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.     

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.     

An evaluation of the clinical, cytological, infectious and histopathological features of feline acne

Clinical, cytological, microbial and histopathological features of feline acne were investigated in 22 cats referred or volunteered to a veterinary dermatology practice in the south-west region of the USA. For comparison, same parameters were evaluated in five unaffected pet cats. Additionally, all cats were evaluated by immunohistochemistry (IHC) for the presence of feline calicivirus (FCV) and feline herpes virus (FHV-1) in acne lesions. The age of onset of acne in affected cats ranged from 6 months to 14 years with a median of 4 years. The most common dermatologic lesions were comedones (73%), alopecia (68%), crusts (55%), papules (45%) and erythema (41%). Pruritus was reported in 35% of the affected cats. Cytological evidence of Malassezia pachydermatitis was present on 4/22 (18%) of affected cats. Microsporum canis was isolated from a single affected cat. Bacteria were isolated from 10 of the 22 (45%) affected cats; coagulase-positive staphylococci and alpha-haemolytic streptococci were most common. Histopathological features included lymphoplasmacytic periductal inflammation (86%), sebaceous gland duct dilatation (73%), follicular keratosis with plugging and dilatation (59%), epitrichial gland occlusion and dilatation (32%), folliculitis (27%), pyogranulomatous sebaceous adenitis (23%) and furunculosis (23%). In one affected cat from a household with five cats, simultaneously having feline acne, FCV antigen was detected in the biopsy of the chin by IHC. Chin tissue samples from all other affected cats, as well as the five healthy cats, were negative by IHC for FCV and FHV-1 antigens.     

Montenegro's skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area

In this study, humoral (circulating anti-Leishmania antibodies) and cellular (Montenegro's skin test) immune responses of dogs from an endemic area of visceral leishmaniosis were tested using Leishmania chagasi, Leishmania amazonensis and Leishmania braziliensis antigens. The antibody response was tested in three animal groups, selected according to their anti-L. chagasi antibody activity, as measured by ELISA in the serum: 19 negative (O.D. below 0.30), seven with undefined (O.D. between 0.40 and 0.70) and 12 positive (O.D. above 1.0) ELISA result. In the group of animals with positive ELISA, the antibody activity against L. chagasi antigens (mean O.D.=1.31) was significantly higher (ANOVA, P<0.01) than against L. amazonensis (mean O.D.=0.88) or L. braziliensis (mean O.D.=0.87) antigens. The Montenegro's skin test results obtained with L. chagasi and L. braziliensis antigens showed a fair agreement (kappa=0.309). The same was observed when antigens from L. braziliensis and L. amazonensis were compared (kappa=0.374), whereas a moderate agreement between the results from tests performed with L. chagasi and L. amazonensis antigens was observed (kappa=0.530). The induration areas obtained with L. braziliensis antigen were smaller than those obtained with the other antigens. The data presented herein indicate that the use of antigens from different Leishmania species may interfere with the results of the immunological tests performed in dogs in an endemic area of visceral leishmaniosis.     

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.     

Canine leishmaniosis in Central Europe: retrospective survey and serological study of imported and travelling dogs

Canine leishmaniosis is a common parasitic disease in Central Europe affecting dogs imported or returning from endemic countries around the Mediterranean basin. Through an internet discussion forum owners of dogs with suspected or proven leishmaniosis living in Central Europe (D, A, CH), were questioned about the dog's history. Additionally, serologic examinations of the dogs for anti-Leishmania antibodies (ELISA using antigen of promastigote stages) were offered to the participants. From February to October 2003, 291 dogs imported or returning from Southern Europe (Spain, Italy, Greece, Turkey, France, Malta, Portugal and others) were analysed. Serologically, 111 dogs (38%) were classified positive; 103 being imported and eight travelling dogs. The majority of seropositive dogs originated from Spain (67%). No significant correlation could be established between race, sex and age and the incidence of the disease. The clinical symptoms in the seropositive dogs varied widely and ranged from mild general symptoms to visceral manifestations with chronic renal failure. Skin disorders were found in 78% of the seropositive, symptomatic animals. In the country of origin or after import or return, 174 out of 291 dogs (60%) had been tested for the presence of anti-Leishmania antibodies by different immunofluorescence antibody tests (IFAT). Discrepancies between the ELISA and the various IFATs used were noted in 55 cases (32%), especially in cases of low IFAT titers. Most of the seropositive dogs (80%) had been treated against leishmaniosis. In 91% of these cases, Allopurinol as monotherapy or in combination with Glucantime had been used. For diagnostics and therapy, dog owners had spent an average of 1,100 euros (median 900 euros, maximum 5,800 euros).     

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.     

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.     

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.     

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.     

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.     

Atypical forms of canine leishmaniosis

Canine leishmaniosis is a common disease in the Mediterranean area, but sporadic cases in dogs having travelled through endemic regions are also reported. The disease's evolution is usually chronic and symptoms are either non-specific (fever, weight loss, lethargy, enlarged lymph nodes), dermatological, renal or ocular. The purpose of this article is to review the literature and to describe our own experience of certain atypical forms of canine leishmaniosis. These include specific skin lesions, monoclonal gammopathy, renal failure (without any other signs), chronic colitis, haemostatic problems and disorders of the cardiovascular, respiratory and musculo-skeletal systems.     

Sporotrichosis: the main differential diagnosis with tegumentary leishmaniosis in dogs from Rio de Janeiro, Brazil

Seventy-four dogs from the State of Rio de Janeiro with ulcerated cutaneous lesions were submitted to clinical, dermatological, parasitological, mycological, histopathological and cytopathological exams, a leishmanin skin test, an indirect immunofluorescence (IIF) test for leishmaniosis, and nonspecific laboratory tests such as blood count and serum biochemistry. Sporothrix schenckii was isolated from 41 dogs and Leishmania (Viannia) braziliensis was isolated from 33 animals. Most dogs with sporotrichosis were from the municipality of Rio de Janeiro (53.7%) and presented ulcerated cutaneous lesions on the head (68.3%). Laboratory alterations in these animals included anemia (58.5%), hypoalbuminemia (83%) and hyperglobulinemia (75.6%). Histopathology revealed the predominance of a chronic granulomatous inflammatory infiltrate (70.7%), and yeast-like structures were detected in 17% of the histopathological exams and in 32% of the cytological exams. Three of 41 dogs with sporotrichosis were seropositive by IIF for leishmaniosis and 2 of 20 animals tested within this group had a positive leishmanin skin test. Similarly, most of the 33 dogs with leishmaniosis were from the municipality of Rio de Janeiro (69.7%) and had ulcerated cutaneous lesions on the head (84.8%). Laboratory alterations in these animals included anemia (66.7%), hypoalbuminemia (100%) and hyperglobulinemia (91%). Histopathology showed the predominance of a chronic granulomatous inflammatory infiltrate (63.6%) and amastigote forms were detected in 30.3% of the histopathological exams and in 31.8% of the 22 cytological exams performed. About 72.7% of the dogs were seropositive by IIF and five of seven animals had a positive skin test. Due to the clinical similarities, histopathological and nonspecific laboratory results similarities, the serological and skin tests for leishmaniosis positive in dogs with sporotrichosis, and the overlapping endemic areas in Rio de Janeiro, the differential diagnosis between the two diseases requires the demonstration of their respective etiological agents.     

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.     

Feline leukemia virus detection by immunohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissue from cats with lymphosarcoma.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feline leishmaniosis due to Leishmania infantum in Italy

A case of leishmaniosis in domestic cats (Felis catus domesticus) is described. The subject showed a nodular lesion on the eyelid. The diagnosis was achieved by serological, parasitological, and light and electron microscopic investigations. By molecular techniques the aetiological agent was identified as belonging to Leishmania infantum, the species implicated in human and canine leishmaniosis in southern Europe. A preliminary study on the prevalence of asymptomatic feline leishmaniosis, performed in the areas where the infected cat was identified, revealed a low seroprevalence of infection: only 1 (0.9%) of the 110 cat sera examined by indirect fluorescent antibody test was positive for anti-Leishmania antibodies. Because clinical signs in feline leishmaniosis are unspecific and similar to those observed in other diseases commonly found in this species, leishmaniosis must be added to the differential diagnosis by feline veterinary practitioners and adequate serologic and histopathologic investigations must be performed in endemic areas.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Coinfection of Leishmania chagasi with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in cats from an endemic area of zoonotic visceral leishmaniasis

The aim of the present study was to determine the coinfection of Leishmania sp. with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in a population of cats from an endemic area for zoonotic visceral leishmaniasis. An overall 66/302 (21.85%) cats were found positive for Leishmania sp., with infection determined by direct parasitological examination in 30/302 (9.93%), by serology in 46/302 (15.23%) and by both in 10/302 (3.31%) cats. Real time PCR followed by amplicon sequencing successfully confirmed Leishmania infantum (syn Leishmania chagasi) infection. Out of the Leishmania infected cats, coinfection with FIV was observed in 12/66 (18.18%), with T. gondii in 17/66 (25.75%) and with both agents in 5/66 (7.58%) cats. FeLV was found only in a single adult cat with no Leishmania infection. A positive association was observed in coinfection of Leishmania and FIV (p<0.0001), but not with T. gondii (p>0.05). In conclusion, cats living in endemic areas of visceral leishmaniasis are significantly more likely to be coinfected with FIV, which may present confounding clinical signs and therefore cats in such areas should be always carefully screened for coinfections.     

Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas

Canine visceral leishmaniasis (VL), caused by Leishmania infantum (Leishmania chagasi in the New World), is a zoonotic, endemic disease in Western Europe and Latin America. The potential spreading to new regions was suggested by the appearance of canine VL among foxhounds in the US. Although the sand fly vectors in the major foci of transmission have been described, no information exists on other sand flies that could propagate the infection outside endemic areas. We evaluated the capacity of Lutzomyia shannoni (Dyar) and Lutomyia youngi (Feliciangeli & Murillo), which are widely distributed in the New World, to acquire L chagasi (Cunha and Chagas) infections. A high proportion of L youngi were infected after feeding on an oligosymptomatic dog (51 per cent) or a polysymptomatic individual (95 per cent), but the intensity of infection was low (< 200 promastigotes/fly). L shannoni became infected only by feeding on the polysymptomatic dog, and the infection rate was lower (9 per cent) than in Lutzomyia longipalpis (36 per cent), and Lutzomyia evansi (Nunez-Tovar) (Lutz and Neiva) (38 per cent), but the intensity of infection (200 to > 500 promastigotes/fly) was comparable (L longipalpis) or higher (L evansi) than in the New World vectors. It is hypothesised that the presence of infected dogs in areas where L shannoni or L youngi occur could initiate new endemic cycles of VL in both South and North America.