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

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

Paraoxonase activity as a tool for clinical monitoring of dogs treated for canine leishmaniasis

This study was designed to determine if the activity of paraoxonase (PON1), an antioxidant enzyme that works as a negative acute phase reactant, is a better predictor for the clinical recovery of leishmaniotic dogs receiving standard treatments compared with inflammatory markers such as C reactive protein (CRP) and electrophoretic fractions. For this purpose we tested 20 healthy dogs (controls) and 39 leishmaniotic dogs classified as sick (group A, n = 23) or severely sick (group B, n = 16) and tested at admission and after 3, 7, 14, 21, 28, 35 and 42 days.At admission, CRP and electrophoresis were altered in both groups, while PON1 activity was abnormal only in group B. There were no differences related to the outcome (mortality, complications or time of recovery). PON1 activity normalized in about 2 weeks in dogs that had abnormal values at admission and a final positive outcome; CRP normalized in 4–6 weeks and electrophoretic fractions were still altered after 6 weeks. The results show that, at admission, inflammatory markers did not predict the outcome of leishmaniasis. PON1 activity decreased only in some dogs with systemic inflammation but not in those with mild leishmaniasis: when decreased, PON1 normalized earlier than other markers in dogs that responded to treatment. This finding most likely depends on the rapid decrease in oxidative phenomena. PON1 activity should therefore be tested on admission: if low values are recorded, severe inflammation may be suspected and PON1 measurement may be repeated during treatment to early identify responsive dogs.     

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.     

Development of two immunochromatographic tests for the serodiagnosis of bovine babesiosis

In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.     

Detection of canine transitional cell carcinoma using a bladder tumor antigen urine dipstick test

Canine transitional cell carcinoma (TCC) carries a poor prognosis in part due to late disease detection. The measurement of specific tumor markers shed in the urine may aid in sensitive, early disease detection and therefore improved prognosis. A 1-year prospective clinical trial was designed to assess the efficacy, sensitivity and specificity of the first generation Bard BTA test to diagnose TCC in dogs. This test is a qualitative, rapid, latex agglutination, dipstick test run on voided urine, which measures a glycoprotein antigen complex associated with bladder cancer in human patients. Sixty-five dogs were entered in the study: 20 TCC confirmed patients, 19 healthy controls and 26 urologic controls with a variety of conditions including urinary tract infection, crystalluria and proteinuria. Overall test sensitivity was 90% and specificity was 78%. False positive test results were noted in the presence of significant glucosuria (4+), proteinuria (4+), and pyuria or hematuria (> 30-40 WBC or RBC per hpf). Urine parameters that had no effect on efficacy included collection method (cystocentesis or free catch), pH, specific gravity, crystalluria, bilirubinuria, bacteriuria and casts. These data indicated that the Bard BTA test was sensitive for the detection of the bladder tumor-associated antigen complex in canine TCC. As evaluated, this test may serve as a useful adjunct to diagnosis, especially when cytology or biopsy is questionable or impractical. Furthermore, because of the high sensitivity of the test, it may be a practical screening test to rule out TCC in geriatric patients or patients with clinical signs related to the lower urinary tract, particularly before pyuria and hematuria develop which may interfere with test results.     

Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs

A recombinant LipL 32 antigen-based dipstick ELISA was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95.9 per cent, 93.8 per cent and 94.8 per cent, respectively.     

Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).     

Evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis

The efficacy of specific immunochemotherapy against Leishmania infantum infection in dog was studied. The effects on transmission of the disease, as well as the cellular and humoral immune response were examined. The treated animals showed a significant reduction in the infection rates that were detected in Phlebotomus perniciosus females fed on the dog. The humoral immune response, assayed with an indirect immunofluorescence antibody test (IFAT), did not show significant variations under the influence of the therapy. The characterisation of the peripheral blood mononuclear cells (PBMC) using flow cytometry indicated a significant increase in the proportion of T lymphocytes, especially of CD4/TcR(alpha)(beta)(+) and CD4/CD45RA(+) cells, without showing evidence for modifications in the other leukocyte subsets. Cellular lymphoproliferation studies indicated a lack of a specific response to soluble leishmanial antigen (SLA), but the non-specific lymphoproliferative capacity assayed with phytohemagglutinin (PHA) was maintained.     

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province,South Africa

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved.     

Treatment of canine Old World visceral leishmaniasis: a systematic review

Canine visceral leishmaniasis is a systemic disease caused by Leishmania infantum. The aim of this systematic review was to identify and evaluate the evidence of efficacy of interventions for treatment or prevention of canine visceral leishmaniasis, and to propose recommendations for or against their use. Forty-seven articles describing clinical trials published between 1980 and 2004 fulfilled selection criteria. The evaluation of clinical trials provided good evidence for recommending the use of meglumine antimoniate at a minimum dosage of 100 mg kg(-1) daily for at least 3-4 weeks, combined with allopurinol in order to obtain a good clinical efficacy and a reduced relapse rate. The evaluation of the articles also provided fair evidence for recommending the use of pentamidine (4 mg kg(-1) twice weekly) and aminosidine (5 mg kg(-1) twice daily) for 3-4 weeks. There was insufficient evidence for recommending the use of allopurinol alone, amphotericin B, buparvaquone, ketoconazole, enrofloxacin, and the combinations of metronidazole with spiramicyn or metronidazole with enrofloxacin. Fair evidence against the use of aminosidine at high dosages (20-80 mg kg(-1) per day) was proposed due to its side effects. Evaluation of articles on repellent measures against sand fly vectors of leishmaniasis provided good evidence for recommending deltamethrin collars and fair evidence for recommending spot-on permethrin.     

Use of domperidone in the treatment of canine visceral leishmaniasis: a clinical trial

The aim of this study was to evaluate the effects of domperidone, a dopamine D2 receptor antagonist, in dogs naturally infected by Leishmania infantum. Ninety-eight dogs were treated with single-agent domperidone at 1mg/kg twice a day orally for 1 month. Clinical, serological, biochemical and immunological examinations were conducted for the following 12 months. Domperidone was effective in controlling and reducing clinical signs and antibody titre. Significant decreases in reciprocal serum antibodies were seen in 74.3% of the dogs with mild clinical signs and 40% of the dogs became seronegative. In dogs with several clinical signs and high antibody titres, clinical improvement occurred in 86% of animals and the reciprocal serum antibody titres decreased in 38% of these dogs. A significant increase was noted in the immune cellular status, as measured by the leishmanin skin test and a lymphocyte proliferation assay.     

Evaluation the Clinitek status automated dipstick analysis device for semiquantitative testing of canine urine

The aim of this prospective study was to evaluate the Clinitek status™ analyser using Multistix10SG™/Microalbustix™ dipsticks (all: Siemens Dx) for canine urine (n = 101) compared to reference methods: visual reading (Combur9 dipstick, Roche), refractometry, microscopy, and quantitative protein/creatinine analysis (Pentra400, AxonLab). The automated analyses were done twice and visual tests were performed by two examiners.An excellent to good concordance was demonstrated between the first/second analysis with the Multistix10SG and the Combur9 dipstick, respectively with Cohen’s κ-values ranging from 0.776 to 1.000. Agreement between both dipsticks was good for glucose (κ = 0.753), blood (κ = 0.793), protein (κ = 0.788), and moderate for bilirubin (κ = 0.431) and ketones (κ = 0.540). In 6/101 specimens, false positive ketone reactions were obtained with the Multistix10SG™. Multistix10SG™ could not be used for determination of pyuria or specific gravity. Semiquantitative/quantitative protein results correlated well (ρ = 0.90) and creatinine measurements moderately (ρ = 0.76). Due to automated data transmission to the laboratory information system, the Clinitek status™ is of advantage in veterinary laboratories/clinics.     

Assessment of serological tests for the diagnosis of canine visceral leishmaniasis

An immunoenzymatic assay (ELISA), an indirect immunofluorescence antibody test (IFAT) with different antigens (ELISA-Leishmania chagasi, ELISA-L. major-like, IFAT-L. chagasi and IFAT-L. major-like), and an immunochromatographic test were assessed for the diagnosis of canine visceral leishmaniasis (CVL). Serum samples from 144 dogs from an endemic area for visceral leishmaniasis in the municipality of Rio de Janeiro were tested. The sensitivities of the serological tests were 93%, 100%, 73%, 60% and 93%, with specificities of 87%, 92%, 77%, 96% and 92% for the ELISA-L. major-like, ELISA-L. chagasi, IFAT-L. major-like, IFAT-L. chagasi and the immuno chromatographic test, respectively. ELISA-L. chagasi was the best test for the diagnosis of CVL, but the immunochromatographic test could be a useful alternative as it offers simple and rapid diagnosis without the need for a specialized laboratory.     

Value of Western blotting in the clinical follow-up of canine leishmaniasis.

Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.     

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.     

鹿布鲁菌病免疫胶体金检测试纸条的研制

利用SUMO-BCSP31重组大肠杆菌表达并纯化了布鲁菌BCSP31重组蛋白,并利用该蛋白研制了布鲁菌免疫胶体金检测试纸条。以BCSP31蛋白作为检测抗原,通过间接法研制胶体金试纸条检测鹿血清抗体。该试纸条与大肠杆菌、沙门菌、小肠结肠炎耶尔森菌和绿脓杆菌无交叉反应;检测阳性血清最大稀释倍数为1∶640,证明试纸条具有良好的特异性和敏感性。临床上对420只鹿进行检测,比对试纸条与虎红平板凝集试验(RBT)检测结果:阳性符合率为100%,阴性符合率为96%,Kappa值为0.73,两种方法符合率较高;比对试纸条与cELISA检测结果:阳性符合率为81%,阴性符合率为100%,Kappa值为0.88,两种方法符合率高。该试纸条简便快捷,具有较高准确性,可用于鹿布鲁菌病的现场初筛检测。