Intestinal Protozoa in Domestic Cats (Carnivora: Felidae, Felis catus) in Northwestern Iran: A Cross-Sectional Study with Prevalent of Microsporidian and Coccidian Parasites.

BACKGROUND: In this study, some microsporidial and coccidian parasites were isolated from 103 domestic cats in the Meshkin Shahr area, northwestern Iran during the Jun 2014 to Jun 2015, and their genera were identified using parasitological methods with emphasis on their zoonotic importance. METHODS: One hundred and three fecal samples of domestic cats were collected and preserved in formalin (10%) and conserved in phosphate buffer saline solution, finally examined by microscopy after formalin-ether concentration and specific staining. Preservation in dichromate potassium (2.5%) was performed for all coccidian positive samples and then sporulated coccidian oocysts were investigated. RESULTS: The detected parasites were Isospora spp. 6/103(5.8%). Microsporidian spores were identified in 46/103 (44.6%) of all samples post-stained by the aniline blue staining method. CONCLUSION: Microsporidial infections were more prevalent in domestic cats. Further studies are needed in the identification of microsporidial spores isolated from infected cats.

Detection of Pseudocyst Forms of Trichomonas muris in Rodents from Iran.

BACKGROUND: Trichomonas muris is one of the most common protozoa diagnosed in rodents. The trichomonads are generally described as presenting only trophozoite form while pseudocyst is another morphological form of trichomonads identified among gastrointestinal and genitourinary trichomonads. We identified and described different shapes of T. muris pseudocysts and trophozoite in stool samples were collected from rodents including Merinos persicus, Mus musculus and Cricetulus migratorius. METHODS: In this cross-sectional study, stool samples from 204 trapped rodents were collected from Meshkin Shahr during Mar to Dec 2014. Samples were preserved in formalin 10% and PVA solution and transferred to Department of Medical Protozoology and Mycology, School of Public Health, Tehran University of Medical Sciences. Formalin-ether concentration method was used for the samples. The slides were stained with tri-chrome staining method and observed under light microscope. RESULTS: The trophozoites were classified as T. muris based on size (18 to 24 µm), presence of three anterior flagella, recurrent flagellum, undulating membrane, and axostyle in direct examination and stained slides with trichrome staining method. Fifty-five out of 204 (27%) rodents were infected with T. muris in which 51(25%) samples pseudocysts form were observed. The spherical bodies of pseudocyst with almost 8 µm size, contained internalized flagella, an undulating membrane with recurrent flagellum, axostyle, and costa were seen. The pseudocysts were more prevalent than trophozoite form and pseudocysts were found with different shapes in this study. CONCLUSION: T. muris pseudocysts were found in stool samples of caught rodents for the first time in northwestern Iran.

Seroprevalence of Visceral Leishmaniasis in Children up to 12 Years old Among Nomadic Tribes from Rural Areas of Pars Abad, Northwestern Iran: an Observational Study in 2015.

BACKGROUND: Since Pars Abad district had been known as a focus of visceral leishmaniasis (VL) in Ardabil Province but the prevalence of the disease in nomadic tribes has not been determined, thus, this study was conducted. METHODS: This descriptive cross-sectional study was conducted on children up to 12yr old of nomadic tribes from Pars Abad County, Ardabil Province, Iran in 2015. For each individual, a questionnaire including age, sex, clinical manifestations, history of disease, and contact with reservoir hosts of VL were completed, separately. To determine VL seroprevalence, blood samples were collected from the children and after centrifugation, the plasma samples were tested using Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. Statistical analyses were performed using SPSS16. RESULTS: From 776 children up to 12yr old, 2 (0.25%) showed anti-L. infantum antibodies at titers 1:1600 and only one case (0.13%) showed anti-Leishmania antibodies at titers 1:3200. The child with anti-L. infantum antibodies titers of 1:3200 showed mild fever for more than 2 months period, paleness, weakness and mild splenomegaly. After physical examination and confirmation of VL (kala-azar), the patient was treated with antileishmanial drugs. CONCLUSION: The findings indicated that L. infantum infection is being circulated with low prevalence in nomadic tribes of Pars Abad but it is necessary that the surveillance system is regularly monitored among physicians and public health managers in the studied areas.

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

BACKGROUND: Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius. METHODS: This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. RESULTS: About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. CONCLUSION: Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.

Canine Visceral Leishmaniasis in Wild Canines (Fox, Jackal, and Wolf) in Northeastern Iran Using Parasitological, Serological, and Molecular Methods.

BACKGROUND: Although many studies had been conducted on various aspects of canine visceral leishmaniasis (CVL) in domestic dogs in the endemic areas of Iran, investigations on CVL in wild canines are rare. METHODS: This is a cross-sectional study was conducted from December 2012 to 2013 in northeast of Iran where human VL is endemic. Wild canines were trapped around the areas where human VL cases had been previously identified. Wild canines were collected and examined both clinically and serologically using direct agglutination test (DAT). Microscopically examinations were performed in all the seropositive wild canines for the presence of the amastigote form of Leishmania spp. Some Leishmania sp. which had been isolated from the spleens of wild canines, were examined analyzed by conventional PCR and sequencing techniques using α-tubulin and GAPDH genes. RESULTS: Altogether, 84 wild canines including foxes (Vulpes vulpes, n=21), Jackals (Canis aureus, n=60) and wolves (Canis lupus, n=3) were collected. Four foxes and seven jackals showed anti-Leishmania infantum antibodies with titers of 1:320-1:20480 in DAT. Furthermore, one fox and one jackal were parasitologically (microscopy and culture) positive and L. infantum was confirmed by sequence analysis. CONCLUSION: The present study showed that sylvatic cycle of L. infantum had been established in the studied endemic areas of VL in northeastern Iran.

Helminth Infections of Meriones persicus (Persian Jird), Mus musculus (House Mice) and Cricetulus migratorius (Grey Hamster): A Cross-Sectional Study in Meshkin-Shahr District, Northwest Iran.

BACKGROUND: Rodents have important role as reservoirs of different parasites. The aim of this study was to determine helminth parasites of abundant rodents in Meshkin-Shahr, Ardabil Province northwest Iran. METHODS: From April 2014 to March 2015; 205 rodents including 118 Meriones persicus, 63 Mus musculus and 24 Cricetulus migratorius were collected, using live traps. All rodents were dissected and their different tissues examined for infectivity with helminth parasites. RESULTS: Overall, 74.2% of rodents were infected with helminth parasites. The rate of infectivity in M. persicus, M. musculus and C. migratorius was 82.2%, 61.9%, 66.7%, respectively. In general, among all 205 rodents, the species and infection rates of helminthes were as follows: Nematoda: Trichuris sp. (46.8%), Capillaria hepatica (18.1%), Syphacia frederici (14.2%), Aspicularis tetraptera (3.4%), Trichuris rhombomidis (2%), Heligmosomom sp. (2%), Streptopharagus kuntzi (0.5%), Spiruridae gen. sp. (0.5%); Cestoda: Hymenolepis nana fraterna (16.6%) Hymenolepis diminuta (7.3%) tetratiridium of Mesocestoides sp. (1%), Paranoplocephala sp. (0.5%), Cysticercus fasciolaris (0.5%), Taenia endothoracicus larva (0.5%), and Acanthocephala: Moniliformis moniliformis (18.5%). CONCLUSIONS: Variable species of helminthes circulate in the rodents of the study area. Presence of several zoonotic species highlights the potential risk of infections for public health.

Age and sex distribution of Dirofilaria immitis among dogs in Meshkin-Shahr, northwest Iran and molecular analysis of the isolates based on COX1 gene.

Dirofilaria immitis is an important filarial nematode in dogs. In this study, age and sex distribution of this zoonotic nematode among dogs were investigated in northwest of Iran in Meshkin-Shahr city. Molecular characteristics of the isolates, based on cytochrome oxidase subunit 1 (COX1) gene were compared to the isolates from other areas of the world.Blood samples were collected from 91 dogs which were selected by simple classified accidental sampling. Thin and thick blood smear examinations were used to find out infectivity with D. immitis. DNA extraction was performed from adult D. immitis recovered from heart of infected dogs. The COX1 gene was amplified and sequenced. Phylogenetic analysis was carried out using sequences obtained in this study along with relevant sequences deposited in the GenBank. Phylogenetic analysis and sequence variation was performed using MEGA software in comparison with those COX1 sequences deposited in GenBank. Out of 91 dogs, 19 (20.87%) were found positive for infection with D. immitis. There was no statistically significant difference between males and females of dogs in terms of D. immitis infection. However, the rate of infection in dogs more than 2 years old was significantly higher than those with lower age. Both sequences analyzed in this study showed 100% homology to each other. Intra-species variation of these isolates with those from other areas of the world amounted to 0 to 0.50%. Phylogenetic analysis of the COX1 gene suggested that it is conserved, and can be used for study on genetic diversity and classification of filarial nematodes.

Canine visceral leishmaniasis: seroprevalence survey of asymptomatic dogs in an endemic area of northwestern Iran.

Canine visceral leishmaniasis is a major public health problem that is endemic in tropical and sub tropical countries and is fatal in humans and dogs. In addition to symptomatic dogs, asymptomatic ones seem as source of Leishmania infantum infection. Thus surveillance and control programs of reservoir hosts are essential. This study aimed to evaluate the sero-prevalence of visceral leishmaniasis in asymptomatic domestic dogs from in an endemic area of north west, Iran. A cross sectional study was carried out in Meshkin-Shahr district during 2011-2012. Blood samples collected from 508 asymptomatic domestic dogs were tested by direct agglutination test. In this study 508 dogs (397 males and 111 females, mean age, 3.24 years) from western and eastern parts of the Meshkin-Shahr were examined. A total of 508 dogs examined 119 dogs (23.4 %) had antibodies (titers of ≥1:320) against L. infantum. Statistically significance was occurred between male (25.4 %) and female (16.2) sero-prevalence (P = 0.042). No statistically significance was observed between age groups (P = 0.22). Compared with previous studies it seems to increase sero-prevalence of visceral leishmaniasis in dogs in the studied areas caused by ecological changes. High proportion of asymptomatic but seropositive dogs emphasizes the importance of dogs without clinical signs in the epidemiology of zoonotic leishmaniasis. Thus, the necessity of using serological tests in asymptomatic dogs is recommended for disease control strategy.

Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran.

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

Cutaneous and post kala-azar dermal leishmaniasis caused by Leishmania infantum in endemic areas of visceral leishmaniasis, northwestern Iran 2002-2011: a case series.

Visceral leishmaniasis (VL) is endemic in Northwest and southern Iran. Reports of cutaneous leishmaniasis (CL) in Northwest areas are rare, and its etiological agents are unknown. In the current study, we report six CL and two post kala-azar dermal leishmaniasis (PKDL) cases caused by Leishmania infantum from endemic areas of VL in the Northwest. Smears were made from skin lesions of 30 suspected patients in 2002-2011, and CL was determined by microscopy or culture. Leishmania spp. were identified by nested-PCR assay. The disease was confirmed in 20 out of 30 (66%) suspected patients by parasitological examinations. L. infantum was identified in eight and Leishmania major in 12 CL cases by nested-PCR. Cutaneous leishmaniasis patients infected with L. major had the history of travel to CL endemic areas. L. infantum antibodies were detected by direct agglutination test (DAT) at titers of 1:3200 in two cases with history of VL. Results of this study indicated that L. infantum is a causative agent of CL as well as PKDL in the VL endemic areas.

First report on natural infection of the Phlebotomus tobbi by Leishmania infantum in northwestern Iran.

Visceral leishmaniasis (VL) is an important health problem in Ardebil, where it borders Azerbaijan in the northwestern Iran. In spite of the presence of both cutaneous and visceral leishmaniasis (CL and VL) in northwestern Iran, previous researches have consistently revealed the etiologic agent of VL in the region to be Leishmania infantum. This is the first report of natural infection of Phlebotomus tobbi with L. infantum in Bilesavar district in the northern part of Ardebil province bordering Azerbaijan. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses revealed presence of L. infantum in six out of 433 tested female sand fly specimens. Although sand flies of P. tobbi were infrequent, two out of 32 (6.25%) females captured in the area were found infected with the parasite. Phlebotomus perfiliewi transcaucasicus, the known vector of VL in the area, were the most dominant species but only four out of 273 (1.47%) tested were infected with L. infantum. This study showed that P. tobbi similar to P. perfiliewi transcaucasicus could play a significant role in the transmission of the L. infantum. However more investigations are needed to demonstrate that L. infantum is the only species circulating in the focus.

Ecological study and risk mapping of visceral leishmaniasis in an endemic area of Iran based on a geographical information systems approach.

Between 1998 and 2001, a total of 1,062 human cases of visceral leishmaniasis were reported from the rural district of Meshkin-Shahr in the mountainous, north-western Iranian province of Ardabil. In the summer of 2008, a cross-sectional study of dogs was conducted in this endemic area by randomly selecting 384 animals from 21 villages and testing them serologically for leishmaniasis. Villages, in which more than 10% of investigated dogs showed anti- Leishmania titres ≥ 1/320, were considered to be high-risk environments. Regression analysis showed no statistically significant correlation between topographic conditions and the prevalence of positive cases. However, when the results were compared with past meteorological records, a statistically significant positive correlation (P = 0.007) was found between the number of infected dogs with anti-Leishmania titres ≥ 1/640 and the number of days in a year with temperatures below 0 °C. While humidity showed an inverse correlation (P = 0.009) with the anti-Leishmania titres, a positive correlation (P <0.001) was found in relation to the amount of rainfall. Mapping of the areas at risk for kala-azar in the Meshkin-Shahr district supports the impression that the low temperatures prevalent in the Ardebil province constitute an important factor influencing the distribution of leishmaniasis there.

Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infantum in northwest Iran.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general.