Genetic analysis of H9N2 avian influenza viruses circulated in broiler flocks: a case study in Iraq in 2014-2015.

H9N2 avian influenza viruses (AIVs) have been recorded in Eurasian for several years. Since 2004-2005, the disease has become endemic in Iraq, causing serious economic losses in the poultry industry. The hemagglutinin (HA) and neuraminidase (NA), two out of eight protein-coding genes, play an important role during the early stage of infection and hinder virus assembling. Little is known about the genetic information of the H9N2 viruses currently circulating in Iraq; thus, gene sequences of six AIVS of the H9N2 subtype have been detected and analyzed in the period of 2014-2015 from different outbreaks of broiler flocks in five provinces situated in the middle and southern parts of Iraq. Genetic comparison of the partial sequences of HA gene indicated that all Iraqi viruses are related to each other and could be divided into two subgroups. Viruses of the first and the second subgroups demonstrated a high similar identity with Pakistani and Iranian viruses, respectively. The nucleotide sequences of the NA protein of the all studied Iraqi viruses were very similar (95.2-100% identity), and shared high nucleotide sequence identity with Iranian, Pakistani, and Lebanese strains. All six recent viruses possessed histidine, alanine, and leucine at positions 183, 190, and 226, respectively, which are the key residues in receptor-binding sites. The Iraqi viruses were closely related to viruses of G1-like lineage isolated from poultry flocks of Iran and Pakistan, suggesting that possible epidemiological links could be derived from a common origin. Further investigations are required and should include the viral isolation and full-length molecular characterization of H9N2 AIVs in this area.

Role of CD271 enrichment in the isolation of mesenchymal stromal cells from umbilical cord blood.

Isolation of mesenchymal stromal cells (MSCs) from the umbilical cord blood (UCB) has a success rate of 25% and is frequently contaminated by osteoclast-like cells (OLCs). CD271 is a well-known marker for the enrichment of bone marrow (BM) MSCs. We have assessed the effect of CD271 isolation on the isolation rate of MSCs from UCB. Twenty-one samples of UCB were collected. Ten samples of UCB and five of BM underwent CD271 isolation using magnetic activated cell sorting. The other 11 UCB samples were used as the control. The isolated cells were cultured and MSC isolation was confirmed with respect to morphology, flow cytometry, adipogenic and osteogenic differentiation potentials. CD271-positive UCB cells did not show outgrowth despite 54.5% MSCs isolation in the non-enriched portion. No OLC was noted in the CD271-enriched group, but 66% of the non-enriched samples were contaminated. All the CD271-positive BM cells formed MSC colonies. Although the per cent of CD271+ cells showed no difference between BM-mononuclear cells (MNCs) and UCB-MNCs, the haematopoietic marker, CD45, was found in a higher percentage of CD271-positive UCB-MNCs. The results of our study indicate that, although CD271 is a valuable marker for enrichment of MSCs from BM, it does not contribute to isolation of MSCs from UCB. In this source, most of the CD271+ cells are from haematopoietic origin, and possibly the process of isolation may eliminate the very low frequent MSCs and the isolation therefore fails.

High prevalence of antibiotic resistance and biofilm formation in Salmonella Gallinarum.

Background and Objectives: Antibiotic resistance is an indicator of the passively acquired and circulating resistance genes. Salmonella Gallinarum significantly affects the poultry food industry. The present study is the first study of the S. Gallinarum biofilm in Iran, which is focused on the characterization of the S. Gallinarum serovars and their acquired antibiotic resistance genes circulating in poultry fields in central and northwestern Iran. Materials and Methods: Sixty isolates of S. Gallinarum serovar were collected from feces of live poultry. The bacteria were isolated using biochemical tests and confirmed by Multiplex PCR. Biofilm formation ability and the antibacterial resistance were evaluated using both phenotypic and genotypic methods. The data were analyzed using SPSS software. Results: According to Multiplex PCR for ratA, SteB, and rhs genes, all 60 S. Gallinarum serovars were Gallinarum biovars. In our study, the antibiotic resistance rate among isolated strains was as follows: Penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). All isolates were susceptible to imipenem, ertapenem, ceftriaxone, ceftazidime, and ceftazidime+clavulanic acid. All sixty isolates did not express the resistance genes IMP, VIM, NDM, DHA, blaOXA48, and qnrA. On the other hand, they expressed GES (85%), qnrB (75%), Fox M (70%), SHV (60%), CITM (20%), KPC (15%), FOX (10%), MOXM (5%), and qnrS (5%). All S. Gallinarum isolates formed biofilm and expressed sdiA gene. Conclusion: Considering that the presence of this bacteria is equal to the death penalty to the herd, the distribution of resistance genes could be a critical alarm for pathogen monitoring programs in the region. This study showed a positive correlation between biofilm formation and 50% of tested resistance genes. Also, it was found that the most common circulating S. gallinarum biovars are multidrug-resistant.

Molecular detection of SARS-CoV-2 in domestic cats in close contact with positively-infected owners in Tehran, Iran in 2021.

Objectives: In 2019, COVID-19 emerged in China and has since spread worldwide. Owing to the virus's ability to adhere to specific receptors, cats are susceptible to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The popularity of pet cats in Iran has sparked fears of human-cat-human transmission of the virus. This study aimed to identify positive cases in cats owned by people infected with SARS-CoV-2, to determine if they remained positive for >3 weeks and to examine the virus genome isolated from a number of cats and one of their owners. Methods: A total of 30 cats were sampled approximately 3 days after their owners tested positive (day 1), and 3 weeks later, in strict accordance with health regulations. Rectal and oropharyngeal samples were collected. All samples were subjected to a qualitative PCR and reverse transcription PCR. The S-gene region was partially sequenced in positive samples and the results were used to create a phylogenetic tree. Results: SARS-CoV-2 was detected in 7/30 (23.3%) cats examined. In the third week, every cat tested negative. The sequence data of positive cats and one of their owners revealed that the retrieved RNAs belonged to the alpha variation. The genetic distance between the samples and the reference sequence (20I/B.1.1.7: OM003849, MZ344997) was minimal, with a 99% similarity. Positive samples of cats had four mutations in gene S. Amino acid substitutions in the spike glycoprotein at positions N501Y, A570D, D614G and P681H were recorded in the isolates compared with 780 other sequences of Iranian strains. Conclusions and relevance: This study confirmed the presence of SARS-CoV-2-infected cats living in close contact with infected owners. Despite cats' susceptibility to COVID-19, the risk of severe infection in these animals is low, as evidenced by the lack of clinical signs in positive cats.

Investigation of the target genes of BLV miRNAs and the expression levels of miR-B4-3p and miR-B2-5p in cattle infected with Bovine Leukemia Virus.

Bovine Leukemia Virus (BLV) is an oncogenic retrovirus of the genus Deltaretrovirus. The genome of BLV encodes a cluster of 10 mature microRNAs (miRNAs). Considering the importance of miRNAs in regulating gene expression, it seems that each of the miRNAs of BLV plays a vital role in the process of pathogenesis and tumorigenesis of the virus. First, sequences of each of the miRNAs of BLV were selected and downloaded from the miRBase database. The sequences were then investigated using TargetScan and miRWalk to identify target genes of each of the mature miRNAs of the virus. Second, the expression levels of the two miRNAs with the highest number of target genes in B lymphocytes and lymphoid tissues were evaluated using qPCR and were compared between cattle with different forms of BLV infection: PL form was compared to aleukemic (AL) form (Group 1) and BLV+ with normal lymph nodes were compared to lymphosarcoma form (Group 2). We identified a total of 1595 target genes of the micro RNAs. The miRNAs with the highest target genes included miR-B4-3p with 760 and B2-5p with 102 target genes. In the second phase, miRNA expression in BLV-infected animals was investigated. The Fold Change (FC) values for miR-B4-3p and miR-B2-5p in group 1 were 22 and 67, respectively. In the second group, the FCs for miR-B4-3p and miR-B2-5p were 47 and 133, respectively. The expression was significantly higher in persistent lymphocytosis (PL) cattle in group one and lymphosarcoma cattle in group two.

Molecular characterization of a pigeon paramyxovirus type 1 virus isolated from Eurasian collared doves in Iran, 2017.

In September 2017, an outbreak with high mortality, which showed the typical signs of ND, occurred among a flock of more than 2000 Eurasian collared doves in Konarak, southeast of Iran. A confirmed pigeon paramyxovirus type 1 strain was isolated from the brain tissues of the dead doves. The isolate, which was called Pigeon/Iran/Konarak/Barin/2017, was classified as a highly velogenic NDV. Complete genome sequencing and phylogenetic analysis showed that the isolate belonged to subgenotype XXI.2, which has never been reported from Iran before. The isolate had the highest homology (96.15%) with early 2010s Italian isolates. Further studies will be required to understand the diversity better.

Complete genome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) in Iran.

OBJECTIVE: Trueperella pyogenes has been considered a major causative agent of metritis, abortion, and death in a broad range of domestic and wild animals, including cattle, swine, sheep, goats, camels, buffalo, deer, antelopes, reptiles, and birds. DATA DESCRIPTION: Here, we report the complete chromosome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) died due to the infection caused by this pathogen. The genome assembly comprised 2,338,282 bp, with a 59.5% GC content. Annotation of the genome showed 46 tRNA genes, 6 rRNA, 1 CRISPR and 2059 coding sequences. Also, several genes coding for antimicrobial resistance such as tetW and virulence factor including plo, nanH, nanP, cbp and 4 fimbrial proteins were found. This study will advance our knowledge regarding the metabolism, virulence factors, antibiotic resistance and evolution of Arash114 strain and serve as an appropriate template for future researches.

Pathogenicity study of Iranian genotype of avian infectious bronchitis virus (IR-1).

Avian infectious bronchitis (IB) is a major cause of economic losses in poultry industry. The IB virus primarily affects respiratory tract, but various strains differ in their tropism for other target organs such as kidney and alimentary tract. The objective of this study was to estimate the pathogenicity of Iranian IBV variant (IR-1), which is limited exclusively to Iran. Specific pathogen free chicks were inoculated intranasally. Sera, fecal swabs and different tissue samples were collected on different days post infection (DPI). Clinical signs, gross pathology and histological changes were recorded. The viral load was quantified in the RNA extractions from different tissue samples using real-time PCR. Anti-IBV antibodies were detected in serum samples. The IgG antibody were found on 21 and 28 DPI. Severe histological lesions were observed in the trachea and lung while the lesions in kidney were appeared to be milder. Viral RNA was detected in all tested tissues from 1 DPI to the last day of the experiment. The highest viral load was measured in the trachea and feces on 1st and 5th DPI, respectively. It can be concluded the IR-1 had broad tropism for respiratory tract, digestive system, and renal tissue, reflecting its epitheliotropic nature, but it caused the most severe lesions in the respiratory tract. This was the first pathogenicity study of Iranian IR-1 IBV. Further knowledge of IBV pathogenesis provides the groundwork to inform more effective prevention practices.

Molecular identification and phylogenetic analysis of chronic bee paralysis virus in Iran.

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.

The effect of Allium sativum (Garlic) extract on infectious bronchitis virus in specific pathogen free embryonic egg.

OBJECTIVE: Garlic is a plant has been used as a flavor, and anti-microbial and anti-diarrheal agent. Infectious bronchitis virus (IBV) is a coronavirus. The available vaccines against IBV cannot cover new variants. This study evaluated the inhibitory effects of garlic extract on IBV. MATERIALS AND METHODS: The constituents of garlic extract were detected by gas chromatography. This study was done in four groups of embryonic SPF eggs; first group was used for virus titration; second group received the mixture of different virus titration and constant amount of garlic extract; third group received 10(-3) titration of virus and after 8 hr received garlic extract and the last group received different dilutions of garlic extract. RESULTS: Based on our results, in the second group, IBV vaccine strain (4/91) at all titration and M41 in 10(-2) and 10(-3) titration and in the third group both variants of virus the embryonic Index (EI) was significantly increased. CONCLUSION: The garlic extract had inhibitory effects on IBV in the chickens embryo.

Prevalence of avian infectious bronchitis virus in broiler chicken farms in south of Iraq, 2014 - 2015.

Avian infectious bronchitis (IB), caused by a gammacoronavirus, is an OIE-listed (List B) disease and characterized by respiratory and renal involvements, causing high mortality, and economic loss in both layers and broilers. In comparison with other diagnostic methods, real-time polymerase chain reaction (RT-PCR) and conventional RT-PCR are potent, more sensitive and faster techniques for infectious bronchitis virus (IBV) detection. This research was conducted to detect IBV using specific primers of IB in three governorates (Basra, Thi-Qar and Muthana) in the south of Iraq. Tracheal specimens were collected from 46 IB suspected commercial broiler flocks. XCE2+ and XCE2- Primers, which amplify all IBV serotypes, were used. Primers MCE1+, BCE1+ and DCE1+ were used to amplify the specific nucleotide sequences of Massachusetts, 793/B and D274 genotypes, respectively. The results of real-time RT-PCR of this study showed that 34 (74.00%) out of 46 infected flocks were positive to IBV. The results of nested PCR showed that 50.00% and 5.89% of positive samples were belonged to genotypes 793/B and Massachusetts, respectively, and the remaining positive (44.11%) were unknown. The results indicate presence of Massachusetts and 793/B IBV strains in commercial broilers in southern Iraq.

Pathogen and Host Response Dynamics in a Mouse Model of Borrelia hermsii Relapsing Fever.

Most Borrelia species that cause tick-borne relapsing fever utilize rodents as their natural reservoirs, and for decades laboratory-bred rodents have served as informative experimental models for the disease. However, while there has much progress in understanding the pathogenetic mechanisms, including antigenic variation, of the pathogen, the host side of the equation has been neglected. Using different approaches, we studied, in immunocompetent inbred mice, the dynamics of infection with and host responses to North American relapsing fever agent B. hermsii. The spirochete's generation time in blood of infected mice was between 4-5 h and, after a delay, was matched in rate by the increase of specific agglutinating antibodies in response to the infection. After initiating serotype cells were cleared by antibodies, the surviving spirochetes were a different serotype and, as a population, grew more slowly. The retardation was attributable to the host response and not an inherently slower growth rate. The innate responses at infection peak and immediate aftermath were characterized by elevations of both pro-inflammatory and anti-inflammatory cytokines and chemokines. Immunodeficient mice had higher spirochete burdens and severe anemia, which was accounted for by aggregation of erythrocytes by spirochetes and their partially reversible sequestration in greatly enlarged spleens and elsewhere.

Molecular and clinical study on prevalence of feline herpesvirus type 1 and calicivirus in correlation with feline leukemia and immunodeficiency viruses.

Upper respiratory tract diseases (URTD) are common clinical problem in cats worldwide. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the main primary pathogens. Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) are also among the most common infectious diseases of cats which suppress the immunity. Oropharyngeal and conjunctival swabs and blood samples were taken from 16 cats with clinical signs of URTD and 26 clinically healthy cats. PCR and RT-PCR were used to detect FHV/FIV or FCV/FeLV infections, respectively. Feline calicivirus was detected in all cats with URTD and 87.00% and 93.00% of them were positive for FIV and FeLV, respectively. Feline herpesvirus rate of infection was 43.00% in sick cats. In clinically normal cats, prevalence rates of FCV and FHV were about 50.00%, but FIV and FeLV rates (42.00% and 65.00% respectively) were higher compared to other studies. Stomatitis was observed in 50.00% of cats with URTD. The main causative agent of corneal ulcers is FHV-1, but in 50.00% of cats with corneal ulcers, FCV was detected alone. It seems new variants of Caliciviruses are the main causative agents to attack uncommon tissues like cornea, although retroviral infections may be in the background of these various signs. The high retroviral prevalence may be due to existence of large population of stray cats. This is the first molecular study of FeLV and FCV in Iran and seems that FCV and FHV prevalence rates in FIV or FeLV infected cats is more than other non-infected ones.

Chromosome Sequence of Borrelia miyamotoi, an Uncultivable Tick-Borne Agent of Human Infection.

Borrelia miyamotoi is a newly recognized agent of human disease. B. miyamotoi strain LB-2001, an isolate from the tick Ixodes scapularis, was propagated in mice. The sequence of the chromosome was determined by next-generation sequencing of DNA isolated from whole blood. The sequence established that B. miyamotoi is a relapsing fever group species.

Full-length characterization and phylogenetic analysis of hemagglutinin gene of H9N2 virus isolated from broilers in Iran during 1998-2007.

H9N2 avian influenza A viruses (AIV) have become panzootic in Eurasia over the last decade and are endemic in Iran since 1998, and inactivated vaccine has been used in chickens to control the disease. The hemagglutinin (HA), one of eight protein-coding genes, plays an important role during the early stage of infection. To study their evolution and zoonotic potential, we conducted an in silico analysis of H9N2 viruses that have infected broiler in Tehran Province, Iran between 1998 and 2007. The complete coding region of HA genes from nine H9N2 subtypes isolated from chicken flocks in Tehran Province during 1998-2007 was amplified and sequenced. Sequence analysis and phylogenetic studies of H9N2 subtype viruses on the basis of data of 9 viruses in this study and 30 selected strains are available in the GenBank. Sequence and phylogenetic analyses revealed a large number of similar substitution mutations and close evolutionary relation among sequences of HA. The isolates possessed two types of amino acid motif -R-S-S-R/G-L- and -R-S-N-R/G-L- at the cleavage site of HA. The results showed that all nine representative H9N2 isolates belong to low pathogenic AIVs since none of the amino acid sequences at the cleavage site of the HA of the isolates possessed the basic motif required for highly pathogenic viruses (R-X-R/K-R). Six out of these nine isolates possessed leucine at position 226, which prevails in the sequences found in human strains. Phylogenetic analysis showed that all our isolates belonged to the G1-like sublineage. Also, these isolates showed some degree of homology with other H9N2 isolates, e.g., 89.46-93.93.39% with qu/HK/G1/97 and 93.39-98.39% with pa/Narita/92A/98. The available evidence indicates that HA genes of H9 influenza virus circulating in Iran during the past years were not well conserved. Our finding emphasizes the importance of reinforcing AIV surveillance, especially after the emergence of high pathogenicity in poultry in Iran.

Elevated carbon monoxide in the exhaled breath of mice during a systemic bacterial infection.

Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ∼10(7) per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8-5.6), when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management.