A zymographic study of metalloproteinase activities in whole cell extracts and extracellular secretions of Leishmania (L.) , L. major and L. infantum from Iran.

Leishmaniosis encompasses a group of diseases that is transmitted by sand flies and caused by different species of Leishmania. The skin is the initial organ to be infected by the Leishmania in cutaneous, mucocutaneous and visceral forms of leishmaniosis. The matrix metalloproteinases (MMPs) are capable of degrading all kinds of extracellular matrix (ECM) proteins. The aim of this study was to investigate the protease activity through zymography in cell extracts and extracellular secretions of L. major, L. tropica and L. infantum as three prevalent Leishmania spp. in Iran. The three Leishmania spp. were cultured in RPMI-1640 medium supplemented with fetal calf serum. Promastigotes and axenic amastigotes were harvested and lysed at various phases, and extracellular secretions and cell extracts were collected. Leishmania spp. were proved by targeting kDNA gene. Enzymes were characterized according to gelatin zymography and sensitivity to distinct proteinase inhibitors. We observed proteinase bands with molecular weights (MWs) between 66 to 180 kDa in cellular extracts of axenic amastigotes of L. infantum, L. tropica, and L. major, and from 66 to 92 kDa in extracellular secretions of L. infantum. No proteinase activities were observed in extracellular secretions of axenic amastigotes and in cellular extracts of promastigotes in logarithmic and stationary phases of L. major and L. tropica. Using specific inhibitors, we determined that these proteolytic activities are due to metalloproteases. Our study demonstrated that amastigotes of all three Leishmania spp. have distinct amounts of proteinase activities and therefore can cause various types of lesions and outcomes of the disease.

Molecular Genotyping of Toxoplasma gondii in Sheep Aborted Fetuses Reveals Predominance of Type I Infection in Southwest of Iran.

BACKGROUND: We aimed to detect Toxoplasma gondii in ovine aborted fetuses and evaluate its genetic variations in the southwest of Iran. METHODS: This cross-sectional study was performed on 100 aborted ovine fetuses collected from the different region of Kohgiluyeh and Boyer-Ahmad Province, Iran, in lambing season during 2017 and 2018. DNA was extracted from the brain samples of all of the aborted fetuses and PCR amplified, targeting a 529 bp repetitive element gene of T. gondii. Moreover, to find out the heterogeneity of the positive samples, PCR-DNA amplification of the two main genetic markers, B1 and GRA6, of T. gondii were performed. Nucleotide sequencing and phylogenetic analysis were performed, using the BLAST program and MEGA-X software. RESULTS: The 529 bp gene of T. gondii was detected in 2 out of 100 (2%) of the ovine aborted samples. The sequences analysis of GRA6 and B1 genes revealed that both isolates from the aborted fetuses of sheep belonged to type I of T. gondii. Intra-divergence was more seen in GRA6 gene whereas less divergence was observed in B1 gene. CONCLUSION: Congenital infection with Type I of T. gondii during the neonatal period is associated with abortion in ovine. Evaluation of more aborted samples from broader geographical areas is needed to elucidate the molecular epidemiology and also the genotypes of T. gondii associated with abortion.

First molecular subtyping and phylogeny of Blastocystis sp. isolated from domestic and synanthropic animals (dogs, cats and brown rats) in southern Iran.

BACKGROUND: Blastocystis sp. is a common intestinal protist that infects humans and many animals globally. Thus far, 22 subtypes (STs) have been identified in mammalian and avian hosts. Since various STs are common to humans and animals, it was suggested that some human infections might arise from zoonotic transmission. Therefore, the aim of this study was to assess the presence of Blastocystis sp. in domestic (dogs and cats) and synanthropic animals (rats) of Fars Province, Iran, and to genetically characterize the samples. METHODS: A total of 400 fresh faecal samples from 154 dogs, 119 cats, and 127 rats were inspected by direct microscopy, Wheatley's trichrome staining, in vitro culture, and 18S rRNA gene nested-PCR. Finally, sequencing and phylogenetic analyses were performed. RESULTS: Out of 400 samples, 47 (11.8%) and 61 (15.3%) samples were detected as positive by direct wet mount and culture, respectively. Molecular analysis detected a larger number of positive samples (n = 70, 17.5%): nested-PCR showed that 29 (18.8%) dogs, 21 (17.7%) cats, and 20 (15.8%) rats were infected by Blastocystis sp. Sequence analysis of positive samples indicated the presence of zoonotic STs in all investigated host species. Specifically, ST2 (allele 9), ST3 (allele 34), ST4 (allele 94), ST7 (allele 99), ST8 (allele 21), and ST10 (allele 152) were detected in dogs; ST1 (allele 2), ST3 (allele 34), ST4 (allele 94), ST10 (allele 152), and ST14 (allele 159) were detected in cats; and ST1 (allele 2), ST3 (allele 34), and ST4 (allele 92) were detected in rats. CONCLUSIONS: Our data suggest that domestic dogs and cats can serve as possible reservoirs for in-contact humans, especially those who handle shelter-resident and client-owned animals. Moreover, rats as synanthropic animals can function as a potential source of human infections. Conversely, humans can act as a source of infections to animals. These results should be reinforced in future molecular epidemiological studies.

Alarming: high prevalence of Leishmania infantum infection in cats from southern Iran based on molecular and serological methods.

Recently, Leishmania infantum has increasingly been detected in stray cats in endemic regions of the world. Cats have been considered playing a role in the epidemiology of visceral leishmaniosis, an endemic zoonosis in Iran. The studies concerning feline leishmaniosis (FeL) allow the hypothesis that cats can be considered as potential reservoirs. The investigations on Leishmania infection in cats are very few in Iran and therefore we aimed to assess the L. infantum infection in stray cats and its possible role in transmission of the disease to human by direct agglutination test (DAT), ELISA, nested-PCR and confirmation via sequencing and phylogenetic analysis in Fars province, Iran. Whole blood samples were obtained from 174 stray cats. Anti-Leishmania antibodies were detected in the sera using DAT and ELISA. DNA was extracted from the buffy coat of each subject and PCR amplified, targeting Leishmania kDNA gene. PCR results were confirmed by sequence analysis. Prevalence of clinical signs in positive cats was 19.0%. Anti-Leishmania antibodies with different titers were detected in 48 (27.59%) and leishmanial DNA in 36 (20.69%) of the cats. The sequencing of PCR-positive cats revealed the parasite as L. infantum. A high seroprevalence of L. infantum was revealed, with higher levels in males, adult cats, and those living in rural districts and southern zones. Despite the reservoir task of cats in nature is still ambiguous, the high serological and molecular detection of L. infantum in stray cats indicates that cats are regularly bitten by infected sand flies in Fars province, southern Iran, and may have a potential reservoir role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran. Anyway, Leishmania infection must be appraised in the differential diagnosis of cutaneous or systemic clinical signs in cats.

DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran.

BACKGROUND & OBJECTIVES: Leishmania major and L. tropica are the main pathogens of cutaneous leishmaniasis (CL) in several rural and some urban regions of Iran, respectively. The aim of this study was to detect Leishmania species, and update the distribution data of these species in humans suspected to CL in two endemic foci in southern Iran. METHODS: From March 2016 to March 2017, 276 positive samples from of 350 suspected cases were diagnosed and compared by different diagnostic methods, viz. microscopy, culture, and PCR. In PCR assay, four different gene identifications were performed including minicircle kDNA, and cysteine protease B genes for Leishmania detection, and glyceraldehyde-3-phosphate dehydrogenase, and internal transcribed spacer 1 genes for Crithidia detection. RESULTS: In total, 68% (235/350) and 65.3% (177/271) of patients suspected of leishmaniasis were positive by microscopy and cultivation methods. In PCR assay, L. major, and L. tropica were detected in 86.2% (238/276), and 13.1% (36/276) of CL cases, respectively. Also, dermal L. infantum strain was isolated from 0.7% (2/276) of post-kala-azar dermal leishmaniasis patients. In addition, Crithidia fasciculata was detected in two CL patients chronically infected with L. major. INTERPRETATION & CONCLUSION: It appears that the epidemiology of CL has changed during the last decades and can complicate the control strategy aspects of CL in southern Iran. Therefore, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province, Iran.

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon's and crow's samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.

Leishmania cytochrome b gene sequence polymorphisms in southern Iran: relationships with different cutaneous clinical manifestations.

BACKGROUND: Cutaneous leishmaniasis (CL) caused by Leishmania species, is a geographically extensive disease that infects humans and animals. CL is endemic in half of the 31 provinces of Iran, with 29,201 incidence cases reported in Fars province from 2010 to 2015. CL is polymorphic and may result in lesions characterized by different clinical features. Parasite genetic diversity is proposed to be one of the factors affecting the clinical outcome and lesion characteristics in CL patients. However, there is still very limited data regarding the genetic variation of Leishmania spp. based on the sequencing of Cytochrome b (Cyt b) gene. METHODS: All patients originated from endemic regions in Fars province. The amplification of the Cyt b gene from isolates of 100 patients with disparate clinical forms of CL was accomplished using Nested-PCR. Sequence analysis of the amplified Cyt b was used to scrutinize the genetic variations among Leishmania isolates and connect the results with clinical pictures. The clinical demonstrations were basically of two types, typical and atypical lesions. Molecular phylogenetic tree was constructed using the Neighbor-Joining method, with species/strains from this study compared to species/strains from other geographical regions. RESULTS: Leishmania major was identified as the predominant infecting Leishmania spp. (86% of cases), with the remainder of cases being infected by Leishmania tropica. Clinical examination of patients revealed 12 different clinical CL forms. Among Leishmania samples analyzed, five distinct haplotypes were recognized: three in L. major and two in L. tropica. We found a correlation between clinical outcomes and Cyt b sequence variation of Leishmania spp. involved. Moreover, we observed a higher presence of polymorphisms in L. major compared with L. tropica. This difference may be due to the different eco-epidemiologies of both species, with L. tropica being an anthroponosis compared to L. major, which is a zoonosis. CONCLUSIONS: The sequence analysis of Cyt b gene from 25 L. major and L. tropica strains demonstrated genetic variability of L. major and L. tropica causing CL in southern Iran, and a feasible connection amid the genetic heterogeneity of the parasite, geographical source and clinical appearance of the disease in human was detected.

Molecular and Serological Detection of Toxoplasma gondii in Stray Cats in Shiraz, South-central, Iran.

BACKGROUND: Toxoplasmosis is a global zoonotic disease that causes critical medical complications in neonates and immunocompromised persons. Infection rates in cats, specifically stray cats, are believed to be the best sentry of the level of Toxoplasma gondii in the environment. Therefore, in this study, we surveyed T. gondii infection in stray cats of Shiraz, one of the metropolises of Iran. METHODS: The appearance of antibodies and DNA of T. gondii in samples from 145 stray cats was determined in order to appraise the prevalence of T. gondii infection, by MAT and Nested-PCR. RESULTS: The rate of T. gondii infection in the cats was 69% by PCR and 82.8% by MAT. Besides, the highest rate of infection was discerned in diaphragm (37.9%) and intercostal muscle (34.5%), while the lowest rate was related to ileum (6.9%). Moreover, the similarity between MAT with titers 1:20, 1:40 and PCR were 79.2% and 86.2%, respectively (P=0.02 and P=0.0001). CONCLUSION: Nested-PCR and MAT are valuable techniques for molecular and serological detection of T. gondii. The prevalence of T. gondii infection in stray cats in Shiraz is high.

Co-detection and isolation of Leishmania and Crithidia among naturally infected Tatera indica (Rodentia: Muridae) in Fars province, southern Iran

Objective:To explore the co-detection of natural infection of Trypanosomatidae parasites such as Leishmania and Crithidia in reservoir hosts of leishmaniasis. Methods: Rodent populations were monitored in two endemic foci of cutaneous leishmaniasis of Fars province, southern Iran from March to October 2016. Rodents were trapped alive in several parts of Shiraz and Kharameh cities. Afterwards, their organs were prepared for detection of Leishmania and Crithidia species by molecular, microscopic, and culture methods. Results: Totally, 115 rodents of five species; Tatera indica (T. indica) (85), Rattus rattus (12), Meriones libycus (9), Mus musculus (7), and Rattus norvegicus (2), were trapped alive and their tissue samples were examined using microscopic, cultivation, and molecular assays. Overall, 59 (51.3％) rodents were positive for Leishmania or Crithidia parasites. The highest rate (61.2％; 52/85) of Leishmania infection was related to the T. indica population. The cultivation, and molecular observations showed that two (2.4％; 2/85) of T. indica (foot-pad, and spleen samples) were positive to Crithidia. Conclusions: This is the first report of Crithidia infection in T. indica in Iran. Consequently, more epidemiological and ecological studies are needed to understand the role of Crithidia and Leishmania in T. indica.

Lip leishmaniasis: a case series with molecular identification and literature review.

BACKGROUND: Mucocutaneous leishmaniasis (MCL), a protozoan infectious disease, is very rare in Iran despite the endemicity of both cutaneous and visceral forms. It is transmitted by the Phlebotomus sand fly. The lip is considered one of the extraordinary sites. Lesions usually initiate with erythematous papules, slowly enlarges and then it ulcerates. The diagnosis of MCL encompasses epidemiological, clinical and laboratory aspects. Usually, the combination of some of these elements is necessary for the final diagnosis. So, lip leishmaniasis lesions can be challenging to diagnose. CASE PRESENTATION: We presented seven rare cases of lip leishmaniasis. Tissue impression smear, culture, PCR and phylogenetic analysis were carried out for explicit diagnosis. Skin scraping investigation showed several Leishmania spp. amastigotes in the cytoplasm of macrophages. Culture examination was positive for Leishmania spp. PCR was positive for L. major, L. tropica, and L. infantum. Differential diagnosis includes orofacial granulomatosis, basal cell carcinoma, squamous cell carcinoma, and mesenchymal tumors. The cases were treated with systemic meglumine antimoniate (Glucantime®). No relapses were observed during 1 year of follow-up. Early detection of the infection are necessary in order to start effective treatment and prevent more serious complications. CONCLUSIONS: In this paper, we reported seven rare cases of lip leishmaniasis in Iran, emphasized the importance of clinical and diagnostic features of lesions, characterized the phylogenetic kinship of isolated parasites, and reviewed the literature on lip leishmaniasis.

Molecular characterization and phylogenetic analysis of Microsporidia and Cryptosporidium spp. in patients with multiple bowel biopsies from Fars Province, Iran

Microsporidia and Cryptosporidium species are prominent agents of enteritis, capable of causing severe chronic diarrhoea in children, immunocompetent and immunocompromised individuals around the world. It is not possible to identify the parasites at species level solely on the basis of microscopy. The aim of the present study was to identify and characterize the species of Microsporidia and Cryptosporidium in immunocompetent humans with GI disturbances by nested PCR-RFLP, sequencing, and phylogenetic analysis. Fresh frozen and fresh paraffin-embedded biopsy specimens of the duodenum, jejunum, ileum and the cecum of 110 patients were examined. Genomic DNA was extracted from all bowel biopsies. Nested PCR targeting the 18S rRNA gene was performed by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing and phylogenetic analysis. A total of three patients with chronic diarrhoea were positive for Microsporidia and Cryptosporidium spp. Species analysis showed the presence of C. parvum and E. bieneusi in two and one samples, respectively. This is the first PCR confirmation of the presence of E. bieneusi and C. parvum in a bowel biopsy of immunocompetent individuals in Iran. This study revealed that PCR, sequencing, and phylogenetic analysis are very powerful tools for the precise species identification of these pathogens.

Cutaneous Leishmaniasis of the Eyelids: A Case Series with Molecular Identification and Literature Review.

Cutaneous leishmaniasis (CL) is a protozoan disease which is endemic in Iran. It is transmitted by the Phlebotomus sand fly. The eyelid is rarely involved possibly because the movement of the lids impedes the sand fly from biting the skin in this region. Here, we report 6 rare cases of eyelid CL. The patients were diagnosed by skin scraping, culture, and PCR from the lesions. Skin scraping examination showed Leishmania spp. amastigotes in the cytoplasm of macrophages. Culture examination was positive for Leishmania spp. PCR was positive for Leishmania major and Leishmania tropica. The lesions were disguised as basal cell carcinoma, chalazion, hordeolum, and impetigo. The patients were treated with intramuscular meglumine antimoniate (20 mg/kg/day) for at least 3 weeks. They showed a dramatic response, and the lesions almost completely disappeared. We emphasized the importance of clinical and diagnostic features of lesions, characterized the phylogenetic relationship of isolated parasites, and reviewed the literature on ocular leishmaniasis.

Molecular and Serological Evaluation of Toxoplasma gondii Infection in Reared Turkeys in Fars Province, Iran.

BACKGROUND: Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii. This parasite infects most of warm-blooded animals, including birds. Turkeys are one of these animals which might be infected by this parasite. Little is known about the prevalence of T. gondii in turkeys in Iran. OBJECTIVES: The current study aimed to evaluate the rate of Toxoplasma infection in turkeys in Fars Province, Southern Iran. MATERIALS AND METHODS: Sera and tissues (brain, neck and tongue) of 54 turkeys were collected from Shiraz slaughterhouse in Fars province. Anti-Toxoplasma antibodies were assessed in the collected sera using modified agglutination test (MAT), while tissues were evaluated by polymerase chain reaction (PCR) and bioassay methods. RESULTS: T. gondii antibodies (MAT titer: ≥ 1:40) were found in 89.8% of turkeys. T. gondii DNA was detected in 61.6% of turkey tissues and brain had the highest rate of infection. Brain tissues from each animal were bioassayed and Toxoplasma tissue cysts were found in 11.5% and Toxoplasma DNA in 62% of inoculated mice. CONCLUSIONS: Results of this study validated a relatively high level of Toxoplasma infection in reared turkeys and turkey meat might be considered as an infection sources for human.

Molecular Survey on Detection of Leishmania Infection in Rodent Reservoirs in Jahrom District, Southern Iran.

BACKGROUND: Zoonotic Cutaneous Leishmaniasis (ZCL) is endemic in many parts of Iran. Recently its incidence is considerable in different parts of Jahrom district, in Fars Province, southern Iran. The aims of our study were to investigate the prevalence of leishmania infection, and identify and characterize the Leishmania species present, among the rodents by molecular methods in a new endemic focus of ZCL, in an urban and rural area of the Jahrom district, Fars Province, southern Iran. METHODS: From May to November 2010), 55 rodents in four regions of Jahrom focus were caught and checked for leishmania infection by the microscopical examination of liver, spleen, ears, and footpads' smears. RESULTS: Overall 18 Meriones persicus, 15 Tatera indica, 14 Mus musculus, and 8 Rattus rattus were caught. Totally, four (16.5%) and two (13.3%) of the Me. persicus and Ta. indica, but only one of Mu. musculus and Ra. rattus were found smear-positive for leishmania amastigotes, respectively. In the nested-PCR assay 8 (14.6%) smears were found positive for Leishmania major, none was found positive for any other Leishmania species. Sequencing based detection of Leishmania confirmed the microscopic and PCR findings. All positive specimens were shown 95-96% similarity with L. major Friedlin. CONCLUSION: Tatera indica and Me. persicus are incriminated as the main 'reservoir' hosts of L. major in the rural area of Jahrom, moreover, Mu. musculus and Ra. rattus have the minor but remarkable role in the maintenance of the disease in the urban regions of Jahrom focus.

Molecular Genotyping of Toxoplasma gondii in Human Spontaneous Aborted Fetuses in Shiraz, Southern Iran.

BACKGROUND: Congenital toxoplasmosis is associated with variable complications including encephalitis, microcephaly, hydrocephaly, hepatitis, lymphadenopathy and even intrauterine death. Presence of Toxoplasma gondii in human placenta may induce congenital infection. The aim of this study was to determine the genotypes of Toxoplasma gondii infection in human spontaneous aborted fetuses in Shiraz, south of Iran. METHODS: Five hundred and forty two paraffin-embedded blocks of aborted placenta were collected, from two university-affiliated hospitals in Shiraz. Occurrence of spontaneous abortion was confirmed by examine of the slides. After re-cutting of the blocks and dewaxing, semi-nested PCR assay was used to detect the fragments of T. gondii B1 gene in the samples. Also direct molecular genotyping was performed on positive samples with Restriction Fragment Length Polymorphism-PCR analysis on the SAG2 gene. RESULTS: Among the 542 tissue samples, the B1 gene was amplified from 78 (14.4%) of cases with the semi nested PCR and typed by RFLP. The genotype of Toxoplasma strains of 65 (out of 78) PCR-positive samples were evaluated and 54 out of 65 (83.1%) were found to be type II and 11 out of 65 (16.9%) were type I. CONCLUSION: Considering the high level of Toxoplasma infection in aborted fetuses in this study, Toxoplasma might largely contribute to spontaneous abortion in this area of Iran.