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.     

Osteolytic osteomyelitis associated with visceral leishmaniasis in a dog

A dog was examined with a history of weight loss and lameness of the left hind limb. A painful response to examination of the left hip joint, and lymphadenopathy were noted. Amastigote forms of Leishmania sp. were observed by cytology in samples from the popliteus lymph node, and anti-Leishmania sp. antibodies at a titer of 1:640 were detected in serum by indirect immunofluorescence. Radiological changes included osteolysis and a periosteal proliferative reaction in the left femoral greater trochanter. These changes were histologically characterized as an osteolytic granulomatous osteomyelitis associated with amastigotes within macrophages. Non-decalcified fragments of the periosteum were processed for immunohistochemistry, observed with prominent immunolabelling of amastigotes of Leishmania sp. within macrophages. The diagnosis was further confirmed by positive PCR for Leishmania sp., belonging to the Leishmania donovani complex.     

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.     

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.     

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Serum profile of IL-1β and IL-17 cytokines in patients with visceral leishmaniasis

Leishmania is an intracellular protozoan parasite, mainly infects macrophages of mammalian tissues. Inflammatory related cytokines have a crucial role in the pathogenesis of leishmaniasis. The aim of the present study was to evaluate serum concentrations of IL-1β and IL-17 in patients with active visceral leishmaniasis (VL) and control group. Serum concentrations of both IL-1β and IL-17 cytokines were assessed by ELISA in Leishmania infantum infected patients (n = 25) and healthy individuals (n = 25) from Meshkin-Shahr, northwest of Iran. Mean serum concentrations of IL-1β in the patients and control groups were 47.34 ± 23.82, and 20.49 ± 9.38, respectively, which was statistically significant (p < 0.001). Furthermore, mean IL-17 concentration in patients with VL (243.96 ± 73.46) was twice higher comparing to control group (106.38 ± 129.06) (p < 0.001). Several cytokines are involved in the regulation of immunity against VL. The present data has shown that, increased serum concentrations of IL-1β and IL-17 are present in the patients with VL. Further investigations are needed to enhance our knowledge about the regulatory role of these cytokines in leishmaniasis.     

PCR technique detection of Leishmania spp. but not Mycobacterium spp. in canine cutaneous 'sterile' pyogranuloma/granuloma syndrome

Cutaneous 'sterile' pyogranuloma/granuloma syndrome (SPGS) is an uncommon canine skin disorder of unknown aetiopathogenesis. Histopathological findings and failure to demonstrate an aetiologic agent are suggestive of this syndrome. Nevertheless, it has been hypothesized that SPGS may be related to an immune response against persistent endogenous or exogenous antigens. The presence of Leishmania and Mycobacterium organisms was investigated by polymerase chain reaction (PCR) techniques in 46 canine skin samples histopathologically diagnosed as SPGS. Concomitantly, an immunohistochemical technique for Leishmania detection was applied on the same samples and the results were compared with those from PCR. The PCR technique yielded positive results for Leishmania spp. in 21 out of 46 skin samples. The results of immunohistochemical techniques were identical to those obtained by PCR. The PCR technique gave negative results for Mycobacterium spp. in all the samples examined. These results suggest the importance of looking for Leishmania spp. in skin biopsies with histopathological findings consistent with the diagnosis of SPGS.     

Subclinical leishmaniasis associated with infertility and chronic prostatitis in a dog

A stud dog was presented for acquired infertility. Haematospermia and teratozoospermia were found on two ejaculates 2 weeks apart. A presumptive diagnosis of prostatitis was made follo-wing ultrasound examination. An ultrasound-guided needle core biopsy was performed under general anaesthesia, revealing a mild chronic macrophagic and plasma cell prostatitis with intracytoplasmic amastigotes consistent with Leishmania spp. infection. Presence of Leishmania infantum, Leishmania donovani or Leishmania chagasi was confirmed by polymerase chain reaction in seminal plasma. Serology and serum protein electrophoresis confirmed the diagnosis of a subclinical active systemic leishmaniasis. A meglumine antimoniate and allopurinol treatment was given which clearly improved within 3 months both general condition and the quality of sperm. To the authors' knowledge, this is the first reported case of a prostatitis secondary to a Leishmania spp. infection. Subclinical systemic leishmaniasis should be considered in the differential diagnosis of infertility in dogs suffering from semen alterations.     

Comparative pathogenicity of different Actinobacillus suis O/K serotypes.

The pathogenicity of Actinobacillus suis serotypes O1/K1 (strain SO4), O1/K2 (strain C84), and O2/K2 (strain H91-0380) was evaluated in specific-pathogen-free (SPF) piglets challenged by intraperitoneal inoculation with approximately 1 x 10(7) colony-forming units per mL. All 3 strains produced peritonitis, but differences were observed in the composite histopathologic scores (P = 0.001) and in their ability to spread (P = 0.008) at 7 h post challenge. The O2/K2 strain caused the most severe peritonitis and disseminated most widely to other tissues. Moderate lesions were seen with the O1/K2 strain while the O1/K1 strain caused mild lesions and remained largely localized to the peritoneum. In an attempt to explain the basis of observed differences, the serum sensitivity of 9 A. suis strains with different O and K types was assessed. Regardless of the O/K type, all of the isolates tested were serum resistant. Moreover, most A. suis isolates grew as well or better in complement-replete sera as they did in complement-depleted sera. These observations indicate that although 02 and K2 strains had a greater propensity to cause a disseminating septic inflammatory response in pigs, they were no more resistant to complement-mediated killing than O1 strains.     

Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.     

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

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

On the reliability of the magnesium serum value as an indicator of body magnesium status

The interrelationships between serum Mg and HCR, and between serum Mg and K were investigated in pigs fed on a low Mg diet. A positive dependence was found between serum Mg and HCR, and between serum Mg and K. It was concluded, that in Mg-deficient pigs, Mg ions originating from soft tissues or erythrocytes can cause an increase of serum Mg level. The serum Mg value, consequently, can not be considered as a reliable parameter indicating the body Mg status. Some difficulties in diagnosing the body Mg status and the effects of the spontaneous increase of serum Mg level on Mg deficiency symptoms were discussed.     

Polymerase chain reaction amplification for direct detection of chicken anemia virus DNA in tissues and sera.

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.     

Performance of commercially available serological diagnostic tests to detect Leishmania infantum infection on experimentally infected dogs

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of a widespread serious zoonotic disease that affects both humans and dogs. Prevalence and incidence of the canine infection are important parameters to determine the risk and the ways to control this reemergent zoonosis. Unfortunately, there is not a gold standard test for Leishmania infection. Our aim was to assess the operative validity of commercial tests used to detect antibodies to Leishmania in serum samples from experimental infections. Three ELISA tests (LEISCAN^® Leishmania ELISA Test, INGEZIM^® LEISHMANIA, and INGEZIM^® LEISHMANIA VET), three immunochromatographic tests (INGEZIM^® LEISHMACROM, SNAP^® Leishmania, and WITNESS^® Leishmania), and one IFAT were evaluated. LEISCAN^® Leishmania ELISA test achieved the highest sensitivity and accuracy (both 0.98). Specificity was 1 for all tests except for IFAT. All tests but IFAT obtained a positive predictive value of 1, while the maximum negative predictive value was achieved by LEISCAN^® Leishmania ELISA Test (0.93). The best positive likelihood ratio was obtained by INGEZIM^® LEISHMANIA VET (30.26), while the best negative likelihood ratio was obtained by LEISCAN^® Leishmania ELISA Test (0.02). The highest diagnostic odds ratio was achieved by LEISCAN^® Leishmania ELISA Test (729.00). The largest area under the ROC curve was obtained by LEISCAN^® Leishmania ELISA Test (0.981). Quantitative ELISA based tests performmed better than qualitative tests (“Rapid Tests”), and the test best suited to detect Leishmania in infected dogs and to provide clinically useful information was LEISCAN^® Leishmania ELISA Test. This and other results point also to the need of revising the status of IFAT as a gold standard for the diagnosis of leishmaniasis.     

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.     

Identification of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining

This paper describes the demonstration of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining. An indirect immunoperoxidase method was applied to the organs of 20 dogs in which leishmaniasis was previously diagnosed. Haemosiderin pigment was eliminated with 5 per cent oxalic acid. Amastigotes of L donovani appeared as dark brown stained bodies which contrasted with haematoxylin stained host cells. No positively stained amastigotes could be seen in any of the sections incubated with control serum. The organs which more frequently showed leishmanids were: skin (macrophages and fibroblasts), liver, spleen, lymph nodes and bone marrow. In a few cases amastigotes were seen in kidneys, gut, adrenal glands, eyes and testicles. This technique is simple to perform, gives consistent results and allows unequivocal histopathological diagnosis of canine leishmaniasis.     

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.