Leishmania infantum infection rate in dogs housed in open-admission shelters is higher than of domiciled dogs in an endemic area of canine visceral leishmaniasis. Epidemiological implications.

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum and is endemic in many areas of southeastern Brazil. We have hypothesized that the prevalence of infection by L. infantum in dogs housed in open-admission animal shelters is beyond the range of 3.4 - 9.6% reported among dogs domiciled in similar CVL-endemic areas. Hence, this study aimed to determine the rate of L. infantum infection among dogs maintained in shelters and to investigate the epidemiology of CVL in such environments by analyzing hematological and biochemical parameters. A total of 627 dogs from 17 different shelters across the State of Minas Gerais were screened using the Dual-Path Platform test and enzyme-linked immunosorbent assay and 211 (33.6%) were found to be seropositive in both tests. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on skin, bone marrow and lymphoid tissues of 118 seropositive dogs with inconclusive CVL clinical diagnosis and, of these, 78 (66.1%) were PCR+ for L. infantum and 7 (5.9%) were PCR+ for L. amazonensis. One dog presented a PCR-RFLP profile that was consistent with co-infection by both parasites. Leishmania amazonensis DNA was detected in skin samples of six single-infected dogs and this constitutes a novel finding. Dogs infected only with L. amazonensis were less debilitated than those infected by L. infantum, which showed typical clinical manifestations of CVL. The co-infected dog showed only mild clinical signs. The results presented herein not only support our original hypothesis but also suggest that dogs are potential reservoirs of L. amazonensis. Public health authorities should acknowledge their responsibility towards animals in collective shelters, recognize that they are potential foci of zoonotic diseases, and establish proper functioning directives to minimize transmission to humans and to other dogs.

Implications of the use of serological and molecular methods to detect infection by Leishmania spp. in urban pet dogs.

The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that amplify a 300-350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4 (4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania amazonensis, and their serum samples tested positive by IFAT and DPP. These findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the first record of Leishmania (Leishmania) amazonensis confirmed by a specific molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.

Important frequency of Anaplasma phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil / Importante frequência da infecção por Anaplasma phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil

Anaplasma phagocytophilum is responsible for granulocytic anaplasmosis in humans and various animal species. The aim of the present study was to determine the prevalence of A. phagocytophilum-infected dogs in a residential area of Belo Horizonte, Minas Gerais state, Brazil. A total of 62 dogs were submitted to serological (indirect fluorescent-antibody -IFI) and molecular (PCR) tests. Anti-A. phagocytophilum antibodies were detected in 43.8% of the dogs. Seven dogs (10.9%) were PCR-positive for the msp4 gene, six and four of these were positive for the for the msp2/p44 gene of A. phagocytophilum and 16S rRNA region of granulocytic Anaplasmataceae respectively. This study confirms a relatively high frequency of A. phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil and highlights the need for further studies on the role of Rhipicephalus sanguineus sensu lato ticks in the transmission of this bacterium to dogs in urban Brazilian areas.(AU)

Anaplasma phagocytophilum é responsável pela anaplasmose granulocítica, doença que acomete seres-humanos e várias espécies de animais. O objetivo do presente estudo foi determinar a prevalência de cães acometidos por A. phagocytophlium em uma área residencial de Belo Horizonte, MG, Brasil. Sessenta e dois cães foram submetidos a testes sorológicos (reação de imunofluorescência indireta - IFAT) e moleculares (PCR). Anticorpos anti-A. phagocytophilum foram detectados em 43,8% dos cães. Sete cães (10,9%) foram positivos no PCR para o gene msp4 de A. phagocytophilum, seis para o gene msp2/p44 A. phagocytophilum e quatro para a região 16S rRNA de Anaplasmataceae granulocíticas. Esse estudo confirma a frequência relativamente alta da infecção por A. phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil e destaca a necessidade de pesquisas para determinar o papel do carrapato Rhipicephalus sanguineus sensu lato na transmissão desse microrganismo para cães de áreas urbanas brasileiras.(AU)

Isolation of Rickettsia rickettsii from the tick Amblyomma sculptum from a Brazilian spotted fever-endemic area in the Pampulha Lake region, southeastern Brazil.

Brazilian spotted fever (BSF) is a tick-borne disease caused by the bacterium Rickettsia rickettsii, the deadliest spotted fever of the world, transmitted in southeastern Brazil mainly by the tick Amblyomma sculptum, a member of the Amblyomma cajennense species complex. In the present study, over 5000 adults of A. sculptum ticks were collected by dry ice traps in the Municipal Ecological Park, alongside the Pampulha Lake region, a BSF-endemic area of Belo Horizonte city, state of Minas Gerais, southeastern Brazil. Ticks were taken alive to the laboratory, where a sample of 2100 specimens was processed for isolation of R. rickettsii. For this purpose, ticks were macerated and intraperitoneally inoculated into guinea pigs. Only one out of 21 inoculated guinea pigs presented high fever within 21days post inoculation with tick homogenates. This febrile animal was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). A spleen sample from a febrile guinea pig was used to inoculate Vero cells, resulting in a successful isolation and in vitro establishment of rickettsiae. Rickettsia-infected Vero cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB), which were all 100% identical to corresponding sequences of R. rickettsii from GenBank. The present R. rickettsii isolate was designated as strain Pampulha. A minimal infection rate of 0.05% R. rickettsii-infected ticks was estimated for A. sculptum population of the Pampulha Lake region. Our results, coupled with epidemiological evidences, suggest that R. rickettsii strain Pampulha, isolated from A. sculptum ticks in the present study, is the strain responsible for human clinical cases of BSF in the Pampulha Lake region of Belo Horizonte city.

Dynamics of Ctenocephalides felis felis (Siphonaptera: Pulicidae) Infestations on Urban Dogs in Southeastern Brazil.

The cat flea, Ctenocephalides felis felis (Bouché, 1835), is an important ectoparasite of dogs and cats throughout the world, causing annoyance to the animals and acting as a vector of infections and a cause of allergic dermatitis in dogs and cats. Although climatic variability and seasonality are known to influence the diversity and abundance of fleas, few investigations of seasonal prevalence of cat flea infestation have involved the same group of dogs being examined regularly over an extended period. The aim of this study was to determine the effects of temperature, rainfall, and relative humidity on the infestation by C. felis felis on 88 outdoor dogs in southeastern Brazil. The dogs, which were of mixed breed, sex, and age, were examined for ectoparasites every month during the period August 2011 to July 2012, and samples of fleas were randomly collected and identified. Meteorological data, comprising mean temperature, total rainfall, and mean relative humidity, were recorded for the calendar month prior to that in which the examinations were performed. Dogs were found to be infested only with C. felis felis, with a higher prevalence in the months with lowest rainfall (July, August, and September). The data obtained in this investigation can be used in control programs in order to establish an efficient strategy for environmental management and the application of insecticides, particularly during the driest months of the year based on the seasonal pattern of infestation of dogs by C. felis felis.

Ectoparasites and anti-Leishmania antibodies: association in an observational case-control study of dogs from a Brazilian endemic area.

It has been proposed that the transmission of canine visceral leishmaniasis might involve the participation of mechanical vectors, including ticks of the family Ixodidae, in particular the brown dog tick Rhipicephalus sanguineus, and the cat flea Ctenocephalides felis felis. Here, the association between the infestation by R. sanguineus and C. felis felis and the occurrence of anti-Leishmania antibodies was evaluated in an observational case-control study of dogs living in a Brazilian endemic area for canine visceral leishmaniasis. Blood samples were taken once every three months for one year from 96 initially seronegative domestic dogs, and submitted to indirect immunofluorescence antibody assay. All dogs were evaluated for the presence of ticks and fleas, and the results were expressed qualitatively as infested or non-infested, irrespective of the intensity of infestation. At the end of follow-up, twenty dogs had turned seropositive, while 68 remained seronegative and 8 were excluded because of incomplete data. All the dogs were asymptomatic. The odds of infection was significantly greater (OR=3.54, CI95%=1.10-12.53) for dogs infested by C. felis felis compared to their non-infested counterparts. In contrast, the odds of infection showed no significance difference between non-infested and R. sanguineus-infested groups of dogs (OR=0.31, CI95%=0.03-1.52). This study provides further evidence for the potential role of C. felis felis in mechanically transmitting Leishmania among the canine population.

Association between the prevalence of infestation by Rhipicephalus sanguineus and Ctenocephalides felis felis and the presence of anti-Leishmania antibodies: A case-control study in dogs from a Brazilian endemic area.

The association between the prevalence of infestation by Rhipicephalus sanguineus and Ctenocephalides felis felis and the presence of anti-Leishmania antibodies has been evaluated in dogs located in a city of Brazil endemic for canine visceral leishmaniasis. Blood samples from 5556 domestic dogs domiciliated in the urban area of Belo Horizonte (Minas Gerais state) were submitted to enzyme linked immunosorbent (ELISA) and indirect immunofluorescent antibody (IFAT) assays, and 432 (7.8%) animals tested positive. Seropositive (n=200) and seronegative (n=200) dogs were randomly selected and examined for the presence of ticks and fleas, the results of which were expressed qualitatively as infested or non-infested, irrespective of the intensity of infestation. The prevalence of infestation by R. sanguineus was significantly greater (ρ=0.04) among seropositive dogs (38.5%) compared with their seronegative counterparts (29.0%). Similarly, the prevalence of infestation by C. felis felis was significantly greater (ρ<0.01) within the seropositive group (36.5%) than within the seronegative group (15.0%). Moreover, the probability of seropositivity for Leishmania was 53% higher in tick-infested dogs and 300% higher in flea-infested dogs in comparison with non-infested animals. Our data provide evidence of the vectorial capacity of these ectoparasites in transmitting Leishmania to the canine population, although further studies are needed to confirm or reject this hypothesis.

Ritmo de queda de Rhipicephalus sanguineus (Acari: Ixodidae) de cães artificialmente infestados / Drop off rhythm of Rhipicephalus sanguineus (Acari: Ixodidae) of artificially infested dogs

O ritmo de queda de Rhipicephalus sanguineus (Latreille) foi avaliado para duas populações do Brasil; uma de Monte Negro, Estado de Rondônia, e outra de Belo Horizonte, Estado de Minas Gerais. Infestações artificiais com carrapatos das duas populações foram realizadas em cães domésticos mantidos em laboratório, sob o regime de luz: escotofase de 12:12 h. O padrão de queda de larvas ingurgitadas do hospedeiro se caracterizou pelo desprendimento de quantidades semelhantes de carrapatos em períodos de luz e de escotofase ou com um predomínio de queda durante o período de luz. No caso de ninfas e fêmeas ingurgitadas, a maioria dos carrapatos se desprendeu dos cães durante o período de escotofase, em todos os casos observados. Desta forma, é possível que em condições naturais, a maioria das ninfas e fêmeas adultas de R. sanguineus tendem a se desprender dos cães durante o período noturno, ao passo que larvas ingurgitadas se desprendem em proporções maiores durante o período diurno. Com base nestes dados, sugere-se que os locais onde os cães freqüentam durante a noite (onde possivelmente estará a maior parte da população de vida livre de R. sanguineus) sejam priorizados com os tratamentos ambientais utilizando-se produtos carrapaticidas.

The present study evaluated the drop-off rhythm of Rhipicephalus sanguineus (Latreille) ticks from two populations from Brazil, one from Monte Negro, state of Rondônia, and another from Belo Horizonte, state of Minas Gerais. Artificial infestations with ticks were performed on dogs in the laboratory, held in a light: scotophase regimen of 12:12 h. Larval drop-off rhythm was characterized by similar number of engorged larvae detaching during both periods of light and scotophase, or by a larger number of larvae detaching during the light period. In contrast, most of the engorged nymphs and females detached from dogs during the scotophase period. These results indicate that under natural conditions, most of R. sanguineus engorged nymphs and females detach from dogs during the night period, whereas engorged larvae detach in higher proportions during daytime. Based on these data, tick control measures, encompassing environmental treatments with acaricide, should be indicated. The control measures are especially indicated in places where dogs spend or visit during the night period, since these places possibly harbor most of the free-living stages of R. sanguineus.

[Control of Rhipicephalus sanguineus (Latrielle, 1806) (Acari: Ixodidae) in the kennel of the UFMG Veterinary School, Belo Horizonte, Minas Gerais, Brazil]. / Controle de Rhipicephalus sanguineus (Latrielle, 1805) (Acari: Ixodidae) no canil da Escola de Veterinária da UFMG, Belo Horizonte, Minas Gerais, Brasil.

The present work was aimed at proposing a control measure for Rhipicephalus sanguineus in a naturally infected kennel of 72 m(2) of the UFMG Veterinary School with 25 dogs of different breeds. A sensitivity test to acaricide products was applied in a sample of R. sanguineus. Out of the acaricide products tested: 12.5% Amitraz (product 1); 2% Alfamethrin and 60% Dichlorvos association (product 2); 5% Deltamethrin (product 3) and the association between 77.6% Trichlorfon, 1.0% Coumaphos and 1.0% Cyfluthrin (product 4); only 1 and 4 products showed 100% efficacy. Amitraz (12.5%) was used for controlling R. sanguineus in the surrounding of the kennel, on which four treatments every seven days were carried out in the facilities. For parasitic R. sanguineus, a single topical treatment was used: Flumetrin 1.0% pour-on on all the 25 dogs. This control measure significantly reduced the population of R. sanguineus within the kennel. A slight infestation was observed in the seventh month after treatment. A new application of 12.5% Amitraz was done in the kennel as well as a new topical treatment with Flumetrin 1.0% pour-on on the dogs. The control procedure proposed in the present study was an emergency measure and its effectiveness was checked through environmental surveys throughout a one-year period.

Controle de Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) no canil da Escola de Veterinária da UFMG, Belo Horizonte, Minas Gerais, Brasil / Control of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) in the kennel of the UFMG Veterinary School, Belo Horizonte, Minas Gerais, Brazil

O presente estudo teve como proposta controlar uma infestação natural de Rhipicephalus sanguineus em um canil pertencente à Escola de Veterinária da UFMG, com uma área de 72 m² e 25 cães de diversas raças. Um teste de sensibilidade a produtos acaricidas foi aplicado em uma amostra de carrapatos capturados no canil. Dos produtos testados: Amitraz 12,5 por cento (Produto 1); uma associação de Alfametrina 2 por cento e Dichlorvós 60 por cento (produto 2); Deltametrina 5 por cento (produto 3) e uma associação de Trichlorfon 77,6 por cento, Coumaphós 1 por cento e Cyfluthrin 1 por cento (produto 4); somente os produtos 1 e 4 apresentaram 100 por cento de eficácia. O Amitraz 12,5 por cento foi utilizado no controle dos estádios de R. sanguineus presentes no ambiente do canil, com um esquema de 4 aplicações a cada 7 dias de intervalo. Para os estádios parasitários, um único tratamento com Flumetrina 1.0 por cento pour-on foi empregado em todos os animais. A inspeção e o monitoramento do ambiente e dos animais foram realizados durante um período de um ano. Estas medidas de controle reduziram significativamente a população de carrapatos dentro do canil. O canil não apresentou infestação pelo carrapato nos seis meses seguintes aos tratamentos. Uma infestação leve foi observada no 7º mês pós-tratamento, quando foi realizada uma nova aplicação de Amitraz 12,5 por cento no ambiente do canil e um novo tratamento com Flumetrina 1.0 por cento pour-on nos animais, com resultados satisfatórios.

The present work was aimed at proposing a control measure for Rhipicephalus sanguineus in a naturally infected kennel of 72 m² of the UFMG Veterinary School with 25 dogs of different breeds. A sensitivity test to acaricide products was applied in a sample of R. sanguineus. Out of the acaricide products tested: 12.5 percent Amitraz (product 1); 2 percent Alfamethrin and 60 percent Dichlorvos association (product 2); 5 percent Deltamethrin (product 3) and the association between 77.6 percent Trichlorfon, 1.0 percent Coumaphos and 1.0 percent Cyfluthrin (product 4); only 1 and 4 products showed 100 percent efficacy. Amitraz (12.5 percent) was used for controlling R. sanguineus in the surrounding of the kennel, on which four treatments every seven days were carried out in the facilities. For parasitic R. sanguineus, a single topical treatment was used: Flumetrin 1.0 percent pour - on on all the 25 dogs. This control measure significantly reduced the population of R. sanguineus within the kennel. A slight infestation was observed in the seventh month after treatment. A new application of 12.5 percent Amitraz was done in the kennel as well as a new topical treatment with Flumetrin 1.0 percent pour - on on the dogs. The control procedure proposed in the present study was an emergency measure and its effectiveness was checked through environmental surveys throughout a one-year period.

[Drop off rhythm of Rhipicephalus sanguineus (Acari: Ixodidae) of artificially infested dogs]. / Ritmo de queda de Rhipicephalus sanguineus (Acari: Ixodidae) de cães artificialmente infestados.

The present study evaluated the drop-off rhythm of Rhipicephalus sanguineus (Latreille) ticks from two populations from Brazil, one from Monte Negro, state of Rondônia, and another from Belo Horizonte, state of Minas Gerais. Artificial infestations with ticks were performed on dogs in the laboratory, held in a light: scotophase regimen of 12:12 h. Larval drop-off rhythm was characterized by similar number of engorged larvae detaching during both periods of light and scotophase, or by a larger number of larvae detaching during the light period. In contrast, most of the engorged nymphs and females detached from dogs during the scotophase period. These results indicate that under natural conditions, most of R. sanguineus engorged nymphs and females detach from dogs during the night period, whereas engorged larvae detach in higher proportions during daytime. Based on these data, tick control measures, encompassing environmental treatments with acaricide, should be indicated. The control measures are especially indicated in places where dogs spend or visit during the night period, since these places possibly harbor most of the free-living stages of R. Sanguineus.