Molecular Identification and Genotyping of Babesia canis in Dogs from Meshkin Shahr County, Northwestern Iran.

BACKGROUND: Canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic diseases in dogs. METHODS: A total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of Meshkin Shahr, Ardabil Province, Iran. The presence of Babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18S rRNA gene. RESULTS: Our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with Babesia. All four Babesia-infected dogs were confirmed B. canis by the molecular-based method. Sequence alignments comparison of the B. canis genotypes A and B, it was revealed that all B. canis isolates belonged to genotype B. CONCLUSION: This study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in Iran.

Morphological Description, Phylogenetic and Molecular Analysis of Dirofilaria immitis Isolated from Dogs in the Northwest of Iran.

BACKGROUND: Dirofilariasis is a globally distributed arthropod-borne parasitic disease of mainly canids and felids. We evaluated to extend the knowledge of morpho-molecular characteristics and outer ultrastructure of Dirofilaria immitis isolated from Northwest of Iran. METHODS: Overall, 67 filarial worms including 41 females and 26 males parasites were collected from the cardiovascular system of the 43 stray dogs in Meshkinshar, Ardebil Province, Northwest of Iran in 2017, and subjected to light and scanning electron microscopy (SEM) as well as carmine alum staining for morpho-molecular and identification. Molecular methods were used for confirmation of morphological findings by sequencing of Cyto-chrome c oxidase subunit I (cox1) gene. RESULTS: The partial DNA sequencing of cox1 gene of adult parasites showed considerable homology and close proximity to the previously isolated from Kerman and Meshkinshahr, Iran. The lowest genetic variation and the highest intra-species variability was found in D. immitis and Dirofilaria repens, respectively. No similarity was identified between D. immitis nucleotide sequence and Wolbachia species as its endosymbiont bacteria. CONCLUSION: The SEM technique is an excellent tool for differential recognition of the parasite surface morphology and molecular techniques could differentiate and identify Dirofilaria spp.

Serological Evaluation of Anti-Toxoplasma gondii Antibodies in Patients with Acute Leukemia and Lymphoma through Chemotherapy.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that belongs to the family Coccidae. We aimed to evaluate IgG avidity and the changes of anti-Toxoplasma immunoglobulins M (IgM) and G (IgG) in patients with acute leukemia and lymphoma. METHODS: Ninety eight patients with Acute myeloid leukemia (AML), Acute Lymphoblastic Leukemia (ALL) and lymphoma, selected from patients referring to Imam Reza Hospital of Tabriz (38°04'N 46°18'E), in terms of the presence of anti-Toxoplasma IgM, IgG, IgG avidity antibodies and the major risk factors were evaluated. RESULTS: The results of pre-chemotherapy evaluation showed that of the examined patients, only two cases, one patient with ALL and another patient with lymphoma, had a positive IgM titer. Overall, 46 cases had positive IgG titers, including 20 patients with AML, 15 patients with ALL and 11 patients with lymphoma. Three (3.06%) patients were positive for anti-T. gondii IgM and one of them was with new infection of toxoplasmosis in lymphoma patients. The post-chemotherapy IgG titer evaluation showed 46 [46.9% (95% CI 37.4-56.7)] positive IgG cases that this result was similar to the result of pre-treatment phase. One [1% (95% CI 0.2-5.6)] positive IgG avidity case was detected using ELISA method, in a patient with lymphoma whose IgM was also positive. There was no significant difference between the type of leukemia and the history of contact with cat. CONCLUSION: Performing specialized tests to diagnose toxoplasma infection before starting treatment, in immunodeficiency patients who undergo chemotherapy, is necessary; therefore, these tests should be considered in therapeutic protocols.

Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran.

BACKGROUND: The purpose of this study was molecular detection and phylogenetic analysis of Wolbachia species of Dirofilaria immitis. METHODS: Adult filarial nematodes were collected from the cardiovascular and pulmonary arterial systems of naturally infected dogs, which caught in different geographical areas of Meshkin Shahr in Ardabil Province, Iran, during 2017. Dirofilaria immitis genomic DNA were extracted. Phylogenetic analysis for proofing of D. immitis was carried out using cytochrome oxidase I (COI) gene. Afterward, the purified DNA was used to determine the molecular pattern of the Wolbachia surface protein (WSP) gene sequence by PCR. RESULTS: Phylogeny and homology studies showed high consistency of the COI gene with the previously-registered sequences for D. immitis. Comparison of DNA sequences revealed no nucleotide variation between them. PCR showed that all of the collected parasites were infected with W. pipientis. The sequence of the WSP gene in Wolbachia species from D. immitis was significantly different from other species of Dirofilaria as well as other filarial species. The maximum homology was observed with the Wolbachia isolated from D. immitis. The greatest distance between WSP nucleotides of Wolbachia species found between D. immitis and those isolated from Onchocerca lupi. CONCLUSION: PCR could be a simple but suitable method for detection of Wolbachia species. There is a pattern of host specificity between Wolbachia and Dirofilaria that can be related to ancestral evolutions. The results of this phylogenetic analysis and molecular characterization may help us for better identification of Wolbachia species and understanding of their coevolution.

Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran.

BACKGROUND: Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. METHODS: A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. RESULTS: Twenty-eight patients (84.8%) were seropositive, two (6%) were both IgG and IgM positive, while one (3%) showed IgG avidity <40%. Molecular testing confirmed toxoplasmosis in 27 patients (81.8%). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. CONCLUSION: Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis.

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.