present study provides, for the first time, data supporting the sporogonic development of H. canis in specimens of Rhipicephalus turanicus collected from a naturally infected fox from southern Italy. The epidemiological role of R. turanicus as a vector of H. canis is discussed, along with information on the potential use of cell cultures for the experimental infection with H. canis sporozoites. The in vitro infection of canine leucocytes by sporozoites from ticks is proposed as a potential tool for future in-depth studies on the biology of H. canis.

Ixodes ventalloi: morphological and molecular support for species integrity.

Despite their medical and veterinary importance, some tick species are so poorly studied, that their role within pathogen vector transmission cycles is difficult to assess. The tick Ixodes ventalloi is one such species, and its biology and phylogenetic status remain an issue of debate. In the present study, specimens of adult I. ventalloi (n = 65 females; n = 31 males) infesting cats in the Lipari Island (Aeolian archipelago, Sicily, southern Italy) were characterized morphologically and molecularly, the latter based on mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes. The genetic data and phylogenetic analyses for both mitochondrial genes suggest the existence of two distinct genogroups. The ecological and epidemiological significance of the genetic structure within the I. ventalloi endemic population remains to be determined. The results highlight the need for further analysis of this tick species, including whole mitochondrial genome sequencing and crossbreeding studies, which will be pivotal to complement features of its status as a vector of pathogens.