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.     

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.     

Risk of infection with Leishmania spp. in the canine population in the Netherlands

The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania-endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey. The questionnaire was sent to 1478 at random chosen families in the Netherlands. Of the 59.0% responders 28.0% had one or more dogs and 4.8% of these dogs had visited Southern Europe during the summer period of that year. On a total population of 1,200,000 dogs in the Netherlands, this means that each year some 58,000 dogs are at risk of being exposed to a Leishmania infection in Southern Europe. During the period 1990-1992 blood was collected for serology in 1911 dogs presented to the Utrecht University Clinic because of clinical problems not related to leishmaniasis, of which 434 had been in Southern Europe in the foregoing years. None was serologically positive. From these data it can be deduced that the highest chance to obtain leishmaniasis during a vacation in Southern Europe is mathematically less than 1/434 or less than 0.23%. Serology was also performed during the period 1989-1993 in 597 dogs that had been in Southern Europe and were suspected of leishmaniasis. Titers were positive in 145 of these samples. Sixty-four of these dogs were born in the Mediterranean and had been imported into the Netherlands. Excluding these imported dogs, it was calculated that at least 0.027% of the 58,000 dogs yearly taken to Southern Europe during holidays become infected with Leishmania. In order to establish the risk of disease transmission for people in close contact with an infected dog, serum samples of owners and house mates of 37 dogs with leishmaniasis were tested. All 112 sera tested negative. It was concluded that the risk to get leishmaniasis was between 0.027% and 0.23% for the dog when taken to Southern Europe during vacation, and that the risk for owners in non-endemic areas to get leishmaniasis from an infected dog is minimal.     

Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.     

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 fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals?

In order to investigate the possible role of dog fleas in the transmission of trypanosomatids, ectoparasites were removed from 59 dogs testing positive for canine zoonotic visceral leishmaniasis according to the indirect fluorescent antibody test (IFAT). Of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive polymerase chain reaction (PCR) amplification assays for Leishmania DNA. Fleas (409) from 9 Leishmania chagasi-infected dogs, each hosting more than 20 fleas per animal, were macerated and administered by peritoneal injection or orally to 36 hamsters. After 6 months, the 30 surviving hamsters were sacrificed and liver and spleen fragments were removed for PCA assay and to produce imprint smears, whilst blood samples were subjected to IFAT assay. Sixteen hamsters tested positive for Leishmania infection, 14 on the basis of PCR amplification and four by IFAT assay (two animals testing positive in both assays). Of the infected hamsters, 11/16 (68.7%) had been infected peritoneally and 5/16 (31.2%) orally. The imprint smears for all animals were, however, negative. Since both PCR and IFAT could present cross-reactivity for Leishmania and Leptomonas, the possibility of oral transmission of L. chagasi by fleas cannot be proven unambiguously even though the hamsters developed infection.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis

The aim of this study was to detect Leishmania infantum DNA by real-time PCR in urine from different groups of dogs with clinical leishmaniosis. Urine from 10 clinically healthy dogs and 43 dogs with clinical leishmaniosis diagnosed by positive serology and/or bone marrow PCR were studied. The group of 43 dogs with clinical leishmaniosis was divided into three subgroups: 13 dogs with renal insufficiency and proteinuria (urine protein-creatinine ratio greater than one), 13 dogs with only proteinuria, and 17 dogs with neither renal insufficiency nor proteinuria. The detection of Leishmania DNA was performed by light cycler real-time PCR using hybridization probes in each urine sample. Leishmania positive PCR was found in 47% (20/43) of the urine from leishmaniotic dogs, while all urine from clinically healthy dogs were negative. The percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency and proteinuria, 23% (3/13) in dogs with proteinuria and 35% (6/17) in dogs with neither renal insufficiency nor proteinuria. Dogs with renal insufficiency and proteinuria presented a statistical significant greater percentage of positive Leishmania PCR in urine when compared with the other subgroups (P<0.02). This study demonstrates the presence of Leishmania DNA in urine of dogs with leishmaniosis. Those dogs with severe renal damage present a greater number of Leishmania parasites in urine.     

Canine visceral leishmaniasis in urban and rural areas of Northeast Brazil

The purpose of this study was to determine the clinical and laboratory profiles of canine leishmaniasis in two distinct areas. Dogs from urban and rural areas were examined. The population studied in the metropolitan area included 54 dogs. Of these, 20 (37%) animals did not present with any signs suggestive of visceral leishmaniasis (VL). Among these, only eight were confirmed negative by ELISA (rK39 and CE) and 12 dogs, clinically negative for leishmaniasis, were seropositive by ELISA (rK39 and CE). Thinness, conjunctivitis and onychogryphosis were the most frequent clinical signs in the urban areas, followed by crusty lesions, alopecia, ulcerated lesions, hyperkeratosis and exfoliation. In the metropolitan area human VL cases occurred mainly in 1991, 1992, 1999 and 2000. In the rural areas the ELISA rK39 test detected a seroprevalence of 11.3% and ELISA CE (Leishmania crude extract) of 20.6%. Thirty-nine dogs were examined 6 months after the first visit. Serological exams using rK39 antigen showed seroconversion of only one dog, whereas Leishmania CE showed seroconversion of 13 (33.4%) dogs. In this rural environment 83.3% of the positive dogs were asymptomatic. Lutzomyia intermedia and Lu. longipalpis were the most predominant sandfly vector species. Amastigotes were identified in spleen and liver fragments of symptomatic necropsied animals. PCR amplification of DNA isolated from promastigote culture indicated that the species was Leishmania chagasi. This finding suggests that delayed diagnosis and euthanasia of potentially infectious animals may occur with an increased transmission risk to sandflies and subsequently to humans.     

Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia

Data on Leishmania spp. infection in dogs in Bolivia is scarce. Dogs from an area where 90% of human cutaneous leishmaniasis (CL) cases are due to Leishmania (Viannia) braziliensis were screened for Leishmania infection using established enzyme-linked immunosorbent antibody test (ELISA) protocols. Although none of the 51 dogs surveyed had clinical lesions indicative of CL, 6 out of 51 (11.8%) sampled dogs tested positive by ELISA.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

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.     

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.     

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.     

Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples

Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis.In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use.The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis.The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre > or =200), and 19% by microscopic examination of lymph node aspirates.     

Initial and long-term efficacy of a lipid emulsion of amphotericin B desoxycholate in the management of canine leishmaniasis

Sixteen dogs in which canine leishmaniasis (CL) was diagnosed by positive identification of Leishmania amastigotes in bone marrow samples were treated with a mixture of amphotericin B (AmB) desoxycholate in soybean oil. To prevent the toxicity of AmB, dogs were pretreated with saline (50 mL/kg) and mannitol (2 g/kg). Dogs were treated twice weekly with an increasing dosage of amphotericin (0.8-2.5 mg/kg) for between 8 and 10 sessions. Transient adverse effects (anorexia, vomiting, or both) appeared in 81% of the dogs during therapy. At the end of the course, all dogs were clinically cured, with no parasites observed in bone marrow smears. Six of the 16 dogs (38%) were positive by polymerase chain reaction (PCR) in bone marrow samples at some stage of their follow-up, but only 2 were positive at the first test after treatment, which was performed within 5 months after the end of the therapy. The other 4 dogs were initially negative and became PCR-positive at subsequent examinations. Three of these 6 dogs also experienced a clinical relapse. Four dogs had at least 3 consecutive negative PCR tests during a minimum period of 18 months and were clinically cured. The results of the present study indicate that despite having a high initial effectiveness in the treatment of CL, relapses can occur with the described protocol. Also, a single negative PCR result in a recently treated dog cannot be interpreted as a complete cure.     

Canine leishmaniasis chemotherapy: dog's clinical condition and risk of Leishmania transmission

The aim of this study was to investigate whether treatment against canine leishmaniasis reduced the presence of Leishmania in the healthy skin of dogs, affecting the capacity of parasite transmission. A total of 37 dogs from an endemic region of leishmaniasis were studied. Thirteen symptomatic animals revealed parasites in the bone marrow and eight had also in the skin. Five of the 22 dogs that had been treated with meglumine antimoniate alone, meglumine antimoniate or trifluralin followed by allopurinol or just with allopurinol had the parasite in bone marrow but none showed Leishmania in the skin. One dog that was treated only with aminosidine was polisymptomatic and had parasites in bone marrow and skin. The different treatments used in this study did not completely eliminate the parasite allowing relapses to occur when the treatment is discontinued, but the use of meglumine antimoniate or allopurinol, alone or combined may improve dogs clinical condition and reduce or eliminate the parasite from the skin decreasing the probability of Leishmania transmission.     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.     

Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies

Visceral leishmaniasis (VL) is a growing zoonosis with an increasing number of new cases and a rapid geographical spreading of the disease. In the present study, a canine survey was carried out in the city of Montes Claros (320,000 inhabitants), an endemic area of American visceral leishmaniasis in the state of Minas Gerais, Brazil. A total number of 4795 dogs were examined by serology, which showed a rate of seropositivity of 5%. Isoenzymatic analysis confirmed Leishmania infantum chagasi as the local aetiological agent of CVL. Canine tissues were assayed for the presence of Leishmania parasite DNA using different techniques. The infectivity of asymptomatic, oligosymptomatic and symptomatic seropositive dogs was tested by xenodiagnosis using laboratory reared Lutzomyia longipalpis. Rates of infection of 5.4%, 5.1% and 28.4% were found for the phlebotomine sand flies that fed in asymptomatic, oligosymptomatic and symptomatic dogs, respectively. Our results indicate that, under experimental conditions, symptomatic dogs are about four times more infective to VL vectors than oligosymptomatic or asymptomatic animals. The lower infectivity rates of dogs displaying any of the last two clinical forms of leishmaniasis, however, must be taken into account in the epidemiology of CVL.