hossein modirrousta

In this study, the Israeli acute paralysis virus (IAPV), a single-stranded RNA virus, was investigated in honey bee colonies, which had a history of mortality, population decline, and parasitic diseases. Samples (adult honey bees) were collected from 328 apiaries from three provinces (Tehran, Alborz, and Mazandaran) of Iran to detect IAPV. After sample preparation, RNA was extracted and cDNA was synthesized to perform the reverse transcription polymerase chain reaction (RT-PCR) method using a PCR primer pair, and a 185 bp fragment was amplified. The results showed that out of 328 samples, 103 (31.4%) samples were positive, which were from Mazandaran (14.33%), Tehran (8.84%), and Alborz (8.23%) provinces. Subsequently, some of the positive samples were sequenced and a phylogenetic tree was drawn. The phylogenetic tree showed that the virus isolates were divided into two distinct groups, including one group that had a high similarity to the European acute bee paralysis virus (ABPV) and one group that had a high similarity to the Kashmir bee virus. In addition, the sequences of the samples in three regions were separated in a node from the strains of ABPV from Eastern Europe. Since the length of the branch between the Iranian sequences and the different strains of ABPV from Eastern Europe was short, it can be assumed that the sequences from Iran have a common ancestor with the mentioned strains of ABPV from Eastern Europe.

Reduction of honey bee colonies in recent years has been  a matter of concern in the beekeeping and agricultural industries. One of the main causes of  mortality in colonies is infection with viruses. In the present study, the molecular epidemiology of six honey bee pathogenic viruses, which are deformed wing virus (DWV)و acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), black queen cell virus (BQCV), Sacbrood virus (SBV) and Kashmir bee virus (KBV) and was studied in apiaries of eight provinces of the country (Fars, Sistan and Blouchestan, Kerman, Hormozgan, , Bushehr, Kermanshah, Kohkilouyeh and Boyer Ahmad and Khouzestan). 45 samples of honey bees were collected. Samples were examined by RT-PCR method and the PCR product was electrophoresed on a 1.2% agarose gel. Also, PCR products of positive samples were sent for sequencing. Results were positive for deformed wing virus 17 (37.7%), chronic bee paralysis virus 16 (35.5%), black queen cell virus 6 (13.3%) and acute bee paralysis virus 1 (2.2%) , but no sacbrood virus and keshmir bee virus  were detected. The highest percentage of chronic paralysis virus (100%) was observed in samples of Kermanshah province and the highest percentage of black queen cell virus (62.5%) and deformed wing virus (75%) were observed in samples of Fars province. Acute paralysis virus (14.29%) was observed only in samples of Hormozgan province. The results of this study showed that epidemiological studies of honey bee viruses are vital to know the dynamics of virus prevalence and can provide key information for designing surveillance programs against these beekeeping threats.

American foulbrood disease is the most serious and deadly bacterial disease of bee infants and is caused by the gram-positive bacterium that produces Paenibacillus larvae spores. In this regard, accurate and timely diagnosis of this disease is very important. In the present study, the molecular epidemiology of this disease was studied regionally in eight provinces of the country. Frothy eight samples of bees, honey and debris were collected. After preparation, the samples were tested by nested PCR method and the PCR product was electrophoresed on 1% agarose gel. The results show out of 48 samples of bees examined, number of 14 were positive (29.16%), out of 48 samples of honey were examined, number of 16 were positive (33.33%) and out of 48 samples of debries, number of 5 were positive (10.41%). The results of this study show that for epidemiological and monitoring studies of Paenibacillus larvae infection in bee colonies, collecting honey samples is a more reliable method than other samples. Today in the world, this method is used for continuous monitoring of this disease and according to the results, the strategy of control, prevention and treatment are formulated and implemented. Also, diagnosis of the disease using honey samples and in the next priority of adult bees, has a higher prognostic value compared to the identification of bacteria in the samples of wax, pollen and debris.

The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis in cattle. Vaccination is recommended by administration of attenuated schizont-infected cell lines. The expected protective immunity post-vaccination can be demonstrated by challenge test through inoculation of highly virulent infective sporozoites. The aim of this study was to produce Hyalomma anatolicum anatolicum tick infected with T. annulata (local strain) for preparation of tick-derived sporozoite stabilates for molecular characterization and infectivity test assay.

A local T. annulata strain was used for experimental infection of calves. A field isolate of H. a. anatolicum was isolated, laboratory-reared and infected by blood-feeding on Theileria infected above-mentioned calves. The infectivity of calf, tick and prepared stabilate were confirmed by clinical signs of theileriosis, microscopic inspection, RT-PCR and in vitro cell culture.

The tick stabilate was prepared and cryopreserved in liquid nitrogen. The infectivity of the tick stabilate was verified by in vivo bioassay, in vitro cell culture infection, microscopic inspection in salivary glands and RT-PCR assay. The in vitro produced cell line in this study was characterized by T. annulata Cytochrome b gene analyzing.

The infectivity of a new prepared tick-derived sporozoite stabilate was confirmed in susceptible calves; by microscopically, post mortem, tick microscopic and molecular assays. Moreover, naïve PBMCs were transformed and proliferated by T. annulata infected tick stabilate to immortal T. annulata schizont infected cell line. The potent infective sporozoite tick derived stabilate could be used for vaccine efficacy and challenge test as well as in vaccine development.

European foul brood, is a honey bee larvae disease. EFB is a dangerous disease of the bee colonies that considered to be caused by Gram-positive Melissococcus plutonius. The disease is widely distributed in the world and has created a growing problem in some areas. Because of recent advances in molecular technology, identification of bacteria is very easy and nucleic acid detection technology is rapidly replacing traditional methods of microbiology. European foul brood, is located in the list of dangerous diseases for the health of animals. The disease is endemic in many parts of areas and provide seasonal epidemic. The scenario of this disease varies in different countries.
In this study with standard strain, PCR was developed by specific primers. PCR products electrophpresed on 1 % agarose gel.Larval samples using culture and PCR methode, were tested simultaneously.Eightsamples werepositive by PCRand2 sampleswere positiveby culture.

American foulbrood (AFB)، a severe bacterial disease of honeybee brood، caused by the sporeforming bacterium Paenibacillus larvae subspecies larvae. The agent، infect and kill larvae and produce billions of resistantspores to environmental factors. The spores are distributed in bee colonies and apiaries، thus bee products (honey…) are contaminated. For diagnosis، culture and chemical tests are very timeconsuming and expensive which occasionally give false result. In this study، 121 samples of honey were collected from apiaries in Tehranprovince. The samples wereselected by simple random method. Honey samples were diluted with an equal volume of distilled water and centrifuged، then the pellet used for DNA extraction. DNA was used for culture and PCR. PCR products were electrophoresed on 0. 8%agarose gel. The result showed that out of 121 honey samples which collected from beekeeping of Tehran province، 31 (25/6 %) samples were positive by culture andPCR. To control and preventive measures before the occurrence of any kind of damages، we can monitor orscreen of honey samples on regional and national scale by using culture and PCR method. According to the possibility of Honey contamination with the spores and its importance in global trade and a clear negative impact on exports، monitoring and detection of spores in honeys is very essential.