Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples

Background: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods: The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results: The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. Conclusion: This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China

Characterization of Isolated Hydatid Cyst from Slaughtered Livestock in Yasuj Industrial Slaughterhouse by PCR-RFLP

Background & aim: Given the existence of 10 different genotypes of the parasite Echinococcus granulosus from different hosts and intermediate and final impact of these genotypes in the life cycle of the parasite and its transmission to humans, the purpose of this study was to determine the molecular characterization of isolates of hydatid cysts in industrial slaughterhouses of Yasuj city. Methods: In this study, 93 animal isolates (56 goat, 31 sheep and 6 cattle) were collected from the industrial slaughterhouse of Yasuj city. The genomic DNA corresponding to protoscolices was extracted, using the standard Phenol–Chloroform method. The fragment of DNA-ITSI of each sample was assessed by PCR with designed primers of EgF, EgR and then amplified. Moreover, the PCR products were assessed by electrophoresis and digested by the Alu I and Rsa I enzymes. RFLP products were evaluated by electrophoresis. Results: Using a PCR test, rDNA-ITSI of all isolates of similar size bands and 1000 bp were obtained respectively.The patterns generated by RFLP using Alu I and Rsa I enzymes, showed Echinococcus granulosus G1 genotype in all isolates. Conclusion: This study showed that strain G1 is the predominant strain causing hydatid cysts in different organs of the animal in Yasuj

Role of environmental, climatic risk factors and livestock animals on the occurrence of cutaneous leishmaniasis in newly emerging focus in Iran

Introduction: Occurrence of leishmaniasis is affected by various biological and environmental factors. Kohgiluye and Boyerahmad (K–B) province is an emerging focus of cutaneous leishmaniasis (CL) in Southwest Iran. Subjects and methods: To elucidate some angles of occurrence of CL in this province, climatic and environmental factors and close proximity to livestock were studied by univariate and two multivariate logistic regression models. The dwelling addresses of 275 CL patients were obtained from Health Centers records for a 5 years period. The effect of mean annual temperature, minimum mean annual temperature, maximum mean annual temperature, mean annual rainfall, slope, elevation, land covers and close proximity to cattle and sheep/goat sheds on the occurrence of CL were analysed using geographical information systems (GIS) approach. Results: CL occurred in all counties with the most cases in Northwest, West and South semi-arid and warm regions. Land cover, slope, elevation and close proximity to cattle sheds, were the most effective factors. Urban, dry farm and thin rangeland were found as the most important land covers. Slope and elevation decreased the probability of disease. Close proximity to cattle sheds notably increased the chance of CL. The importance of other factors such as rainfall, temperature and close proximity to sheep/goat sheds only were shown when their effects were evaluated independently from other factors. So regions with urban, dry farm and thin rangeland covers with lower slope and altitude where in close proximity of cattle sheds seems to be potentially most high risk areas. Conclusion: Distribution of CL cases is influenced by combination of environmental, ecological factors and close proximity to livestock sheds but control programs should be focused on cities and villages in the above-mentioned most high risk regions. Keywords: Cutaneous leishmaniasis, GIS, Land cover, Slope, Elevatio

Comparison between Serology and PCR Methods for the Diagnosis of Visceral Leishmaniasis

ABSTRACT: Introduction & Objective: Canine visceral leishmaniasis (CVL) caused by Leishmania Infantum is endemic in most Mediterranean basin and its seroprevalence ranges from 10 to 37%. Diagnosis of Infection is very important especially in asymptomatic dogs for control of human leishmaniasis for control of human visceral leishmaniasis. This study was aimed to compare three methods for detection of canine visceral leishmaniosis. Materials & Methods: In this research process study, 71 dogs were selected from 4 endemic villages in Meshkin-Shahr district. Peripheral blood samples were tested by serologic (DAT and Dipstick rK39) and molecular (PCR) methods. Skin samples were tested by molecular (PCR) methods. Twelve samples of PCR products were sequenced that all of them were identified as Leishmania infantum and 2 nucleotide sequence data submitted to the GenBank database. Results: From 71 dogs that were studied, 21.1% were symptomatic and others were asymptomatic(78.9%). 17 dogs (23.9%) had ≥ 1:320 titer of antibody by direct agglutination test (DAT). Twenty two dogs(31%) were positive by Dipstick rK39 test, 21 dogs (29.6%) were positive by PCR on skin samples, 31 dogs (43.7%) were positive in blood PCR and 38 dogs (53.5%) were positive by skin/blood PCR. The highest correlation was between DAT and Dipstick test (76%). Conclusion: According to the results of this study, we can diagnose infection in symptomatic and asymptomatic dogs by DAT as a suitable method and PCR is suitable to follow parasite DNA in skin and other tissues of dogs. Keywords: Direct Agglutination Test(DAT), Dipstick rK39, PCR, Visceral Leishmaniosis, Do

Characterization of Visceral leishmaniasis in Reservoir Host (dogs) and Determination of Agent by PCR in Boyer-Ahmad District, Iran

Background & Aim: Visceral Leishmaniasis (VL) is an endemic disease in some parts of Iran. Leishmania infantum is the agent of disease in studied areas. The aim of the present study was the characterization of visceral leishmaniasis in reservoir host (dogs) and determination of agent by molecular method in Boyer-Ahmad district, Iran Methods: In this study 15 infected dogs with symptoms of canine visceral leishmaniasis were selected from 5 VL endemic villages of Boyer-Ahmad district in 2010. All cases were tested by DAT for evaluation of anti leishmanial antibodies. After necropsy, parasitological study was conducted by use of impression smear of liver and spleen. Nested PCR was use to detect the parasite DNA in the liver and spleen tissues. Results: From fifteen cases, fourteen dogs had antibody titer above of 1:320 while one of the cases was seronegative. Leishmania amastigotes was seen in 13 smears of liver and spleen (13 cases). The agent of disease in 14 dogs determined as Leishmania infantum by nested PCR. Conclusion: This study confirmed that Leishmania infantum is the causative agent of canine VL in Boyer-Ahmad and the diseases pattern is similar to the rest of country.

Seroepidemiological Study on Canine Visceral Leishmaniasis in Meshkin-Shahr District, Ardabil Province, Northwest of Iran during 2006-2007

"nBackground: This study aimed to determine the seroprevalence of canine visceral leishmaniasis in Meshkin-Shahr district as endemic areas of human visceral leishmaniasis (HVL) for presenting control program of HVL to health authorities. "nMethods: A seroepidemiological study to determine seroprevalence of canine visceral leishmaniasis (CVL) among owner­ship dogs using direct agglutination tests (DAT) in 21 villages of Meshkin-Shahr district, Ardabil Province was carried out from June 2006 to August 2007. Three hundred and eighty four ownership dogs were selected by multi-stage cluster sam­pling. Chi-square and Fisher exact tests were used to compare seroprevalence values relative to gender, age and clinical signs."nResults: Of the 384 serum samples tested by DAT, 17.4 %( 95%C.I, 13.2%-20.8%) were positive (1:320 and higher). No statistical significant difference was found between male (16.5%) and female (20.2%) seroprevalence (P=0.416). The high­est seroprevalence rate (64.2%) was observed among the ownership dogs of three years age and above. Only 25.4% of the seropositive dogs had clinical signs and symptoms. The most clinical signs among symptomatic dogs were cachexia (75%) and alopecia (36.5%)."nConclusion: The majority of seropositive dogs (74.6%) lived in endemic areas of Meshkin-Shahr district were asympto­matic. It seems that all symptomatic and asymptomatic infected dogs are the most important risk factors for human infec­tion in VL endemic areas