Molecular detection of Anaplasma marginale and Anaplasma ovis in sheep and goat in west highland pasture of Iran

Objective To determine the prevalence of Anaplasma marginale (A. marginale) and Anaplasma ovis from sheep and goat in different highland pasture in west of Iran. Methods From July 2015 to October 2015, 370 blood samples of sheep and goat were collected from different regions in Hamedan province, Iran. The DNA extracted from blood and subsequently, 16S rRNA and MSP4 genes were analyzed by nested-PCR, semi nested-PCR and RFLP methods. Results In the PCR assessment, overall 27.5% (102/370) of sheep and goat were positive for Anaplasma ovis and A. marginale infection, which was lower than reports from tropical and subtropical regions of Iran. Statistical analysis (the Chi-square test) did not show any significant relation between infection and variables such as location, tick infestation age and sex (P > 0.05). No significant correlation between the altitude and the Anaplasma species infection was found (Mann–Whitney test: P > 0.05). However, Anaplasma infection in goat significantly is more than the sheep (P = 0.008). Conclusions The ecological changes affect the frequency and distribution of Anaplasma species. Furthermore, our results indicate that sheep as potential reservoirs of A. marginale.

Morphological and genetic characteristics of the liver hydatid cyst of a donkey with Iran origin

No data is available on morphology and genetic characteristics of Echinococcus granulosusderived from donkeys of Iran, despite of its existence in donkeys. In the present study morphometric variations of the rostellar hooks of protoscoleces and genotype characteristics of hydatid cyst of donkey from Iran were determined. Protoscoleces prepared from hydatid cyst of donkey of Iran were morphometric and genetic analyzed. The genetic analysis was done using Cox 1 gene by comparative sequence analysis. Our morphometric results showed that donkey of Iran shares 6 out of 7 determined parameters with donkeys of Jordan and 4 out of 7 with 4 available data with Switzerland donkeys. Morphological similarities and dissimilarities were observed with sheep-dog [G1] and camel-dog strains [G6] of Iran. The nucleotide sequence alignment showed that the partial sequence of Cox 1 from donkey had 91% homology with query coverage of 99% to the corresponding sequence of E. equinus, 90% homology to the E. felidis, 90% homology to E. ortleppi, 89% homology to the E. shiquinus, 89% homology to the E. vogeli, 89% homology to the E. oligarthrus, 88% homology to the E. Canadensis and 83% homology to the Taenia solium. Additionally, the amino acid sequence of this gene has also some differences between this strain and all known strains of E. granulosus even with E. equinus [G4]. Despite of common morphological characteristics of Iranian donkey hydatid cyst with those of donkeys of other parts of the world, genetically it has its own entity

Comparison of internal transcribed spacers and intergenic spacer regions of five common Iranian sheep bursate nematodes

Accurate identification of sheep nematodes is a critical point in epidemiological studies and monitoring of drug resistance in flocks. However, due to a close morphological similarity between the eggs and larval stages of many of these nematodes, such identification is not a trivial task. There are a number of studies showing that molecular targets in ribosomal DNA [Internal transcribed spacer 1, 2 and Intergenic spacer] are suitable for accurate identification of sheep bursate nematodes. The objective of present study was to compare the ITS1, ITS2 and IGS regions of Iranian common bursate nematodes in order to choose best target for specific identification methods. The first and second internal transcribed spacers [ITS1and ITS2] and intergenic spacer [IGS] of the ribosomal DNA [rDNA] of 5 common Iranian bursate nematodes of sheep were sequenced. The sequences of some non-Iranian isolates were used for comparison in order to evaluate the variation in sequence homology between geographically different nematode populations. Comparison of the ITS1 and ITS2 sequences of Iranian nematodes showed greatest similarity among Teladorsagia circumcincta and Marshallagia marshalli of 94% and 88%, respectively. WhileTrichostrongylus colubriformis and M. marshalli showed the highest homology [99%] in the IGS sequences. Comparison of the spacer sequences of Iranian with non-Iranian isolates showed significantly higher variation in Haemonchus contortus compared to the other species. Both the ITS1 and ITS2 sequences are convenient targets to have species-specific identification of Iranian bursate nematodes. On the other hand the IGS region may be a less suitable molecular target

Isolation of small number of cryptosporidium parvum oocyst using immunochromatography

Cryptosporidium parvum causes severe gastroenteritis in immunocompromised human and new borne animals. The organism can be transmitted through water. Since small number of C. parvum is infectious, the aim of the present study was to develop a chromatography method for the isolation of C. parvum oocyst in samples with limited number of oocysts. Antibody was prepared against whole antigen from C. parvum oocysts, the achieved Ab bound to the sepharose 4B and used for the isolation of oocysts. Antibody against P23 bound to the sepharose 4B, used also for the isolation of C. parvum oocyst. In comparison to these both methods, 2 traditional methods [Salt floatation and 55% sucrose floatation] were also performed. Both chromatography methods could bind oocysts with capacity depends on the column size. The isolated oocysts were free of bacteria. Our results showed that the traditional methods are useful for the isolation of oocysts from feces, in its smear stained with ziehl-nelsen, at least 3 oocyts are detectable in each microscopic field under 1000 X magnification. In contrast to the chromatography methods, the bacterial contamination was always observed in oocysts isolated with traditional methods. Immunochromatography could be used for the successful isolation of C. parvum oocysts from the samples containing limited number of oocysts

Molecular detection and identification of theileria species by PCR-RFLP method in sheep from Ahvaz, Southern Iran

The present study was carried out to investigate the accurate status of ovine Theileria infection in sheep from Ahvaz and surrounding region, a tropical area southwest Iran. A PCR-RFLP method based on 18S ribosomal RNA gene was designed which could detect and differentiate Theileria and Babesia spp. and also differentiate main Theileria species in sheep at the same time. 119 sheep blood samples were collected from Ahvaz and surroundings. Microscopic examination of blood smears revealed 69.7% [83/119] infection with Theileria spp. Of the total samples subjected to PCR, 89% [106/119] were found to be positive, all of which were identified as Theileria by RFLP analysis using enzyme Hind II. In enzymatic digestion of PCR products by Vsp I, 91.5% [97/106] of Theileria positive samples were identified as T. ovis while mixed Theileria infections were found in 9 samples. The samples with mixed infections were analyzed with an additional nested PCR-RFLP method, by Hpall enzyme digestion. 3 samples with T, lestoquardi infection, 1 sample with T. ovis and T. annulata., 1 sample with T. lestoquardi and T. annulata, and 4 samples with T. ovis, T. lestoquardi and T. annulata mixed infections were detected. Ovine theileriosis caused by T. ovis is highly prevalent in southwest Iran while T. lestoquardi and T. annulata infection can be detected in a lesser proportion of sheep in this region. The new PCR-RFLP method that was designed in this study, can serve as a beneficial diagnostic tool, especially in T. ovis prevalent regions

Epidemiological study of hydatidosis in the dromedaries (Camelus dromedarius) of different regions of Iran

Objective: To determine the prevalence of hydatidosis in dromedaries.Methods:2011. The relationship between host age and the mean number of hydatid cysts, and prevalence and fertility rates was analyzed using chi-square test.Results:438 dromedaries were examined in five regions of Iran from 20 March, 2010 to 19 March, Echinococcus granulosus. Number of cysts was 700 with 72.5% lung cyst. The highest rate of infection was that 54 (40%) of camels was found in the Khorasan Razavi region (in the north-east part of Iran) while the lowest 6 (4.4%) of camels was found in Semnan province. Infection was higher in >15 years age group. The most commonly infected organs were lungs (72.5%) followed by liver (12.6%). Both liver and lungs together constituted 14.8% of infection. A comparison found that hydatid cysts of liver had a higher fertility rate (32.57%) than that of lung (19%); while most of cysts of lung were calcified (24.42%). The mean number of protoscoleces per mL in the lung fertile cysts was higher than that of liver cysts. Fertile or sterile might be due to the different species or genotypes. The mean number of cysts in infected liver and lungs was 1-5 cysts. The intensity of infection increased with age.Conclusions:The results of current study can make a background data for implementing hydatid One hundred and thirty five out of 438 (30.82%) camels harboured hydatid cysts of control programs and warrant the importance of camel in public health.

Molecular diagnostic of anaplasma marginale in carrier cattle

Anaplasmosis belongs to the complex of several tick-borne diseases and can cause diseases in the livestock with high economical losses. Cattle that recover from acute infection become carriers and the parasite can persist most probably for the lifetime in the blood. The aim of the present study was the determination of the persistently infected cattle in a region of Iran with the previous history of acute anaplasmosis. One hundred and fifty blood samples and corresponding blood smears of cattle without any signs of diseases were prepared from a region in Isfahan/ Iran with the previous history of acute anaplasmosis from March 2007 to July 2007 for cross sectional study of carriers of Anaplasma. The blood smears were first screened by Giemsa staining, the extracted DNA from blood cells were analyzed by Anaplasma marginale specific nested PCR, and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst 1107 I. Anaplasma like structures could be identified in the limited amount of erythrocytes of 75 blood smears. In these samples, the percentage of erythrocytes harboring Anaplasma like structures varied from 10[-3]% to 10[-2]%. Nested-PCR and PCR-RFLP analysis showed 58 A. marginale positive cases within 75 Anaplasma suspected blood samples. In 150 total blood samples, 50% were A. marinale positive. Our results revealed that the traditional Giemsa staining method is not applicable for the determination of the persistently infected cattle. In addition, the results showed that the carrier animals must be widespread in the Anaplasma endemic areas in Iran

role of GM-CSF in the generation of CD1a positive cells in vitro by JMML patients

Juvenile myelomonocytic leukemia [JMML] is a rare myeloproliferative disorder of childhood. It was recently demonstrated that mononuclear cells from spleen, peripheral blood and bone marrow of the patients with JMML proliferate in vitro and population of the cells differentiate within 7 days to the CD1a positive dendritic cells. In the present study, the role of GM-CSF in the proliferation and differentiation of the JMML cells in vitro was investigated. MNC from patients with JMML was cultured for 7 days in RPMI/10%FCS and 1% penicillin-streptomycin. The phenotype of the cultured cells was determined at day 7 using antibodies against Ki-67, CD1a, CD14 and CD68. GM-CSF concentration was first measured at days 1, 3, 5 and 7 in the supernatants of the cultured mononuclear cells from two patients with JMML. To study the roll of GM-CSF in JMML cultures, GM-CSF activity was neutralized using monoclonal antibody [Ab] against human GM-CSF in different concentrations [0.05 micro g/ml, 0.5 micro g/ml, 5 micro g/ml and 10 micro g/ml]. The effect of neutralizing antibody was determined at day 7 in treated and untreated cultures by immunostaining using antibodies against Ki-67, CD68, CD1a and CD14. The expression of GM-CSF, GM-CSFR, TNF-alpha, IFN-alpha and SCF was also determined at day 7 in mononuclear cells treated and untreated cultures by RT-PCR. GM-CSF neutralization led to decrease of the total cell number. Additionally, neutralization of GM-CSF decreased in dose dependent manner the number of CD1a, CD14 and Ki-67 positive cells in JMML cultures, whereas the percentage of CD68 remained unchanged. The expression of GM-CSF, GM-CSFR, TNF-alpha and SCF could be demonstrated at the mRNA level in the antibody treated and untreated culture as well. GM-CSF plays an important role in the proliferation and differentiation of JMML cells into the CD1a and CD14 positive cells

Juvenile myelomonocytic leukemia [JMML] cells spontaneously differentiate into dendritic cell like populations in vitro

Juvenile myelomonocytic leukemia [JMML] is a rare myelodysplastic/ myeloproliferative malignancy of early childhood, characterized by monocytosis, hepatosplenomegaly and an aggressive clinical course. semi-solid culture JMML progenitor cells proliferate spontaneously into colony forming units. In order to study the mechanisms of proliferation and differentiation of JMML cells we developed a suspension culture system without additional exogenous growth factor supplement. Mononuclear cells [MNC] from peripheral blood, bone marrow or spleen of 14 patients with JMML and 24 controls were studied. JMML cells expressed higher levels of the proliferation marker Ki67 [median 24% [7-39%] vs a median of 3.5% in controls]. 90% of JMML cells were CD68-positive [vs 35% in controls] and by day 7 all JMML samples contained CD1a- positive cells. Electron microscopy demonstrated cytoplasmic vesicular structures resembling multilamellar MHC II compare-timents, which together with the expression of CD1a - support a dendritic cell [DC]-phenotype. Differentiation into CD1a-positive DC seems to be a frequent phenomenon in cultured JMML MNC, which in vivo may contribute to clinical characteristics such as skin and organ infiltration

Status of Haemaphysalis tick infestation in domestic ruminants in Iran

The geographical distribution and ecological preferences of Haemaphysalis in domestic animals in Iran were studied 4 times a year from April 2003 to March 2005. A total of 1,622 ixodid tick specimens were collected from 3 different zones. Among them, 108 (6.7%) Haemaphysalis ticks, consisting of 6 species, were identified; H. punctata (3.4%), H. parva (0.5%), H. sulcata (0.6%), H. choldokovskyi (1.7%), H. concinna (0.06%) and Haemaphysalis sp. (0.6%). H. punctata was the most abundant species, whereas H. concinna was the rarest species collected in humid and sub-humid zones on cattle, sheep and goats. H. choldokovskyi was principally collected from sheep and goats grazed in cold mountainous areas. The infested areas consisted of Caspian Sea (Guilan, Mazandaran, Golestan, and central provinces), mountainous (Azarbaiejan, Ardebil, Kohgilouyeh, and Kordestan) and semi-dessert (Khorasan, Semnan, Kerman, Sistan, and Baluchestan) zones. The Caspian Sea zone (23.6%) was the most highly infested region. The results show that various species of Haemaphysalis ticks infest domestic ruminants in Iran and each tick species show characteristic geographical distributions.