Live attenuated Leishmania major p27 gene knockout as a novel vaccine candidate: A study on safety, protective immunity and efficacy against canine leishmaniasis caused by Leishmania infantum.

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27-/-) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27-/- candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27-/- vaccinated group showed no clinical signs after inoculation with Lmp27-/- mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27-/- parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum.

Seroprevalence of Toxoplasma gondii infection in domestic dogs in an area from northwest of Iran: a cross-sectional study using immunodominant surface antigen 1 (SAG1).

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects humans and animals. T. gondii surface antigen 1 (SAG1) is an appropriate antigen with high specificity and sensitivity for the detection of T. gondii infection in humans and animal hosts. The aim of this study was to determine the seroprevalence of T. gondii infection using SAG1 antigen (P30) in ownership dogs in Meshkin-Shahr district in the northwestern Iran. The sera samples were collected from 171 domestic dogs and tested using indirect ELISA (SAG1 antigen). The data were analyzed using SPSS software version 13. From a total of 171 dogs, 82 (48 %) of them were sero-positive. No statistical significant difference was seen between T. gondii infection and gender (P = 0.995). The highest sero-prevalence of rate was observed in >5 years animals; but no statistical significant difference was seen between T. gondii infection and age (P = 0.589). Our findings indicate that Toxoplasma seropositivity rate is high in ownership dogs in northwest of Iran. This is probably due to high exposure to contaminated food, soil, or water sources with sporulated Toxoplasma oocysts.

Emergence of a new focus of visceral leishmaniasis due to Leishmania infantum in Golestan Province, north-eastern of Iran.

Over the last decade, a few cases of visceral leishmaniasis (VL) have been reported in some districts of the province of Golestan, in north-eastern Iran. The aim of the present study was to investigate the prevalence of Leishmania infantum infection among humans and domestic dogs by using direct agglutination test (DAT) and PCR assays in the eastern zone of the province. Between 2011 and 2012, blood samples were randomly collected from 450 humans and 50 domestic dogs, in the eastern zone of Golestan Province including 7 villages from Marave-tappeh district where new cases of human VL had been recorded there. Each of these samples was tested for anti-Leishmania antibodies, in DAT, and for L. infantum kinetoplast DNA on whole blood, in PCR-based assays. A total of 450 human samples, 6 (1.33 %) were found seropositive and 13 (2.8 %) was found PCR-positive. Of the 50 dog samples, 16 (32 %) were found seropositive and 15 (30 %) were PCR-positive. All PCR-positive dogs were found seropositive except one as well as 6 (46.2 %) PCR-positive humans were also found seropositive. Moreover, the species of L. infantum was detected in all PCR-positive samples. The high prevalence of VL in the study areas offer it has emerged as an endemic focus in the province. Further investigations on the vectors, reservoirs and human population are recommended.

Seroepidemiologcal investigation of visceral leishmaniasis in stray and owned dogs in alborz province, central iran using direct agglutination test.

BACKGROUND: The aim of present study was to determine the seroprevalence of canine visceral leishmaniasis (CVL) among stray and owned dogs in Kouhsar district of Alborz Province, central Iran. METHODS: The study was performed from March 2011 to July 2011 using Direct Agglutination Test (DAT). Three hundred and thirty seven dogs including 257 stary and 80 owned dogs were selected by random sampling. The agreement between serological data and sex, age, life style of dogs and clinical signs were assessed by Chi-square. RESULTS: DAT showed that from 337 serum samples collected from owned and stray dogs, 12sera (3.6%) were positive. The seroprevalance was 10% (8/80) among owned dogs and 1.6% (4/257) among stray dogs. A significant difference in seroplevalance was seen between owned and stray dogs (P = 0.01). The highest seroprevalence rate (14%) was observed among the ownership dogs of 5 years old and above. Statistical analysis revealed significant relation between seroprelvalence and age (P= 0.02). There was no statistically significant relation between male (6.3%) and female (2.2%) seroprevalence (P= 0.085). CONCLUSION: This survey indicates the importance and necessity of serologic screening of visceral leishmaniasis in human and dogs in Kouhsar district.

Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection.

Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available.

Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples.

Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (P<0.05). The highest agreement was obtained between real-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively.

Human visceral leishmaniasis in kermanshah province, Western iran, during 2011-2012.

BACKGROUND: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. It is endemic in some parts of provinces of Iran. According to the reported cases of VL in Kermanshah Province in recent years, this study was conducted to determine the seroprevalence of VL in high risk villages of the province. METHODS: Totally, 1622 serum samples obtained from children under 15 years old and 178 from adults in 22 villages of studied areas. Serum samples were examined by direct agglutination test (DAT) for the detection of anti-Leishmania antibodies. Data were analyzed using SPSS software ver.11.5. RESULTS: Only 6 serum samples (0.33%) showed anti-Leishmania antibodies against L.infantum at titers ≥ 1/3200. Four of the seropositive cases had a history of kala-azar and Leishman bodies were seen in their bone marrows. The highest (0.5%) and lowest (0.29%) seroprevalence was seen in the age groups of 5-9 and 10-14 years old, respectively. None of the adults were seropositive. There were not any significant differences between the rate of seropositivity in males (0.36%) and females (0.31%). 66.7% of seropositive individuals showed clinical manifestations. The most important symptoms in Kala-azar patients were fever, hepato-spleenomegally and anemia. CONCLUSION: Kala-azar is occurred sporadically in Kermanshah Province. But presence of significant number of positive sera confirms the necessity for attention of people and clinicians to kala-azar.

Canine visceral leishmaniasis in boyer ahmad district, kohgiluyeh & boyer ahmad province, southwest of iran.

BACKGROUND: Mediterranean type of visceral leishmaniasis (VL) is present in different parts of Iran. Several studies have identified dogs as the main reservoirs of the VL caused by Leishmania infantum in Iran and other Mediterranean regions. This study aimed to determine the seroprevalence of canine visceral leishmaniasis as animal reservoir host for human visceral leishmaniasis in Boyer Ahmad district in southwest of Iran. METHODS: A seroepidemiological study was carried out to determine the seroprevalence of canine visceral leishmaniasis (CVL) among ownership dogs by using direct agglutination test (DAT) in 23 of 182 villages of Boyer Ahmad district, during August 2009 to August 2010. One hundred and seventy serum samples from ownership dogs were selected by multi-stage cluster sampling in villages of Boyer Ahmad district. All samples were tested by DAT and anti-Leishmania antibodies titers at ≥ 1:320 was considered as positive. RESULTS: Of the 170 serum samples, 10% were positive by DAT at titers of 1:320 and higher. No statistical significant difference was found between male (10.7%) and female (8.3%) seroprevalence. The highest seroprevalence rate (15.1%) was observed among the ownership dogs of four to seven years age. Altogether, seventeen (25.4%) of the seropositive dogs had clinical signs and symptoms. CONCLUSION: It seems that Boyer Ahmad district is an endemic area for canine visceral leishmaniasis in Iran.

Neospora caninum and Leishmania infantum Co-Infection in Domestic Dogs (Canis familiaris) in Meshkin-Shahr District, Northwestern Iran.

BACKGROUND: Mediterranean visceral leishmaniasis (MVL) is an infectious disease that affects both human and animals. Domestic dogs (Canis familiaris) are principal reservoir hosts of MVL caused by Leishmania infantum. Dogs are definitive hosts for Neospora caninum and a risk factor for infecting intermediate hosts. The immunosuppression caused by visceral leishmaniasis (VL) can promote the occurrence of co-infections with other agents such as neosporosis. This study aimed to determine the frequency of co-infection of the both protozoan parasites in the endemic areas of VL from Meshkin-Shahr District, north-west of Iran. METHODS: Altogether, 171 serum samples were collected from domestic dogs of Meshkin-Shahr District by multistage cluster sampling from October 2008 to August 2009. The collected serum samples were tested for the detection of simultaneous infection of L. infantum and N. caninum using direct agglutination test (DAT) and indirect ELISA, respectively. RESULTS: Of the 171 domestic dogs, 27 (15.8%) and 52 (30.4%) were showed antibodies against L. infantum and N. caninum, respectively. Simultaneous infections of N. caninum and L. infantum was found in 16 (9.4%) of the dogs. In VL-positive and VL-negative dogs, N. caninum infection was found in 59.3% and 25.0%, respectively. A statistically significant difference was found between VL-positive and VL-negative dogs with N. caninum infection (P= 0.001). CONCLUSION: These findings indicate that Meshkin-Shahr District in northwestern Iran is an active focus of canine visceral leishmaniasis (CVL). Neospora caninum and L. infantum co-infection is prevalent in the area and infection by L. infantum seems to enhance susceptibility to N. caninum infection in domestic dogs.

Epidemiological aspects of visceral leishmaniasis in baft district, kerman province, southeast of iran.

BACKGROUND: Visceral leishmaniasis (kala-azar) is an endemic disease in some areas of Iran. A cross- sectional study was conducted for sero-epidemiological survey of visceral leishmaniasis (VL) in Baft district from Kerman Province, southeast of Iran. METHODS: Blood samples were collected from children up to 12 years old and 10% of adult population from Baft villages with a multi-stage randomized cluster sampling. In addition, blood samples were collected from 30 domestic dogs from the same areas. All the collected blood samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in both human and dog using the cut-off value of ≥1:3200 and ≥1:320, respectively. Parasitological, molecular, and pathological were performed on infected dogs. Chi-square and Fisher exact tests were used to compare sero-prevalence values. RESULTS: From 1476 collected human serum samples, 23 (1.55%) showed anti-Leishmania antibodies at titers of 1:800 and 1:1600 whereas 14 (0.95%) showed anti-Leishmania infantum antibodies at titers of ≥1:3200. No statistically significant difference was found between male (1.18%) and female (0.69%) sero-prevalence (P=0.330). Children of 5-8 years showed the highest sero-prevalence rate (3.22%). Seven out of 30 domestic dogs (23%) showed anti-Leishmania antibodies at titers ≥1:320. Leishmania infantum was identified in five infected dogs by nested - PCR assay. CONCLUSION: It seems that visceral leishmaniasis is being endemic in southern villages of Baft district, southeast of Iran.

Disseminated Leishmaniasis Caused by Leishmania tropica in a Puppy from Karaj, Central Iran.

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT). DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis among Humans and Domestic Dogs in Mazandaran Province, North of Iran.

BACKGROUND: New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmaniainfantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province. METHODS: In this cross-sectional study, blood samples were randomly collected from 402 humans and forty-nine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species. RESULTS: None of 402 collected human (402) and dog (49) blood samples showed anti Leishmaniainfantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1%) were found PCR-positive corresponding to L.infantum. CONCLUSION: This study confirms the circulation of L. infantum at least among domestic dogs and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding to sand flies fauna and wild canines as reservoir hosts of the disease, are recommended.

Seroepidemiological study of visceral leishmaniasis in Booyerahmad district, south-west Islamic Republic of Iran.

Visceral leishmaniasis (VL) is endemic in parts of Islamic Republic of Iran. A cross-sectional seroprevalence study was carried out in children in Booyerahmad district in the south-west of the country. Serum samples were taken from 1628 children up to 10 years old from different areas in Booyerahmad in 2005-06. AntiLeishmania antibody was detected in 50 out of 1628 children (3.1%) by direct agglutination test (antibody titre > or = 1:3200). There was no significant difference in seropositivity between the sexes (2.8% males and 3.3% females). The highest rate of infection (5.2%) was in the age group 10 years. Further studies are needed to explore the reservoirs and vectors of the disease in this region.

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran.

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged < or = 12 years in the primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 before (1985-2000) to 0.77 per 1000 child population after the intervention (2001-07). In a control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests and timely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas.

Seroepidemiological study of visceral leishmaniasis in Booyerahmad district, south-west Islamic Republic of Iran

Visceral leishmaniasis [VL] is endemic in parts of Islamic Republic of Iran. A cross-sectional seroprevalence study was carried out in children in Booyerahmad district in the south-west of the country. Serum samples were taken from 1628 children up to 10 years old from different areas in Booyerahmad in 2005-06. Anti-Leishmania antibody was detected in 50 out of 1628 children [3.1%] by direct agglutination test [antibody titre > 1:3200]. There was no significant difference in seropositivity between the sexes [2.8% males and 3.3% females]. The highest rate of infection [5.2%] was in the age group 10 years. Further studies are needed to explore the reservoirs and vectors of the disease in this region

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged ≤ 12 years inthe primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic ofIran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referredfor physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88before (1985–2000) to 0.77 per 1000 child population after the intervention (2001–07). In a control area with nosurveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests andtimely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas

En 2001, un système de surveillance de la leishmaniose viscérale a été mis en place pour les enfants âgésde 0 à 12 ans dans le système de santé primaire du district de Meshkin-Shahr, province d’Ardebil, nord-ouest dela République islamique d’Iran. Tous les cas présentant des signes cliniques et des symptômes de leishmanioseviscérale ainsi qu’une réaction positive au test d’agglutination directe étaient orientés en vue d’un examen physiqueet d’un traitement. L’incidence annuelle moyenne de la leishmaniose viscérale a nettement diminué, passant de1,88 avant l’intervention (1985-2000) à 0,77 pour 1 000 enfants après l’intervention (2001-2006). Elle a augmentédans une zone témoin sans surveillance, passant de 0,11 à 0,23 pour 1 000 enfants. Un dépistage précoce dela leishmaniose viscérale à l’aide de tests sérologiques pratiques et une prise en charge rapide des cas permettraientde réduire les taux de mortalité et de morbidité de la leishmaniose viscérale dans les zones endémiques

Comparison of miltefosine and meglumine antimoniate for the treatment of zoonotic cutaneous leishmaniasis (ZCL) by a randomized clinical trial in Iran.

This study was a randomized, open label comparison that was designed to determine efficacy and safety of miltefosine as the first oral drug for the treatment of zoonotic cutaneous leishmaniasis caused by Leishmania major in comparison with meglumine antimoniate. Complete clinical response was defined as 100% re-epithelialization of the lesion. Definitions of lesion cure and failure were based on both clinical and parasitological criteria two weeks after the end of treatment and clinical recovery three months after this period. Of 32 patients enrolled for miltefosine treatment 28 patients completed treatment, of which 26 were cured at three months, corresponding to a cure rate of 92.9% on a per protocol analysis, and 81.3% according to intention to treat analysis. There was one failure (3.1%), one relapse (3.1%) and four dropouts due to lack of tolerability (12.5%) during the first week of treatment. Of 31 patients who received intramuscular meglumine antimoniate (20mgSb(5)/kg body weight daily for 14 days) 25 were cured (83.3% on a per protocol basis, 80.6% on intention to treat basis), five failed (16.1%) and one was lost (3.2%) at 3-month follow-up. At 6-month follow-up after the end of treatment, no relapse was observed. Both regimens were tolerated but averages of nausea (32.2%) and vomiting (21.5%) were observed in patients during two weeks after initiation of miltefosine treatment. Other gastrointestinal, musculoskeletal, and total adverse events were not statistically different in the two groups during one to four weeks after therapy initiation. No relevant changes were observed in levels of liver enzymes, creatinine and hematological tests before and after end of treatment in both groups. In conclusion, miltefosine is apparently at least as good as meglumine antimoniate for the treatment of cutaneous leishmaniasis caused by L. major in Iran, based on parasitological as well as clinical criteria two weeks, three months, and six months after end of treatment.