Molecular detection and analysis of beak and feather disease viruses in Iran.

The beak and feather disease virus (BFDV) is one of the few pathogens capable of causing extinction of psittacines. To determine the prevalence and the nature of BFDV mutation, this study investigated the presence of the BFDV among 1,095 individual birds of the 17 psittacine species in Iran followed by analyzing the DNA sequences of seven replication-associated protein (rep) and 10 capsid (cap) genomes of the virus. The BFDV was found to be the foremost pathogen among more than 12 psittacine species, and phylogenetic analysis showed that the BFDV GenBank-published sequences from Poland, Saudi Arabia, South Africa, Taiwan, and Thailand were most similar to those of this study. Evolutionary analysis concluded that arginine, leucine, and glycine were the amino acids frequently involved in the least-conserved substitution patterns of BFDV, and conversely, methionine, glutamine, and tryptophan were the amino acids that exhibited ultra-high conservation through the substitution patterns. The high substitution rate of arginine to lysine and glycine to serine also made greater contribution to the BFDV gene mutation. The relative synonymous codon usage between two genes revealed that the cap genome encoded proteins frequently used fewer codons, while the rep genome encoded proteins used more codons only at moderate frequency, explaining the broader divergence of the cap compared to the rep sequence. The data analysis also introduced a new variant of BFDV that exists in the rep and cap sequences of budgerigars. While the existence of more new variants was suspected, more solid evidence is required to substantiate this suspicion.

An Integrated Perspective on Virulence-Associated Genes (VAGs), Antimicrobial Resistance (AMR), and Phylogenetic Clusters of Pathogenic and Non-pathogenic Avian Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is an important bacterial pathogen that causes avian colibacillosis and leads to huge economic losses in the poultry industry. Different virulence traits contribute to pathogenesis of APEC infections, and antimicrobial resistance (AMR) has also been an overwhelming issue in poultry worldwide. In the present study, we aimed to investigate and compare the presence of virulence-associated genes (VAGs), AMR, and phylogenetic group's distribution among APEC and avian fecal E. coli (AFEC) strains. E. coli from birds with colisepticemia and yolk sac infection (YSI) (APEC) plus E. coli strains from the feces of healthy birds (AFEC) were compared by the aforementioned traits. In addition, the clonal relatedness was compared using Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Although all strains were susceptible to fosfomycin, ceftriaxone, and cefixime, almost all strains (98%) were multi-drug resistant (MDR). All strains (except two) harbored at least three or more VAGs, and the virulence scores tended to be higher in pathogenic strains especially in the colisepticemic group. All phylogenetic groups were found in isolates from YSI, colisepticemia, and the feces of healthy birds; however, the frequency of phylogroups varied according to the source of the isolate. B1 and C phylogroups were statistically more likely to be found among APEC from YSI and colisepticemic E. coli groups, respectively, while phylogroup A was the most frequently occurring phylogroup among AFEC strains. Our findings also revealed that AMR and VAGs are not essentially co-evolved traits as in some instances AMR strains were more prevalent among AFEC. This reflects the divergent evolutionary pathways of resistance acquisition in pathogenic or non-pathogenic avian E. coli strains. Importantly, strains related to phylogenetic group C showed higher virulence score and AMR that requires further attention. To some extent, ERIC-PCR was able to group strains by isolation source, phylogroup, or virulence genes. Further integrated studies along with assessment of more detailed genotypic and phenotypic features could potentially lead to better understanding of virulence, resistance, and evolution of ExPEC.

High prevalence of clonally related multiple resistant Salmonella Infantis carrying class 1 integrons in broiler farms.

The poultry industry in Iran is the main supplier of protein in the food chain. In the present study, we showed the importance of the possible dissemination of clonally related multiple drug resistant (MDR) Salmonella Infantis in broiler farms in Iran. In total, 156 fecal samples belonging to 23 poultry farms in Razavi Khorasan province, northeast of Iran, were examined for the presence of Salmonella serovars. Molecular serotypes and serogroups, class 1 and 2 integron types, colistin resistance genes ( mcr1 and mcr2) and antimicrobial susceptibility patterns were determined on the recovered Salmonella isolates. Based on PCR analysis, 30 recovered Salmonella isolates were identified asS. Infantis (23 isolates; 76.6%),S. Enteritidis (six isolates; 20%), and one isolate (3.3%) was not serotyped by the applied method. Class 1 integrons were detected in 22 isolates (95.6%) and class 2 integrons were not detected in any of the isolates. Although colistin resistance was prevalent in disc diffusion test, mcr-1 and mcr-2 genes were not detected. All class 1 integrons carried the cassette aadA1 gene. All Salmonella isolates were resistant to colistin and amoxicillin/clavulanic acid and MDR patterns were observed in most (96.6%) isolates. This study revealed a high prevalence rate of S. Infantis and the presence of class 1 integrons in broiler farms. The presence of the same integron cassettes in the sequenced isolates suggests that strains are clonally related. Stringent monitoring programs are required to prevent the spreading of MDR Salmonella serovars into food chain via poultry products.

Isolation of infectious laryngotracheitis virus in broiler chicken in Iran: First report.

In February 2019, a severe respiratory distress with co-infection of infectious laryngotracheitis (ILT) and Newcastle disease accompanied with Salmonella Enteritidis occurred in a broiler flock in the western region of Iran. Clinical signs included paralysis, torticollis, nasal discharge, conjunctivitis, gasping and respiratory rale with high mortality. At necropsy, caseous diphtheritic membrane adherent to the larynx and trachea was observed. Microscopically, syncytial cells formation with dense eosinophilic intranuclear inclusion bodies were main histopathological findings in tracheal tissues. Conventional polymerase chain reaction (PCR) for ICP4 gene amplification as a definitive diagnosis was utilized for the detection of ILT virus nucleic acid in suspected tracheal samples inoculated on to the chorioallantioc membrane of 11-day-old specific pathogen free (SPF) chicken eggs. Tracheal tissues taken from these SPF birds were positive by nested ILT PCR. In conclusion, because of no vaccination policy against ILT in broilers, the most probable scenario is that virus-laden dust or other fomites can be vectors and virus persistence and disease outbreak can be a sequel of wild virus introduction to the farm.

Antimicrobial and antibiofilm effects of Satureja hortensis essential oil against Escherichia coli and Salmonella isolated from poultry.

BACKGROUND AND OBJECTIVES: Escherichia coli and some Salmonella serovars cause various disease manifestations in poultry leading to significant economic losses. The widespread and imprudent use of antibacterial agents in poultry flocks have increased resistant to many antibacterial agents which has become a major public health concern. Some medicinal plants may be alternative to antibacterial agents. The purpose of this study was to investigate the antibacterial and anti-biofilm activity of summer savory essential oil against E. coli and Salmonella isolated from poultry. MATERIALS AND METHODS: The essential oil was extracted using a Clevenger apparatus and subsequently its compounds were determined using GC-MS. Antibacterial properties of essential oil were determined by disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To evaluate the anti-biofilm properties the Microtiter plate test was used. Herbal essential oil was extracted and its compounds were identified correctly. RESULTS: The major components of Satureja hortensis essential oil were thymol (41.28%), γ-terpinene (37.63%), p-cymene (12.2%) and α-terpinene (3.52%). The inhibition zone diameter in the disc diffusion test for E. coli and Salmonella were 32 ± 3 and 38 ± 4 mm, respectively, which was confirmed by MIC and MBC values. Regarding anti-biofilm activity, the MIC/2 concentration of S. hortensis significantly inhibited biofilm formation of E. coli. However, inhibition of biofilm formation of Salmonella was shown at concentration of MIC/2 and MIC/4. CONCLUSION: Based on our results, S. hortensis essential oil showed the growth inhibition and bactericidal activity against E. coli and Salmonella. Moreover, this study demonstrated anti-biofilm activity of S. hortensis essential against both tested bacteria.

Osteofibrous Dysplasia in a Cockatiel (Nymphicus hollandicus).

Osteofibrous dysplasia is a rare and benign nonneoplastic condition of unknown etiology in humans and mammals. An adult female cockatiel (Nymphicus hollandicus) was presented with the following problems: a soft tissue mass on the left frontal area of the head that extended over the left eye, anorexia, lethargy, and emaciation. Computed tomographic imaging and ultrasonography revealed a soft tissue opacity in the left cranial area of the head with multifocal heterogeneous opacities and foci of mineralization. Cytological, histopathological, and immunohistochemistry examinations were performed on biopsy samples from tissue collected from the mass. Following surgical removal of the cranial mass the patient was treated with a nonsteroidal anti-inflammatory drug and antibiotic medication. The surgical site healed with no complications and through follow-up examinations there were no signs of reoccurrence of the abnormal tissue for 2 months. Through cytological and histopathological examination and immunohistochemistry testing of the submitted tissue, a diagnosis of osteofibrous dysplasia was determined.

Survey of Salmonella infections in broiler farms in Iran during 2013-2014: a cross-sectional study.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the prevalence of Salmonella at broiler breeder farms of Iran and investigate the factors underlying salmonellosis in these farms. This is a cross-sectional investigation conducted in 23 provinces of Iran. MATERIALS AND METHODS: Fecal samples were collected from 139 broiler breeder farms in the country and standard bacteriological tests were carried out on the samples for the isolation of Salmonella. The serological tests were then applied for the samples that were positive in the bacteriological test. The information on the sampled farms extracted from the Iran GIS-VET Monitoring and Surveillance System was used for the analysis of the risk factors. RESULTS: A total of 11 farms out of the 139 sampled farms were infected with Salmonella with the largest number of infected cases related to Tehran and Fars Provinces. CONCLUSION: The statistical analysis results showed that flocks with older ages and farms with larger number of houses are at greater risk of Salmonella infection.

Detection of a Newly Described Bacteriocin, Perfrin, Among Clostridium perfringens Isolates from Healthy and Diseased Ostriches and Broiler Chickens in Iran.

Necrotic enteritis due to Clostridium perfringens strains harboring the netB gene is a well-known disorder in poultry. The aim of this study was to investigate the association of a novel bacteriocin, perfrin, with netB among isolates from healthy and diseased ostriches and broiler chickens. Forty-six C. perfringens isolates from broiler chickens and ostriches collected from 2010 to 2014 were included in this study and subjected to PCR to detect netB and perfrin genes. Six (60%) and 9 (25%) isolates were positive for both netB and perfrin genes in broilers and ostriches, respectively. Statistical analysis found a significant difference between healthy and diseased flocks for perfrin both in broilers and ostriches. For netB, the significant difference was only found between healthy and diseased ostrich flocks. This is the first report of the presence of perfrin in netB-positive C. perfringens strains in ostriches.

Preparation and Characterization of Three Tilmicosin-loaded Lipid Nanoparticles: Physicochemical Properties and in-vitro Antibacterial Activities.

Tilmicosin (TLM) is an important antibiotic in veterinary medicine with low bioavailability and safety. This study aimed to formulate and evaluate physicochemical properties, storage stability after lyophilization, and antibacterial activity of three TLM-loaded lipid nanoparticles (TLM-LNPs) including solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and lipid-core nanocapsules (LNCs). Physicochemical parameters such as particle size-mean diameter, polydispersity index, zeta potential, drug encapsulation efficiency (EE), loading capacity, and morphology of the formulations were evaluated and the effects of various cryoprotectants during lyophilization and storage for 8 weeks were also studied. The profiles of TLM release and the antibacterial activities of these TLM-LNPs suspensions (against Escherichia coli and Staphylococcus aureus) were tested in comparison with their corresponding powders. TLM-LNPs suspensions were in nano-scale range with mean diameters of 186.3 ± 1.5, 149.6 ± 3.0, and 85.0 ± 1.0nm, and also EE, 69.1, 86.3, and 94.3% for TLM- SLNs, TLM-NLCs, and TLM- LNCs respectively. TLM-LNCs gave the best results with significantly low particle size and high EE (p<0.05). Mannitol was the most effective cryoprotectant for lyophilization and storage of TLM-LNPs. The drug release profiles were biphasic and the release times were longer at pH 7.4 where TLM-NLCs and TLM-LNCs powders showed longer release times. In microbiological tests, S. aureus was about 4 times more sensitive than E. coli to TLM-LNPs with minimum inhibitory concentration ranges of 0.5-1.0 and 2-4 µg/mL respectively, and TLM-LNCs exhibited the best antibacterial activities. In conclusion, TLM-LNP formulations especially TLM-LNCs and TLM-NLCs are promising carriers for TLM with better drug encapsulation capacity, release behavior, and antibacterial activity.

Molecular clonality and antimicrobial resistance in Salmonella enterica serovars Enteritidis and Infantis from broilers in three Northern regions of Iran.

BACKGROUND: Multidrug-resistant Salmonella strains are frequently encountered problems worldwide with considerable increased occurrences in recent years. The aim of this study was to investigate the occurrence and frequency of antimicrobial resistance and associated resistance genes in Salmonella isolates from broiler farms in different regions of Iran covering a time period of four years. RESULTS: From 2007 to 2011, 36 Salmonella strains were isolated from broiler farms located in three northern provinces of Iran. The isolates were serotyped, antimicrobial susceptibility tested, and characterized for antimicrobial resistance genes associated to the phenotype. Pulsed-field gel electrophoresis (PFGE) was applied for comparison of genetic relatedness.Two serovars were detected among the isolates; Salmonella enterica serovar Infantis (75%) and S. Enteritidis (25%). Thirty-four (94%) of the isolates exhibited resistance to nalidixic acid and ciprofloxacin caused by a single mutation in the quinolone resistance-determining region (QRDR) of gyrA. For all strains this mutation occurred in the codon of Asp87 leading to a Asp87-Tyr, Asp87-Gly or Asp87-Asn substitutions. All S. Infantis (n = 27) were resistant to tetracycline, spectinomycin, streptomycin, and sulfamethoxazole and harbored the associated resistance genes; tetA, dfrA14, aadA1, and sulI together with class 1 integrons. The isolates revealed highly similar PFGE patterns indicating clonal relatedness across different geographical locations. CONCLUSION: The data provided fundamental information applicable when launching future control programs for broilers in Iran with the aim to conserve the effectiveness of important antimicrobials for treatment in humans.

Drug resistance, plasmid profile and random amplified polymorphic DNA analysis of Iranian isolates of Salmonella enteritidis.

The aim of this study was to characterize 49 Salmonella Enteritidis (SE) isolates from different sources (poultry, human, cow, poultry house environment) in Iran with respect to drug resistance, plasmid profile, and random amplified polymorphic DNA (RAPD) analysis. Antimicrobial susceptibility test to 29 agents found 33 resistance patterns among the isolates. No resistance was observed to danofloxacin, levofloxacin, norfloxacin, ceftriaxone, imipenem, and amikacin. The highest resistance (38.8%) was observed to flumequine. Thirty (61.2%) isolates were multidrug-resistant. Six plasmid profiles were detected and a 68-kb plasmid was found in 98% of isolates. Two different primers, MK22 and P1254, were used for RAPD analysis which each produced six profiles. For MK22 and P1254 primers, 83 and 86% of the isolates, respectively, belonged to one profile only and the rest distributed among other 5 patterns. The findings of the present study showed that SE isolates from poultry-related sources were closely related to human SE isolates. This study confirmed previous evidence that molecular techniques such as RAPD-PCR or plasmid profile alone do not demonstrate sufficient discriminatory power in epidemiological studies and a combination of patterns obtained by several techniques will provide more discriminatory power.

Ossifying fibroma in a canary (Serinus canaria).

A 1-year-old male canary (Serinus canaria, Jibber breed), with a protuberance on the left tibia was referred to the clinic. The bird was not able to use the affected foot to stand on its perch. Results of radiographs revealed a radio-opaque hard tissue mass. The canary was euthanatized and submitted for necropsy. On histopathologic examination, the mass was diagnosed as an ossifying fibroma tumor. To our knowledge, ossifying fibroma has not been reported in birds so far, and this is the first known report of this tumor in birds.