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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.)

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.)

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.)

Visceral leishmaniasis caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae) is a major neglected tropical disease and Brazil is the responsible for most cases reported in the Americas. In this region, L. infantum is primarily transmitted by Lutzomyia longipalpis and Migonemyia migonei (França) (Diptera: Psychodidae) is considered a permissive vector. We evaluated the susceptibility of Lu. longipalpis and Mg. migonei to Beauveria bassiana and to Eucalyptus globulus (Myrtales: Myrtaceae) essential oil. A spore suspension of B. bassiana was prepared and sand flies divided into five groups: test 1 (107 spores/ml of B. bassiana with E. globulus essential oil at 4 mg/ml), test 2 (107 spores/ml of B. bassiana), test 3 (E. globulus essential oil at 4 mg/ml), positive control (cypermethrin 0.1%), and negative control (sterile distilled water). Scanning electron microscopy (SEM) was performed on specimens from each group. A 50% reduction was recorded in the survival time of Lu. longipalpis in test 1 and 2, where hyphal adhesion and cuticle damage were observed by SEM. No significant differences in the survival time of Mg. migonei were found, probable due to the high mortality rate observed in the negative control group, which may be a result of the greater sensitivity of this species to laboratory conditions. The results obtained herein suggest that B. bassiana may be a potential biological control agent against Lu. longipalpis, the main vector of L. infantum in the Americas., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

With the aim of evaluating the presence of Fusarium spp . in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles ( Caretta caretta ) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp . were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 ( Fusarium keratoplasticum ), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp . Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

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.)

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 study compared the level of agreement between two commercially available rapid serological tests and the official screening test used to detect Leishmania seropositive dogs in Brazil. Ninety-five canine sera from a visceral leishmaniasis endemic area were tested by using the official immunochromatographic test (T1; rK28 antigen) based on dual path platform technology, a rapid ELISA (T2; purified Leishmania antigens) and an immunochromatographic test (T3; rK28 antigen) based on lateral flow. There was substantial agreement (Kappa 0.77; 95% confidence interval, CI, 0.62-0.91; P<0.001; observed agreement 90.5%) between T1 and T2, and a fair agreement (Kappa 0.26; 95% CI 0.08-0.43; P<0.001; observed agreement 74.7%) between T1 and T3. Sixteen dogs positive at T1 and T2 were negative at T3. T2 may be a reliable alternative to T1, while T3 could lead to an underestimation of the actual number of seropositive dogs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.)

Outbreaks of cutaneous leishmaniasis are relatively common among soldiers involved in nocturnal activities in tropical forests. We investigated the population dynamics of sand flies in a military training camp located in a remnant of Atlantic rainforest in northeastern Brazil, where outbreaks of cutaneous leishmaniasis have sporadically been described. From July 2012 to July 2014, light traps were monthly placed in 10 collection sites, being nine sites located near the forest edge and one near a sheep and goat stable. Light traps operated from 5:00 pm to 6:00 am, during four consecutive nights. Leishmania infection in sand flies was assessed using a fast real-time PCR assay. Cases of cutaneous leishmaniasis among soldiers were also investigated. In total, 24,606 sand flies belonging to 25 species were identified. Males (n = 12,683) predominated over females (n = 11,923). Sand flies were present during all months, being more numerous in March (n = 1,691) and April 2013 (n = 3,324). Lutzomyia choti (72.9%) was the most abundant species, followed by Lutzomyia longispina (13.8%), Lutzomyia complexa (5.3%), representing together >90% of the sand flies collected. Forty cases of cutaneous leishmaniasis were recorded among soldiers from January 2012 to December 2014. Leishmania isolates were obtained from eight patients and were all characterized as Leishmania braziliensis. Soldiers and anyone overnighting in Atlantic rainforest remnants should adopt preventative measures such as the use of repellents on bare skin or clothes and insecticide-treated tents.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Essential oils (EOs) and entomopathogenic fungi such as Beauveria bassiana (Bb) strains have the potential to be used as alternative insecticides and acaricides for controlling ectoparasites as Dermanyssus gallinae. These compounds have some limitations in their use: the acaricidal effect of EOs is rapid, but short-lived, whilst that of Bb is delayed, but long-lived. To evaluate the effect of both compounds combined against D. gallinae, the non-toxic dose of Eucalyptus globulus, Eucalyptus citriodora, Thymus vulgaris and Eugenia caryophyllata essential oils were firstly calculated for "native" strains of Bb. Subsequently, the effects of the combination of selected EOs with Bb against nymph and adult poultry red mites (PRMs) was assessed. EO concentrations ranging from 0.0015 to 8% v/v (i.e., nine double dilutions) were used to evaluate their effect on germination, sporulation and vegetative growth rates of native strains of Bb. A total of 1440 mites (720 nymphs and 720 adults) were divided into three-treated group (TGs) and one control group (CG). In TGs, mites were exposed to Bb in combination with the selected EO (TG1), EO alone (TG2) or Bb (TG3) alone. In the CG, mites were exposed to 0.1% tween 80 plus EO solvent (CG). E. globulus and E. citriodora were toxic for Bb in concentrations higher than 0.2% and 0.003% respectively, whilst E. caryophyllata and T. vulgaris were toxic at all concentrations tested against Bb. Based on the results of the toxicity assays against Bb, E. globulus was chosen to be tested as acaricide resulting non-toxic for Bb at concentration lower than 0.4%. Increased mortality of D. gallinae adults was recorded in TG1 than those in other TGs from 4days post-infection (T+4DPI). A 100% mortality of D. gallinae was recorded in adults at T+9DPI and at T+10DPI in nymphs in TG1 and later than T+11DPI in the other TGs. Used in combination with E. globulus, Bb displayed an earlier acaricidal effect towards both haematophagous D. gallinae stages. The combination of B. bassiana and E. globulus at 0.2% might be used for controlling arthropods of medical and veterinary importance as D. gallinae., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Rhipicephalus sanguineus sensu lato ticks are widespread worldwide due to their adaptability to survive under different environmental conditions. They may act as vectors of a wide range of pathogens to humans and animals and their control is based on the use of chemical products on dogs and in the environment. Alternative control strategies, such as the use of entomopathogenic fungi as bio-control agents have also been investigated. The ability of native strains of Beauveria bassiana sensu lato in causing mortality in different tick species (e.g., Amblyomma cajennense and Rhipicephalus microplus) has been demonstrated. However, limited studies have assessed the use of B. bassiana for the control of R. sanguineus s.l. and none of them have employed native strains of this fungus. Here we investigated the pathogenicity of a native strain of B. bassiana (CD1123) against all developmental stages of R. sanguineus s.l.., Methods: Batches of eggs, larvae, nymphs and adult ticks were immersed in a suspension of 10(7) conidia/ml of B. bassiana s.l., isolated from a R. sanguineus s.l. engorged female. All treatment and control groups were observed for 20 days, and the biological parameters (i.e., mortality, hatching, moulting percentage, pre-oviposition period, oviposition period and rate, eggs production efficiency, reproductive efficiency and fitness indexes) were assessed., Results: The effect of the B. bassiana strain tested herein on eggs, larvae, nymphs and adults showed a significantly higher mortality than those of the control groups (p < 0.05) at 5 days post-infection. No infected eggs hatched and no infected larvae moulted. Only 15% of infected nymphs moulted into adults. All biological parameters of treated groups differed significantly (p < 0.001) from those of control groups., Conclusions: This study demonstrates that a suspension containing 10(7) conidia/ml of a native B. bassiana strain is highly virulent towards all life-cycle developmental stages of R. sanguineus s.l. and may be of potential interest as a biological control agent against these ticks.

Fungaemia caused by Malassezia spp. in hospitalized patients requires prompt and appropriate therapy, but standard methods for the definition of the in vitro antifungal susceptibility have not been established yet. In this study, the in vitro susceptibility of Malassezia furfur from bloodstream infections (BSIs) to amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), posaconazole (POS) and voriconazole (VRC) was assessed using the broth microdilution (BMD) method of the Clinical and Laboratory Standards Institute (CLSI) with different media such as modified Sabouraud dextrose broth (SDB), RPMI and Christensen's urea broth (CUB). Optimal broth media that allow sufficient growth of M. furfur, and produce reliable and reproducible MICs using the CLSI BMD protocol were assessed. Thirty-six M. furfur isolates collected from BSIs of patients before and during AMB therapy, and receiving FLC prophylaxis, were tested. A good growth of M. furfur was observed in RPMI, CUB and SDB at 32 °C for 48 and 72 h. No statistically significant differences were detected between the MIC values registered after 48 and 72 h incubation. ITC, POS and VRC displayed lower MICs than FLC and AMB. These last two antifungal drugs showed higher and lower MICs, respectively, when the isolates were tested in SDB. SDB is the only medium in which it is possible to detect isolates with high FLC MICs in patients receiving FLC prophylaxis. A large number of isolates showed high AMB MIC values regardless of the media used. In conclusion, SDB might be suitable to determine triazole susceptibility. However, the media, the drug formulation or the breakpoints herein applied might not be useful for assessing the AMB susceptibility of M. furfur from BSIs., (© 2014 The Authors.)

Among diseases of horses caused by fungi (=mycoses), dermatophytosis, cryptococcosis and aspergillosis are of particular concern, due their worldwide diffusion and, for some of them, zoonotic potential. Conversely, other mycoses such as subcutaneous (i.e., pythiosis and mycetoma) or deep mycoses (i.e., blastomycosis and coccidioidomycosis) are rare, and/or limited to restricted geographical areas. Generally, subcutaneous and deep mycoses are chronic and progressive diseases; clinical signs include extensive, painful lesions (not pathognomonic), which resemble to other microbial infections. In all cases, early diagnosis is crucial in order to achieve a favorable prognosis. Knowledge of the epidemiology, clinical signs, and diagnosis of fungal diseases is essential for the establishment of effective therapeutic strategies. This article reviews the clinical manifestations, diagnosis and therapeutic protocols of equine fungal infections as a support to early diagnosis and application of targeted therapeutic and control strategies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Antifungal resistance has been associated with biofilm formation in many microorganisms, but not yet in Malassezia pachydermatis. This saprophytic yeast can cause otitis and dermatitis in dogs and has emerged as an important human pathogen, responsible for systemic infections in neonates in intensive care units. This study aims to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains, in both their planktonic and sessile forms, to fluconazole, miconazole, ketoconazole, itraconazole, posaconazole, terbinafine and voriconazole using the XTT assay and Clinical and Laboratory Standards Institute (CLSI) microdilution method. The minimum inhibitory concentration (MIC) values recorded for each drug were significantly higher for sessile cells relative to planktonic cells to the extent that ≥ 90% of M. pachydermatis strains in their sessile form were classified as resistant to all antifungal agents tested. Data suggest that M. pachydermatis biofilm formation is associated with antifungal resistance, paving the way towards investigating drug resistance mechanisms in Malassezia spp.

The few studies attempting to specifically characterize dermatophytes from hair samples of dogs and cats using PCR-based methodology relied on sequence-based analysis of selected genetic markers. The aim of the present investigation was to establish and evaluate a PCR-based approach employing genetic markers of nuclear DNA for the specific detection of dermatophytes on such specimens. Using 183 hair samples, we directly compared the test results of our one-step and nested-PCR assays with those based on conventional microscopy and in vitro culture techniques (using the latter as the reference method). The one step-PCR was highly accurate (AUC > 90) for the testing of samples from dogs, but only moderately accurate (AUC = 78.6) for cats. A nested-PCR was accurate (AUC = 93.6) for samples from cats, and achieved higher specificity (94.1 and 94.4%) and sensitivity (100 and 94.9%) for samples from dogs and cats, respectively. In addition, the nested-PCR allowed the differentiation of Microsporum canis from Trichophyton interdigitale (zoophilic) and geophilic dermatophytes (i.e., Microsporum gypseum or Trichophyton terrestre), which was not possible using the one step-assay. The PCRs evaluated here provide practical tools for diagnostic applications to support clinicians in initiating prompt and targeted chemotherapy of dermatophytoses.

Yeasts of the genus Malassezia are commensals of the normal skin microbial flora of humans and animals. These yeasts may become pathogenic under certain circumstances and their pathogenic role may be related to host immune system as well to yeast virulence factors (e.g., phospholipase production and biofilm formation). This study aims to evaluate the in vitro ability of M. pachydermatis strains to produce biofilm, and its relationship with phospholipase activity and the genetic make-up of isolates from lesioned (n=32) and healthy (n=30) dog skin. The production of biofilm was determined by crystal violet staining and scanning electronic microscopy (SEM). Biofilm was produced by almost all M. pachydermatis isolates (95.2%) from dogs with and without skin lesions at variable level and different structure. At the SEM, biofilm matrix presented adhering blastoconidia clustered in multi- or monolayer structures with variable quantity of extracellular production. Of the three genotypes detected, genotype B showed the lowest ability to produce biofilm. Of the 59 isolates producing biofilm, 33 (55.9%) produced phospholipase, with a higher biofilm formation (p<0.05) in strains collected from animals with skin lesions. It is here suggested that phospholipase production might act in synergism with the biofilm formation by inducing or exacerbating skin lesions in dogs. The results provide evidences for a better understanding of the interactions between yeasts and host immune system, toward revealing the pathogenicity of M. pachydermatis in animals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dermatitis caused by Malassezia spp., one of most common skin disease in dogs, requires prolonged therapy and/or high doses of antifungal agents. In the present study, the antifungal susceptibility of M. pachydermatis to ketoconazole (KTZ), fluconazole (FLZ), itraconazole (ITZ), posaconazole (POS) and voriconazole (VOR) was evaluated in vitro using both CLSI reference broth microdilution (CLSI BMD) and E-test. A total of 62 M. pachydermatis strains from dogs with and without skin lesions were tested. M. pachydermatis strains were susceptible to ITZ, KTZ and POS using both test methods, with the highest MIC found in tests of FLZ. Essential agreement between the two methods ranged from 87.1% (VOR) to 91.9% (ITZ), and categorical agreement from 74.2% (FLZ) to 96.8% (ITZ). Minor error discrepancies were observed between the two methods, with major discrepancies observed for KTZ. A higher MIC(50) value for FLZ was noted with M. pachydermatis genotype B. The MICs(50) of M. pachydermatis genotype B for KTZ, VOR and POS were higher in isolates from dogs with skin lesions than those in isolates from animals without skin lesions. The results suggest a link between genotypes of M. pachydermatis and in vitro drug susceptibility. The categorical agreement for both E-test and CLSI BMD methods found in this investigation confirms the E-test as a reliable diagnostic method for routine use in clinical mycology laboratories.

The microdilution antifungal method (CLSI BMD, M27-A3) was used for testing the antifungal susceptibility of Malassezia species. However, optimal broth media that allow sufficient growth of M. pachydermatitis and produce reliable and reproducible MICs using the CLSI BMD protocol are yet to be established. In this study, the susceptibility of M. pachydermatis isolates to ketoconazole (KTZ), itraconazole (ITZ) and fluconazole (FLZ) was evaluated in vitro by the CLSI BMD test using Christensen's urea broth (CUB) and mRPMI 1640 containing lipid supplementation, Sabouraud dextrose broth with 1% tween 80 (SDB), and Dixon broth (DXB). A FLZ-resistant M. pachydermatis was generated in vitro and tested under the same conditions. A good growth of M. pachydermatis incubated for 48 and 72 h, respectively, was observed in CUB, SDB and DXB and not in mRPMI 1640 (p<0.001). No statistically significant differences were detected between the MIC values registered after 48 h and 72 h incubation. ITZ displayed lower MIC values than KTZ and FLZ regardless of the media employed. A large number of FLZ-resistant Malassezia strains (86.6%) was observed using DXB. A MIC>64 mg/L was observed only when the FLZ-resistant M. pachydermatis isolate was tested in SDB. Based on the results obtained herein, culture in SDB, stock inoculum suspensions of 1-5 × 10(6)CFU/ml, and an incubation time of 48 h are proposed as optimal conditions for the evaluation of the in vitro antifungal susceptibility of M. pachydermatis using a modified CLSI BMD protocol., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Canine cutaneous leishmaniasis by Leishmania braziliensis is a neglected, but widespread disease of dogs in South America. This paper describes clinical and hematological alterations in 17 L. braziliensis-infected dogs from Brazil. The most common hematological findings were thrombocytopenia (82.4%), anemia (70.6%), low packed cell volume (52.9%) and eosinophilia (41.2%). Twelve (70.6%) dogs displayed at least one evident physical alteration; 11 dogs (64.7%) presented skin lesions, four (23.5%) had weight loss and two (11.8%) onychogryphosis. L. braziliensis-infected dogs present clinical and hematological signs often observed in dogs infected by other pathogens. This indicates that veterinarians and public health workers should not consider the presence of non-specific clinical signs as diagnostic criteria for visceral leishmaniasis in dogs living endemic areas to avoid misdiagnosis and subsequent elimination of dogs infected by L. braziliensis.

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Little information is available on the molecular epidemiology of dermatophytoses in rabbit farms and farm workers. A total of 117 isolates belonging to the Trichophyton mentagrophytes complex and 21 isolates of Microsporum canis were collected from rabbits with or without skin lesions, air samples of farms known to harbour these pathogens, and from farm workers with skin lesions, and molecularly characterized. Sequencing of amplicons from the T. mentagrophytes complex and M. canis isolates revealed the presence of one sequence-type for both partial chitin synthase-1 gene (pchs-1) and ribosomal internal transcribed spacer (ITS+), respectively. On the basis of comparative sequence analyses, isolated representing the T. mentagrophytes complex were molecularly identified as Trichophyton interdigitale (zoophilic) Priestley. The M. canis and T. interdigitale pchs-1 sequences herein analysed were 100% homologous to known sequences from different hosts (i.e., cats, dogs, humans and rabbits). Conversely, the ITS+ sequences of T. interdigitale from dogs, pigs and mice were identical, but displayed up to 8.6% difference with those from humans, guinea pigs and rabbits. The results of this study suggest that environmental and clinical isolates of T. interdigitale (zoophilic) and M. canis might share a common origin. Interestingly, the close phylogenetic relationship between T. interdigitale (zoophilic) strains and isolates from dogs, pigs and mice might indicate that these animals represented a reservoir of dermatophyte infection in rabbit farms. These animal species should therefore be considered when setting up control protocols to prevent infections by dermatophytes and their zoonotic transmission., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis., (© 2011 Blackwell Verlag GmbH.)

Three lipid-dependent Malassezia isolates (here named 114A, 114B and 114C) recovered from a dog with skin lesions were phenotypically and genotypically characterized. All presented ovoid cells and buds formed on a narrow base. Most of the results from physiological tests were consistent with those of Malassezia furfur. The phylogenetic analysis of ITS-1 and LSU nucleotide sequences was concordant in placing all three clinical Malassezia isolates close to M. furfur. However, the phylogenetic data on the chs-2 sequence revealed that clinical isolate 114A is distinct from M. furfur and was closely affiliated to the sequence of M. pachydermatis with high nodal support. In particular, lipid-dependent isolates 114A displayed chs-2 sequences similar (100%) to that of the non-lipid dependent species Malassezia pachydermatis. The presence of the genetic and physiological polymorphisms detected in these three isolates of M. furfur could have resulted from a process of adaptation of this anthropophilic species to a new host.

Objective: Rhipicephalus sanguineus is the most widespread tick in the world and a vector of many pathogens infecting dogs and humans. To date, there have been no investigations on the biology of R. sanguineus under natural Mediterranean climatic conditions., Methods: The biology of R. sanguineus was studied under laboratory and natural climatic conditions in southern Italy. Several biological parameters were compared in different seasons, and their correlation with climatic data was assessed., Results: The life cycle of R. sanguineus lasted for a mean of 101·4 and 116·2 days under laboratory and environmental conditions respectively. Reproductive parameters of wild-collected females kept in the environment in May were significantly different from first-generation females maintained in September-October, and the correlation between reproductive parameters and climate data varied according to season., Conclusion: These results indicate that the Mediterranean climate affects the biology of R. sanguineus, which compensates its losses during the initial phases of its cycle in spring with high feeding and moulting rates of larvae and nymphs during summer as well as with high egg production during autumn. These results advance our knowledge of the biology of R. sanguineus and will be useful for the understanding of the eco-epidemiology of tick-borne diseases that this tick transmits.

Fungi of the genus Geotrichum are commonly found in the environment and, in some circumstances, they may cause diseases in humans and animals. Although these fungi have been isolated from skin lesions of some animal species, their pathogenic role in horses remains uncertain. With the aim to investigate the role of Geotrichum candidum as etiological agent of dermatomycoses, a retrospective study of 64 horses presenting skin lesions and suspected to have fungal infections was carried out. For each animal, anamnestic data were recorded and fungal culture were performed using hair. Out of 64 cases, 18 (28.1%) were positive for G. candidum and only two (3.1%) for dermatophytes (i.e., one for Microsporum equinum and the other for Microsporum canis). Alopecia, desquamation, and pruritus localized mainly on head and neck were frequently observed in G. candidum infected animals. Most of G. candidum infections were recorded during spring (44.4%). Out of the 18 animals presenting G. candidum infections, eight were treated using a disinfectant with antifungal properties. After one month of treatment, the clinical lesions were healed and fungal cultures resulted negative. The high prevalence of G. candidum in skin lesions of horses and the clinical recover following antifungal treatment indicated that these yeast-like fungi might play a role as etiological agents of horse cutaneous mycoses., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Members of the genus Malassezia are lypophilic and/or lipid-dependent, unipolar budding yeasts that can become pathogenic under the influence of particular predisposing factors (e.g., changes in the cutaneous microenvironment and/or alterations in host defences). This genus comprises at least 14 species, which have been identified traditionally based on their morphology and biochemical features. However, phenetic characteristics often do not allow the identification or delineation of closely related Malassezia spp., such that molecular tools need to be used to assist in fundamental studies of the epidemiology and ecology of Malassezia as well as aspects of the pathogenesis and disease caused by members of this genus. This article briefly reviews the morphological and biochemical methods commonly used for the identification of Malassezia as well as DNA technological methods that have been established for the specific identification of members of this genus and the diagnosis of their infections. New avenues for the development of improved molecular-diagnostic methods to overcome diagnostic limitations and to underpin fundamental investigations of this interesting group of yeasts are proposed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

While dermatophytoses of several animal species have been extensively investigated, information on their occurrence and epidemiology in rabbits is limited. We carried out a study from October 2006 to February 2007 of 23 rabbit farms in Apulia and Basilicata regions (southern Italy) in order to investigate the occurrence and risk factors associated with dermatophytoses in breeding rabbits. Dermatophytes were isolated from 86.9% (20/23) of the farms and from 51.8% (420/810) of the animals sampled. Trichophyton mentagrophytes (47.9%) and Microsporum canis (3.2%) were isolated from diseased (71.7%) and healthy (48.4%) animals as well from the surrounding environment (7.5%). The occurrence of lesions, the age of rabbits, and farm management (e.g., temperature, humidity and methods and frequency of disinfection practices) were identified as the most significant risk factors (P < 0.05) for the occurrence of dermatophytes. Animals in fattening and finishing stages were the most frequently infected (i.e., 58.2 and 61.6% respectively). Dermatophyte prevalence was significantly (P < 0.05) higher in areas with higher temperature (>20°C) and relative humidity ranging from 62-65%. The results of the present investigation suggest that zoonotic dermathophytes are present in rabbit farms and highlight the importance of correct management procedures for the control of the infections.

The widespread geographical distribution of the brown dog tick Rhipicephalus sanguineus (Acari: Ixodidae) is related to the cosmopolitan distribution of its primary host and to its adaptability to different environments, under variable climate conditions. Field studies have suggested that temperature is the most important factor driving the population dynamics of this tick species. In order to investigate the effects of prolonged exposure to low temperature on eggs of R. sanguineus, nine groups (II-X) of five egg batches each were maintained at 8+/-2 degrees C (70+/-10% RH, and scotophase) for 15, 30, 45, 60, 75, 90, 105, 120, and 135 days. One group (group I) was maintained in the incubator (26+/-1 degrees C, 70+/-10% RH, and scotophase) and used as control. The mean egg incubation period from group I was 11.6+/-0.5 days, with an egg hatch rate of 99.5+/-0.5%. Conversely, no egg hatched at 8 degrees C. Exposure to cold showed a strong positive correlation with egg incubation period (r=0.99) as well as a strong negative correlation with egg hatch rate (r=-0.95) and larval longevity (r=-0.99). Overall, the present results points out that R. sanguineus eggs are sensible to prolonged exposure to low temperature, which is definitely a major limiting factor for the establishment of populations of this tick in cold temperate regions., (Copyright 2010 Elsevier B.V. All rights reserved.)

A community-based epidemiological study was carried out in a rural area in northeastern Brazil, where visceral leishmaniasis is endemic, but the primary vector Lutzomyia longipalpis has never been found. Forty-one dogs were screened by an indirect immunofluorescence antibody test (IFAT) for the presence of anti-Leishmania antibodies and 12 (29.3%) of them were positive. One of the IFAT-positive dogs was also positive for Leishmania amastigotes in bone marrow cytology and for Leishmania infantum by real-time polymerase chain reaction (PCR) on blood. One IFAT-negative dog was positive for L. infantum by PCR on bone marrow and other for Leishmania amastigotes in skin stained-smears. When tested for L. braziliensis by PCR, 20 dogs were positive. Considering all diagnostic tests, the estimated prevalence of Leishmania spp. infection in the studied rural dog population was 58.5%. There was no significant difference in IFAT-positivity in relation to age, gender, and clinical status of the dogs. When tested for L. infantum by real-time PCR, 20 ticks collected from IFAT-positive dogs were all negative. This study shows a high level of exposure to Leishmania spp. infection in dogs from a rural community in northeastern Brazil. In general, the results do not support the participation of ticks as vectors of L. infantum in this area, which is likely to be transmitted by Lutzomyia spp. other than L. longipalpis. Finally, this study highlights that the use of IFAT in areas where both L. infantum and L. braziliensis are present should be withdraw in order to avoid the unnecessary culling of dogs that are actually infected only by L. braziliensis., ((c) 2010 Elsevier B.V. All rights reserved.)

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 microl of L. infantum promastigotes (1x10(6) cells per ml) was injected into the hemocel through the coxa I of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L. infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.

This article presents the results of a study on ectoparasites infesting rural dogs in Pernambuco, northeastern Brazil. Forty-one dogs from a rural community of Pernambuco were examined and 70.7% were infested by ectoparasites. The prevalence rates of infestation by ticks, fleas, and lice were 58.5, 43.9 and 22%, respectively. Of 24 dogs parasitized by ticks, 15 were exclusively infested by Rhipicephalus sanguineus (Latreille) (32 females, 66 males, 9 nymphs; prevalence, 48.8%; mean intensity, 5.4), four by Amblyomma ovale Koch (33 females, 19 males; prevalence, 22%; mean intensity, 5.8), and five were coinfested by both species. Ctenocephalides felis felis (Bouché) (25 females, 8 males; prevalence, 43.9%; mean intensity, 1.9) and Heterodoxus spiniger (Enderlein) (16 females, 11 males, 10 nymphs; prevalence, 22%; mean intensity, 4.1) were the only species of flea and louse identified. Tick infestation was more frequent than flea or lice infestations (p < 0.05). No statistical difference was found by sex and age group of the dogs studied and prevalence of infestation. Overall, 48.8% (20/41) of the dogs were infested by more than one ectoparasite species.

A serological survey was carried out to assess the occurrence of anti-Neospora caninum antibodies in dogs from the State of Pernambuco. A total of 625 serum samples of dogs (289 from Paulista, 168 from Amaraji and 168 from Garanhuns) were tested by an immunofluorescence antibody assay for the detection of anti-N. caninum antibodies. A total of 177 (28.3%; IC 95%, 24.9-32.1) samples were positive. The seropositivity rates found in Paulista, Amaraji and Garanhuns were 26% (IC 95%, 21-31.4), 26.2% (IC 95%, 19.7-33.5) and 34.5% (IC 95%, 27.4-42.2), respectively. Of the 177 serum samples positive to anti-N. caninum antibodies, 170 were additionally tested for the presence of anti-Toxoplasma gondii antibodies and out of these 57.6% (IC 95%, 49.8-65.2) were positive. The results indicate that dogs from Amaraji, Paulista and Garanhuns are exposed to both N. caninum and T. gondii infections. The presence of dogs infected by N. caninum in Pernambuco represents a potential risk factor for the occurrence of outbreaks of abortion in cattle and small ruminants in this state. This study is the largest serological survey on the presence of anti-N. caninum antibodies in dogs carried out in Brazil and reports for the first time the exposure to N. caninum and T. gondii in dogs from Pernambuco.

Canine babesiosis is highly endemic in Brazil, caused by Babesia canis and Babesia gibsoni, both transmitted by Rhipicephalus sanguineus ticks. The present review argues for a more adequate method of characterizing the Babesia species infecting dogs and cats in different Brazilian endemic zones. It advocates for a comprehensive understanding of the biology of R. sanguineus ticks under Brazilian conditions in order to define the more effective preventive strategies against canine babesiosis and calls for partnerships between the public and private sectors for research on canine babesiosis and other vector-borne diseases in Brazil.

The objective of this paper is to describe four cases of human parasitism by Rhipicephalus sanguineus (Latrielle) in Brazil. During an investigation regarding the species of ectoparasites of domestic dogs from the metropolitan region of Recife, Pernambuco state, four dog owners were found to be parasitized by ticks. The ticks were collected from these individuals and their dogs. All the ticks were identified as Rhipicephalus sanguineus . These are, to our knowledge, the first four cases of human parasitism by this tick species in Brazil. The possible implications of this finding are discussed here.