Veterinary Research Forum

Abstract The present study was planned to confirm the clinical diagnosis of canine distemper in dogs with reverse transcriptase polymerase chain reaction (RT-PCR) and its comparison with lateral flow assay based immuno-chromatographic (IC) technique. Fifty clients owned dogs having clinical signs suggestive of respiratory, skin or nervous form of canine distemper were included in the study. An immuno-chromatography-based test was applied using serum to screen each of the suspected dog. In addition to serum, nasal discharges of 15 cases and ocular discharges of 10 samples were used to diagnose canine distemper. Screening with IC kit revealed 72.00% serum samples positive, 66.66% ocular and 50.00% nasal samples were found positive for antigen. The RT-PCR targeting N gene of canine distemper virus was used for the molecular diagnosis of canine distemper. Out of 50 blood samples tested with RT-PCR assay, 38 (76.00%) samples were positive showing characteristics band of 287bp. Statistical comparison of IC Kit (serum samples) results with RT-PCR results comparison showed that IC kit was 52.00% accurate with 36.84% sensitivity, 100% specificity, positive predictive value of 93.33% and negative predictive value of 31.43%. In the current study it was observed that the IC test was rapid, quick and specific but was found to be less sensitive compared to RT-PCR.

Abstract In November 2021, an investigation was conducted into an outbreak of abortion, stillbirth, and the birth of calves with congenital abnormalities (arthrogryposis and hydranencephaly) at a dairy farm in Dasht-e-Mughan city, Ardabil province. A total of 70 cows experienced these issues. To determine the cause of the outbreak, post-mortem brain tissue samples were collected from two calves affected by hydranencephaly, which occurred shortly after their birth. Polymerase chain reaction (PCR) testing was conducted for multiple viruses, including bovine viral diarrhea (BVD), border disease, Akabane, Schmallenberg, and bluetongue viruses (BTVs). The samples were positive only for Akabane virus. Serum samples were collected from a group of 60 cattle, consisting of 45 adult cows and 15 younger calves aged between 8 to 10 months. These samples were analyzed to detect the presence of antibodies against the Akabane and Schmallenberg viruses. Both of these viruses are known to be responsible for causing abortion, stillbirth, and congenital abnormalities in calves. Among 45 cows that tested by competitive enzyme-linked immunosorbent assay (cELISA), 26.66% and 33.33% exhibited antibodies against Akabane and Schmallenberg viruses, respectively. Notably, 20.00% of cows co-exhibited antibodies for both viruses. Despite PCR evidence implicating Akabane virus as the principal etiology of clinical signs observed in the affected herd, the high co-seropositivity to Schmallenberg virus, warrants a thorough investigation into potential viral interactions. Further research is required to determine the source of the virus and their transmission routes. This information could facilitate the refinement of disease control strategies and improving the management of reproductive challenges in such affected herds.

Abstract The torsion model of testis in a rat was adopted for evaluation of possible effects of propolis (Prop) on ischemia-reperfusion (IS/REP) injury. The healthy male Wistar rats (totally 24 animals) were randomized into four groups (n = 6) and animals experienced bilateral testicular torsions as follows: In sham group just, laparotomy was performed and in IS group, animals experienced a 3 hr period testicular IS. In IS/REP group, a 3 hr period of IS followed by a 3 hr period of testicular REP for left testis and a one-week testicular REP for right testis were done. In this group animals were gavaged by 1.00 mL normal saline 1 hr before the onset of IS. In IS/REP/ Prop group, the same procedures for IS/REP animals were followed as well as gavage of 1.00 mL Prop extract solution 1 hr before the onset of IS. Analyses of biochemistry, histology, inflammatory biomarkers and sperm parameters were carried out. In IS/REP/Prop group, nitric oxide synthase malondialdehyde, myeloperoxidase and 8-hydroxy-2 deoxyguanine in IS/REP/Prop group were significantly decreased and, superoxide dismutase, total glutathione, glutathione peroxidase, glutathione reductase and glutathione S-transferase were significantly increased compared to the other animals. In IS/REP/Prop group, seminiferous tubules (with normal spermatogenesis) showed all stages of spermatogenic cells with plentiful spermatozoa. Tubular deterioration and atrophy and spermatogenic cell loss in were seen in a limited extent. The mean concentrations of Interleukin-1 beta and tumor necrosis factor alpha in IS/REP/Prop were significantly decreased. Sperm quality was significantly improved by Prop in IS/REP/Prop group. It was concluded that Prop could be supportive in diminishing IS/REP injury in testicular tissue exposed to ischemia.

Abstract Canine distemper virus (CDV) is responsible for high morbidity and mortality in dogs worldwide. Epidemiological study of canine distemper can help to control and treat the disease in any area. This study aimed to investigate the prevalence of CDV in dogs referred to the Veterinary Hospital from September 23, 2018 to September 22, 2019. Dogs with at least two clinical signs of canine distemper underwent blood tests, rapid test kit from the eye and cerebrospinal fluid (CSF), and RT-PCR from whole blood and/or CSF samples. Out of 1212 referred dogs, 112 dogs were suspected to have canine distemper of which 90 underwent RT-PCR and rapid test kits. The disease prevalence was 4.04% (49/1212) and 7.44% (49/659) according to the total number of referring dogs and number of referring sick dogs, respectively. The distemper fatality rate was 69.57% (32/46). Seventy percent of distemper positive cases were under 12 months old and 52.08% were under 6 months old. Female dogs were more susceptible than males; however, the fatality rate of males was more than females. Of distemper positive dogs, 91.84% were unvaccinated. The highest prevalence (71.43%) of dogs diagnosed with CDV occurred during the cold seasons. It is concluded that canine distemper is endemic in the geographical area of Mashhad and its prevalence rate in dogs referred to the Veterinary Hospital of Ferdowsi University of Mashhad is 4.04% and its fatality rate is 69.57%. This indicates that a significant number of dogs may die if they develop distemper despite treatment.

Abstract Histamine widely involves in local immune responses, physiological function in the gut, and acting as a neurotransmitter in the brain. Scientist also found the importance of histamine in the reproductive systems. The present study aimed to determine the existence of histamine receptor subtypes; H1R, H2R, H3R, and H4R on mouse oocytes through immunofluorescence (IF) staining and reverse transcription- polymerase chain reaction (RT-PCR). These further confirmed by the involvement of histamine receptor antagonists in in vitro fertilization (IVF). In IF staining, mouse oocytes were incubated with primary antibody against histamine receptor, followed by incubation with fluorescence conjugated secondary antibody. Then RT-PCR analysis was carried out for the undetected receptors during IF for confirmation. The RT-PCR used RNA extracted from mice COCs and cumulus free oocytes. In IVF, sperm was cultured in a group of treated histamine receptor antagonists oocytes. This investigation revealed the existance of H1R, H2R, and H3R on mouse oocytes in IF and RT-PCR analyses. The treatment of IVF with histamine receptor antagonists (H1R: pyrilamine; H2R: cimetidine; H3R: thioperamide) led to a significant reduction quantity of 2-cell embryos (4.61 ± 2.44%; 5.83 ± 4.65%; 3.83 ± 1.82%, respectively) as compared with the control group (22.50 ± 6.44%). Therefore, according to the results of this study, the presence of H1R, H2R, and H3R on mouse oocytes possibly will suggest the involvement of histamine in fertilization.

Abstract The extraction of intact RNA from oocyte is quite challenging and time-consuming. A standard protocol using commercial RNA extraction kit, yields a low quantity of RNA in oocytes. In the past, several attempts in getting RNA for gene expression study ended up with a few different modified methods. Extraction of high-quality RNA from oocyte is important before further downstream analyses such as reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, or northern blot analysis. In this review, the efficiency of RNA extraction methods from all species oocytes was compared between published articles and our research to gather all possible methods of RNA extraction. Two different methods of RNA extraction that were proposed from various experiments were reviewed to determine the best method of RNA extraction from the oocyte. Modified TRIzol method can be concluded as an efficient RNA extraction method especially for good RNA from oocytes. Meanwhile, comparing RNA extraction kits to extract the RNA from oocytes or pre-implantation embryos, the micro RNA extraction kit type is the best. Therefore, an appropriate RNA extraction method is important to obtain high quality of total RNA for gene expression profiling analysis.

Abstract Chronic bee paralysis virus (CBPV) is an unclassified polymorphic single-stranded RNA virus. Among the viruses infecting honeybees, CBPV is known to induce significant losses in honeybee colonies. In this study, a total number of eighty-nine suspected apiaries from four regions of Iran (including Mazandaran, Khorasan Razavi, Hormozgan, and Kurdistan) were sampled and submitted for molecular identification. Three positive samples were detected by RT-PCR. All positive samples were confirmed by sequencing. The phylogenetic tree which displays the molecular relationship between the viruses of different Iranian geographic regions and references isolates was constructed. The Iranian isolates formed two distinct phylogenetic groups (Group 1 and Group 2). The IR-CPV-GMG-1, IR-CPV-GMG-2, IR-CPV-GMG-4, and IR-CPV-GMG-6 formed Group 1 and IR-CPV-GMG-3, IR-CPV-GMG-5, and IR-CPV-GMG-7 were in Group 2 as a distinct group. Iranian isolates in group 1 were similar to European and East Asian CBPVs. This research was the first phylogenetic analysis of CBPV in Iran. Further researches are needed to study the other aspects of this virus-like genetic characteristics and pathogenesis in Iran.

Abstract The aim of this study was to provide information on the molecular characteristic and the phylogenic relationship of infectious bronchitis viruses (IBV) strains in Bushehr province in comparison to other strains reported in the Middle East. Samples were collected from broiler flocks in Bushehr province during 2014 - 2015. These flocks had respiratory problems such as gasping, sneezing and bronchial rales. A number of 135 tracheal swabs were taken from fifteen flocks (nine swabs per flock). Each three swabs collected from each flock were pooled in one tube (finally, we had three tubes for each flock). The samples were subjected to reverse transcription polymerase chain reaction (RT-PCR). The PCR products of positive samples were analyzed by sequencing of a (392 bp) segment of the spike gene and the related results were compared with the other IBV sequences in GenBank database. Samples from twelve farms (80.0%) were found to be positive. The viruses from seven farms (46.6%) were identified as field viruses closely related to variant 2. The viruses from three farms (20.0%) were characterized as Mass type and were related to vaccine strains. Two different IB viruses (variant 2 and Mass) were detected in samples from two farms (13.3%). The variant 2 genotype detected in Bushehr had high similarity to variant 2 reported from the Middle East.These variants displayed homologies ranging from 72.9% to 76.5%, and 78.8% to 80.0% with H120 and 4/91, respectively. It is necessary to design vaccination program of poultry farms using IBV strains circulating in the region.

Abstract Avian metapneumovirus (aMPV) causes diseases like rhinotracheitis in turkeys, swollen head syndrome in chickens and avian rhinotracheitis in other birds. Causing respiratory problems, aMPV adversely affects production and inflicts immense economic losses and mortalities, especially in turkey flocks. In recent years, several serological and molecular studies have been conducted on this virus, especially in poultry in Asia and Iran. The purpose of the present study was detecting and subtyping aMPV by reverse transcriptase polymerase chain reaction (RT-PCR) from non-vaccinated, commercial turkey flocks in Iran for the first time. Sixty three meat–type unvaccinated turkey flocks from several provinces of Iran were sampled in major turkey abattoirs. Samples were tested by RT-PCR for detecting and subtyping aMPV. The results showed that 26 samples from three flocks (4.10%) were positive for viral RNA and all of the viruses were found to be subtype B of aMPV. As a result, vaccination especially against subtype B of aMPV should be considered in turkey flocks in Iran to control aMPV infections.

Abstract Toxocara canis (Nematoda: Ascaridae) is an intestinal nematode parasite of dogs, which can also cause disease in humans. Transmission to humans usually occurs because of direct contact with T. canis eggs present in soil contaminated with the feces of infected dogs. This nematode has extraordinary abilities to survive for many years in different tissues of vertebrates, and develop to maturity in the intestinal tract of its definitive host. Survival of parasitic nematodes within a host requires immune evasion using complicated pathways. Morphine-like substance, as well as opioids, which are known as down regulating agents, can modulate both innate and acquired immune responses, and let the parasite survives in their hosts. In the present study, we aimed to find evidences of morphine-like substance and µ-opiate receptor expression in T. canis, using high performance liquid chromatography (HPLC) and reverse transcription polymerase chain reaction (RT-PCR). The results indicated that T. canis produced morphine-like substances at the level of 2.31± 0.26 ng g^-1 wet weight, and expressed µ-opiate receptor as in expected size of 441 bp. According to our findings, it was concluded that T. canis, benefits using morphine-like substance to modulate host immunity.

Abstract Rapid detection and differentiation of infectious bronchitis virus (IBV) involved in the disease outbreak is very important for controlling disease and developing new vaccines. In the present study, three regions of the genome of IBV vaccine and field isolates including S1 gene, gene 3 and nucleocapsid (N) gene along with 3' untranslated region (3' UTR) were amplified and subjected to restriction fragment length polymorphism (RFLP) using three different endonucleases. Amplicons from S1 gene and N-3’UTR generated four RFLP patterns, grouping IBV strains into four similar groups, while amplicons of gene 3 generated three RFLP patterns classifying examined IBVs in different groups from those of S1 and N-3' UTR. 4/91 strain and MNS-7862-1field isolate both belong to 793/B serotype were differentiated from each other based on gene 3, N-3’UTR and S1gene. IBVs belonged to different serotypes showed different RFLP patterns based on RFLP patterns of all three regions. S1 gene and N-3’UTR RFLP analysis differentiated IB88, MNS-7862-1 and 4/91 from each other. This is the first report on the molecular analysis of the gene 3 for IBV strain differentiation. Our results revealed that RFLP analysis of N-3’UTR and S1 gene had the higher discriminatory power than gene 3. None of the RFLP patterns of different regions differentiated 4/91 vaccine strain from its field isolate.