Intestinal Parasitic Infections in People Referring to the Central Laboratory of Meshkin Shahr County, Ardabil Province, Iran.

Background: Intestinal parasitic infections are still a considerable global public health problem. We aimed to determine the frequency of intestinal parasitic infections among people referring to the central laboratory of Meshkin Shahr City, Ardabil Province, Iran. Methods: In this cross-sectional survey, 460 fecal samples were collected randomly from persons referred to the central laboratory of Meshkin Shahr City, from January to June 2022. The samples were examined by direct wet-mount, Trichrome and modified Ziehl-Neelsen staining, formalin ethyl acetate sedimentation, and agar plate culture. Results: The frequency of intestinal parasites was 15.7% (72 out of 460 cases), with some people with numerous intestinal parasites. The frequency of protozoan infections (13.9%) was higher than the helminthic infections (2.6%). Blastocystis spp. (8.1%) was the most prevalent detected intestinal protozoan. Entamoeba coli (5.7%), Dicrocoelium dendriticum (2.2%), Giardia lamblia (1.5%), Fasciola spp. (0.2%), and Hymenolepis nana (0.2%) were other detected parasites. Conclusion: In- spite of betterment of the health condition in Iran and reduction of parasitic infection, intestinal parasitic infections are still a considerable public health issue in some parts of Iran.

Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022.

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022. Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti-Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023. Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti-L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs (Canis familiaris) samples showed anti-L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence. Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant.

Live attenuated Leishmania infantum centrin deleted mutant (LiCen-/-) as a novel vaccine candidate: A field study on safety, immunogenicity, and efficacy against canine leishmaniasis.

This study was designed to evaluate the safety, immunogenicity, and efficacy of a single dose of L. infantum (LiCen-/-) live attenuated candidate vaccine against canine leishmaniasis (CanL). Eighteen healthy domestic dogs with no anti-Leishmania antibodies and negative leishmanin skin test (LST) were randomly inoculated intravenously with either L. infantum (LiCen-/-) vaccine candidate in 10 dogs or phosphate-buffered saline (PBS) in 8 dogs. The safety, immunogenicity, and efficacy rate of L. infantum (LiCen-/-) vaccine candidate against CanL were evaluated by different criteria, including clinical manifestations, injection-site lesion, hematology and biochemistry values, anti-Leishmania antibodies using direct agglutination test (DAT), delayed-type hypersensitivity (DTH) using LST, and CD4+ and CD8+ T-cells subsets, as well as by measuring interferon (IFN-γ), interleukin (IL-23), IL-17, and IL-10 cytokines. Spleen aspiration and detection of Leishmania parasite using parasitological examinations (microscopy and culture) were performed in both vaccinated and control groups. Two months after intervention, each dog was challenged intraperitoneally (IP) with wide type (WT) L. infantum. Two-month follow-up post vaccination showed no clinical signs and serious side effects associated with the vaccination. A significant increase was found in the expression of IL-17, CD4+, and CD8+ gene transcripts in PBMCs, as well as increased levels of Th1 cytokines, and reduction of Th2 cytokine. The efficacy of the vaccine candidate was calculated to be 42.85%. While the time window for assessing the vaccine's effectiveness was too limited to draw any real conclusions but the preliminary results showed a moderate efficacy rate due to inoculation a single dose of L. infantum (LiCen-/-) vaccine candidate. Further investigations with more sample sizes and multiple doses of the vaccine candidate using natural challenges in the endemic areas of CanL are recommended.

Molecular Characterization of Mesocestoides litteratus (Cestoda: Cyclophyllidea: Mesocestoididae) Tetrathyridium Isolated from Two Species of Rodents from Iran.

PURPOSE: Mesocestoides spp. are Cyclophyllidean tapeworms with zoonotic importance. The current study aimed to investigate the molecular characteristics of Mesocestoides larvae (tetrathyridium) isolated from the abdominal cavity of persion jird, Meriones persicus, and from the liver of grey hamster, Cricetulus migratorius, in Ardabil Province, northwest Iran. METHODS: Genomic DNA of the isolates of Mesocestoides tetrathyridium were extracted, and mitochondrial gene of cytochrome-c oxidase subunit1 (cox1) was amplified. Sequencing of PCR products were performed and phylogenic analysis was run using MEGA 6.0 software. RESULTS: Both isolates were identified as Mesocestoides litteratus, showing high identity with M. litteratus sequences available in GenBank. Also, they had 100% homology to each other. Intra-species variation within isolates of M. litteratus were 0-2.4%. The phylogenetic reconstruction based on the partial sequence of the cox1 gene showed that our sequences of M. litteratus were clustered with M. litteratus isolates from Slovakia, Netherlands, Germany and Italy. CONCLUSION: This is the first molecular description of M. litteratus from M. persicus and C. migratorius. Phylogenetic analysis illustrated that M. litteratus isolates of the current study had very high identities with the isolates of this species from other countries.

In Vitro Study on Four Types of Commercial Lectins on Leishmania infantum, L. major and L. tropica with Stage-Specific Binding and Leishmania Species Identification.

Background: We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. Methods: The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Results: Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 µg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Conclusion: Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

Indirect immunofluorescence assay using embryonated eggs of Toxocara in human toxocariasis diagnosis is unreliable due to autofluorescence nature.

Toxocariasis is caused by infection with the nematode species Toxocara canis and Toxocara cati. Serological methods using eggs, larvae and adult worms of Toxocara spp. as antigen have been used for the diagnosis of human toxocariasis. The current study aimed to evaluate indirect immunofluorescence assay (IFA) using embryonated eggs of Toxocara for diagnosis of human toxocariasis. A total of 58 sera including twenty sera from patients with toxocariasis, 20 from healthy persons and 18 from patients with other parasitic infections were collected and used for the study. The embryonated eggs of Toxocara were prepared as antigen. Indirect immunofluorescence assay was performed using the frozen section of uterus containing embryonated T. canis eggs and unembryonated T. cati eggs. All serum samples had a positive reaction using IFA. The eggs of Toxocara as antigen exposed to the serum samples of toxocariasis, other parasitic infections and healthy persons, followed by IFA gave a bright greenish-yellow fluorescence. A number of samples such as eggs of Toxocara, Toxascaris, Trichuris and strongyloides larvae, and adult worm of Ancylostoma exhibited the bright greenish-yellow autofluorescence under fluorescent microscope. IFA using cryocut of embryonated eggs of Toxocara cannot be used for the diagnosis of human toxocariasis due to the existence of autofluorescence of the unembryonated and embryonated eggs, the second stage larva and adult worms of Toxocara spp.

A Convenient and Sensitive kDNA-PCR for Screening of Leishmania infantum Latent Infection Among Blood Donors in a Highly Endemic Focus, Northwestern Iran.

BACKGROUND: Recent global evidences showed that asymptomatic blood donor carriers of Leishmania infection will appear as a threat for blood transfusions recipients in endemic areas. As yet, there is no appropriate diagnostic procedure for detecting infection of blood donors in blood banks. SUBJECTS AND METHODS: The present study was aimed to apply various current diagnostic tests among blood donors in an endemic area of visceral leishmaniasis (VL), Ardabil Province, northwestern Iran. Blood samples were gathered from 860 blood donors in endemic areas of the province between 2017 and 2018, at eight blood donation centers. These samples was assessed using microculture, serological (DAT and rK39-ICT) and molecular based (conventional kDNA-PCR and HRM-PCR) tests. RESULTS: Of 860 eligible donors, 24 (2.8%) were seropositive for VL by DAT, and 388 (45%) were positive by kDNA-PCR. Moreover, 19 (19/860) were positive for both of them. Out of 19 subjects, 5.3% (1/19) was positive by rK39-ICT, 10.5% (2/19), and 79% (15/19) were detected positive in microculture and HRM-PCR methods, respectively. Nineteen donors were followed up for 2 years, of which 16 (84.2%) had a serological conversion, and 4 (21%) were positive by kDNA-PCR. The sensitivity of kDNA-PCR, and HRM-PCR procedures in detecting Leishmania parasite was found to be 98.7%, and 79%, respectively. CONCLUSIONS: Our findings justify the use of kDNA-PCR as a convenient and sensitive tool for screening subjects with leishmanial latent infection in blood banks at least in endemic regions. In these areas, however, a PCR-based test should be used to validate Leishmania infection among seropositive donors.

SARS-CoV-2 in domestic cats (Felis catus) in the northwest of Iran: Evidence for SARS-CoV-2 circulating between human and cats.

This study aimed to investigate the prevalence of COVID-19 in domestic cats, focusing on the disease in the northwest of Iran and then showing the natural transmission of SARS-COV-2 circulating between domestic cats and humans. After receiving ethic codes from Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1399.303) and confirmed by the Center of Communicable Diseases Control (CDC) of Iran, 124 domestic cats were collected from the homes and only one hospital of Meshkin -Shahr district from northwestern Iran where SARS-CoV-2 patients were hospitalized and quarantined during 2020. Samples were prepared from fluid materials of oropharynx and nasopharynx. All samples were tested by real-time PCR (RT-PCR) using specific genes N and ORF1ab in Pasteur Institute of Iran, and then partial sequence analyses of S gene were performed. All collected cats were kept in separated cages until SARS-COV-2 infection was confirmed with the RT-PCR. RT- PCR Ct values of 123 collected cats were ≥40; thus, all of them showed negative results, but one of the collected cats with close contact with its owner, whom confirmed SARS-CoV-2 showed positive results with gene N(Ct=30) and gene ORF1ab (Ct=32). Furthermore, the positive pet cat showed respiratory and gastro-intestinal clinical manifestations, and its owner was infected with SARS-CoV-2 two weeks ago. Cats are susceptible animals to SARS-CoV-2 infection. Epidemiological evidence showed that SARS-COV-2 is able to transmit to healthy cats due to having close contact with its owner as a reverse zoonosis.

Leishmania spp. infection in Rhombomys opimus and Meriones libycus as main reservoirs of zoonotic cutaneous leishmaniasis in central parts of Iran: Progress and implications in health policy.

Rodents from the subfamily Gerbillinae are the principal reservoir of cutaneous leishmaniasis (CL) caused by Leishmania major in the center and northeast of Iran. This study was conducted to determine both naturally occurring Leishmania infection rates and the distribution of Leishmania species in the central parts of Iran during 2019-2020. In this regard, presence of Leishmania parasites were confirmed by microscopic examination and the species were identified by nested-PCR using the Internal Transcribed Spacer2- Ribosomal DNA (ITS2-rDNA). Finally, some samples were sequenced and, the blast analysis of L. major samples, showed a 92.45-100% homology to the L. major sequence. Of the 181 wild gerbils collected (Rhombomys opimus=157 and Meriones lybicus=24), 88 (48.6%) tested positive for Leishmania sp. by microscopic examination whereas 162 (89.5%) were positive by nested-PCR. Of the 162 infected gerbils, 103 showed single strain infections (30 L. major, 28 L. gerbilli and 45 L. turanica), 43 showed dual infections with only the non-human species (L. gerbilli and L. turanica), and 16 were mixed infections of L. major and L. turanica (n = 14) or L. gerbilli (n = 2). All single or mixed L. major infections were detected in gerbils from areas with reports of human CL during the last decade. These findings suggest that Rhombomys opimus and Meriones libycus have a potential role in the maintenance of human and non-human transmission of Leishmania species in the CL foci.

Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mechanism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and preserved in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infantum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran.

BACKGROUND & OBJECTIVES: Visceral leishmaniasis (VL),a protozoan disease caused by Leishmania infantum is a major public health problem and cause of death among infants aged under 1 year and the elderly in endemic foci of Iran. The aim of this study is to determine the status of L.infantum infection in stray dogs from Meshkin-Shahr, a typical endemic area of VL in Iran. METHODS: Sixty-eight randomly trapped stray dogs in Meshkin-Shahr area were tested for L. infantum infection using the direct agglutination test (DAT) from June to October 2016. The confirmation of seropositive samples was performed by Microscopic slides of spleen, culture and then PCR. The molecular methods performed by ITS1-PCR, RFLP-PCR and kDNA-PCR. The allof kDNA -PCR products were sequenced. RESULTS: Out of 68 examined stray dogs, 17 (25.0%) were positive for L. infantum by DAT (1:320 titers or higher). Parasite test showed that all of seropositive samples have amastigote forms in their spleens but only 3 out of them could be cultured. The kDNA-PCR confirmed all of seropositive samples but ITS1-PCR and RFLP-PCR only confirmed 3 out of 17 (17.6%) seropositive samples. The sequenced products showed 94% homology with L. infantum. INTERPRETATION & CONCLUSION: The results showed a high prevalence of L. infantum infection in dogs in an endemic area of CVL and it provided key information for designing control programs against canine and human leishmaniasis.

Molecular characterization of Ribosomal DNA (ITS2) of hard ticks in Iran: understanding the conspecificity of Dermacentor marginatus and D. niveus.

OBJECTIVES: Hard ticks (Acari: Ixodidae) are ectoparasites of medical and veterinary importance. They are obligate blood-feeding vectors with the ability to transmit a wide variety of pathogens. Standard morphological keys are normally used for the identification of tick species. However, considering the importance of accurate species identification and the determination of bio-ecological characteristics of species, relying on morphological keys alone can be questionable. In this study, two DNA fragments (ITS2 and COI) were selected for phylogenetic evaluation of Iranian hard tick species belonging to the genera Dermacentor, Hyalomma, and Rhipicephalus. RESULTS: 1229 specimens of Dermacentor marginatus, D. niveus, Hyalomma anatolicum, Rhipicephalus bursa, and R. sanguineus s.l constituting 11 populations were collected from three different climatic and zoogeographical zones in Iran. Morphological studies revealed notable differences in important morphological characteristics between different populations of D. marginatus. The results of ITS2 sequence analysis provided additional evidence which supports the conspecificity of D. niveus and D. marginatus. Contrary to this finding, the sequence analysis of COI and phylogeny favored the separation of the two species. Given the greater importance of COI in identifying and discriminating species, a possibility heterospecificity between the two species should be considered.

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis in Owned Dogs (Canis familiaris) in New Foci of Rural Areas of Alborz Province, Central Part of Iran: A Cross-Sectional Study in 2017.

BACKGROUND: Mediterranean form of visceral leishmaniasis (VL) is endemic among some provinces of Iran. The present study was designed to determine the prevalence of canine visceral leishmaniasis (CVL) in the owned dogs of the rural areas of Alborz Province near Tehran as the capital of Iran. METHODS: This study conducted on 303 owned dogs that selected using a stratified random sampling method. The direct agglutination test (DAT) was used to determine the frequency of Vl. The spleen biopsy was taken from the serology-positive dogs for the confirmation of CVL in the suspected dogs. Nested PCR and sequencing methods were used to determine the type of Leishmania species in the dogs which were parasitological positive. RESULTS: Overall, the DAT results of 9 dogs (2.97%, CI: 1.57-5.55) showed anti Leishmania antibodies at titers ≥ 1:320 indicating VL infection. One dog (0.33%, CI 95%: 0.06-1.85) showed clinical signs and symptoms of VL. There was a significant correlation between the positive cases of CVL and rural area (p< 0.001). The Leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs. Leishmania infantum were confirmed in all them using nested-PCR assay. The sequence analysis of all five isolates was 95% similar to L. infantum. CONCLUSION: This study shows that domestic cycle of L. infantum has been established in rural areas of Alborz province where located near Tehran as capital city of Iran. It is necessary to increase the awareness and monitoring of the disease periodically.

Serological Survey of Neospora caninum and Toxoplasma gondii Co-Infection in Rodents in Northwestern Iran.

BACKGROUND: Our knowledge of the epidemiology of rodents' parasitic agents in Iran is scarce, although some of these pathogens play an important role in human and veterinary medicine, such as Toxoplasma gondii and Neospora caninum. The purpose of this study was to determine the seroprevalence of Toxoplasma gondii and Neospora caninum in rodents of northwestern Iran between Mar and Dec 2015. METHODS: Overall, 157 serum samples from rodents (101 Meriones persicus, 41 Mus musculus, and 15 Cricetulus migratorius) were assayed by the indirect fluorescence antibody test (IFAT) for antibodies to T. gondii and N. caninum. RESULTS: We found a prevalence of 20.38% (32/157) for N. caninum, 35% (55/157) for T. gondii. Co-presence of antibodies to N. caninum and T. gondii was found in 10 (6.36%) rodents. A significant association was found between the rodents species and seropositivity to N. caninum (P<0.05) but there was no association with rodents species for T. gondii. The overall prevalence of the aforementioned parasites was higher in male versus female rodents. CONCLUSION: The high seroprevalence of toxoplasmosis and neosporosis in rodents in the study area has implications for translocation of these infections across wider geographical regions since these rodents are mostly preyed on by cats or dogs; hence, which can transfer the parasite to other hosts.

Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA.

BACKGROUND: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. METHODS: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013-2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. RESULTS: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0-0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0-0.2% within E. multilocularis isolates for cox1 and nad1, respectively. CONCLUSIONS: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher.

Spironucleus muris and Eperythrozoon coccoides in Rodents from Northwestern Iran: Rare Infections.

BACKGROUND: Rodents perform a crucial role in dispersal of zoonosis causes globally. We aimed to investigation about infection levels of parasitic agents in rodents' population in Meshkinshahr areas, northwest of Iran from Apr to Sep 2014. METHODS: Two hundred four rodents were trapped and anaesthetized. A sample of blood was collected via cardiopuncture from each one. Thin and thick blood smears were prepared and stained with Giemsa. All stained smear were examined under light microscopy with high magnification by two expert microscopists. Every suspected unicellular observed were measured microscopically and compared with key references to diagnose. RESULTS: Captured rodents were identified as three genera including Meriones persicus, Mus musculus, Cricetulus migraturius. Protozoa identified in this study were included of Spironucleus muris and Eperythrozoon coccoides, these parasites were observed in blood smear of 0.98% of rodents. S. muris and E. coccoides were seen in M. musculus and C. migraturius, respectively. CONCLUSION: The present study increases awareness about Eperythrozoonosis in rodents and its potential transmission to domestic animals and even to human in rural districts in Iran. Moreover, the attack of Spironucleus on the mucus of colon and its systemic risk was confirmed.

Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK (rDgK) antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test. Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned into pET-24a(+) expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni²+-charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays (ELISA) with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test. Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement (0.764) with molecular identification. Conclusion: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test.

Wild Rodent Ectoparasites Collected from Northwestern Iran.

BACKGROUND: Rodents play an important role as reservoir of some pathogens, and the host of some ectoparasites as well. These ectoparasites can transmit rodents' pathogens to human or animals. The aim of this study was to assess the distribution and infestation load of ectoparasites on rodents in Meshkin-Shahr District, northwestern Iran. METHODS: Rodents were captured using baited live traps in spring 2014 from Meshkin-Shahr District and were transferred to the laboratory for identification to the species level. Their ectoparasites were collected, mounted and identified. RESULTS: Three rodent species including Meriones persicus (74%), Mus musculus (16.9%) and Cricetulus migratorius (9%) were identified. Among all rodents, 185 specimens (90.69%) were infested with a total of 521 ectoparasites. Overall, 10 arthropods species were collected, including fleas (97.6%), one mite (1.6%) and one louse species (0.6%) as follows: Xenopsylla nubica, X. astia, X. buxtoni, X. cheopis, Nosopsyllus fasciatus, N. iranus, Ctenocephalides felis, Ctenophthalmus rettigismiti, Ornithonyssus sp and one species of genus Polyplax. The most prevalent ectoparasites species was X. nubica (89%). CONCLUSION: Nearly all rodent species were infested with Xenopsylla species. Monitoring of ectoparasites on infested rodents is very important for awareness and early warning towards control of arthropod-borne diseases.

Feline leishmaniosis due to Leishmania infantum in Northwest Iran: The role of cats in endemic areas of visceral leishmaniosis.

Visceral leishmaniosis caused by Leishmania infantum (L. infantum) is an important zoonotic disease endemic in the Mediterranean region. Domestic dogs and other wild canines are the main reservoir hosts for the parasite, while domestic cats (Felis catus) may be carriers of L. infantum, and play a role as secondary reservoirs for the parasite. In the present study, serological (DAT), parasitological (microscopic smears and culture) and molecular methods (nested PCR) were used to evaluate infection with L. infantum in 103 stray cats collected from villages of Meshkin Shahr district, located in Ardabil province which is a well-known endemic region of human and canine visceral leishmaniosis in Iran. Overall, 25 out of 103 cats (24.27%) displayed anti-Leishmania antibodies with different titers. Amastigote forms of the parasite were detected in microscopic smears of the spleen of a cat with high anti-Leishmania antibodies using DAT. L. infantum was identified on microscopic slides by nested PCR, and the results were confirmed by sequence analysis. Based on the high rate of seropositive cats in this study, we conclude that cats may have an important role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran.

Transmission of Leishmania infantum by Rhipicephalus sanguineus (Acari: Ixodidae) in Dogs.

BACKGROUND: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs. METHODS: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests. RESULTS: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative. CONCLUSION: L. infantum-positive R. sanguineus ticks were unable to transfer L. infantum from infected dogs to healthy ones. The detection of L. infantum DNA in ticks collected from naturally infected dogs by semi-nested PCR does not prove their vectorial competence.