Veterinary Research Forum

Abstract Physalopteridae nematodes pose a threat to a broad spectrum of animals including amphibians, reptiles, birds and mammals. The current study was the first molecular and phylogenetic characterization of Physaloptera clausa parasitizing long-eared hedgehogs (Hemiechinus auritus) in Iran. A male road-killed H. auritus was collected from Shahriar City, Tehran province in May 2022 and it was subjected to necropsy. After isolating parasites, they underwent morphological analysis using a light microscope and an identification key. For molecular analysis, the genomic DNA was isolated using the FavorPrepTM Tissue Genomic DNA Extraction Mini Kit. The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. For phylogenetic analysis, these nucleotide sequences were aligned in MEGA 11 based on the lowest bayesian information criterion score. A cluster of parasites was found on the mucosa of the hedgehog stomach. All the nematodes were identified as P. clausa. The sequence obtained in this study has been submitted to GenBank^® with the accession number OR088573. The phylogeny analysis revealed that the genus Physaloptera formed a major clade where P. clausa was clustered with various Physaloptera species, closely related to Turgida, Physalopteroides and Skrjabinoptera genera. Our study specified the necessity for additional taxonomic and phylogenetic research on Physaloptera species and related genera to fully understand the evolutionary dynamics and ecological significance of these parasites.

Abstract This study was conducted in West Azerbaijan province, Iran (37°27'18.022" N, 45°0'0" E) to investigate the genotyping and phylogenetic characterization of Mannheimia haemolytica in cattle and buffaloes from November 2022 to January 2024. Mannheimia haemolytica is a bacterium known to cause pasteurellosis pneumonia, a respiratory disease in ruminants, such as cattle and sheep. This is one of the main causes of economic losses in the feedlot industry. In addition to the deaths, treatment costs are also significant. The lung and nasal swab samples were collected from 378 cattle and buffaloes. The M. haemolytica was detected in 32 (8.46%) of the samples, with a notably higher isolation rate from lung tissue (56.25%; n = 18) compared to the nasal swabs (43.75%; n = 14). Interestingly, the study also revealed a seasonal pattern, with the highest isolation rates observed during January, February, and March. Multi-locus sequence typing demonstrated that all isolates belonged to sequence type 1 (ST1) within clonal complex 28. This finding is consistent with the global prevalence of ST1 in bovine isolates, indicating widespread distribution. Phylogenetic analysis revealed a strong correlation between ST1 and STs 30 and 54, highlighting the prevalence of ST1 in M. haemolytica among ruminants in West Azerbaijan, Iran. Further research is needed to investigate its potential for causing disease and its transmission pattern.

Abstract Fowl adenovirus (FAdV) is a DNA virus causing significant diseases, like inclusion body hepatitis, hydropericardium-hepatitis syndrome (HHS), and gizzard erosion. These diseases lead to severe economic losses in the poultry industry. Recent increases in HHS outbreaks in Iran, particularly among broilers, prompted this study to analyze FAdV isolates in Kashan, Iran. In December 2021, a high-mortality HHS outbreak in a Kashan broiler flock led to liver and heart samples being sent for analysis. Histopathological investigations revealed mononuclear hepatitis and intra-nuclear viral inclusion bodies in hepatocytes. Polymerase chain reaction and phylogenetic analyses confirmed the presence of FAdV-4 (accession number: PP856395), showing 99.99% identity with strains from Japan, the United Arab Emirates, Pakistan, and the United States. These findings highlight the genetic similarity and potential common origin of FAdV-4 strains. This study emphasizes the need for heightened biosecurity measures and effective vaccination strategies to mitigate the spread of FAdV-4. The confirmed presence of FAdV-4 in central Iran poses a significant threat to the poultry industry, necessitating prompt action to prevent substantial economic losses.

Abstract African swine fever (ASF) is considered as one of the most threatening diseases for the pig farming industry all over the world. Due to the lack of an effective vaccine, organized farms and backyard rearing must strictly enforce control measures in order to combat the disease. The present report describes the ASF epidemic in a piggery in Uttar Pradesh state, India. The pathological samples were collected from the affected pigs and processed for histopathological and molecular studies. Gross lesions comprised of cyanosis of ear pinna, multi-focal hemorrhagic spots on ventral abdomen and inner aspect of thigh, highly congested mesenteric lymph nodes with marbling, marked congestion, hemorrhages and splenomegaly, interstitial pneumonia, and multi-focal endocardial hemorrhages on papillary muscles and wall of ventricle in heart. Histopathological investigation revealed marked congestion and hemorrhages of mesenteric lymph node, liver and spleen. Depletion of lymphocytes from the splenic white pulp was visible in the splenic parenchyma. The virus was confirmed by polymerase chain reaction and phylogenetic analysis revealed a distinct clustering of the Uttar Pradesh virus isolates from Vietnam with other Ib group isolates, indicating a close genetic relationship between these samples. Additionally, the mutant Chinese virus isolate showed clear genetic differences with the Vietnamese Ib group, confirming its suitability as an out-group for comparison. The study represents the first report of ASF outbreak in North India, establishing the phylogenetic relationship between ASF virus circulating in the study area and other regions.

Abstract Bovine clinical mastitis is an economically important disease in dairy industry worldwide resulting in reduction of milk yield and quality. Among mycotic mastitis, Candida spp. are commonly occurring opportunistic mycosis in immunocompromised animals. The micro-organism’s causing mastitis has high zoonotic potential and has been linked with rapid growth and introduction of antimicrobial resistance between animals and humans. The present study was conducted to isolate and identify the common pathogenic Candida spp. from bovine mastitis cases in India. The isolates were phenotypically characterized by culturing on Sabouraud’s dextrose agar, Hichrome Candida differential agar and germ tube production test. Antibiogram was also performed to determine their antifungal activities. The phenotypically positive isolates were confirmed by polymerase chain reaction (PCR) and genetically analyzed by targeting 18S-ITS1-5.8S-ITS2-28S region specific for Candida spp. and identified the yeast at the species level. The antibiogram showed the isolates were highly sensitive with ketoconazole, clotrimazole and miconazole. The PCR assay identified C. lusitaniae and C. tropicalis based on the two distinctive amplicon sizes (592bp and 737bp) respectively. Also, the sequence analysis and phylogeny confirmed C. lusitaniae in six sequences and C. tropicalis in one sequence. It is worth noting that in this study, the species identification was consistent among PCR and genetic analysis. Therefore, the PCR based identification system of the fungal species performed in this study could be an efficient and time saving tool for early diagnosis of clinical mastitis in milch animal, which allows prompt control and application of speedy effective treatment.

Abstract An internationally recognized syndrome that leads to deaths among domestic and ornamental pigeons, particularly after racing, is young pigeon disease syndrome (YPDS). Pigeon circovirus (PiCV) is regarded as one of the potential factors contributing to the occurrence of YPDS. This survey was conducted to determine the prevalence of PiCV infection and molecularly characterize the PiCV in pigeons suspected of YPDS. Eighty fecal samples were collected from 80 diseased pigeons (exhibiting symptoms such as lethargy, weight loss, crop stasis, vomiting and diarrhea) from 20 lofts in different areas of Ahvaz, Iran. Also, 20 fecal samples were obtained from 20 clinically healthy pigeons. The nested broad spectrum polymerase chain reaction test was done to identify the circovirus, using primers targeting part of the replication-associated protein gene with 350 bp, and several positive samples were sequenced. This study showed that PiCV was detected in 86 out of the 100 samples (86.00%). Two types of circoviruses were determined in the samples. One type of the detected circoviruses was PiCV which based on phylogenetic analysis had high genetic similarity with A, B, G and H genotypes of PiCV. The other type of detected circoviruses was closely related to beak and feather disease virus (BFDV) which causes one of the most significant viral diseases in psittacine birds. This is the first report of BFDV identification in pigeons.

Abstract Histoplasma capsulatum is a dimorphic fungus that is traditionally classified in three varieties: Hc var. capsulatum, Hc var. duboisii, and Hc var. farciminosum (HCF). Cytology, hematology, pathology, polymerase chain reaction (PCR), sequencing, and phylogenetic analyses were applied on samples collected from the blood and the eye of a horse with pustular lesions and ocular discharge. Physical examination and cytopathological tests showed H. capsulatum infection. Additionally, the results of two PCR tests confirmed H. capsulatum infection. The phylogenetic tree of the internal transcribed spacer sequence of Iranian H. capsulatum showed homology with the HCF variety. For the first time, H. capsulatum infection in the eye of a horse from Iran was detected and phylogenetically analyzed. This study revealed that H. capsulatum could establish infection in Iranian animals in addition to people, and indicated the role of soil enriched with bird dropping in the preparation of a favorable environment for H. capsulatum propagation. Further investigations are required to clarify the natural history and risk factors associated with histoplasmosis in Iran.

Abstract Fowl adenoviruses (FAdVs) associated with inclusion body hepatitis (IBH) was identified in commercial broiler chickens in Palestine. Investigated birds showed primary clinical signs and lesions of IBH including growth retardation, congested and enlarged liver with necrosis, petechial hemorrhage and basophilic intra-nuclear inclusion bodies. The mortality rate was from 15.00%. The FAdV was detected and sequenced in the liver samples of infected chicken by polymerase chain reaction using hexon gene-specific primers. Phylogenetic analysis revealed that FAdVs belong to FAdV-D serotype 10, clustered within the European highly pathogenic isolates. The highest nucleotide sequence similarity was 99.48% with highly pathogenic FAdV-D serotype 10 detected from infected chicken in Poland (GenBank: LN907532.1) and FAdV-D from infected chicken in Sweden (GenBank: HE961828.1). The lowest similarity was 93.46% with Canadian FAdV-D (GenBank: EF685576.1). In conclusion, this is the first report describing the presence of IBH revealing that the causative virus is closely similar to the highly pathogenic FAdV-D serotype 10 of IBH in broiler chickens in Palestine.

Abstract Foot-and-mouth disease (FMD) is endemic in Iraq. The current study can be considered as the first molecular characterization of serotype O in Iraq. The present investigation reported the determination of FMDV serotype O from local farms in Sulaimani districts in 2016 outbreaks. Samples were collected from suspected cattle. The virus was primarily detected with RT-PCR directly from mouth epithelial samples. The direct sequencing and subsequent analysis of amplified PCR products for the VP1 gene indicated the circulation of serotype O of FMD in studied areas. Moreover, phylogenetic analysis revealed that the field isolated serotype O belonged to topotype ME-SA and lineage PanAsia II and clustered with Pakistan and Iran isolates (KU365843 and KY091283) with identity (96.00%, 95.00%) respectively. Furthermore, according to the phylogenic tree, the field isolates had different lineage with the three O/Manisa vaccine strains and Iraq/2000 strains. These findings highlighted the continuous circulation of serotype O of FMD in the region.

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 Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees’ gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products.

Abstract Canine distemper virus (CDV) creates a very contagious viral multi-systemic canine distemper (CD) disease that affects most species of Carnivora order. The virus is genetically heterogeneous, particularly in section of the hemagglutinin (H) gene. Sequence analysis of the H gene can be useful to investigate distinction of various lineages related to geographical distribution and CDV molecular epidemiology. Since vaccination program is conducted only in large cities of Iran, CD still remains as one of the major causes of death in dogs in this country. In order to monitor H gene, CDV has been detected in 14 out of 19 sampled dogs through the amplification of nucleoprotein (NP) gene in nested-PCR assay. In the next step 665 bp of H gene was amplified in 9 out of 14 NP-gene positive dogs. Phylogenetic analysis distinguished two distinct CDV genotypes in Iran. JN941238 has been embedded in European cluster and JN941239 has been embedded in Arctic cluster. Nucleic analysis has been shown high difference among both Iranian CDV lineages with CDV vaccine strains.

Abstract Despite routine vaccination programs against Newcastle disease (ND), sporadic cases have occasionally occurred that remain a constant threat to commercial poultry. Ten isolates of Newcastle disease viruses (NDV) from infected broiler chicken cases were obtained from various locations in Fars province during 2009-2011 and genetically analyzed using reverse transcription polymerase chain reaction (RT- PCR) with primers specific to the viral fusion (F) protein- gene. The viruses were confirmed as NDV by hemagglutination inhibition assay and RT- PCR. The isolates based on the sequence and phylogenetic analyses of partial F gene were genotypically analyzed by RT PCR. In the present investigation, the pathogenicity of NDV strains was determined by internationally recognized test mean death time (MDT). Analysis based on F gene showed that characterized isolates possess three different types of protease cleavage site motifs and appear to show maximum identities with isolates in the region. The subsequent phylogenetic analysis was implemented using MEGA and the phylogenetic tree. The results of RT-PCR and MDT showed that 10 isolates were positive for NDV, (60% velogenic, 30% mesogenic and 10% lentogenic). The results of the phylogenetic analysis showed that 10 NDV isolates from Iran belong to the class II, genotype III viruses. This information is fundamental to improve the efficacy of controlling strategies and vaccine development for NDV.