Prevalence and antimicrobial susceptibility of Salmonella enteritidis and Salmonella typhimurium isolated from hen eggs and quail eggs in Karaj, Iran.

BACKGROUND AND AIM: Different Salmonella serotypes are considered one of the most important food pathogens in the world. Poultry meat and eggs are the primary carriers of Salmonella in human populations. This study aimed to estimate the Salmonella enteritidis and Salmonella typhimurium contamination rates of retail hen and quail eggs in Karaj, Iran. Moreover, the antimicrobial resistance patterns of the strains were evaluated, and the efficiency of the standard culture method and multiplex polymerase chain reaction (m-PCR) were compared. MATERIALS AND METHODS: In this descriptive cross-sectional study over 1 year (Jan-Dec 2022), 150 commercial and 150 backyard hen eggs and 300 commercial quail eggs, without cracks and fractures, were collected randomly from best selling groceries in Karaj city. All samples were examined for Salmonella contamination independently by standard culture and m-PCR approaches. A standard disc diffusion method was employed to assess the antimicrobial susceptibility of the strains against 18 antimicrobial agents. RESULTS: Out of 300 examined eggs, 2 S. enteritidis strains were isolated from the shell of backyard hen eggs. The same serotype was also detected in the contents of one of these two eggs. One S. typhimurium was isolated from the shell of a commercial hen egg. Overall, the Salmonella contamination of the shell and contents was 1% and 0.3%, respectively. Salmonella was not isolated from the eggshells or the contents of the quail eggs. There was complete agreement between the results of m-PCR and the standard culture methods. Among the 18 tested antibiotics, the highest resistance was recorded for colistin (100%), followed by nalidixic acid (75%). CONCLUSION: As most Salmonella spp. are associated with human food poisoning, continuous surveillance is required to effectively reduce the risk posed by contaminated poultry eggs. Furthermore, mandatory monitoring of antimicrobial use on Iranian poultry farms is recommended.

Evaluation of the Newcastle disease virus genotype VII-mismatched vaccines in SPF chickens: A challenge efficacy study.

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine-B1, Clone.12IR, and La Sota-or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms.

BACKGROUND: Multi-causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. OBJECTIVES: The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi-causal respiratory disease (MCRD) from 2017 to 2020. METHODS: Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. RESULTS: Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out-group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. CONCLUSIONS: The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.

Complete genome sequencing and molecular characterization of SARS-COV-2 from COVID-19 cases in Alborz province in Iran.

Iran was among countries which was hard hit at the early stage of the coronavirus disease 2019 (COVID-19) pandemic and dealt with the second wave of the pandemic in May and June 2020; however, there are a very limited number of complete genome sequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Iran. In this study, complete genome sequences of the virus in the samples obtained from three patients in Alborz province in May and June 2020 were generated and analyzed using bioinformatic methods. The sequenced genomes were positioned in a cluster with B.4 lineage along with the sequences from other countries namely, United Arab Emirates and Oman. There were seven single nucleotide variations (SNVs) in common in all samples and only one of the sequenced genomes showed the D614G amino acid substitution. Three SNVs, 1397 G > A, 28688T > C, 29742 G > T, which had already been reported in February, were found with high frequency in all the sequenced genomes in this study, implying that viral diversity reflected in the early stages of viral transmission in Iran were established in the second wave. Considering the importance of molecular epidemiology in response to ongoing pandemic, there is an urgent need for more complete genome sequencing and comprehensive analyses to gain insight into the transmission, adaptation and evolution of the virus in Iran.

Evolution of H9N2 avian influenza viruses in Iran, 2017-2019.

Since its first detection in 1998, avian influenza virus (AIV) subtype H9N2 has been enzootic in Iran. To better understand the evolutionary history of H9N2 viruses in Iran, we sequenced 15 currently circulating H9N2 viruses from domestic poultry during 2017-2019 and performed phylogenetic analysis of complete genome sequences. Phylogenetic analyses indicated that the Iranian H9N2 viruses formed multiple well-supported monophyletic groups within the G1-lineage of H9N2 virus. Our analysis of viral population dynamics revealed an increase in genetic diversity until 2007, corresponding to the multiple introductions and diversification of H9N2 viruses into multiple genetic groups (named Iran 1-4 subgroups), followed by a sudden decrease after 2008. Only the Iran 4 subgroup has survived, expanded, and currently circulates in Iran. The H9N2 viruses possessed many molecular markers associated with mammalian adaption in all gene segments, except neuraminidase gene. Considering the presence of mammalian host-specific markers, the public health threat of H9N2 viruses continues. Molecular analysis showed that Iranian H9N2 strains have continued to evolve and recent strains have multiple amino acid changes and addition of potential N-glycosylation on the antigenic sites of haemagglutinin. Continued antigenic and molecular surveillance of H9N2 viruses in poultry and mammals would be required to monitor further increments in viral evolution and their potential threat to public health.

Ongoing genetic evolution of H9N2 avian influenza viruses in Iranian industrial poultry farms.

Despite the use of wide-scale vaccination programmes against the H9N2 virus, enzootic outbreaks of H9N2 avian influenza (AI) have often occurred and caused serious nationwide economic losses, particularly in broiler chickens. In this study, the haemagglutinin (HA) and neuraminidase (NA) genes of nine recent H9N2s and a common vaccine strain were fully sequenced and compared with other representative Iranian viruses. Phylogenetic analysis revealed that all Iranian viruses were grouped into the G1 sub-lineage with different clusters in which recent isolates (2014-2017) formed a distinct cluster compared to the vaccine group (1998-2004). All Iranian H9N2s exhibited low pathogenicity AI connecting peptide feature with an R/KSSR motif. Amino acid 226, located in the 220 loop of the receptor binding site, was leucine among the recent Iranian viruses, a characteristic of human influenza viruses. With an overall gradual increase in the genetic diversity of H9N2s, Bayesian skyline plots of Iranian HA and NA genes depicted a fluctuation and a relative stable situation, respectively. It is recommended to apply constant surveillance to assess any increase in viral human adaptation and evolutionary changes in circulating field H9N2s. Moreover, antigenic characterisation of the prevailing H9N2 viruses seems to be necessary for evaluating the possible antigenic drift from the vaccine strain.

Complete Sequence-Based Genotyping of mgc2/pvpA Genes in Chicken-Derived Mycoplasma gallisepticum Isolates of Iran.

Mycoplasma gallisepticum (MG) is a major pathogen of the poultry industry throughout the world. MG causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite constant improvements in the biosecurity of the poultry industry in Iran, MG infection still occurs and causes significant economic issues. To evaluate genetic variability, 10 Iranian MG isolates along with 17 available sequences were characterized by gene-targeted sequencing (GTS) analysis of complete mgc2/pvpA genes. According to the findings, 21 different sequence types within the sample set of 27 strains were typed by this method. The discriminatory power of this typing assay was established to be 0.97. Although no insertions and deletions of nucleotides were observed in the mgc2 gene among the Iranian strains, different lengths of pvpA genes with 1086, 1095, and 1101 nucleotides were detected within direct repeats (DRs) 1 and 2. Generally, eight tetrapeptides Pro-Arg-Pro-Met/Gln/Asn were found in the DRs of PvpA. Analysis of the carboxyl ends of PvpA proteins exhibited various repeats of prolines. In the phylogenetic tree of partial and complete mgc2/pvpA genes, all Iranian MG isolates were clustered into two distinct groups. Because this typing assay could provide a higher discriminatory power than the previously reported GTS scheme of partial mgc2/ pvpA genes, these results can be considered a blueprint for future national control and diagnostic strategies. Furthermore, consistent surveillance with larger datasets will be needed to clarify the epidemiologic characteristics of MG outbreaks in different poultry hosts.

NDV subgenotype VII(L) is currently circulating in commercial broiler farms of Iran, 2017-2018.

BACKGROUND: Based on our previous work, it was discovered that some Newcastle disease virus (NDV) isolates from backyard poultry between 2011 and 2013 in Iran formed a new separate cluster when phylogenetic analysis based on the complete F gene sequence was carried out. The novel cluster was designated subgenotype VII(L) and published. AIM: In the current study, for further validation, we initiated a comprehensive epidemiological study to identify the dominant NDV genotype(s) circulating within the country. Collection of samples was executed between October 2017 and February 2018 from 108 commercial broiler farms which reported clinical signs of respiratory disease in their broilers. RESULT: We report that 38 of the farms (> 35%) tested positive for NDV. The complete F gene sequences of seven of the isolates are shown as representative sequences in this study. According to the phylogenetic tree constructed, the recent broiler farm isolates clustered into the newly designated cluster VII(L) together with the older Iranian backyard poultry isolates in our previous work. All the sequences shared the same virulence-associated F cleavage site of 112RRQKR↓F117. CONCLUSION: Our phylogenetic analysis suggested that the NDV subgenotype VII(L) may have been derived from subgenotype VIId, and contrary to popular belief, subgenotype VIId may not be the dominant subgenotype in Iran. Tracking of the subgenotype on BLAST suggested that the NDV subgenotype VII(L), although previously unidentified, may have been circulating in this region as an endemic virus for at least a decade. Other NDV genotypes, however, have also been reported in Iran in recent years. Hence, ongoing study is aimed at determining the exact dominant NDV genotypes and subgenotypes in the country. This will be crucial in effective mitigation of outbreaks in Iranian broiler farms.

Characterization of a novel VIIl sub-genotype of Newcastle disease virus circulating in Iran.

Newcastle disease is an economically important and highly contagious disease affecting wild and domestic avian species. Despite extensive vaccination efforts within the poultry industry, Newcastle disease virus (NDV) outbreaks causing significant economic losses still occur. Rural chickens may act as a potential reservoir of NDVs for commercial poultry due to poor biosecurity and inadequate vaccination. The aim of this study was to investigate the phylogenetic relationship and molecular characterization of eight NDVs isolated from backyard poultry in Iran during 2011-2013. The complete coding sequence of fusion (F) and haemagglutinin-neuraminidase (HN) genes of eight NDVs were determined and compared with other published NDVs. Based on inter-population distances and phylogenetic topology between available NDV categories, Iranian isolates formed a novel VIIl sub-genotype distinct from previous groups designated in genotype VII. Furthermore, both F and HN genes of the Iranian isolates shared high nucleotide sequence similarity with viruses isolated in China. All viruses analysed contained a polybasic cleavage site motif (111G/RRRQKR↓F117), indicating that all isolates could be categorized as a virulent pathotype. No mutation was observed in the neutralizing epitopes of the F protein. Analysis of amino acids associated with neutralizing antigenic sites within the HN protein revealed that all isolates exhibited a unique amino acid (Q) at position 347. These results emphasize the need for strengthening the biosecurity measures implemented on village flocks and practicing a mandatory vaccination programme for local poultry. Moreover, continuous monitoring of NDVs in different species of birds can help to gain more knowledge about the evolution of this virus and prevent future panzootics.

Phylogenetic analysis of neuraminidase gene of H9N2 avian influenza viruses isolated from chicken in Iran during 2010-2011.

BACKGROUNDS AND OBJECTIVES: Classified as low pathogenic avian influenza (LPAI) viruses, the H9N2 subtype causes severe respiratory disease in poultry farms and occasional respiratory disease in humans. In this study, the neuraminidase (NA) gene of three Avian Influenza (AI) H9N2 strains isolated from poultry farms in Iran during 2010-11, as well as other reported Iranian H9N2 isolates, were genetically analyzed and their nucleotide changes were evaluated. MATERIALS AND METHODS: The NA gene of three AIVs were sequenced and evaluated for genetic characteristics and phylogenetic relationship. RESULTS: One new potential glycosylation site (PGS) at amino acid position 306 was observed in one of the studied isolates (A/Chicken/Iran/N102/2011). Antigenic sites of NA in Iranian H9N2 isolates have varied in a yearly manner. The Iranian isolates can be divided into 2 main subgroups; 11-T like subgroup viruses isolated mainly during 1998-2004 and second subgroup viruses isolated during 2004-2009. Interestingly, the three studied isolates fell into a third subgroup. The nucleotide sequences of the three studied isolates showed high identity to recent Pakistani H9N2 isolates (94.5-97%) compared to former Iranian AIV isolates (89-94%). CONCLUSION: High frequency of substitutions in the NA gene of studied isolates in recent years and effects of those substitutions on the pathogenicity of AI virus highlight the need to continue surveillance of genetic characteristics of AIV H9N2 in Iran.

Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran.

Infection with avian influenza H9N2 virus is widespread in the Asian poultry industry, resulting in great economic losses due to mortality and a severe decline in egg production. To obtain more-comprehensive genomic data from circulating H9N2 viruses in Iran, we sequenced the whole genomes of early (Ck/IR/ZMT-101/98) and recent (Ck/IR/EBGV-88/10) isolates of this virus in Iran. The M and NS genes of Ck/IR/EBGV-88/10 shared a high level of similarity with a highly pathogenic H7N3 virus isolated from Pakistan. The cleavage site within the HA protein of these viruses contained two different motifs, RSSR and KSSR, which are similar to those found in low-pathogenic viruses. The deduced amino acid sequence of the new isolate contained the mutation Q226L, which is a characteristic of human-type sialic acid influenza receptor binding. An analysis of the viral amino acid sequence of the M2 protein of the recent strain revealed a V27A mutation, which is associated with amantadine resistance in avian influenza virus. The present results emphasize the need for continuous surveillance of H9N2 viruses in poultry and the human population to obtain more information about the nature and evolution of future pandemic influenza viruses.

Comparison of the activities of four antifungal agents in an in vitro model of dermatophyte nail infection.

BACKGROUND: Onychomycosis is a difficult condition to treat and cure rates are disappointing. Moreover fungicidal action of antifungal agents in NCCLS assays and their rapid accumulation in nails in vivo are not compatible with the duration of treatment. AIMS: This study aimed to find the effectiveness of 4 different antifungal agents in an in vitro model with some similarities to in vivo conditions. MATERIALS AND METHODS: Strains of Trichophyton rubrum I-III, Trichophyton mentagrophytes (usual form), Trichophyton mentagrophytes 73, Epidermophyton Flucosom, Microsporum Canis, and Trichophyton Schoenleini which were isolated from the nails of patients, were hired. Inocula suspensions were prepared from 7 to 14 day-old cultures of dermatophytes. Antifungal agents including fluconazole, ketoconazole, terbinafine, and griseofulvin were obtained as standard powders. For each antifungal agent, initial MIC was calculated by registering the optical density for 10 two-fold serially diluted forms which was incubated with diluted fungal suspensions with RPMI 1640. Human nail powder inoculated with different strains and incubated in RPMI 1640 and different concentrations of antifungal drugs for 4 weeks. Final MIC at different steps of 1, 2, 3 and 4 weeks were investigated. RESULTS: The final MIC that resulted from the incubation of dermatophytes with nail powder was much more than the initial which was concluded from conventional MIC assay. Terbinafine had the lowest rate of initial and final MICs. CONCLUSION: The model described here may present more similar conditions to clinical fungal infections; therefore the results such as MIC may be more helpful for hiring the most effective antifungal agent.