Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus

Visceral leishmaniasis is a disease caused by Leishmania (Leishmania) chagasi and represents a serious public health problem. The dog is the main urban reservoir of the disease; however, investigations regarding the occurrence and epidemiological importance of leishmaniasis in cats have recently been initiated. This study aimed to detect cats seropositive for Leishmania spp. using different antigens. Additional studies were performed using sera from cats with Toxoplasma gondii (n=15) to evaluate cross-reactivity. Serum samples (n=113) from cats living in the town of Ara?atuba, State of S?o Paulo, Brazil, an endemic area for human and canine visceral leishmaniasis, were tested by indirect ELISA using different antigens: crude (CAG-ELISA), fucose-mannose ligand (FML-ELISA) and K39 (rK39-ELISA). Anti-Leishmania spp. antibodies were detected in 23.0% of samples evaluated by CAG-ELISA, 13.3% by FML-ELISA and 15.9% by RK39-ELISA. Only reactive sera in all three tests were considered truly positive. No disagreement occurred among the tests (p<0.05). Serum samples seropositive for toxoplasmosis tested by CAG-ELISA were negative, but one sample (6.7%) was positive for FML-ELISA and rK39-ELISA suggesting a cross-reaction between these antigens and anti-T. gondii antibodies. These findings indicate the occurrence of feline leishmaniasis in Ara?atuba. Further studies are required to clarify the role of cats in the epidemiological cycle of leishmaniasis.     

Infection by Leishmania infantum in cats: epidemiological study in Spain

More than 40 cases of feline leishmaniasis have been reported in the scientific literature. The influence of some immunodepressive conditions of viral origin, such as leukemia and feline immunodeficiency, are still unknown. The purpose of this study was to assess the prevalence of Leishmania infection in cats and possible relations with these viral infections. Markers of Leishmania infection were searched in 183 cats from Southern Spain by IFAT, PCR, Giemsa stain and culture, with a follow-up of positive cats. Seropositivity was 60.0% (Ab titer > or =10) and 28.3% of animals presented Ab titers > or =40. Around 25.7% of the cats studied were parasitemic and some of them remained positive for months. Combining both data, 70.6% of the feline population was, or could be, infected. We observed a negative association between seropositivity to Leishmania and infection by FeLV. Hence, production of antibodies against the parasite appears to be compromised in cats with leukemia, which have a prevalence of 36% in our study. In contrast, we found no association with feline immunodeficiency. The results makes us doubt the value of conventional serological methods to detect active Leishmania infection in cats.     

Seroprevalence of Antibodies Against the Excreted Antigen Superoxide Dismutase by Trypanosoma Cruzi in Dogs From the Yucatan Peninsula (Mexico)

Numerous studies have shown the role of dogs as a reservoir for the American trypanosomiasis, as the bridge connecting sylvatic and peridomestic cycles. The objective of this study was to determine the prevalence of American trypanosomiasis in the dog population (630 sera) from seven localities in the Yucatan Peninsula (city of Mérida and the towns of Molas, Playa del Carmen, Akumal, Xcalacoop, Xcalac and Xahuachol). These data are key for developing control measures for the disease. The sera were analysed to detect antibodies against Trypanosoma cruzi, using Fe‐SOD excreted as the antigenic fraction by ELISA and Western blot as confirmation. The total prevalence found in the Yucatan Peninsula was some 14.76%, with 10.74% in the state of Yucatan (city of Mérida, towns of Molas and Xcalacoop) and 21.34% in the state of Quintana Roo (towns of Playa del Carmen, Akumal, Xcalac and Xahuachol). However, a more thorough epidemiological study of the dog population, both wild and urban, in the Yucatan Peninsula will be required to design a control strategy for these diseases, paying particular attention to the population affected and even broadening the study to other Mexican states as well as neighbouring countries. These results again confirm that iron‐superoxide dismutase excreted by T. cruzi constitutes a good source of antigen for serodiagnosis in epidemiological studies.     

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.     

Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs

Although visceral leishmaniasis is primarily transmitted by a biological invertebrate vector, transmission in the absence of the vector has been reported, including venereal transmission in humans. Considering the possibility of venereal transmission, we studied genital lesions in dogs naturally infected with visceral leishmaniasis and shedding of Leishmania sp. in the semen. Approximately 200 dogs were serologically tested for anti-Leishmania antibodies and divided into three groups: 1) serologically negative dogs (n = 20), 2) asymptomatic serologically positive dogs (n = 20), and 3) symptomatic serologically positive dogs (n = 20). Samples from both testes, all segments of both epididymes, prostate gland, glans penis, and prepuce were histologically evaluated and processed for immunodetection of Leishmania sp. Semen samples were obtained from 22 symptomatic serologically positive dogs and processed for detecting Leishmania DNA by polymerase chain reaction. A significantly higher frequency of inflammation was observed in the epididymes, glans penis, and prepuce of dogs with visceral leishmaniasis, which was associated with a high frequency of immunohistochemically positive tissues (up to 95% of tissues from symptomatic dogs were positive by immunohistochemistry). Leishmania DNA was detected in eight of 22 semen samples from symptomatic dogs. Together these findings indicate that genital lesions and shedding of Leishmania sp. (donovani complex) in the semen are associated with visceral leishmaniasis. Additional studies should address the possibility of venereal transmission of the disease in the dog.     

Feline leishmaniasis due to Leishmania (Leishmania) amazonensis in Mato Grosso do Sul State, Brazil

A case of leishmaniasis in a domestic cat (Felis domesticus) is described. The animal showed a single, nodular lesion on the nose and many nodules of different size on the ears and digital regions of all the paws. Diagnosis was made by microscopic detection of amastigotes in Giemsa-stained smears from the lesions. By monoclonal antibodies the aetiological agent was identified as Leishmania (Leishmania) amazonensis, one of the seven species implicated in human leishmaniasis in Brazil. The clinical signs in feline leishmaniasis are unspecific and similar to those observed in other diseases such as cryptococcosis and in sporotrichosis, commonly found in cats. Leishmaniasis should therefore, be added to the differential diagnosis by feline veterinary practitioners and adequate investigations should carried out for dermal leishmaniasis in the area where the feline infection is detected.     

Visceral leishmaniasis in a New York foxhound kennel

Although endemic throughout much of the world, autochthonous visceral leishmaniasis has been reported on only 3 previous occasions in North America. After diagnosis of visceral leishmaniasis in 4 foxhounds from a kennel in Dutchess County, New York (index kennel), serum and ethylenediamine-tetraacetic acid (EDTA)-anticoagulated blood were collected from the remaining 108 American or cross-bred foxhounds in the index kennel and from 30 Beagles and Basset Hounds that were periodically housed in the index kennel. Samples were analyzed for antibodies to or DNA of tickborne disease pathogens and Leishmania spp. Most dogs had antibodies to Rickettsia spp., Ehrlichia spp., Babesia spp., or some combination of these pathogens but not to Bartonella vinsonii (berkhoffi). However, DNA of rickettsial, ehrlichial, or babesial agents was detected in only 9 dogs. Visceral leishmaniasis was diagnosed in 46 of 112 (41%) foxhounds from the index kennel but was not diagnosed in any of the Beagles and Basset Hounds. A positive Leishmania status was defined by 1 or more of the following criteria: a Leishmania antibody titer > or = 1:64, positive Leishmania polymerase chain reaction (PCR), positive Leishmania culture, or identification of Leishmania amastigotes by cytology or histopathology. The species and zymodeme of Leishmania that infected the foxhounds was determined to be Leishmania infantum MON-1 by isoenzyme electrophoresis. Foxhounds that were > 18 months of age or that had traveled to the southeastern United States were more likely to be diagnosed with visceral leishmaniasis. Transmission of Leishmania spp. in kennel outbreaks may involve exposure to an insect vector, direct transmission, or vertical transmission.     

The first record in the Americas of an autochthonous case of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felix catus) from Cotia County, São Paulo State, Brazil

A case of feline cutaneous leishmaniasis is reported in a domestic cat (Felis catus) as an apparently natural infection in a non-endemic area. Amastigotes were seem in smears of a nodular lesion on the cat's nose. No parasite could be seen in cytological preparations of liver or spleen but DNA obtained from a sample of the spleen produced the expected fragment in a Leishmania specific rDNA based PCR assay. The PCR product, a 520 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania) infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported from this region. This case suggests that natural transmission of this disease is occurring in this area, and that cats could act as a reservoir of L. (L.) infantum chagasi.     

The susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis

Over the last few years, several cases of feline leishmaniasis (FL) with cutaneous and visceral forms have been reported around the world. Nonetheless, the real susceptibility of cats to infection with Leishmania spp. and the outcome of leishmaniasis in these animals are poorly understood. Experimental studies on feline models will contribute to the knowledge of natural FL. Thus, in order to determine the susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis, 13 stray cats were infected with 10(7) promastigotes by the intradermal route in the ear and nose simultaneously and followed up for 72 weeks. Soon after infection, the earliest indication of a lesion was a papule on the ear at 2 weeks post-infection (w.p.i.). The emergence of satellite papules around the primary lesion was observed about 4 w.p.i. Two weeks later these papules coalesced and formed a huge and irregular nodule. Thereafter, there was lesion dissemination to the external and marginal surface of the ipsilateral ear, and later to the contralateral ear. At 10 w.p.i., some nodules became ulcerated. Nose lesions presented a similar evolution. At both sites, the largest lesion sizes occurred at 10 w.p.i. and started to decrease 15 days later. Ear and nose nodules healed at 32 and 40 w.p.i., respectively. Specific L. braziliensis IgG antibody titers (optical density> or = 0.01 as positive result) were detected as early as 2 w.p.i. (0.09 +/- 0.02) in only three animals (23%), and all cats had positive titers at 20 w.p.i. (0.34 +/- 0.06). Only three animals (38%) continued to show positive serology at 72 w.p.i. (0.08 +/- 0.02). Up to that time, none of the cats had lesion recurrence. In a feline model of cutaneous leishmaniasis, it seems that there is no correlation between active lesions and positive serology. The implications of these data are discussed.     

Seroepidemiological survey on canine leishmaniasis among dogs from an urban area of Brazil

A cross-sectional seroepidemiological survey on canine leishmaniasis among pet dogs was carried out in an urban area in the State of Pernambuco (Brazil) where human cases of visceral leishmaniasis have sporadically been reported. Using an indirect fluorescent antibody test, anti-Leishmania antibodies were detected in 130 out of 322 dogs, confirming previous exposure to Leishmania parasites. The overall seroprevalence found was 40.3% (95% confidence interval: 34.9-45.9). Data analysis revealed that serological positivity was statistically associated with male (chi2=20.60, P-value=0.000) and juvenile dogs (chi2=4.24, P-value=0.039). Furthermore, it was observed that 85.3% of all seropositive dogs showed no clinical signs of leishmaniasis. The results showed a high seroprevalence of anti-Leishmania antibodies among dogs from an urban area of Pernambuco - with a large proportion of asymptomatic seropositive dogs - indicating that the prevalence of Leishmania infection in this area has been underestimated.     

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.     

American trypanosomiasis infection in fattening pigs from the south-east of Mexico

American Trypanosomiasis (AT) is an infectious parasitic disease produced by the protozoa Trypanosoma cruzi (T. cruzi). Infection is acquired by vectorial via but can also be transmitted congenitally, by ingestion of an infected host, by transfusion with contaminated blood or transplant of organs from an infected donor. Currently, AT is widely distributed from the South of the United States to South America. In Mexico, the presence of the parasite has been reported throughout the country where several reservoirs such as dogs, opossums, rats and cats have been identified. Yucatan is in the south-east of Mexico where AT is endemic and has been reported since 1940s. There is little information about the role of pigs as reservoirs of T. cruzi. The frequency of specific antibodies against T. cruzi was determined in fattening pigs from Yucatan, Mexico. After sampling in the 3 main areas of pig production in the state, IgG ELISA and Western blot were performed to identify seropositive cases. Association of farm size, farm area and production system with infected pigs was evaluated. From 273 sampled pigs, 5.4% (n = 15) positive cases were found. No association with evaluated factors and infected pigs was found. Pigs are also reservoirs of T. cruzi in the studied area. These findings are considered important to improve vectorial control in the area in order to avoid the parasite infection in animal populations destined for human consumption and avoid further transmission to humans.     

Feline leishmaniasis in Jerusalem: serological investigation

Visceral leishmaniasis caused by Leishmania infantum is an endemic zoonosis, present in the Mediterranean area and well recognized in Israel and Palestine for human and dog disease. A serological study using an ELISA technique was performed on 104 cats living in the Jerusalem area. Seroprevalence was 6.7% (7/104). Significant correlation between seropositive cat results and altitude > 2500 ft was observed (p = 0.02). This is the first serological survey of feline leishmaniasis (FL) in the Middle East. To prove cat involvement as a secondary host, more investigations are still needed. The study concludes that cat involvement in Leishmania host studies should not be ignored.     

Genetic diversity of human zoonotic leishmaniasis in Iberian Peninsula

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous.     

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.     

Seroprevalence of antibodies against Leishmania spp among dogs in the United States

OBJECTIVE: To determine seroprevalence of antibodies against Leishmania spp among dogs other than Foxhounds in the United States. DESIGN: Cross-sectional study. SAMPLE POPULATION: 957 serum samples from dogs throughout the United States submitted between January 2000 and August 2001 to the Diagnostic Center for Population and Animal Health at Michigan State University for serologic testing for tick-borne diseases. PROCEDURE: Samples were tested for antibodies against Leishmania spp with an immunofluorescent antibody (IFA) assay. Samples with positive results were submitted to the Centers for Disease Control and Prevention for confirmatory testing. RESULTS: Results of the IFA assay were negative for 939 of 957 samples. For 16 samples, titers were from 1:16 to 1:64, and titers in these dogs were considered likely to be a result of cross-reactivity with antibodies directed against other organisms. For the remaining 2 samples, the titers were > or = 1:128. One of these samples was from a blood donor dog that had never had any clinical signs of leishmaniasis. Follow-up samples from both dogs also had Leishmania IFA titers > or = 1:128. Both dogs had antibodies against Trypanosoma cruzi, as determined with a radioimmunoprecipitation assay. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the seroprevalence of antibodies against Leishmania spp in dogs in the United States was low. However, results further suggested that leishmaniasis may not be limited to Foxhounds in the United States.     

Canine visceral leishmaniasis in São José de Ribamar, Maranhão State, Brazil

Here, we describe the situation of canine visceral leishmaniasis in two villages of S?o José de Ribamar in Maranh?o State/Brazil, where human cases have been registered. Blood samples of 36 household crossbred dogs from Sergio Tamer village and 43 dogs from Quinta village were collected and the serum used for serological diagnosis. An Indirect Fluorescent Antibody Test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were used to detect antibodies against Leishmania. The clinical examination showed that 25% of the canine population of Quinta presented a poor body condition and in 39%, ectoparasites (ticks and fleas) were detected. In both tests, serology revealed that 21% (9 out of 43) of the dogs presented antibodies against Leishmania (55% were asymptomatic and 45% were symptomatic). In the Vila Sérgio Tamer, 25% (9 out of 36) of the dogs were seropositive for Leishmania (66.67% were asymptomatic and 33.33% were symptomatic), 33% presented poor body condition, and 22% have ectoparasites. The clinical signs more frequent were skin lesions. The statistical analysis showed that there was no statistical difference (p>0.05) between the seropositivity of the dogs from the two villages. The same was observed when the clinical signs were compared (p>0.05). Both villages have favorable conditions to maintain the cycle of leishmaniasis.     

Endemic Visceral Leishmaniasis in a Dog from Texas

Visceral leishmaniasis was diagnosed by cytology and positive indirect immunofluorescent antibody titers to Leishmania donovani in a 7-month-old female Basenji dog from Texas. Clinical and laboratory findings included weight loss, hematochezia, hyperglobulinemia, hypoalbuminemia, anemia, and neutrophilic leukocytosis. Evidence of response to treatment with diminazene aceturate and ketoconazole included improvement in the abnormal clinical, hematologic, and biochemical findings, decreased serum globulin concentration and antibody titer to Leishmania donovani, and absence of organisms in examined tissues. Several foci of endemic leishmaniasis have been reported in the United States. Because of its zoonotic potential and the lack of approved treatments for dogs with leishmaniasis in the United States, the development of effective treatment strategies is needed.     

Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model

For experimental infections with viscerotropic strains of Leishmania, a suitable animal model is not yet defined. In the present work, we have reappraised the use of golden hamster (Mesocricetus auratus) as an experimental model for infection with Leishmania infantum. Groups of hamsters were challenged by the intracardial route with doses ranging from 10(3) to 10(5) infectious promastigotes and the animals were monitored for 1-year follow-up period. The outcome of the infection was assessed by clinical symptoms of leishmaniasis, parasite loads in both liver and spleen, humoral response to Leishmania antigens and antibody levels in kidneys. The humoral response was analysed using either crude antigens (by ELISA and Western blotting) or several recombinant Leishmania antigens (Hsp70, Hsp83, LiP2a, LiP2b, H2A, H3 and KMP-11). From the analysis of all these parameters, we established the existence of three groups of animals: symptomatic or susceptible, oligosymptomatic, and resistant. Given the parallelism existing between the outcomes of Leishmania-infection in hamsters, dogs and humans, we believe that our data illustrate that the hamster is an excellent experimental model to study visceral leishmaniasis and for the design of vaccine development.     

A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva

In this study, we compare the development of infection and/or disease in Beagle dogs intradermally infected with Leishmania chagasi, in the presence or absence of Lutzomyia longipalpis saliva, with those of intravenously infected animals.Spleen samples of all the animals inoculated with parasites had positive polymerase chain reaction tests for Leishmania DNA. Positive spleen cultures for Leishmania were detected earlier (P < or = 0.018) and were more frequent (five out of the five animals) in intravenously infected animals than in the intradermally infected animals, in presence (two out of the six animals) or absence (three out of the five animals) of salivary gland lysate of L. longipalpis. Significant increase in serum antibodies against Leishmania was observed only in the intravenously infected group (P = 0.004). In addition, dogs with infection confirmed by isolation of amastigotes or detection of parasite DNA were, nevertheless, negative for anti-Leishmania antibodies up to 5 months or more after infection. Only animals of the intravenously infected group developed progressive decreases in hematocrit (Pearson r = -0.8076, P = -0.0026) and hemoglobin (Pearson r = -0.8403, P = 0.0012) during the infection period. No significant difference in the course of infection was observed between groups of intradermally infected animals. The data presented herein confirms that the intradermal inoculation of dogs with Leishmania produces an asymptomatic form of infection. It also fails to show an advantage in using L. longipalpis saliva as an infection-enhancing agent in experimental canine leishmaniasis.