Description of the Occurrence of Canine Dirofilariasis in the State of Rio de Janeiro, Brazil

In order to evaluate the prevalence of canine heartworm in the State of Rio de Janeiro, a multicenter survey was carried out in two phases. The survey involved 1376 dogs from two cities: Rio de Janeiro and Niterói, and its surroundings, including the eastern shore and mountain resorts, which were further divided into sections. In the first phase, 795 dog blood samples were examined by the modified Knott test for the detection of microfilariae. A total of 134 samples (16.85%) were microfilaremic: 8.61% from Rio de Janeiro, 21.76% from Niterói and its surroundings, 33.33% from the eastern shore and 30.43% from the mountain resorts. In the second phase, 595 dog blood samples were examined first by the modified Knott test and the amicrofilaremic samples were subsequently examined by an immunoenzymatic test (ELISA) for antigen detection. In summary, 83 samples (13.95%) were microfilaremic and 44 (7.98%) of the amicrofilaremic samples were positive for heartworm antigen (occult infections). In Rio de Janeiro, 13.68% of the dogs were infected (i.e., antigen-and/or microfilaria-positive) and 8.51% of the dogs had microfilaremic infections. In comparison, Niterói and its surroundings showed values of 24.46% and 17.30% and the eastern shore showed values of 52.46% and 31.15%. In contrast the mountain resorts showed 20% microfilaremic only

Mosquito Frequency and Feeding Habits in an Enzootic Canine Dirofilariasis Area in Niterói, State of Rio de Janeiro, Brazil

Heartworm, a chronic fatal mosquito-borne canine disease, is frequently diagnosed in dogs from the State of Rio de Janeiro, where its prevalence is 29.7% in the city of Niterói. Nevertheless it is rarely detected in cats (0.8%) from the same state. Dogs are the primary source of infection to mosquitoes, because cats either do not demonstrate microfilaremia or it is too low and transient for transmission. A mosquito survey was conducted in Itacoatiara, Niterói, from March 1995 to February 1996, using canine, feline and human baits. A total of 21 mosquito species (3,888 females) was collected and biting frequency was highest at dusk. The four species collected most frequently (88.9%) were: Aedes taeniorhynchus (30% of the total catch; with the peak in May/June); Culex quinquefasciatus (22.5%; August/October); Aedes scapularis (19.4%; August, October/November and January) and Culex declarator (17%; November/January). Human baits were attractive to these species and dogs were significantly more attractive to them than cats. Ae. taeniorhynchus, Cx. quinquefasciatus, Ae. scapularis, Cx. declarator and Cx. nigripalpus are the most likely mosquito species to transmit Dirofilaria immitis parasites to dogs and may transmit the parasite to humans. It is also suggested that the vector to cats belongs to the genus Culex

Survey of canine heartworm in the city of Recife, Pernambuco, Brazil

Six hundred and eleven random-source dogs (338 male, 273 female) one year of age or older, from six sections of the city of Recife, Pernambuco, were examined antemortem for circulating microfilariae Dirofilaria immitis and Dipetalonema reconditum adult heartworm (D. immitis) antigen, and examined postmortem for adult heartworms. The prevalence of heartworm infection was 2.3% (14/611), as determined by necropsy for adult worms, and 1% (6/611) had circulating microfilariae of D. immitis; thus, 57.1% of the heartworm-infected dogs had occult infections. The results of serological testing indicated that 1.3% (8/611) of the dogs were positive for adult heartworm antigen. A total of 42 (6.9%) of the dogs had microfilariae of D. reconditum; 40 of these had only D. reconditum and two additional dogs had microfilariae of both species, D. immitis and D. reconditum.

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Force of infection and evolution of lesions of canine tegumentary leishmaniasis in Northwestern Argentina

A clinical-serological follow-up was carried out in a canine population in endemic foci of Leishmania braziliensis spread in northwestern Argentina. Each dog was studied in at least two visits, 309±15 days (X±SE) apart. Some initially healthy dogs (n=52) developed seroconversion or lesions. The clinical evolution of the disease in dogs resembles in many aspects the human disease. Similarities include the long duration of most ulcers with occasional healing or appearance of new ones and the late appearance of erosive snout lesions in some animals. Yearly incidence rates of 22.7% for seroconversion and of 13.5% for disease were calculated as indicators of the force of infection by this parasite upon the canine population.

Comparison between precipitin and ELISA tests in the bloodmeal detection of Aedes aegypti (Linnaeus) and Aedes fluviatilis (Lutz) mosquitoes experimentally fed on feline, canine and human hosts

The identification of arthropod bloodmeals is important in many epidemiological studies, as, the understanding of the life cycle of vectors and the patogens they transmit, as well as helping to define arthropods' control strategies. The precipitin test has been used for decades, but ELISA is slowly becoming more popular. To compare the two tests for sensitivity, specificity and accuracy to detect small insect bloodmeals, Aedes aegypti or Ae. fluviatilis mosquitoes were fed either on feline, canine or human hosts. Mosquitoes were frozen at 6, 12, 24, 48 or 72 h after feeding. Precipitin test showed better specificity and accuracy and ELISA test showed higher sensitivity. Better results with both tests were achieved when mosquitoes were frozen within 48 h from feeding.

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

Epidemiological and Serological Aspects in Canine Toxoplasmosis in Animals with Nervous Symptoms

The presence of anti-Toxoplasma gondii IgM and IgG antibodies was studied in samples of blood serum taken from eighty dogs with nervous symptoms at the Serviço de Enfermidades Infecciosas dos Animais, Faculdade de Medicina Veterinária e Zootecnia, Unesp, Botucatu, São Paulo, Brazil. The frequency of IgG titers were 16 (13.7%), 64 (13.7%), and 256 (5%), and for IgM titers were 16 (7.5%), 64 (15%), and 256 (8.7%). Positive reactions were more frequent in the older animals, males, from a rural environment, in constant contact with small animals, principally birds and rodents. There was a higher frequency of a positive reaction in dogs fed with kitchen food, especially in those fed with raw ingredients. The most common neurological pictures were alterations in consciousness, in movement, and in the hand-cart test. The percentage of reagents with specific IgM antibodies was high, indicating active infections, but the possibility of co-infection with the distemper virus can not be discarded, and this may be a predisposing factor for toxoplasmosis infection, once the distemper virus has a potent immunosupressive action.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages

The yield as well as phenotypic and functional parameters of canine peripheral blood monocyte-derived macrophages were analyzed. The cells that remained adherent to Teflon after 10 days of culture had high phagocytic activity when inoculated with Leishmania chagasi. Flow cytometric analysis demonstrated that more than 80% of cultured cells were positive for the monocyte/macrophage marker CD14.

Study of sand fly fauna in an endemic area of American cutaneous leishmaniasis and canine visceral leishmaniasis in the municipality of Espírito Santo do Pinhal, São Paulo, Brazil

Canine American visceral leishmaniasis and American cutaneous leishmaniasis (ACL) cases have been recorded in Espírito Santo do Pinhal. The aim of this study was to gather knowledge of the sand fly community and its population ecology within the municipality. Captures were made weekly over a period of 15 months in the urban, periurban and rural areas of the municipality, using automatic light traps. A total of 5,562 sand flies were collected, comprising 17 species. The most abundant species were Nyssomyia whitmani and Pintomyia pessoai in the rural area, Lutzomyia longipalpis and Ny. whitmani in the periurban area and Lu. longipalpis in the urban area. The highest species richness and greatest index species diversity were found in the rural area. The similarity index showed that urban and periurban areas were most alike. Lu. longipalpis was found in great numbers during both dry and humid periods. The presence of dogs infected with Leishmania infantum chagasi in the urban area indicates a high risk for the establishment of the disease in the region. A high abundance of Ny. whitmani and Pi. pessoai in the rural and periurban areas indicates the possibility of new cases of ACL occurring in and spreading to the periurban area of Espírito Santo do Pinhal.

The distribution pattern of Lutzomyia longipalpis (Diptera: Psychodidae) in the peridomiciles of a sector with canine and human visceral leishmaniasis transmission in the municipality of Dracena, São Paulo, Brazil

The specimen distribution pattern of a species can be used to characterise a population of interest and also provides area-specific guidance for pest management and control. In the municipality of Dracena, in the state of São Paulo, we analysed 5,889 Lutzomyia longipalpis specimens collected from the peridomiciles of 14 houses in a sector where American visceral leishmaniasis (AVL) is transmitted to humans and dogs. The goal was to analyse the dispersion and a theoretical fitting of the species occurrence probability. From January-December 2005, samples were collected once per week using CDC light traps that operated for 12-h periods. Each collection was considered a sub-sample and was evaluated monthly. The standardised Morisita index was used as a measure of dispersion. Adherence tests were performed for the log-series distribution. The number of traps was used to adjust the octave plots. The quantity of Lu. longipalpis in the sector was highly aggregated for each month of the year, adhering to a log-series distribution for 11 of the 12 months analysed. A sex-stratified analysis demonstrated a pattern of aggregated dispersion adjusted for each month of the year. The classes and frequencies of the traps in octaves can be employed as indicators for entomological surveillance and AVL control.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.