Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros Municipality,Minas Gerais State,Brazil

The Montes Claros City is located in an endemic area for visceral leishmaniosis in the Minas Gerais State, Brazil. With the implementation of a program for the control of visceral leishmaniosis in 1994, a sectional study was carried out to evaluate the infection by viscerotropic Leishmania in the population of dogs from Montes Claros, basically using indirect immunofluorescence antibody test (IFAT). Blood samples were collected on filter paper from 33,937 dogs, representing 96.1% of the canine local population. The prevalence for visceral leishmaniosis was found to be 9.7% in the municipality, being 9.9% in the urban area and 8.8% in the rural area. The annual incidence showed to be 64.3/1000 dogs. Prevalence of infection was not correlated with dogs age. The most affected breeds were: Boxer (24.6%) and Cocker (26.9%); Mongrel dogs had a prevalence of 7.8%. Short-hair animals had a prevalence of 11.9%, while long-furred animals had a prevalence of 8.9%. The isoenzymatic profile indicated that Leishmania (Leishmania) chagasi was the visceral leishmaniosis etiological agent in Montes Claros City, Minas Gerais State, Brazil. The main geographical areas for the parasite transmission were identified, and control measures were immediately started. The role of the dog as a reservoir for L. chagasi was confirmed. It was demonstrated that short-furred animals are at a higher risk of acquiring visceral leishmaniosis than the long-furred dogs.     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.     

PCR identification of Leishmania in diagnosis and control of canine Leishmaniasis

Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding "in vitro" cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.     

Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals

The purpose of this study was to evaluate the safety of spleen aspiration as a sampling technique for the parasitological detection by culture and microscopy of Leishmania (chagasi) infantum. Two hundred and nine domiciled dogs from an endemic area for visceral leishmaniasis in Bahia State, Brazil, were studied. Most dogs (87%) were seropositive for anti-L. chagasi antibodies by ELISA. Clinical signs of disease were recorded and the animals monitored during and after spleen puncture in order to detect possible complications associated with the procedure. From a total of 257 splenic punctures in the 209 animals, only three minor events occurred, with no significant consequence for the animals and no association with risk factors. Leishmania was isolated from 149/180 (83%) seropositive dogs, and from 6/26 (23%) seronegative animals. The procedure did not cause adverse side effects or unnecessary suffering and confirmed the diagnosis in a large percentage of dogs. We conclude that spleen aspiration can be considered an effective and safe procedure for the definitive diagnosis of canine visceral leishmaniosis.     

Post mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil

A parasitological study was conducted on 66 dogs seroreactive for Leishmania captured as a control measure of visceral leishmaniasis in the State of Rio de Janeiro, Brazil. Biological samples from different anatomical sites were collected during autopsy of the animals and cultured on biphasic medium (NNN/Schneider). The Leishmania isolates were characterized by isoenzyme electrophoresis. Leishmania was isolated from 80.3% of the animals: 12 animals with Leishmania (Viannia) braziliensis isolated exclusively from cutaneous lesions, 39 with L. (L.) chagasi isolated from different sites in the same animal, and 2 with simultaneous isolation of L. (V.) braziliensis from cutaneous lesions and L. (L.) chagasi from different sites. Isolation in culture revealed the absence of Leishmania parasites in 13 animals. The results obtained confirm the existence of mixed infections in dogs in Rio de Janeiro and indicate the need to complement the investigation of seroreactive dogs using methods for the parasitological diagnosis and identification of Leishmania species.     

Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program

In 2008, in the west zone of Rio de Janeiro municipality-Brazil, the leishmaniasis control program identified 155 dogs with titers ≥ 40 by Indirect ImmunoFluorescence (IIF) on blood collected onto filter paper. The objective of this study was to describe the laboratory test findings performed in dogs euthanized by the leishmaniasis program control of Rio de Janeiro municipality. Dogs were examined, subjected to euthanasia and collection of clinical specimens. Parasite isolation was obtained in 29 animals: Leishmania chagasi was isolated in 14 dogs; Leishmania braziliensis was isolated in five dogs; Trypanosoma caninum was obtained in seven animals and one dog had mixed infection (L. braziliensis and L. chagasi). By Polymerase Chain Reaction, seventeen animals were positive in intact skin fragments. In the serological reassessment of serum samples, 28% and 22% were positive for IIF and enzyme immunoassay, respectively. Ninety-one (59%) dogs were negative for all tests performed in this study. The findings indicate that the visceral leishmaniasis control program needs to be adjusted in order to avoid non-infected dogs from being removed or permit that dogs infected with L. chagasi to remain undetected in endemic areas.     

Canine Visceral Leishmaniosis in Anastácio,Mato Grosso do Sul State,Brazil

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed as Leishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.     

Use of ELISA employing Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens for the detection of IgG and IgG1 and IgG2 subclasses in the diagnosis of American tegumentary leishmaniasis in dogs

American tegumentary leishmaniasis (ATL) shows a reduced humoral response in dogs and levels of specific antibodies may therefore not be detected by indirect immunofluorescence. Although the sensitivity of enzyme-linked immunosorbent assay (ELISA) is higher than that of indirect immunofluorescence, the best antigen for the diagnosis of ATL in dogs has not been defined. The detection of IgG subclasses represents an alternative to increase the efficiency of the serological diagnosis. In Rio de Janeiro, sporotrichosis is the main differential diagnosis of ATL in dogs, and a sensitive, specific and little invasive method that permits the discrimination of the two diseases is desired. In the present study, 69 serum samples, 34 obtained from dogs with ATL and 35 from dogs with sporotrichosis, all of them with a confirmed etiological diagnosis, were tested. The samples were analyzed by ELISA using Leishmania (Viannia) braziliensis and L. (L.) chagasi antigens for the detection of anti-Leishmania IgG, IgG1 and IgG2. The use of L. (V.) braziliensis antigens for the detection of IgG and IgG2 yielded the best results. Using L. (L.) chagasi antigen, the sensitivity and specificity for the detection of IgG were 82.4% and 100%, respectively, whereas both sensitivity and specificity were 97.1% with the L. (V.) braziliensis antigen. No improvement in the performance of the test was observed when IgG2 was analyzed separately. The IgG1 assays presented low accuracy, irrespective of the antigen used: sensitivity and specificity of 58.8% and 60% for L. (V.) braziliensis and of 64.7% and 77.1% for L. (L.) chagasi, respectively. The present results suggest that IgG ELISA using the L. (V.) braziliensis shows the best performance for the diagnosis of ATL, permitting the discrimination between cases of ATL and sporotrichosis in dogs.     

Leishmania chagasi infection in cats with dermatologic lesions from an endemic area of visceral leishmaniosis in Brazil

Although dogs are considered the main domestic reservoirs for Visceral Leishmaniosis (VL), which is caused in the Americas by Leishmania chagasi, infected cats have also been recently found in endemic areas of several countries and became a public health concern. Accordingly, the purpose of this study was to evaluate cats with dermatologic lesions from an endemic area of VL and the natural infection of L. chagasi. A total of 55 cats were selected between April 2008 and November 2009 from two major animal shelters of Ara?atuba, Southeastern Brazil. All cats underwent general and dermatologic examinations, followed by direct parasitological examination of lymphoid organs, immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT). In addition, detection of amastigotes was performed by immunohistochemistry (IHC) in skin lesions of all cats. VL was diagnosed in 27/55 (49.1%) cats with dermatological problems. Amastigotes were found in lymphoid organs of 10/27 (37.0%) cats; serology of 14/27 (51.9%), 6/27 (22.2%) and 5/27 (18.5%) cats was positive for ELISA, IFAT and both, respectively. The IHC identified 9/27 (33.3%) cats; 5/27 (18.5%) were positive only for IHC and therefore increased the overall sensitivity. Specific FIV antibodies were found in 6/55 (10.9%) cats, of which 5/6 (83.3%) had leishmaniosis. Real time PCR followed by amplicon sequencing successfully confirmed L. chagasi infection. In conclusion, dermatological lesions in cats from endemic areas was highly associated to visceral leishmaniosis, and therefore skin IHC and differential diagnosis of LV should be always conducted in dermatological patients in such areas.     

The first records of Leishmania (Leishmania) amazonensis in dogs (Canis familiaris) diagnosed clinically as having canine visceral leishmaniasis from Araçatuba County, São Paulo State, Brazil

Two cases of Leishmania (Leishmania) amazonensis are reported in the domestic dog (Canis familiaris). These are the first records of this parasite in this species. The animals lived in the endemic visceral leishmaniasis area of Ara?atuba, S?o Paulo State, Brazil and were initially diagnosed, on clinical grounds, as having visceral leishmaniasis. Attempted parasite isolation from inguinal lymph node aspirates was unsuccessful and the indirect immunofluorescent test for visceral leishmaniasis was negative in both cases. Parasites were seen in cytological preparations of their lymph nodes and the DNA obtained from these same tissues produced the expected fragment in a Leishmania specific rDNA based PCR assay. The products only hybridized with the L. (L.) amazonensis specific probe S8. No human cases of L. (L.) amazonensis have been reported in this region. These results suggest that L. (L.) amazonensis is being transmitted in the peridomestic habitat and that this parasite is responsible for a clinical condition that is similar to visceral leishmaniasis caused by L. (L.) i. chagasi that is present in the same area.     

Clinical and serological aspects of visceral leishmaniasis in northeast Brazilian dogs naturally infected with Leishmania chagasi

Human visceral leishmaniasis is endemic in the northeast of Brazil, where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. In this study, we evaluated the clinical signs of canine visceral leishmaniasis (CVL), serum protein profile and the antileishmanial IgG antibody production in 86 dogs living in northeast endemic areas of leishmaniasis. Thirty dogs from a leishmaniasis-free area were used as a control group. The major clinical signs of CVL seen were emaciation and skin ulcers (80%), followed by onychogryphosis and conjunctivitis (73%). Depilation was observed in 60% of animals while lymphadenomegaly, splenomegaly, liver enlargement or kidney involvement was less frequent (< or =20%). VL seropositive dogs presented with serum hyperproteinemia, hypoalbuminemia, hypergammaglobulinemia and decreased albumin/globulin ratio. A lower sensitivity and higher specificity was observed for promastigote indirect fluorescent antibody test (IFAT) (83 and 100%, respectively) compared with enzyme-linked immunosorbent assay (ELISA) (94 and 90%), which uses a crude extract of Leishmania. There was a positive correlation between IFAT and ELISA titers of antileishmanial IgG antibodies (Spearman test, P < 0.05), which was augmented in CVL dogs. This study found that the determination of serum protein, A/G ratio and the use of two different leishmanial serological tests like IFAT and ELISA are essential in CVL screening.     

Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.     

Eco-epidemiology of visceral leishmaniasis in the urban area of Paracatu, state of Minas Gerais, Brazil

The present study was developed in the urban area of Paracatu, an endemic city for the American visceral leishmaniasis in Brazil. A six-month canine survey was performed with 6295 domiciled dogs in 28 districts in that area and showed that 4.2% of those (267 dogs) were positive for VL by ELISA and IFAT serum assays. Prevalence ratios for canine VL varied between 1.2% and 16.1%, depending on the district under investigation. Fifteen dogs - 80% of which were clinically asymptomatic for VL - were submitted to a more detailed study that comprised direct parasitological examination and Leishmania kDNA amplification of tissue samples as well as two PCR-RFLP methods using myelocultures. Leishmania amastigotes or Leishmania DNA were detected in all dogs but one. The infecting species of Leishmania was identified in about 50% (7/15) of the sample dogs: Leishmania (Leishmania) chagasi in two of them and, unexpectedly, Leishmania (Leishmania) amazonensis in the remaining five. Three months after the end of confiscation and elimination of the VL-seropositive dogs in the 28 districts of Paracatu, a systematic entomological survey was performed in five of them. Six hundred and sixty five (665) phlebotomine sand flies were captured in total, from which 89.5% were identified as Lutzomyia longipalpis. The population density of that species increased during the rainy season. Other thirteen (13) species of phlebotomine sand flies were captured at varying percentages from 0.2 to 5.0%. It is worth noting that L. longipalpis females were predominantely intradomicile when compared to males, suggesting that the VL transmission cycle in Paracatu may be occurring inside home.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.     

Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations

Three serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n=20), and from dogs with other parasitic diseases including Trypanosoma cruzi (n=7), Leishmania braziliensis (n=5), Toxoplasma gondii (n=5) and Ehrlichia canis (n=3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi, L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from an E. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.     

Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi

Visceral leishmaniasis (VL) presents vigorous Th2 immune response, which is mainly characterized in human by augmented expression of Il-4, polyclonal B cell activation, intense hypergammaglobulinemia and production of antileishmanial IgE antibodies. However, few aspects of this type of immune response have been demonstrated in studies of canine visceral leishmaniasis (CVL). This work investigated by ELISA and western immunoblotting the production of antileishmanial IgE antibodies (IgE Ab) in symptomatic and asymptomatic dogs naturally infected by Leishmania chagasi, and also compared this IgE immune response with those of IgG, IgG1 and IgG2 antibodies. Three groups of dogs were evaluated: 12 VL dogs with positive Leishmania biopsies (GI), 44 dogs with a positive leishmanial indirect fluorescent antibody test (IFAT), 30 of them presenting clinical signs of VL and 14 asymptomatic (GII) and 21 healthy dogs living in kennels located in leishmaniasis endemic areas (GIII), which were seronegative in the IFAT. Eighteen dogs from an area free of CVL were used as controls (GIV). Antileishmanial IgE antibodies were detected in 4 of 12 VL dogs from group I (33%) and 14 of 30 symptomatic dogs from group II (47%). While all asymptomatic dogs from group II (100%) were seronegative for antileishmanial IgE Ab, 7 of 21 healthy animals from group III (33%) had these immunoglobulins. A strong correlation was verified between antileishmanial IgG and IgG2 antibody titers in all symptomatic dogs, but only 15 of these 42 animals (36%) produced simultaneously IgE, IgG, IgG1 and IgG2 antibodies to Leishmania. IgE antibodies recognized leishmanial antigens of 12, 36, 61, 81 and 118 KDD, while a more complex pattern of immunoblotting was verified mainly for IgG and IgG2 antibodies from symptomatic animals. IgG1 and IgG2 antibodies shared the recognition of L. chagasi polypeptides of 118, 81, 61, 36, 18, 14 and 12 KDD, being more intense the immune reactions between IgG1 Ab and the leishmanial polypeptides of 61 and 36 KDD, and also between IgG2 antibodies and the antigens of 26, 21, 18, 14 and 12 KDD. Our results suggest that the polyclonal production of antileishmanial antibodies that includes IgE Ab could characterize a Th2 immune response in CVL and can help the laboratory diagnosis of this disease.     

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.     

Oxidative stress and non-enzymatic antioxidative status in dogs with visceral Leishmaniasis

Leishmaniasis is a potentially fatal chronic protozoan disease in human, canine and rodent species. The infection by Leishmania is endemic in the Mediterranean Sea region, Africa, Asia and South America. Canine visceral leishmaniasis (CanVL) is a systemic disease caused by Leishmania infantum and Leishmania chagasi from the Leishmania donovani complex group. The blood glutathione (GSH), plasma malondialdehyde (MDA), ascorbic acid (AA), beta-carotene, retinol and ceruloplasmin levels of dogs with CanVL were investigated to establish the status of the antioxidant defense mechanism in the infected animals. Dogs diagnosed as CanVL with amastigotes in lymph node smear examination and/or antibody titers > or = 128 were used as subjects, while those with no serological response against leishmaniasis were used as healthy controls. The glutathione and retinol amounts were decreased although not significantly (p > 0.05), but the MDA levels were significantly higher in dogs with VL, suggesting increased lipid peroxidation.