The effects of allogeneic and xenogeneic lyophilized leukocyte-and platelet-rich fibrin on bone healing in rat.

BACKGROUND: Critical size defects are one of the challenges in the treatment of fractures in humans and animals. Blood products such as leukocyte-SAand platelet-rich fibrin (L-PRF) are one of the alternatives to bone autograft to solve this challenge. This study aims to evaluate the effects of allogeneic and xenogeneic lyophilized L-PRF on bone healing in a critical defect of radius bone in rat. METHODS: A defect with a diameter of 5 mm was created in the radius bone of 60 rats in four groups. The defect was left empty in the untreated group, and it was filled with autogenous bone graft, allogeneic, and xenogeneic lyophilized L-PRF, respectively, in the other three groups. Radiographic evaluation was done every two weeks, and histopathological evaluation in the 14th, 28th, and 56th days after surgery. RESULTS: The radiographic scores of allogeneic and xenogeneic lyophilized l-PRF groups were significantly higher than the untreated group in all times (P<0.05). In connection with histopathological Emery's scoring system, the score of allogeneic lyophilized L-PRF was significantly higher than the untreated group (P<0.05) in the 14th and 28th days after surgery. The score of the xenogeneic lyophilized L-PRF group was also higher than the untreated group, but the difference was not significant (P>0.05). The allogeneic and xenogeneic lyophilized L-PRF scores were significantly higher than the untreated group (P < 0.05) on the 56th day. CONCLUSION: The results of the present study showed that the allogeneic and xenogeneic lyophilized L-PRF can improve bone healing in the critical radius bone defect in rat model of study.

Zoledronate loaded polylactic acid/polycaprolactone/hydroxyapatite scaffold accelerates regeneration and led to enhance structural performance and functional ability of the radial bone defect in rat.

Abstract. Background: One of the most common concerns in the regeneration of massive bone defects necessitating surgery and bone grafts is the application of tissue engineering using drug delivery. Zoledronate is a well-known effective drug for the healing bone fractures in osteoporotic patients. Aims: An attempt was made to design a more efficient bone scaffold with polycaprolactone, polylactic acid, and hydroxyapatite. Methods: The scaffold was fabricated by freeze-drying and indirect 3D printing approaches. X-ray diffraction, Fourier transform infrared spectroscopy, rheometry, scanning electron microscopy, and neutral red tests were performed to characterize the scaffold. qRT-PCR was also done to define the osteoinductivity and angiogenic induction capacity of this scaffold. Forty rats were selected and randomly divided into four groups: the control group, which received no treatment, the autograft group, scaffold group, and Zol-loaded scaffold group (n=10 in each group). The injured area was studied by radiology, biomechanical analysis, histopathology, histomorphometry, immunohistochemistry, and CT scan analyses. Results: The qRT-PCR results demonstrated significantly higher expression levels of OPN, OCN, and CD31 markers in the scaffold group when compared to the control group (P<0.05). Histopathologically, the newly formed bone tissue was significantly detected in the Zol-loaded scaffold and autograft groups in comparison with the non-treated group (P<0.001). The immunohistochemistry (OC marker), biomechanical, and histomorphometric results indicated a significant improvement in the regeneration of the injured area in the groups treated with autologous bone and Zol-loaded scaffold compared to the non-treated group (P<0.05). Conclusion: The Zol-loaded scaffold accelerated bone regeneration, and led to enhanced structural performance and functional ability of the injured radial bone in rats.

Efficacy of amiodarone and voriconazole combination therapy in cutaneous leishmaniasis in the mice experimentally infected with Leishmania major.

INTRODUCTION: The aim of the present study was to evaluate in vitro and in vivo efficacy of combination therapy of amiodarone and voriconazole against Leishmania major and investigating immune and wound healing responses of cutaneous leishmaniasis to this combination therapy. METHODS: For in vitro study, replication of L. major promastigotes and intracellular amastigotes were investigated in the presence and absence of amiodarone and voriconazole. Isobologram construction and calculation of the Fractional Inhibitory Concentration (FIC) were performed. After the appearance of ulcers on the base of tails of BALB/c mice, treatment was initiated by a combination of amiodarone at 40 mg/kg plus voriconazole at 30 mg/kg orally and glucantime at 60 mg/kg intraperitoneally for 28 consecutive days. RESULTS: According to the concave isobologram and fractional inhibitory concentration <1, combination of amiodarone plus voriconazole had synergistic effects against L. major promastigotes and intracellular amastigotes. There were less inflammatory cells, more fibroblasts and more collagen deposition in tissue sections in the mice treated with combined drugs compared to the vehicle and untreated mice. Increased glutathione peroxidase activity and decreased malondialdehyde, Interleukin-6, and Tumor necrosis factor-α levels were detected in the combination therapy group in comparison to the vehicle and untreated groups. CONCLUSIONS: It seems a combination of amiodarone plus voriconazole can be a rational and promising therapeutic approach in the treatment of cutaneous leishmaniasis.

Effectiveness of mesenchymal stem cell-seeded onto the 3D polylactic acid/polycaprolactone/hydroxyapatite scaffold on the radius bone defect in rat.

PURPOSE: The importance of regeneration in large bone defects forces the orthopedic surgeons to search for a proper methodology. The present experiment evaluated the capability of polylactic acid/polycaprolactone/hydroxyapatite (PLA/PCL/HA) scaffold loaded with and without mesenchymal stem cells (MSCs) on bone regeneration. METHODS: Fourier transform infrared spectrometry, X-ray diffraction, scanning electron microscopy, and rheology methodologies were used to characterize the scaffold. Forty Wistar rats were randomly divided into the four groups including the untreated defects as the control group and three other groups in which the bone defects were treated with autologous bones (autograft group), the PLA/PCL/HA scaffolds (PLA/PCL/HA group), and the MSCs-seeded scaffolds (MSCs-seeded PLA/PCL/HA group). RESULTS: Based on the qRT-PCR results, significantly higher expression levels of osteocalcin, osteopontin, and CD31 were seen in the cell-seeded scaffold group compared to the control group (P < 0.05). The CT scanning and radiographic images depicted significantly more newly formed bonny tissue in the MSCs-loaded scaffold and autograft groups than the untreated group (P < 0.001). The immunohistochemistry, biomechanical, histopathologic, and histomorphometric evaluations demonstrated significantly improved regeneration in the autograft and MSCs-loaded scaffold groups compared to the non-treated group (P < 0.05). There were significant differences between the scaffold and untreated groups in all in vivo evaluations (P < 0.05). CONCLUSION: The MSCs enhanced bone healing potential of the PLA/PCL/HA scaffold and the MSCs-seeded scaffold was comparable to the autograft as the golden treatment regimen (P > 0.05).

Effectiveness of amiodarone in treatment of cutaneous leishmaniasis caused by Leishmania major.

Development of new chemotherapeutic agents is an essential issue in the treatment and control of a disease. This study aimed to evaluate the anti-leishmanial activity of amiodarone, an antiarrhythmic class III drug, against Leishmania major, the most prevalent etiological agent of cutaneous leishmaniasis in the old world. The proliferation of promastigotes and intracellular amastigotes in the absence or presence of amiodarone was estimated, in an in vitro study. For in vivo study, five weeks after infection of BALB/c mice with L. major, when the lesions appeared at the injection site, the mice were divided into four groups (n = 6 each); treatment was conducted for 28 consecutive days with vehicle, amiodarone at 40 mg/kg orally and glucantime at 60 mg/kg intraperitoneally. Therapy with amiodarone reduced the size of lesions compared to the untreated group after 12 days. Amiodarone decreased the parasite load and inflammatory responses, particularly the macrophages containing amastigotes, and enhanced granulation tissue formation in the dermis and subcutaneous area. The Tumor necrosis factor-α and Interleukin-6 levels were significantly lower in the cell culture supernatants of the inguinal lymph node in the amiodarone treated group compared to the vehicle and untreated groups. Amiodarone significantly increased the activity of glutathione peroxidase in comparison to the vehicle and untreated groups but did not affect the plasma levels of superoxide dismutase, malondialdehyde, adiponectin, and ferric reducing ability of plasma. Therefore, the anti- L. major activity and immunomodulatory effects of amiodarone reduced the parasitic load and enhanced wound healing in cutaneous leishmaniasis in BALB/c mice. Amiodarone reduced the lesion surface area, but it did not cure it completely.

Emerging role of amiodarone and dronedarone, as antiarrhythmic drugs, in treatment of leishmaniasis.

Leishmaniasis is a group of human and animal diseases causing 20,000-40,000 annual deaths and its etiological agents belong to the Leishmania genus. The most current treatment against leishmaniasis is chemotherapy. Pentavalent antimonials such as glucantime and pentostam have been administrated as the first-line drugs in treatment of various forms of leishmaniasis. The second-line drugs such as amphotericin B, liposomal amphotericin B, miltefosine, pentamidine, azole drugs and paromomycin are used in resistant cases to pentavalent antimonials. Because of drawbacks of the first-line and second-line drugs including adverse side effects on different organs, increasing resistance, high cost, need to hospitalization and long-term treatment, it is necessary to find an alternative drug for leishmaniasis treatment. Several investigations have reported the effectiveness of amiodarone, the most commonly used antiarrhythmic drug, against fungi, Trypanosomes and Leishmania spp. in vitro, in vivo and clinical conditions. Moreover, the beneficial effects of dronedarone, amiodarone analogues, against Trypanosoma cruzi and Leishmania mexicana have recently been demonstrated and such treatment regimens resulted in lower side effects. The anti- leishmanial and anti- trypanosomal effectiveness of amiodarone and dronedarone has been attributed to destabilization of intracellular Ca2+ homeostasis, inhibition of sterol biosynthesis and collapse of mitochondrial membrane potential. Because of relative low cost, excellent pharmacokinetic properties, easy accessibility and beneficial effects of amiodarone and dronedarone on leishmaniasis, they are proper candidates to replace the current drugs used in leishmaniasis treatment.

Effects of chitosan scaffold along with royal jelly or bee venom in regeneration of critical sized radial bone defect in rat.

The aim of this study was to compare the efficacy of honey bee venom (BV) and royal jelly (RJ) alongside chitosan scaffold (CS) in improving radius bone defect in rats. A total of 60 full thickness radial bone defects with a length of 5 mm were created in 60 male Wistar rats. Six healthy radial bones (3 rats) were also assigned as normal control for biomechanical studies. The defects were left empty (untreated group) or were filled by the autograft (autograft group), CS (CS group), CS alongside the BV solution (CS-BV group), and CS alongside the RJ solution (CS-RJ group). Healing of the bone defects were evaluated clinically and radiologically on days 0, 28, 42 and 56 after operation while the biomechanical testing and histopathological examination were performed on the 56th day after surgery. The autograft was more radiopaque than the untreated and CS groups at the 28th, 42nd and 56th postoperative days (P<0.05). The CS-BV and CS-RJ groups showed significantly higher radiographic outcomes than the untreated and CS groups at the 56th post-operative day (P<0.05). The density of osseous tissue (DOT) and the osteocytes and osteoblasts count of the CS-RJ and CS-BV groups were significantly higher than the CS and autograft groups (P<0.05). The biomechanical results of the CS-RJ group were significantly superior to the autograft, while the biomechanical properties of CS-BV group were not significantly different with the autograft group (P>0.05). The scaffolds in CS group were observable in the surgical site after 56 days. There was no significant difference in radiographs, DOT, cartilage tissue and fibrous tissue, and also biomechanical performances of the CS-BV and CS-RJ groups at the 42nd and 56th day after surgery. The untreated and CS groups showed weakest biomechanical results among all groups. It could be concluded that both treatment strategies in the CS-BV and CS-RJ groups were appropriate and useful in treating critical bone defects.

Burn wound healing: present concepts, treatment strategies and future directions.

Burns are the most extensive forms of soft tissue injuries occasionally resulting in extensive and deep wounds and death. Burns can lead to severe mental and emotional distress, because of excessive scarring and skin contractures. Treatment of burns has always been a difficult medical problem and many different methods have been used to treat such injuries, locally. Biofilms are a collection of microorganisms that delay wound healing. One of the new methods of prevention and treatment of burn wound infections is application of antimicrobials, which act on biofilms and prevent the wound infection. Biofilm initiates a persistent, low-grade, inflammatory response, impairing both the epithelialisation and granulation tissue formation. Skin grafts have been shown to dramatically reduce deaths from infection. However, grafting has considerable limitations. Such injuries are long-lasting and many patients suffer from chronic pain for a long time. Tissue engineering is a new approach in reducing the limitations of conventional treatments and producing a supply of immunologically tolerant artificial tissue, leading to a permanent solution for damaged tissues; such criteria make it a cost-effective and reliable treatment modality. To overcome the present limitations of burn wound healing, knowledge about the latest findings regarding healing mechanisms is important. Here the authors discuss the most important events regarding burn wound healing and review the latest treatment strategies that have been used for burn wounds from in vitro to clinical levels. Finally, we discuss the role of tissue engineering and regenerative medicine in the future of burn wound healing, modelling and remodelling.

Worldwide risk factors in leishmaniasis.

Recently, vector-borne parasitic diseases such as leishmaniasis have been emerged or re-emerged in many geographical areas and resulted in global health and economic concerns that involve humans, domestic animals and wild life. The ecology and epidemiology of leishmaniasis are affected by the between host, reservoir and vector (human, animal and sandfly) and the environment. Important drivers for the emergence and spread of leishmaniasis include environmental factors such as alterations in temperature and water storage, irrigation habits, deforestation, climate changes, immunosuppression by HIV or organ transplant, development of drug resistance, increase traveling to endemic regions and dog importation. War, poor socio-economic status and low level household are also major contributors to the spread of this disease. Health education via the public media and training should be implemented by international organizations and governmental agencies in collaboration with research institutions. Fully protection during transmission season, using bednets and insecticides and reservoirs' control should be also mentioned in the planning. Based on the findings of the recent studies and high prevalence of leishmaniasis, it is concluded that serious public health monitoring should be considered.

Optimization of murine model for Besnoitia caprae.

It has been shown that mice, particularly the BALB/c ones, are susceptible to infection by some of the apicomplexan parasites. To compare the susceptibility of the inbred BALB/c, outbred BALB/c and C57 BL/6 to Besnoitia caprae inoculation and to determine LD50, 30 male inbred BALB/c, 30 outbred BALB/c and 30 C57 BL/6 mice were assigned into 18 groups of 5 mice. Each group was inoculated intraperitoneally with 12.5 × 10(3), 25 × 10(3), 5 × 10(4), 1 × 10(5), 2 × 10(5) tachyzoites and a control inoculum of DMEM, respectively. The inbred BALB/c was found the most susceptible strain among the experienced mice strains so the LD50 per inbred BALB/c mouse was calculated as 12.5 × 10(3.6) tachyzoites while the LD50 for the outbred BALB/c and C57 BL/6 was 25 × 10(3.4) and 5 × 10(4) tachyzoites per mouse, respectively. To investigate the impact of different routes of inoculation in the most susceptible mice strain, another seventy five male inbred BALB/c mice were inoculated with 2 × 10(5) tachyzoites of B. caprae via various inoculation routes including: subcutaneous, intramuscular, intraperitoneal, infraorbital and oral. All the mice in the oral and infraorbital groups survived for 60 days, whereas the IM group showed quicker death and more severe pathologic lesions, which was then followed by SC and IP groups. Therefore, BALB/c mouse is a proper laboratory model and IM inoculation is an ideal method in besnoitiosis induction and a candidate in treatment, prevention and testing the efficacy of vaccines for besnoitiosis.

A rare case of cerebral hydatidosis caused by a G1 genotype of Echinococcus granulosus in a cow from Iran.

Hydatidosis is a medically and veterinary important parasitic disease that is endemic in many parts of the world. Unilocular hydatid cysts may develop in almost any part of the body. Up to 70% of hydatid cysts are located in the liver, followed by 25% in the lungs. Cerebral hydatidosis is an uncommon manifestation of the disease, occurring in less than 1/1000 infected hosts, yet diagnosis does pose a problem. We have reported an exceptionally rare case of cerebral hydatidosis in cattle. This is the first report to describe the characteristic pathological features of the cerebral hydatidosis in cattle caused by the G1 genotype of Echinococcus granulosus. Genotypic analysis was performed on a hydatid cyst from a cow originating from southern Iran, based on the sequence analysis of the cox1 mitochondrial gene.

Suspension culture of Besnoitia caprae by murine macrophage.

Besnoitia caprae is a tissue cyst-forming protozoan that infects goats and has considerable economic importance in certain regions of Asia and Africa. Murine macrophage J774 cell line was inoculated with tachyzoites of Besnoitia caprae (BC-Pars isolate) collected from mice. A significant growth of tachyzoites was observed in J774. Mice were inoculated with tachyzoites harvested from J774 cell culture. Skin samples from the mice infected with tachyzoites of BC-Pars were PCR positive. One mouse showed alopecia and skin lesions on 45 DPI. Dermal lesions started from around right eye and gradually developed more and more. After euthanasia on 60 DPI, histopathological evaluation of skins around the eye showed necrosis of the epidermis and follicular adnexa with chronic inflammatory cell infiltration. Histopathological sections of their skin showed the presence of necrosis and mononuclear cell infiltration. To the authors' knowledge, this is the first report of successful production of Besnoitia caprae tachyzoites was achieved in vitro by suspension culture technique. Another interesting finding is the report of the alopecia and skin lesions around the eye in mouse that quite similar to lesions of goats due to infection of Besnoitia caprae.

First identification of Sarcocystis hominis in Iranian traditional hamburger.

Zoonotic concerns of cattle sarcocystosis are of importance, because humans are the final host for Sarcocystis hominis. Therefore the meat products containing beef may encompass sarcocysts which endanger food safety. In this study, we described the first report of molecular identification of S. hominis in Iranian traditional hamburgers using PCR-RFLP. Throughout a pilot research that was carried out to setup a molecular approach to identify the Sarcocystis spp., using PCR-RFLP, a sample of raw Iranian traditional hamburger was purchased from a street food seller located in Yazd, central Iran in May 2013. DNA extraction was done, by salting out method; briefly, the sample was lysed with NET buffer. The DNA purification and precipitation was then performed. Amplicon and digestion results were analyzed, using gel agarose electrophoresis. The results showed a PCR product with 926 bp in length after amplification and 376 and 550 bp in length after digestion. This product was identified as S. hominis. To the best of our knowledge, this is the first report of S. hominis infection in Iranian hamburger.

Correlation between some hematological parameters, acute phase proteins and serum immunoglobulins in experimental caprine besnoitiosis.

The present study was undertaken to investigate correlation between some hematological parameters, acute phase proteins and immunoglobulins in the experimentally infected goats with Besnoitia caprae from the time of infection till 360 days post infection (DPI). Six male goats, approximately 12-16 months old, were inoculated subcutaneously with approximately 1.3 × 10(8) bradyzoites of B. caprae and blood samples were collected at weekly intervals from the jugular vein of the goats. Total leukocyte count and differential leukocyte counts were determined. Acute phase proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), fibrinogen and ceruloplasmin were undertaken at weekly intervals. We evaluated an enzyme-linked immunosorbent assay (ELISA) (using a somatic antigen of bradyzoite) to detect anti-B. caprae antibodies in caprine sera. Cysts were present in the skin biopsies of the distal parts of the leg of the infected goats from 28 DPI. From 30 to 360 DPI, results showed that the APPs concentrations including SAA, Hp, fibrinogen and ceruloplasmin were enhanced in the serum of infected goats. However, there were some variation in hematological parameters; the differences were not significant with those of the normal values. Some variations were seen in the levels of specific antibodies against this parasite and they had correlation with some hematological parameters and acute-phase proteins.

Larval cyathostominosis in a working donkey.

Parasitic infections are one of the most common factors that threaten the health and working performance of donkeys. One of the life threatening parasites is the small strongyles that encyst or burrow into the large intestine and their larvae can initiate severe damage in the lining of the intestine. A 6 years old female donkey with clinical signs of diarrhea and emaciation was necropsied and gross examination of gastro-intestinal tract revealed thin-walled, hyperemic and hemorrhagic cecum. Multifocal petechial hemorrhages were particularly prominent in the submucosa of cecum. Parasitological examination revealed two cyathostomin species included Cylicocyclus elongatus and Cyathostomum pathratum. At microscopic examination, cross sections of cyathostomins larvae associated with parasitic granuloma were observed in the submucosa of cecum. The lesions were associated with non-suppurative enteritis with infiltration of eosinophils, plasma cells, lymphocytes and macrophages in the intestinal mucosa, submucosa and lamina propria.

Plant-derived compounds in treatment of leishmaniasis.

Leishmaniasis is a neglected public health problem caused by the protozoan species belonging to the genus Leishmania affecting mostly the poor populations of developing countries. The causative organism is transmitted by female sandflies. Cutaneous, mucocutaneous, and visceral clinical manifestations are the most frequent forms of leishmaniasis. Chemotherapy still relies on the use of pentavalent antimonials, amphotericin B, paromomycin, miltefosin and liposomal amphotericin B. However, the application of these drugs is limited due to low efficacy, life-threatening side effects, high toxicity, induction of parasite resistance, length of treatment and high cost. Given the fact that antileishmanial vaccines may not become available in the near future, the search for better drugs should be continued. Natural products may offer an unlimited source of chemical diversity to identify new drug modules. New medicines should be less toxic or non-toxic, safe, more efficient, less expensive and readily available antileishmanial agents, especially for low-income populations. In the present review, special focus is on medicinal plants used against leishmanaiasis. The bioactive phytocompounds present in the plant derivatives including the crude extracts, essential oils, and other useful compounds can be a good source for discovering and producing new antileishmanial medicines.

Collagen implants in experimental tendon injury in rabbits: a clinical, ultra-structural and biomechanical investigation.

This study investigated the effects of hybridized micro and nano structured collagen implants on tendon healing in an experimental tendon injury in rabbits. Fifty mature male New Zealand white rabbits were randomly divided into two groups of treated and control. Two cm of the left Achilles tendon were discarded. In the treated group, a 3-dimensional (3D) collagen implant was engineered and implanted in the defect area. No implant was used in the control group. At day 120 after injury, the Achilles tendon of the animals were ultrasonographically (days 0-120 after injury) and radiographically (day 120 after injury) examined, and the animals were euthanized. The tendons were dissected and used for gross pathological, histopathological, ultra-structural and biomechanical investigations. Application of the collagen implant significantly increased the diameter of the newly regenerated tissue in the defect area compared to the control tendons. Treatment also significantly increased the echogenicity and homogeneity of the injured area, the diameter of the collagen fibrils and fibers, maturity of the tenoblasts, number of tenocytes, collagen density, alignment, ultimate and yield load, stiffness, stress and modulus of elasticity. The collagen implants were almost totally absorbed 120 days after surgery. No inflammatory reaction or tissue degeneration or necrosis was evident in the treated tendons compared to the control ones. 3D collagen implants produced a newly regenerated tendinous tissue at the defect area that was morphologically and biomechanically superior to the control group. This collagen implant was biocompatible and biodegradable with high bio-safety in rabbits.

Caprine besnoitiosis: an emerging threat and its relationship to some other infections of ungulates by Besnoitia species.

Caprine besnoitiosis, caused by the cyst-forming protozoal apicomplexan Besnoitia caprae appears to be endemic in Kenya, Nigeria and Iran, but has yet to be detected in other parts of the world. The infection causes an important parasitic disease of goats in affected developing countries. Bovine besnoitiosis, is a widespread disease of cattle in Africa, Asia (but not Iran) and southern Europe. Recent epidemiological data confirm that the incidence and geographical range of bovine besnoitiosis in Europe is increasing, which is why growing attention has been given to the condition during the past decade. This paper reviews pertinent information on the biology, epidemiology, pathology, clinical signs, diagnosis and control of caprine besnoitiosis, together with its similarities to, and differences from, bovine besnoitiosis. The serious economic consequences of besnoitiosis on goat breeding and local meat and hide industries is also considered.

Molecular investigation and cultivation of camelpox virus in Iran.

Camelpox virus (genus Orthopoxvirus, family Poxviridae) is the etiologic agent of camel pox. The clinical manifestations of this virus range from inapparent infection to mild, moderate and, less commonly, severe systemic infection and death. Following an outbreak of camelpox, samples that were collected from camel flocks suspected to have camelpox in Qom Province in central Iran and Khash city, Sistan and Baluchestan Province and South Khorasan Province in eastern Iran were sent to Razi Vaccine and Serum Research Institute in Mashhad. DNA extraction was performed primarily by the phenol-chloroform method, and PCR was carried out using a Bioneer kit. Using the primer pair 5'-AAT-ACA-AGG-AGG-ATC-T-3' and 5'-CTT-AAC-TTT-TTC-TTT-CTC-3', the gene sequence encoding the A-type inclusion protein (ATIP) was amplified. The size of the PCR product, specific for camelpox virus, was 881 bp. The PCR product was purified, and to confirm its sequence, it was sent to the reference laboratory. The sequence was subjected to a BLAST search and then phylogenetically analyzed using CLC software. The results showed that all samples were nearly 100 % identical to each other and to strains CMS and M-96. These isolates also had 99 % and 95 % similarity to the CP-1 strain and isolate FIN/T2000, respectively. In Vero cell culture, inoculation with this virus caused a cytopathic effect (CPE), which appeared 2-5 days post-inoculation. Characteristic CPE showing foci of rounded cells, ballooning, giant-cell formation and syncytia with degenerative changes appeared.