Respiratory disease complex due to mixed viral infections in chicken in Jordan.

The global distribution of avian respiratory viruses highlights the need for effective surveillance programs and international collaboration to monitor viral circulation and implement timely control measures. In the current study, we aim to provide a comprehensive overview of avian respiratory viral infections in the poultry flocks in Jordan, focusing on the major viruses involved, their epidemiology, clinical manifestations, and evolution based on viroinformatics that will be helpful to improve the diagnostic methods, and control strategies including vaccines in the region. In this research, various poultry broiler groups in Jordan experiencing respiratory symptoms were tested for respiratory viral pathogens from January 2021 to February 2022. The mortality rates observed in the examined groups varied between 6% and 40%. The identified strains were authenticated using the RT-qPCR assay. Furthermore, they underwent in-depth characterisation through the sequencing of the complete spike (S1) gene for infectious bronchitis virus (IBV) and the haemagglutinin (HA) gene for avian influenza virus (AIV) subtype H9N2. Co-infection of IBV and AIV H9N2 viruses was detected through molecular analysis. The IBV strains showed affiliation with the variant groups GI-16 (3 strains) and GI-23 (9 strains) and exhibited numerous mutations. Meanwhile, H9N2 avian influenza viruses displayed various changes in amino acids within the HA gene, suggesting the influence of antibody-driven selection pressure. The phylogenetic analysis revealed that the H9N2 viruses identified in this investigation shared close genetic ties with EG3 (3 strains) and the Middle East group (ME1; 8 strains). These strains have been recently found in Jordan and nearby countries in the Middle East. Moreover, their HA genes exhibited similarities to viruses belonging to the G1-like lineage. In conclusion, avian respiratory viral infections remain a significant concern for the poultry industry, requiring constant vigilance and proactive measures to minimise their impact. Continued surveillance, robust diagnostic methods, effective vaccines, and international cooperation are essential components of a comprehensive approach to combat avian respiratory viral infections (AI, IBV, ND and ILT 'viruses) and safeguard avian health and global poultry production.

Evolutionary Trajectories of Avian Avulaviruses and Vaccines Compatibilities in Poultry.

Newcastle disease virus (NDV) causes one of the highly infectious avian diseases in poultry leading to genuine financial misfortunes around the world. Recently, there has been an increasing trend in the number of ND-associated outbreaks in commercial Jordanian poultry flocks indicating a possible complex evolutionary dynamic of NDV infections in the country. To underpin the dynamics of circulating NDV strains and to assess the vaccine-escape potential, a total of 130 samples were collected from different poultry flocks in six Jordanian Governorates during 2019-2021. Twenty positive isolates, based on real-time reverse transcriptase PCR, were used for further genetic characterization and evolutionary analysis. Our results showed that there is a high evolutionary distance between the newly identified NDV strains (genotype VII.1.1) in this study and the commercially used vaccines (genotypes I and II), suggesting that circulating NDV field strains are under constant evolutionary pressure. These mutations may significantly affect flocks that have received vaccinations as well as flocks with insufficient immunity in terms of viral immunity and disease dynamics. To assess this further, we investigated the efficacy of the heterologous inactivated LaSota or homologous genotype VII.1.1 vaccine for their protection against virulent NDV in chicken. Vaccine-induced immunity was evaluated based on the serology, and protection efficacy was assessed based on clinical signs, survival rates, histopathology, and viral shedding. Chickens vaccinated with the inactivated genotype VII.1.1 based vaccine showed 100% protection with a significant reduction in virus shedding, and ameliorated histopathology lesions compared to LaSota vaccinated chicks that showed 60% protection. These results revealed that the usage of NDV inactivated vaccine from the circulating field strains can successfully ameliorate the clinical outcome and virus pathobiology in vaccinated chicks and will serve as an effective vaccine against the threat posed by commonly circulating NDV strains in the poultry industry.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses.

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Congenital abnormalities in dead-in-shell chicks associated with mixed bacterial infections.

Determination of the chick embryonic developmental period at which embryonic mortalities occur could help in establishing the cause of such mortalities. The late stage of embryonic development has particular importance due to its dramatic effect on life after hatching. This study was conducted to investigate the occurrence, frequency and bacterial isolates from dead-in-shell chick embryos in Northern Jordan. A total of 1,000 unhatched eggs were collected at hatching day from 10 hatcheries located in Northern Jordan. Out of 1,000 eggs, 357 (35.7%) were fertile, of which 210 (58.8%) were dead-in-shell embryos. Approximately 50.5% of the dead embryos displayed abnormalities, including neck muscles with subcutaneous petechial haemorrhages (44.3%), beak abnormalities (3.8%), eye deformities (1.9%) and anencephaly (0.5%). Sixty-six bacterial isolates were identified from 82 samples from the dead-in-shell embryos. The isolates were 22 (33.3%) Escherichia coli, 18 (27.3%) Klebsiella pneumoniae, 14 (21.2%) Staphylococcus aureus, 5 (7.6%) Pseudomonas aeruginosa, 4 (6.1%) Salmonella enteritidis, 2 (3%) Bacillus cereus and 1 (1.5%) Proteus vulgaris. Mixed growth was also recorded in 16 (19.5%) samples. There was a significant (P < 0.05) association between Escherichia coli as a bacterial isolate and the occurrence of neck and beak abnormalities. In this study, infection of check embryos with several bacterial species, particularly Escherichia coli, was identified as an important cause of multiple congenital abnormalities involving the neck and beak of unhatched chicks.

Artificial Intelligence-Assisted Loop Mediated Isothermal Amplification (AI-LAMP) for Rapid Detection of SARS-CoV-2.

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.

Resistance of Campylobacter jejuni Isolated from Layer Farms in Northern Jordan Using Microbroth Dilution and Disc Diffusion Techniques.

Campylobacter jejuni is an important pathogen of significant public health importance. This pathogen is associated with human infection and has been isolated from mammals and birds. Ninety-two cloacal C. jejuni isolates were identified from 35 layer farms in Northern Jordan. Antimicrobial susceptibility was determined using minimal inhibitory concentration (MIC) and disc diffusion techniques with variable suggested breakpoints. Using MIC and EUCAST cut-off values, the study revealed a significantly high resistance level (100%) among the layers' isolates against ciprofloxacin and tetracycline. A relatively high resistance (41%) toward gentamicin and amoxicillin and low resistance to nalidixic acid (21%), erythromycin (14%), and florfenicol (6.5%) were also found. This high level of resistance may indicate abuses in the handling of antibiotics, which may require stricter control in the local layer industry. A good agreement between the 2 techniques used was demonstrated and the disc diffusion technique could be used as a rapid screening test for antimicrobial susceptibility of C. jejuni to many antibiotics using specific Campylobacter breakpoints.

Survival and growth of Salmonella Typhimurium, Escherichia coli O157:H7 and Staphylococcus aureus in eggplant dip during storage.

Eggplant dip is an internationally popular appetizer, prepared in some instances under uncertain hygienic conditions with inconsistent refrigeration. This study examined the effects of citric acid on the survival of pathogenic microorganisms (Salmonella Typhimurium, Escherichia coli O157:H7 and Staphylococcus aureus) and naturally present organisms (lactic acid bacteria, LAB, aerobic bacteria, APC and yeast and mold, YM) in eggplant dip during storage. Eggplant dip with 0, 0.2, 0.4, 0.6 or 0.8% citric acid was inoculated with S. Typhimurium, E. coli O157:H7 or S. aureus and stored at 4, 10 and 21 °C for ≤15 d. Throughout the study, the survival of the inoculated microorganisms was monitored, and LAB, APC, YM numbers and pH were determined. There was no significant (p>0.05) effect of citric acid on inoculated S. Typhimurium and E. coli O157:H7. Salmonella and E. coli O157:H7 survived >7d with little reduction in viability. Reduction of S. aureus viability increased with citric acid concentration and reached>3.0 log10 CFU/g by 15 d at 4 °C. Citric acid had no effect (p>0.05) on the background YM during storage at 4, 10 and 21 °C or LAB stored at 4 and 10 °C, while at 21 °C, 0.6 and 0.8% citric acid significantly reduced LAB. Citric acid had no effect (p>0.05) on the APC in samples stored at 4 °C but it had significant effects on samples stored at 10 and 21 °C. Work reported showed that the use of citric acid at 0.4-0.8% can inhibit the growth of S. aureus in eggplant dip, but adequate refrigeration is essential to minimize risk from this and other pathogens in this product.

Molecular characterization of a recent Newcastle disease virus outbreak in Jordan.

Newcastle disease (ND) is a highly contagious viral disease and is a continuous threat to the poultry industry worldwide. In the early months of 2011, several devastating ND outbreaks occurred in Jordan affecting broilers, layers and breeders. The fusion gene of the isolated Newcastle disease virus (NDV) was partially amplified by RT-PCR, then directly sequenced. The NDV isolates were found to have the motif112RRQKRF117. This motif and a mean death time (MDT) of 46 h are indicative of the velogenic nature of these NDV isolates. Phylogenetic analysis showed that the new NDV strain belongs to the lineage 5d (Aldous et al., 2003) and is closely related to the Chinese strain SG/Liaoning/2009. NDV outbreaks in 2010 and 2011 have been noted in neighboring countries. Based on the high nucleotide similarity between our isolated NDV isolates and the Chinese NDV strain, the origin of these recent NDV isolates might be from China.

Escherichia coli O157:H7 facilitates the penetration of Staphylococcus aureus into table eggs.

Fresh eggshells collected from a local farm were subjected to different levels of surface contamination with feces containing different levels (3 to 5 log10) of Escherichia coli O157:H7 or Staphylococcus aureus and incubated at 3 different temperatures (10, 25, and 32 °C). The penetration rates of contaminating bacteria were followed throughout the incubation period by tracing bacterial presence in shell, shell membranes, albumen, and yolk. The study revealed the ability of both E. coli O157:H7 and enterotoxigenic S. aureus to grow on shell in feces, penetrate the shell, and move and multiply within egg contents at different rates and periods depending on bacterial type and incubation conditions. High temperatures (25 and 32 °C) increased penetration rate, whereas storage at 10 °C decreased significantly the rate of penetration. High levels of contamination with E. coli O157:H7 also shortened the time needed for the penetration process. Results showed that when eggshells were contaminated with both organisms simultaneously, the penetration of E. coli O157:H7 preceded that of S. aureus and facilitated the invasion of the latter bacteria.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses.

The triple reassortant H3N2 viruses were isolated for the first time from pigs in 1998 and are known to be endemic in swine and turkey populations in the United States. In 2004, we isolated two H3N2 triple reassortant viruses from two turkey breeder flocks in Ohio and Illinois. Infected hens showed no clinical signs, but experienced a complete cessation of egg production. In this study, we evaluated three triple reassortant H3N2 isolates of turkey origin and one isolate of swine origin for their transmission between swine and turkeys. Although all 4 viruses tested share high genetic similarity in all 8 genes, only the Ohio strain (A/turkey/Ohio/313053/04) was shown to transmit efficiently both ways between swine and turkeys. One isolate, A/turkey/North Carolina/03, was able to transmit from pigs to turkeys but not vice versa. Neither of the other two viruses transmitted either way. Sequence analysis of the HA1 gene of the Ohio strain showed one amino acid change (D to A) at residue 190 of the receptor binding domain upon transmission from turkeys to pigs. The Ohio virus was then tested for intraspecies transmission in three different avian species. The virus was shown to replicate and transmit among turkeys, replicate but does not transmit among chickens, and did not replicate in ducks. Identifying viruses with varying inter- and intra-species transmission potential should be useful for further studies on the molecular basis of interspecies transmission.

Genetic comparison of H9N2 AI viruses isolated in Jordan in 2003.

SUMMARY. During the 1990s, several outbreaks of avian influenza (AI), caused by viruses of the H9N2 subtype, were reported in poultry in various parts of the world. Currently, this infection seems to be endemic in poultry in the Middle and Far East, and the extensive circulation of H9N2 in poultry represents a risk factor for infection of humans, because viruses of this subtype have been sporadically introduced into the human population. Little is known about the gene constellation of the H9N2 viruses that are currently circulating in the Middle East; thus, gene sequences of eight IA viruses of the H9N2 subtype isolated in Jordan in 2003 from poultry were analyzed. The results of this investigation show that all eight Jordanian isolates are closely related to each other and to other H9N2 isolates from the Middle East. Seven of eight genes of the Jordanian strains show a percentage of homology not higher than 95% with the genes of two H9N2 strains, A/HK/1073/99 and A/HK/1074/99, isolated from humans in Hong Kong. The M gene is more closely related to the corresponding gene of the two H9N2 human isolates from Hong Kong (97.7-98.2% homology). This homology suggests that the M gene of the Jordanian and human Hong Kong strains could derive from a common ancestor.

Estimated risk of coronary heart disease in obese adult males in Northern Jordan.

OBJECTIVE: To examine the relationship between obesity, lipid profile and blood pressure, and to quantify the risk of coronary heart disease (CHD) for the next 10 years, using the Framingham risk scoring scheme among Jordanian adult males. METHODS: We conducted this study in Al-Sarieh, Jordan during the period March to May 2001. A total of 306 apparently healthy adult males, aged 30-50 years completed all the study procedures. We selected the participants using a multi-stage cluster sampling design. Dietary history and smoking habits were obtained using a pre-tested questionnaire and interview. Blood samples were obtained and examined for lipid profiles. We measured the blood pressures, as well as the weight and height to calculate the body mass index (BMI). The sample was categorized into 3 groups using the World Health Organization classifications for BMI. The risk of CHD was calculated using a scoring scale according to Framingham scheme. Analyses of data were carried out using the Chi-square test, and the Analysis of Variance. RESULTS: The mean age of the subjects was 39 years with a mean BMI of 28.2 kg/m2. The percentage of current smokers was 44.1%. The mean of serum total cholesterol, triglycerides, low density lipoprotein cholesterol and systolic blood pressure, increased significantly with increasing BMI categories, whereas the mean of high density lipoprotein cholesterol decreased with increasing BMI categories. Prevalence of medium and high risk of CHD significantly increased as BMI categories increases. CONCLUSION: The prevalence of estimated CHD risk for the next 10 years in moderate and high CHD categories increases as the BMI categories increases among Jordanian adult men in Al-Sarieh area.

Sero-prevalence of avian influenza among broiler-breeder flocks in Jordan.

Thirty blood samples were collected randomly from each of the 38 breeder-broiler farms in Jordan. Serum samples were examined using indirect ELISA for specific antibodies to avian influenza virus. The overall true flock-level sero-prevalence of avian influenza was 71% (95% CI: 55,83). Positive flocks had 2-30 sero-positive chickens and half of flocks had >20 sero-positive birds. The number of sero-positive flocks varied in the studied localities with more sero-positives in farms located within the migratory route of migratory wild fowl. The examined broiler-breeder flocks had no clinical signs, or noticeable decrease in egg production; mortalities were within the normal range (0.1-1%). The number of positive sera/flock correlated with flock size. There were a no significant (Pearsons r=0.21, p=0.21) correlation between positive flocks and age. A non-pathogenic AI virus infects broiler-breeder farms in Jordan. Wild local and migrating birds might promote the further spread of this virus in Jordan and other countries.

Flock-level prevalence of Eimeria species among broiler chicks in northern Jordan.

Six chicks (3-6 weeks of age) were taken randomly from each of 200 broiler farms in northern Jordan, these chicks were submitted for post-mortem and parasitological examinations. Seven Eimeria spp. were identified: E. acervulina, E. brunetti, E. maxima, E. necatrix, E. mivati, E. mitis, and E. tenella. Half (50%) of the farms surveyed had all six chicks infected, 23% of the farms were free of the infection. E. tenella was the most prevalent species (39%) followed by E. necatrix (12%), E. brunitti (12%), and E. maxima (10%). Prevalences did not vary by flock size. Also, neither the use of coccidiostat nor previous coccidiosis clinical outbreaks was associated with the prevalence of coccidiosis.