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Leishmaniases are a group of neglected diseases of significant public health concern, with Brazil being the primary focus of this disease in the Americas. The municipality of Sobral, in the state of Ceará, is a historical focus of visceral leishmaniasis in both humans and dogs, but data on Leishmania spp. infections in cats are limited. Between April 2021 and February 2022, 205 cats from a referral hospital population were sampled and tested for Leishmania spp. by real-time PCR. Eight cats (3.9%; 95% CI: 1.7-7.5%) tested positive. Among these, three (37.5%) displayed clinical signs compatible with feline leishmaniosis. Non-domiciled cats showed significantly higher positivity compared to domiciled ones (Fisher's exact test, P = 0.0124). Considering their potential role as reservoirs of L. infantum, it is crucial to conduct further studies to understand the Leishmania spp. circulating among cats in Sobral and to implement measures for reducing their exposure to phlebotomine sand fly vectors in this important focus of leishmaniases., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

Since 2015, the Dengue, Zika, and Chikungunya viruses gained notoriety for their impact in public health in many parts of the globe, including Brazil. In Recife, the capital of Pernambuco State, the introduction of ZIKV impacted human population tremendously, owing to the increase in the number of neurological cases, such as the Guillain−Barré and congenital Zika disorders. Later, Recife was considered to be the epicenter for ZIKV epidemics in Brazil. For arboviral diseases, there are some risk factors, such as climate changes, low socioeconomic conditions, and the high densities of vectors populations, that favor the broad and rapid dispersion of these three viruses in the city. Therefore, continuous arbovirus surveillance provides an important tool for detecting these arboviruses and predicting new outbreaks. The purpose of the present study was to evaluate the circulation of DENV, ZIKV, and CHIKV by RT-qPCR in mosquitoes collected in health care units from the metropolitan area of Recife (MAR), during 2018. A total of 2321 female mosquitoes (357 pools) belonging to two species, Aedes aegypti and Culex quinquefasciatus, were collected from 18 different healthcare units, distributed in five cities from the MAR. Twenty-three pools were positive for ZIKV, out of which, seventeen were of C. quinquefasciatus and six were of A. aegypti. Positive pools were collected in 11/18 health care units screened, with Cq values ranging from 30.0 to 37.4 and viral loads varying from 1.88 × 107 to 2.14 × 109 RNA copies/mL. Nosocomial Aedes- and Culex-borne transmission of arbovirus are widely ignored by surveillance and vector control programs, even though healthcare-associated infections (HAI) are considered a serious threat to patient safety worldwide. Although the results presented here concern only the epidemiological scenario from 2018 in MAR, the potential of hospital-acquired transmission through mosquito bites is being overlooked by public health authorities. It is, therefore, of the ultimate importance to establish specific control programs for these locations.

Ornithodoros mimon is an argasid tick primarily associated with bats that also infest other animals including birds, opossums and humans. In this paper, we report the finding of an argasid species resembling O. mimon, which similarly may be found in human dwellings and parasitize humans in Brazil. We also provide molecular evidence that this argasid tick species may carry a rickettsial organism, whose pathogenicity remains unknown. A total of 16 ticks (two females, two males and 12 nymphs) were collected in the bedroom and in the attic of a human house, where cases of "insect" bites have been recurrent. These ticks were identified morphologically and genetically as Ornithodoros cf. mimon. Upon PCR testing, four of these ticks (one female and three nymphs) were positive for human blood and for a bacterium closely related to "Candidatus Rickettsia paranaensis". In conclusion, we report for the first time in Brazil an argasid tick species morphologically and genetically related to O. mimon, which feeds on humans and carry a rickettsial organism belonging to the spotted fever group. Further studies are needed to formally assess the taxonomic status of this tick species and also to investigate the pathogenicity of its associated rickettsial organism., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Proinflammatory cytokine secretion determines the infection course in leishmaniasis. The immunopathology of canine leishmaniasis (CanL) caused by Leishmania infantum is characterized by low Leishmania-specific IFN-γ and IL-17 production. Mutations in the human IL-17 gene promoter alter cytokine expression and may increase the susceptibility of humans to some infectious diseases. In this study, we correlated canine IL-17 single nucleotide polymorphisms (SNPs) with anti-Leishmania IgG levels, parasite load and external clinical signs in dogs naturally exposed to L. infantum in Brazil. A higher frequency (Chi-square test: X 2 = 5.378, df= 1, P= 0.020) of major alleles was observed among dogs showing no external clinical signs attributable to Leishmania infection. A high proportion of A allele carriers (mutant) were observed among dogs with high antibody levels, although differences were not statistically significant (Chi-square test: X 2 = 4.410, df= 4, P= 0.353), as compared to dogs with low antibody levels. In general, the association of canine IL-17 SNPs with disease expression or disease exasperation did not reach enough statistical power to allow the use of these mutations as prognostic markers. This knowledge may pave the way for further investigations on the genetic aspects of CanL and its immunotherapy., (Copyright © 2022. Published by Elsevier B.V.)

Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new Fraction 1 capsular antigen (F1)-based rapid diagnostic test (RDT) as an alternative method for plague serological diagnosis and surveillance in humans and other mammals. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the plague RDT method. To calculate its performance, results were compared to those obtained by traditional hemagglutination (HA) and ELISA, which are well-established methods in the plague routine serodiagnosis. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohen's Kappa test coefficient was 1.0 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Y. pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Ehrlichia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance of the Plague RDT when compared to HA and ELISA. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results., (Copyright © 2022. Published by Elsevier B.V.)

Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinical form of leishmaniasis in the Americas. Migonemyia migonei is a widely distributed phlebotomine sand fly species in Brazil and has been implicated as a vector for L. (V.) braziliensis. In the present study, we investigated the effects of salivary gland homogenates (SGH) of Mg. migonei on the course of L. (V.) braziliensis infection in BALB/c mice. Mice were separated into four groups (six mice per group): CTRL (uninfected mice); SGH (mice inoculated with Mg. migonei SGH); SGH+LEISH (mice inoculated with Mg. migonei SGH plus L. (V.) braziliensis promastigotes); LEISH (mice inoculated with L. (V.) braziliensis promastigotes). Mice were followed up for 8 weeks and the cellular immune response was evaluated by flow cytometry at the end of the experiment. Analysis of cytokine production by splenic cells stimulated with 0.5 SGH, 0.25 SGH of Mg. migonei or L. (V.) braziliensis soluble antigen stimulation (LSA) demonstrated that upon stimulation with SGH 0.25, the production of IL-17A and TNF was not sustained in the SGH group, with decreasing levels of these cytokines after 5 days compared to 3 days of incubation. Analyzing the production of cytokines after LSA stimulation, we observed lower levels of IL-17A in the SGH group after 5 days compared to 3 days. The same was observed for IFN-γ in the SGH group. Yet, the levels of TNF were significantly higher in the LEISH group after 5 days compared to 3 days. Among SGH+LEISH and LEISH mice, three animals in each group developed skin lesions on the tail, the mean lesion size was significantly higher in the LEISH group. Our study suggests that Mg. migonei SGH may modulate BALB/c immune response, as reflected by the low production or early decrease of pro-inflammatory cytokines in splenic cell cultures following stimulation with L. (V.) braziliensis antigen. Our data also suggest that Mg. migonei saliva may reduce the lesion size in BALB/c mice, but further research with a larger sample size is needed to confirm this hypothesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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.

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.

The genus Amblyomma is the most representative tick genus in Brazil and some species act as vectors of pathogenic organisms to animals and humans. Information on the seasonal dynamics of Amblyomma spp. as well as on rickettsial organisms infecting these ticks in some regions in Brazil is still fragmentary. Herein, we investigated the seasonal dynamics and rickettsial infections in Amblyomma dubitatum ticks collected in the Atlantic forest biome in north-eastern Brazil. Using carbon dioxide traps, ticks were collected monthly for two consecutive years. In total, 15,789 ticks were collected: 69 females (0.4%), 116 males (0.7%), 1,067 nymphs (6.8%), and 14,537 larvae (92.1%). All nymphs, females and males were identified as A. dubitatum, whereas larvae were identified as Amblyomma spp. Larvae were more frequent in summer (77% of the larvae collected), whereas nymphs were collected with similar frequency in summer (32.8%), autumn (30.0%) and spring (28.4%). Adults were more frequent in spring (47.6%). A total of 648 ticks (485 nymphs, 60 females, and 103 males) were tested by PCR for the gltA gene of Rickettsia spp. and 87 (13.4%; 95% CI: 10.9-16.3%) were positive. A consensus sequence (size, 350 bp) of 66 gltA gene sequences indicate that the organism detected herein is similar to Rickettsia tamurae, Rickettsia monacencis and Rickettsia sp. strain Pampulha. One of these positive samples was also positive for the ompA gene of spotted fever group rickettsiae, but attempts to sequence the amplicon were not successful. We also tested this sample by a PCR targeting the rickettsial htrA gene, but no amplification product could be detected. This study indicates that A. dubitatum may be a common tick in areas where capybaras are present in north-eastern Brazil, occurring during the whole year. It also suggests the circulation of a spotted fever group rickettsia in this A. dubitatum population, whose identity has yet to be determined., (Copyright © 2021. Published by Elsevier B.V.)

Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

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.

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 < 0.05), and in G2 (Kruskal-Wallis H-test, P = 0.004) for T1 vs T2 and T1 vs T4 (Dunn's post-hoc, P < 0.05). For 260/280 purity ratios, the only statistically significant difference was found for G5 (Kruskal-Wallis H-test, P = 0.020) between T1 vs T4 (Dunn's post-hoc test, P < 0.05). The cox1 and CAC genes were successfully amplified, regardless of the preservation method and storage time; except in one sample from G2 at T1, for which the CAC gene failed to amplify., Conclusions: The preservation methods and storage times herein evaluated did not affect the concentration and purity of DNA samples obtained from field-collected phlebotomine sand flies, for up to 12 months. Furthermore, these preservation methods did not interfere with PCR amplification of CAC and cox1 genes, being suitable for molecular analyses under the conditions studied herein.

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 10 3 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.

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.

Cutaneous leishmaniasis (CL) by Leishmania braziliensis Vianna (Kinetoplastida: Trypanosomatidae) is a widespread disease in the western hemisphere and Brazil is the country reporting the majority of the human cases each year. Several phlebotomine sand flies have been regarded as putative or proven vectors of L. braziliensis and multiple vectors may be involved in the transmission of this parasite in the same endemic foci. In this study, we tested a subset of female phlebotomine sand flies collected in the frame of a previous study conducted in a military training camp, where outbreaks of CL by L. braziliensis have sporadically been reported. In total, 1,807 female phlebotomine sand flies were tested (individually or in pools) by real-time PCR and a minimum infection rate of 0.9% (95% CI: 0.6-1.5%) was detected. Positive females belonged to the species Psychodopygus complexus (Mangabeira) or Psychodopygus wellcomei Fraiha, Shaw & Lainson (females of these species are morphologically indistinguishable), Trichopygomyia longispina (Mangabeira), and Sciopemyia sordellii (Shannon & Del Ponte) (Diptera: Psychodidae). By restriction enzyme analysis, 13 samples (nine of Ty. longispina, two of Ps. complexus/wellcomei, and two of Sc. sordellii) presented a HaeIII restriction profile identical to L. braziliensis. The results of this study reinforce the notion that multiple vectors may be involved in the transmission of L. braziliensis in a single focus, ultimately making the epidemiological picture more complex than currently recognized., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amblyomma sculptum is the principal vector of Rickettsia rickettsii, the main agent of spotted fever rickettsiosis in Brazil. Little information is available regarding the population dynamics of this tick in some regions, including north-eastern Brazil, where cases of spotted fever rickettsioses are increasingly reported. Herein, we studied the phenology of A. sculptum in a rural area in north-eastern Brazil. Ticks were collected from the environment, using dry ice-baited traps, monthly for two consecutive years. In total, 1500 ticks were collected: 94 females (6.3%), 74 males (4.9%), 468 nymphs (31.2%), and 864 larvae (57.6%). All nymphs and females were identified as A. sculptum. Males were tentatively identified as A. sculptum and larvae as Amblyomma spp. Ticks were more numerous during spring and summer, followed by autumn and winter. Peaks of larvae and nymphs were recorded during summer and spring, respectively, whereas adults were more frequently collected in spring. A total of 380 ticks were tested by PCR for the gltA gene of Rickettsia spp., but none of them were positive. While our results revealed a seasonal pattern for A. sculptum in north-eastern Brazil that is distinct from the seasonal pattern in south-eastern Brazil, we caution that the observed pattern could have been biased by the relatively low number of ticks collected. Finally, the absence of Rickettsia-infected ticks does not rule out the possibility that rickettsial organisms are circulating in the study area and further long-term studies are warranted., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

In the present study, we assessed the annual screening coverage (i.e., the percentage of dogs that are screened for anti-Leishmania antibodies annually) in the municipality of Sobral, Ceará state, Brazil. Data on the number of dogs screened during 2008-2017 (except 2010) were obtained from the Centre for Zoonoses Control of Sobral. The annual screening coverage during 2012-2017 was calculated. Data on human visceral leishmaniasis (VL) cases during 2008-2017 were compiled from the National Disease Notification System. Correlation analyses were performed to assess the correlation between canine and human data. During 2008-2017, 73,964 dogs (range, 0 to 13,980 dogs/year) were serologically screened and 2,833 (3.8%) were positive. The annual screening coverage during 2012-2017 ranged from 11.1% to 45.7%. There were no significant correlations between the number of dogs culled and the number of human VL cases, canine positivity and human VL incidence, number of dogs culled and human VL incidence, or between canine positivity and number of human VL cases. An inconsistent and relatively low annual screening coverage was found in the study area, with no dog being screened in 2010 due to the lack of serological tests. Our results highlight that many dogs potentially infected with Leishmania infantum have been virtually overlooked by public health workers in the study area, perhaps with a negative, yet underestimated, impact on the control of canine and human VL. Hence, the failure of the dog culling strategy in controlling human VL in Brazil may be due to the low screening coverage and low percentage of culled dogs, rather than the absence of associations between canine and human infections., Competing Interests: FDT has received funding from Bayer Animal Health to conduct a clinical trial to access the efficacy of flumethrin plus imidacloprid collar in preventing vector-borne pathogen infections on dogs in Brazil. He also received honoraria for talks in scientific meetings organized by Bayer Animal Health and Boehringer Ingelheim.

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.

This survey was conducted in four Brazilian cities, from three federative units (Pernambuco, Minas Gerais, and Federal District), representing different socioeconomic settings, to determine the presence of antibodies to or antigens and DNA of selected pathogens in privately owned dogs. From January to April 2015, blood and serum samples were collected and assayed using different tests. In particular, antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato, and Ehrlichia spp., and antigens of Dirofilaria immitis were detected using a rapid enzyme-linked immunosorbent assay, whereas antibodies to Babesia spp. were detected by an immunofluorescence antibody assay. Moreover, the presence of Leishmania DNA in blood samples was assessed by real-time polymerase chain reaction. Overall, 208 (69.3%) out of 300 dogs were positive for at least one tested pathogen (intended here as antibodies, antigen or DNA, as abovementioned), with 139 (66.8%) of them being positive to two or more pathogens. No dog presented antibodies to B. burgdorferi s.l., and D. immitis antigens were detected exclusively in dogs from Pernambuco. Among positive dogs, the most common clinical signs were lymphadenomegaly (45.2%), onychogryphosis (41.3%), dermatitis (34.1%), pale mucous membranes (19.7%), weight loss (14.9%), fever (12.5%), alopecia (11.1%), and lethargy (4.8%). Tick and flea infestations were recorded in 41.7% and 29.3% of the dogs, respectively, with 49 (16.3%) dogs being co-infested by both ticks and fleas. Most of the tick- and flea-infested dogs presented high level of infestation (>10 ticks and >20 fleas). The level of tick infestation varied significantly among federative units, being highest in Minas Gerais (68.0%), followed by Pernambuco (36.0%) and Federal District (21.0%). On the other hand, the level of flea infestation was higher in Pernambuco (50.0%), followed by Minas Gerais (29.0%) and Federal District (9.0%). The number of dog owners reporting the use of ectoparasiticides (on dogs and/or in the environment) varied significantly, ranging from 6.0% in Pernambuco to 60.0% in Federal District. This study reveals disparate results in terms of dog exposure to fleas and ticks in the studied communities, which may be related to factors such as the owners' capability/willingness to afford the use of ectoparasiticides on their dogs and the dog's level of restriction. Further research is needed to establish the relationship between dog owners' socioeconomic situation and the level of exposure to ectoparasites and their transmitted pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)