Infectious Laryngotracheitis Research Articles

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Detection of Chicken Respiratory Pathogens in Live Markets of Addis Ababa, Ethiopia, and Epidemiological Implications.

Simple SummaryIt is unclear what microorganisms are associated with respiratory disease in chickens sold in live markets of Addis Ababa, Ethiopia. Identifying microorganisms in diseased animals is the first step to delineating programs to control disease spread. Two Addis Ababa markets were visited weekly for three months to understand better the microorganisms found in chickens with respiratory disease. During this time, 18 sick chickens were acquired and tested for common microorganisms that cause respiratory disease in chickens. Three or more microorganisms, including viruses and bacteria, were detected in 17 of the 18 cases, showing that detection of multiple pathogens is widespread in live markets. These microorganisms possibly cause substantial productive loss in Ethiopia. Further studies are warranted to investigate their contribution to disease and economic losses in the country.A moderate to high seroprevalence of exposure to Newcastle disease (NDV), avian metapneumovirus (aMPV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV) and Mycoplasma gallisepticum (MG) has recently been reported in Ethiopia, but it is unclear to what extent these contribute to clinical cases of respiratory disease. This study investigated the presence of these pathogens in chickens exhibiting respiratory disease in two live markets in Addis Ababa. Markets were visited weekly for three months, and 18 chickens displaying respiratory clinical signs were acquired. Swab samples were taken from the choana, trachea, air sac and larynx for bacteriology and PCR tests targeting these five pathogens. PCR-positive samples were sequenced. All 18 chickens were PCR-positive for aMPV, 50% for each of Mg and NDV, 39% for IBV and 11% for ILTV. Infections with >3 pathogens were detected in 17 of 18 chickens. Potentially pathogenic bacteria such as Escherichia coli, Klebsiella spp., Streptococcus spp. and Staphylococcus were found in 16 to 44% of chickens. IBV-positive samples were of the 793B genotype. The results associate the presence of these organisms with clinical respiratory disease and are consistent with recent serological investigations, indicating a high level of exposure to multiple respiratory pathogens.

Molecular and genetic detection of infectious laryngeotrachitis disease virus in broiler farms after a disease outbreak in Egypt.

Infectious laryngotracheitis (ILT) is a viral respiratory illness in poultry that causes massive financial losses. This research aimed to isolate and identify the ILT virus in suspected outbreaks of broiler flocks in Egypt during 2020-2021, besides investigating its genetic link with other circulating strains. Real-time-PCR was used to test 57 samples taken from unvaccinated broiler farms. Ten samples are positive for ILTV, and the virus is being isolated in SPF chicken embryos. The Sanger sequencing was used to conduct (partial) sequencing of the infected cell protein4 gene (ICP4) for eight isolates. Phylogenetic analysis conducted Maximum Likelihood, comparative sequencing analysis of ICP4 of strains under study with vaccination ILT reference strains reveled that all isolates were clustered into two major groups. The (OM291843and OM291846) clustered together with the chicken embryo origin vaccine strains (IV and V group). The remaining six strains belong to the TCO vaccine(I, II and III group). The total sequence similarity between the strains under study and the various Egyptian strains varied from (97 to 100%) while the similarity with TCO or chicken embryo origin -vaccine strains ranged from (95to 100%). There were no deletions detected in the 272-283-bp region of the ICP4 gene. Detection of arginine to methionine substitutions at position 180 (R180M) and change of Serine to Asparagine at position 227 (S227N) in the (OM291843 and OM291846) which were previously described in chicken embryo origin -vaccine strains. This reveals that field strains may have evolved from vaccine strains, notably identification of non-synonymous substitutions which might be linked to the virulence strains' attenuation. Finally, independent of geographical distribution, both chicken embryo origin-vaccine-like and TCO-Vaccine-like virus strains were circulating in Egyptian non-vaccinated broiler flocks in 2020 and 2021. Despite their genetic differences, both viruses caused significant illnesses in the field.

Molecular Characterization and Phylogenetic Analysis of Marek's Disease Virus in Iran.

Marek's disease (MD) is a highly contagious, lymphoproliferative poultry disease caused by the oncogenic herpesvirus, serotype 1 Marek's disease virus (MDV-1), or Gallid herpesvirus 2 (GaHV-2). MDV strains have shown a continued evolution of virulence leading to immune failure, and MD cases continue to occur or surge. Meq, the major MDV-1 oncoprotein, induces T-cell neoplastic transformation through several mechanisms including inhibition of apoptosis, cell cycle regulation, and serum-anchorage independent growth. There is no current information on the MDV serotypes and pathotypes circulating in vaccinated commercial farms in Iran, where the birds are vaccinated at the hatchery with GaHV-2 and Meleagrid herpesvirus 1 (MeHV-1) vaccines. This study reports the molecular characterization of a GaHV-2 strain detected in 19 flocks of Iranian layer farms exhibiting MDV-1-like clinical signs and visceral lymphomas. Based on sequencing and phylogenetic analysis of the Meq gene, the Iranian GaHV-2 isolates could be divided into two separate clades regarding molecular features. The clade containing strains was closely related to Italian, Indian, and Hungarian virulent isolates, and the clade was related to American very virulent plus (vv+) isolates. For the first time, the MDV-1 virus was characterized by an outbreak in poultry flocks in Iran. Although MDV-1 strains obtained in Iran's present outbreak are presumably related to virulent (v) and vv+ pathotypes based on nucleotide, amino acid, and phylogenetic analysis of the viruses, they are not confirmed so far. Thus, it is highly recommended to perform further analyses to demonstrate the pathotype characteristics in vivo.

Field Application of qPCR Monitoring of Infectious Laryngotracheitis Virus in Settled Chicken House Dust and Its Role in Control of a Major Outbreak.

Population-level sampling based on qPCR detection of infectious laryngotracheitis virus (ILTV) in poultry dust can be used to assess ILT vaccination outcomes following mass administration in drinking water. We report on the field application of this approach to assess the success of vaccine administration and its use in ILT outbreak control in meat chickens. In Study 1, dust samples were collected from 26 meat chicken flocks at 0, 4, 7, 14, and 21 days post drinking water vaccination (DPV) given between 7 to 13 days of age with the Serva or A20 live attenuated ILT vaccines. Unexpectedly, ILTV DNA was detected in dust samples collected prior to vaccination in 22/26 flocks. Typing revealed that the detected ILTV was different from the vaccine virus. To determine whether the detected ILTV DNA was from active infection or carryover of a noninfectious virus, Study 2 was implemented in 14 additional flocks with dust samples collected at 0, 7, 14, and 21 DPV and tracheal swabs collected from 15 birds/flock at 0 and 21 DPV. The results indicated that there was active infection with ILTV in those flocks before vaccination. This approach contributed to a statewide control program resulting in the eradication of ILT from South Australia as confirmed by negative ILTV test results for dust samples from 50 flocks and the absence of clinical ILT. These findings show that ILTV infection prior to vaccination is common in outbreak situations and that dust samples must be collected at 0 and 7 DPV for meaningful interpretation of vaccination outcomes and ILTV status. Comparatively low-cost dust testing during an outbreak, coupled with typing information, greatly assisted with decision making and control strategies during a major outbreak, including confirmation of the absence of infection in the final stages.

Isolation and Molecular Detection of Marek's Disease Virus from Outbreak Cases in Chicken in South Western Ethiopia.

BackgroundMarek’s disease virus is a devastating infection, causing high morbidity and mortality in chickens in Ethiopia.MethodsThe current study was conducted from March to November, 2021 with the general objective of performing antemortem and postmortem, isolation, and molecular detection of Marek’s disease virus from outbreak cases in southwestern Ethiopia. Accordingly, based on outbreak information reported from the study sites namely, Bedelle, Yayo, and Bonga towns in southwestern Ethiopia, 50 sick chickens were sampled. The backyard and intensive farming systems of chickens were included in the sampling and priorities were given for chickens that showed clinical signs that are characteristics of Marek’s disease.ResultsBy clinical examinations, paralysis of legs and wings, gray eye, loss of weight, difficulty in breathing, and depression were recorded on all chickens sampled for this study and death of diseased chickens was observed. In addition, enlargement of the spleen and gross lesions of the liver and heart were recorded during postmortem examination. The death of infected chickens was observed in both vaccinated and non-vaccinated flocks. Out of 50 pooled feather follicle samples, Marek’s disease virus was isolated from 14/50 (28%) by cell culture method and out of six tissue samples, the virus was isolated from 5/6(83.30%). By Real time polymerization chain reaction technique, which was targeted to detect the Meq gene, Marek’s disease virus was detected from 18/50 feather follicles which accounts for 36% of sampled chickens.ConclusionIn general, current study showed that the circulating Marek’s disease virus in southwestern Ethiopia was caused by the oncogenic Gallid herpesvirus-2 (Serotype-1). Further research on molecular characterization of revolving virus in current and other regions is recommended for effective control of the disease through vaccination.

New rapid detection by using a constant temperature method for avian leukosis viruses.

The avian leukemia virus causes avian leukemia (AL), a severe immunosuppressive disease in chickens (ALV). Since the 1990s, the diversity of ALV subpopulations caused by ALV genome variation and recombination, and the complexity of the infection and transmission, with currently no effective commercial vaccine and therapeutic for ALV, has resulted in severe economic losses to the chicken business in various parts of the world. Therefore, as a key means of prevention and control, an effective, rapid, and accurate detection method is imperative. A new real-time reverse transcription recombinase-aided amplification (RT-RAA) assay for ALV with rapid, highly specific, low-cost, and simple operational characteristics have been developed in this study. Based on the amplification of 114 base pairs from the ALV P12 gene, real-time RT-RAA primers and a probe were designed for this study. The lowest detection line was 10 copies of ALV RNA molecules per response, which could be carried out at 39°C in as fastest as 5 min and completed in 30 min, with no cross-reactivity with Marek's disease virus, avian reticuloendothelial virus, Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, infectious laryngotracheitis virus, and avian influenza virus. Furthermore, the kappa value of 0.91 (>0.81) was compared with reverse transcription–polymerase chain reaction (RT-PCR) for 44 clinical samples, and the coefficients of variation were within 5.18% of the repeated assays with three low-level concentration gradients. These results indicate that using a real-time RT-RAA assay to detect ALV could be a valuable method.

Serological Evidence of Infectious Laryngotracheitis Infection and Associated Risk Factors in Chickens in Northwestern Ethiopia.

Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Gallid herpesvirus 1 (GaHV-1), a.k.a infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. Despite the clinical signs on the field suggestive of ILT, it has long been considered nonexistent and a disease of no concern in Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021 in three selected zones of the Amhara region (Central Gondar, South Gondar, and West Gojjam zones), Ethiopia, with the objective of estimating the seroprevalence of ILTV in chickens and identifying and quantifying associated risk factors. A total of 768 serum samples were collected using multistage cluster sampling and assayed for anti-ILTV antibodies using indirect ELISA. A questionnaire survey was used to identify the potential risk factors. Of the 768 samples, 454 (59.1%, 95% CI: 0.56–0.63) tested positive for anti-ILTV antibodies. Mixed-effect logistic regression analysis of potential risk factors showed that local breeds of chicken were less likely to be seropositive than exotic breeds (OR: 0.38, 95% CI: 0.24–0.61). In addition, factors such as using local feed source (OR: 6.53, 95% CI: 1.77–24.04), rearing chickens extensively (OR: 1.97, 95% CI: 0.78–5.02), mixing of different batches of chicken (OR: 14.51, 95% CI: 3.35–62.77), careless disposal of litter (OR: 1.62, 95% CI: 0.49–4.37), lack of house disinfection (OR: 11.05, 95% CI: 4.09–47.95), lack of farm protective footwear and clothing (OR: 20.85, 95% CI: 5.40–80.45), and careless disposal of dead chicken bodies had all been associated with increased seropositivity to ILTV. Therefore, implementation of biosecurity measures is highly recommended to control and prevent the spread of ILTV. Furthermore, molecular confirmation and characterization of the virus from ILT suggestive cases should be considered to justify the use of ILT vaccines.

Seroprevalence of major respiratory diseases of chickens in central Ethiopia in different chicken production systems

In Ethiopia, most chicken disease outbreaks and mortalities are attributed to a respiratory syndrome known as “fengil” with variable clinical signs and undefined etiology. The main goal of this study was to determine whether key respiratory pathogens that could contribute to the fengil syndrome circulate in Ethiopia. Specifically, we aimed to determine the seroprevalence of infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (Mg), and avian metapneumovirus (aMPV). A cross-sectional survey was conducted in 158 scavenging and 42 small and medium-scale intensive chicken holdings in the East, West and North Shewa Zones of central Ethiopia. Blood from 495 chickens was collected and serological tests were used to determine exposure to these pathogens. Vaccination against NDV was the only immunization practiced with a significantly higher vaccination rate in the intensive than the scavenging system. Serological evidence of a high level of exposure to all pathogens was detected, including the first report on the seroprevalence of aMPV, ILTV, and IBV in the East Shewa Zone. The chicken and holding seroprevalence rates were respectively 91% and 94% for IBV, 34% and 57% for aMPV, 47% and 66% for Mg, 27% and 51% for ILTV and in unvaccinated flocks, 39% and 53% for NDV. These pathogens could contribute to the fengil syndrome, commonly ascribed to NDV. The seroprevalence of aMPV and ILTV was higher in chickens under the scavenging system. Exposure to multiple pathogens was common, with more than 50% of chickens positive for three or more pathogens in the scavenging system. This was reflected in significant positive associations between seropositivity to ILTV, Mg, ILTV, and IBV. The role of these pathogens in the causation of respiratory disease in the field requires further investigation.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Molecular detection and isolation of infectious laryngotracheitis virus (ILTV) in layer farms of Waset province, Iraq

Infectious Laryngotracheitis (ILT) is an acute highly contagious respiratory disease of chickens. It has significant economic importance due to mortalities and the decrease in egg production. In this study, twenty nine samples from different layer farms were collected from the outbreaks that occurred in waset province, Iraq, at the period from November 2021 till March 2022 to detect ILTV by molecular test through real time polymerase chain reaction assay (RT-PCR) as well as isolation on embryonated chicken eggs through chorioallantoic membrane (CAM) route. The clinical signs examination of infected birds revealed extend head with expectoration of bloody mucous, gasping with weeping eye (watery eye). Gross pathological lesions examination revealed catarrhal, caseated, fibrinonecrotic, hemorrhagic tracheitis. The RT-PCR revealed amplification specific for glycoprotein G gene of ILT virus. Preparation and Inoculation of tissue samples which giving positive results with RT-PCR on embryonated chicken eggs (ECE) appeared as white pock lesions on the inoculated CAM from the second passage.

Earlier detection of infectious laryngotracheitis virus by development of Dot-ELISA in commercial layer farms

Earlier detection of infectious laryngotracheitis virus by development of Dot-ELISA in commercial layer farms

Biological Characteristics of Infectious Laryngotracheitis Viruses Isolated in China.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens and results in huge economic losses in the poultry industry worldwide. To correlate the genomic difference with the replication and pathogenicity, phenotypes of three ILTVs isolated from chickens in China from 2016 to 2018 were sequenced by high-throughput sequencing. Based on the entire genome, the isolates GD2018 and SH2017 shared 99.9% nucleotide homology, while the isolate SH2016 shared 99.7% nucleotide homology with GD2018 and SH2017, respectively. Each virus genome contained 82 ORFs encoding 77 kinds of protein, 31 of which share the same amino acid sequence in the three viruses. GD2018 and SH2017 shared 57 proteins with the same amino acid sequence, while SH2016 shared 42 and 41 proteins with the amino acid sequences of GD2018 and SH2017, respectively. SH2016 propagated efficiently in allantoic fluid and on chorioallantoic membranes (CAMs) of SPF chicken embryo eggs, while GD2018 and SH2017 proliferated well only on CAMs. GD2018 propagated most efficiently on CAMs and LMH cells among three isolates. SH2016 caused serious clinical symptoms, while GD2018 and SH2017 caused mild and moderate clinical symptoms in chickens, although the sero of the chickens infected with those three isolates were all positive for anti-ILTV antibody at 14 and 21 days after challenge. Three ILTVs with high genetic homology showed significant differences in the replication in different culture systems and the pathogenicity of chickens, providing basic materials for studying the key determinants of pathogenicity of ILTV.

Marek's Disease in an Indian Peafowl (Pavo cristatus) with Clinical Ocular Disease and Paraparesis

Marek's disease (MD) is caused by virulent strains of Gallid alphaherpesvirus type 2 (MD virus serotype 1; MDV 1) and frequently causes a lymphoproliferative disorder in poultry and other galliform birds worldwide. However, within the peafowl (Phasianinae) subfamily, there are only rare confirmed reports of MD. Here we report MD in an Indian peafowl (Pavo cristatus), which clinically presented with hindlimb paraparesis and intraocular swelling of the right eye. Soft, off-white to tan masses within the right eye, sciatic nerves and coelomic cavity were identified at post-mortem examination which effaced the cranial pole of the kidneys and diffusely effaced the testes. Lymphoid neoplasia was identified histologically at all of these sites and there was extensive hepatic lymphoid cell infiltration, which had not been grossly evident. The T-cell origin of the lymphoid cells was confirmed by immunohistochemistry for CD3 antigen. A virulent strain of MDV 1 was detected by real-time polymerase chain reaction in DNA samples extracted from the kidney and testes. As MD is rare in peafowl it should be considered as a differential diagnosis for intraocular and coelomic masses with associated clinical signs.

Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is caused by Gallid herpesvirus-1 (GaHV-1) or infectious laryngotracheitis virus (ILTV) and was first described in Canadian poultry flocks. In Canada, ILTV infection is endemic in backyard flocks, and commercial poultry encounters ILT outbreaks sporadically. A common practice to control ILT is the use of live attenuated vaccines. However, outbreaks still occur in poultry flocks globally due to ILTV vaccine strains reverting to virulence and emergence of new ILTV strains due to recombination in addition to circulating wildtype strains. Recent studies reported that most of the ILT outbreaks in Canada were induced by the chicken-embryo-origin (CEO) live attenuated vaccine revertant strains with the involvement of a small percentage of wildtype ILTV. It is not known if the host responses induced by these two ILTV strains are different. The objective of the study was to compare the host responses elicited by CEO revertant and wildtype ILTV strains in chickens. We infected 3-week-old specific pathogen-free chickens with the two types of ILTV isolates and subsequently evaluated the severity of clinical and pathological manifestations, in addition to host responses. We observed that both of the isolates show high pathogenicity by inducing several clinical and pathological manifestations. A significant recruitment of immune cells at both 3 and 7 days post-infection (dpi) was observed in the tracheal mucosa and the lung tissues of the infected chickens with wildtype and CEO vaccine revertant ILTV isolates when compared to uninfected controls. Overall, this study provides a better understanding of the mechanism of host responses against ILTV infection.

Phylogenetic analyses on Marek's disease virus circulating in Iranian backyard and commercial poultry indicate viruses of different origin.

As neoplastic viruses have been affecting Iranian chicken farms more frequentlyin recent years, the first step in prevention may thereforebe to genetically characterize and systematically identify their source and origin. Recently, we published a phylogenetic analysis based on the meq gene of Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1), that circulated in Iranian backyard and commercial chickens. In the current study, we are reporting for the first time the identification of a 298 aa meq protein containing only two PPPP motifs from an MDV-1-infected unvaccinated backyard turkey. This protein length has never been reported from any turkey species before. According to phylogenetic analysis, a close genetic relationship (0.68%) to several chicken-origin isolates such as the American vv + 648A strain was found. In addition, we identified a standard meq protein from a MDV-1-infected commercial chicken farm. In corroboration with our previous finding from other Iranian provinces, it is likely that the highly identical MDV-1 viruses currently circulating in Iranian chicken farms, which may be indicative of human role in the spread of the virus, have similar Eurasian origin. Our data suggest that regardless of the meq size, MDV-1 circulating in Iran are from different origins. On the other hand,meq sequences from bird species other than chicken have been reported but are very few. Our investigation suggests MDV-1 circulating in turkey do not have species-specific sequences.

PFT-α inhibits gallid alpha herpesvirus 1 replication by repressing host nucleotide metabolism and ATP synthesis

Therapeutics targeting virus–host interactions have been considered promising strategies for treating herpesvirus infection. Our previous study on avian infectious laryngotracheitis virus (ILTV), an avian herpesvirus economically important to the poultry industry worldwide, identified the small molecule Pifithrin-α (PFT-α) as a potential therapeutic agent. However, the underlying mechanisms of its antiviral function remain largely unknown. Using the ILTV-permissive chicken cell line LMH as the model, we found that PFT-α effectively suppressed the transcription and genome replication of ILTV and greatly reduced the level of infectious virions. Genome-wide transcriptome analysis revealed extensive repression of the metabolic processes of infected cells by PFT-α administration. Further metabolome assays of ILTV-infected cells using liquid chromatography coupled with mass spectrometry suggest host nucleotide metabolism and ATP synthesis as the key targets of PFT-α treatment during its repression of ILTV replication, which was experimentally supported by the reduced transcription of many key enzymes essential to nucleotide metabolism and ATP synthesis. The present study provides insights into the mechanisms by which PFT-α inhibits ILTV infection, which may increase the probability of successful clinical application of this molecule.

Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments.

Viral respiratory diseases, such as avian influenza, Newcastle disease, infectious bronchitis and infectious laryngotracheitis, have considerable negative economic implications for poultry. Ensuring the virus-free status of premises by environmental sampling after cleaning and disinfection is essential for lifting a quarantine and/or safely restocking the premises following an outbreak. The objectives of this study were to identify optimal sample collection devices and to determine the locations in poultry housing which are best for poultry respiratory virus sample collection. Chickens exposed to infectious bronchitis virus, which was used as a representative virus for enveloped poultry respiratory viruses, were housed in floor-pens in either a curtain-sided wood framed house or a cement block house. Foam swabs, cellulose sponges, polyester swabs, dry cotton gauze and pre-moistened cotton gauze were evaluated for comparative efficiency in recovering viral RNA. Cotton gauze pre-moistened with the viral transport media had the highest sensitivity among the devices (wood-framed house: 78% positive, geometric mean titre [GMT] of 2.6 log10 50% egg infectious doses [EID50 ] equivalents/ml; cement block houses: 55% positive, GMT of 1.7 log10 EID50 equivalents/ml). Targeting virus deposition sites is also crucial for efficient virus elimination procedures and subsequent testing; therefore, 10 locations within the houses were compared for virus detection. In both housing types, the highest viral RNA loads were recovered from the tops of drinker lines within the pen. Places the chickens could contact directly (e.g., feeder rim) or were contacted by caretaker feet (hallway floor) also yielded higher levels of viral RNA more consistently. These results will facilitate the establishment of efficient environmental sampling procedures for respiratory viruses of poultry.

Deletion of miR-M8 and miR-M13 eliminates the bursa atrophy induced by Marek’s disease virus infection

Marek’s disease (MD) is a neoplastic disease of chickens caused by an avian alphaherpesvirus, Marek’s disease virus (MDV, also known as Gallid alphaherpesvirus 2 [GaHV2]). A total of 14 microRNA (miRNA) precursors and 26 mature miRNAs have been identified in MDV genome, which were grouped in three distinct clusters. In recent years, our studies revealed the role of MDV encoded cluster 3 miRNAs (or miR-M8-M10) and the specific function of its three members, miR-M6, miR-M7 and miR-M10, in regulating MDV replication and pathogenesis. In this study, we characterized the unique function of the other two members, miR-M8 and miR-M13, in cluster 3 miRNAs. Our results show that miR-M8 and miR-M13 are not important for MDV plaque formation and genome replication in vitro. Animal experiment results show that deletion of miR-M8–5p and miR-M13–5p eliminates the bursa atrophy, but not thymus atrophy, of MDV inoculated chickens. In addition, we found that the survival curve and MD incidences were not affected by disruption of miR-M8 and miR-M13. Taken together, this study uncovers the unique role of miR-M8 and miR-M13 in MDV replication and pathogenesis, which filled the gap in the research of MDV encoded miRNAs.

Investigation of Serotype Prevalence of Escherichia coli Strains Isolated from Layer Poultry in Greece and Interactions with Other Infectious Agents.

Colibacillosis is the most common bacterial disease in poultry and it is caused by avian pathogenic Escherichia coli (APEC), which is assigned to various O-serogroups. Previous studies have shown that APEC strains are more often related to certain O-serogroups such asO78, O2 and O1. E. coli has been reported to act either as a primary or secondary agent in complicating other infections. The aim of this study was to investigate the occurrence of and characterize the O-serogroups of E. coli strains isolated from commercial layer and layer breeder flocks showing macroscopic lesions of colibacillosis and increased or normal mortality in Greece. Furthermore, we attempted to assess the interaction between infectious agents such as Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), infectious bronchitis (IBV) and infectious laryngotracheitis (ILT) with E. coli infections in layer flocks with increased mortality. Our study revealed that in addition to the common serogroups (O78, O2), many other, and less common serogroups were identified, including O111. The O78, O111 and O2 serogroups were frequently detected in flocks with lesions of colibacillosis and increased mortality whereas O2, O88 and O8 were reported more commonly in birds with colibacillosis lesions but normal mortality rates. These data provide important information for colibacillosis monitoring and define preventative measures, especially by using effective vaccination programs because E. coli vaccines are reported to mainly offer homologous protection. Finally, concerning the association of the four tested infectious agents with E. coli mortality, our study did not reveal a statistically significant effect of the above infectious agents tested with E. coli infection mortality.