Failure to experimentally infect 10 days-old piglets with a cell culture-propagated infectious stock of a classical genotype 1a porcine epidemic diarrhea virus.

Introduction: Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs of all ages. PEDV can be grouped into G1 (classical strains) and G2 (variant strains) based on sequence differences in the spike gene. Although several pathogenesis studies using contemporary strains of PEDV have been conducted to date, there is limited information on the pathogenesis of historical PEDV strains in contemporary pigs. This study aimed to investigate the clinical disease course of 10 days-old pigs infected with a classical European G1a PEDV strain from the 1980s which was last passaged in pigs in 1994. Methods: Sequencing results confirmed that the virus inoculum was a PEDV strain closely related to the prototype CV777 strain. The PEDV stock was serially passaged three times in Vero cells, and the P3 infectious virus stock was used to inoculate the pigs. A total of 40 pigs were inoculated using the oral route. Results: Pigs showed no enteric disease signs, and PEDV shedding was not detected for 44 days post-inoculation (dpi). At necropsy at 3 (5 pigs) or 7 dpi (5 pigs), no lesions were observed in intestinal sections, which were negative for PEDV antigen by immunohistochemistry. In addition, no IgG or IgA PEDV-specific antibodies in serum or fecal samples for 35 dpi further indicates a lack of infection. Titration of the leftover thawed and refrozen PEDV virus stock inoculum showed that the virus stock retained its infectivity in Vero cell culture and the porcine small intestine enterocytes cell line IPEC-J2. Discussion: The reasons for the loss of infectivity in pigs are unknown. In conclusion, we showed that a classical G1a PEDV strain successfully propagated in cell cultures could not orally infect 40 piglets.

Protection Efficacy of the Infectious Laryngotracheitis (ILT) Serva CEO Vaccine Strain in Broiler Chickens Under Different Vaccination Coverage Conditions.

Mass vaccination against infectious laryngotracheitis virus (ILTV) in drinking water can result in variable initial vaccine take. Partial initial vaccine coverage of 20% with an Australian ILT vaccine (A20) previously resulted in significant protection against virulent ILTV challenge. This follow-up study used the international Serva ILT vaccine strain in a factorial design testing four levels of vaccination coverage (0%, 10%, 20%, or 100% of chicks eye-drop vaccinated with the live vaccine at 7 days of age) and three levels of ILTV challenge (no challenge or challenge at 7 or 21 days postvaccination [DPV]). The increase in ILTV load in choanal cleft swabs detected by qPCR after challenge was significantly reduced by 20% and 100% but not by 10% vaccination coverage. Vaccination reduced weight gain in unchallenged birds. Daily weight gain of birds was not affected by ILTV challenge at 7 DPV in any group, but following challenge at 21 DPV, it was significantly reduced in unvaccinated and 10% vaccinated groups relative to 20% and 100% vaccinated groups. Vaccination of 20% of the chickens provided substantial but incomplete protection (protective index range 44%-70%) against the severity of clinical signs and mortality following challenge while 10% vaccination coverage provided limited or no protection. Clinical signs were more severe and appeared earlier following challenge at 21 DPV than at 7 DPV. Within the vaccination treatments, eye-drop-vaccinated birds were better protected than their in-contact cohorts. In conclusion, partial vaccination of 20%, but not 10% of chickens, induced substantial protection against subsequent challenge. However, the attendant risks of reduced protection against early challenge and the possible reversion to virulence of vaccine virus when transmitted to unvaccinated chickens make it essential that 100% initial vaccine take be the goal of mass vaccination programs.

Eficacia protectora de la cepa vacunal CEO Serva del virus de la laringotraqueítis infecciosa (ILT) en pollos de engorde bajo diferentes condiciones de cobertura vacunal. La vacunación masiva contra el virus de la laringotraqueítis infecciosa (ILTV) en el agua de bebida puede resultar en una cobertura vacunal inicial variable. La cobertura vacunal inicial parcial del 20 % con una vacuna ILT australiana (A20) previamente resultó en una protección significativa contra el desafío virulento con el virus de la laringotraqueítis. Este estudio de seguimiento utilizó la cepa de la vacuna vacunal internacional Serva ILT en un diseño factorial para probar cuatro niveles de cobertura de vacunación (0 %, 10 %, 20 % o 100 % de pollitos vacunados por gota ocular con la vacuna viva a los siete días de edad) y tres niveles de desafío con el virus de la laringotraqueítis (sin desafío o con desafío a los 7 o 21 días después de la vacunación [DPV]). El aumento en la carga viral en hisopos de la hendidura coanal detectados por qPCR después del desafío se redujo significativamente con cobertura de vacunación del 20% y 100%, pero no con el 10%. La vacunación redujo el aumento de peso en las aves no desafiadas. La ganancia diaria de peso de las aves no se vio afectada por el desafío con el virus de la laringotraqueítis a los siete días después de la vacunación en ningún grupo, pero después del desafío a los 21 días después de la vacunación, se redujo significativamente en los grupos no vacunados y con cobertura del 10% en comparación con los grupos con cobertura del 20% y 100%. La vacunación del 20 % de los pollos brindó una protección sustancial pero incompleta (con un rango de índice de protección del 44 % al 70 %) contra la severidad de los signos clínicos y la mortalidad después del desafío, mientras que la cobertura de vacunación del 10 % brindó protección limitada o nula. Los signos clínicos fueron más graves y aparecieron más temprano después del desafío a los 21 días después de la vacunación en comparación con el desafío a los siete días después de la vacunación. Dentro de los tratamientos de vacunación, las aves vacunadas con gota ocular estaban mejor protegidas que sus cohortes en contacto. En conclusión, la cobertura de vacunación parcial del 20%, pero no del 10% de los pollos, indujo una protección sustancial contra el desafío posterior. Sin embargo, los riesgos concomitantes de una protección reducida contra el desafío temprano y la posible reversión a la virulencia del virus vacunal cuando se transmite a pollos no vacunados hacen que sea esencial que la cobertura vacunal inicial del 100% sea el objetivo de los programas de vacunación masiva.

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

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.

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.

Aplicación de campo del monitoreo por qPCR del virus de la laringotraqueítis infecciosa en el polvo de casetas avícolas y su función en el control de un brote importante El muestreo a nivel de población basado en la detección por qPCR del virus de la laringotraqueítis infecciosa (ILTV) en el polvo de instalaciones avícolas se puede utilizar para evaluar los resultados de la vacunación contra esta enfermedad después de la administración masiva en el agua de bebida. Se reporta la aplicación de campo de este enfoque para evaluar el éxito de la administración de vacunas y su uso en el control de brotes por laringotraqueítis infecciosa en pollos de engorde. En el Estudio 1, se recolectaron muestras de polvo de 26 parvadas de pollos de engorda a los 0, 4, 7, 14 y 21 días después de la vacunación en el agua de bebida (DPV) a los 7 a 13 días de edad con las vacunas de laringotraqueítis vivas atenuadas Serva o A20. Inesperadamente, se detectó ADN del virus de laringotraqueítis en muestras de polvo recolectadas antes de la vacunación en 22/26 parvadas. La tipificación reveló que el virus detectado era diferente del virus de la vacuna. Para determinar si el ADN del virus de laringotraqueítis detectado procedía de una infección activa o del remanente de un virus no infeccioso, se implementó el Estudio 2 en 14 parvadas adicionales con muestras de polvo recolectadas a los 0, 7, 14 y 21 días después de la vacunación y de hisopos traqueales recolectados de 15 aves/parvada a los cero y 21 días después de la vacunación. Los resultados indicaron que había infección activa con el virus de laringotraqueítis en esas parvadas antes de la vacunación. Este enfoque contribuyó a un programa de control estatal que resultó en la erradicación de laringotraqueítis del sur de Australia, como lo confirmaron los resultados negativos de las pruebas del mismo virus para muestras de polvo de 50 parvadas y la ausencia de laringotraqueítis infecciosa clínica. Estos hallazgos muestran que la infección por el virus de la laringotraqueítis antes de la vacunación es común en situaciones de brotes y que las muestras de polvo deben recolectarse a los cero y 7 días después de la vacunación para una interpretación significativa de los resultados de la vacunación y el estado de esta enfermedad. Las pruebas de polvo comparativamente de bajo costo durante un brote, junto con la información de tipificación, ayudaron mucho con la toma de decisiones y con las estrategias de control durante un brote importante, incluida la confirmación de la ausencia de infección en las etapas finales.

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.

Circulation and Molecular Characterization of Hemorrhagic Enteritis Virus in Commercial Turkey and Meat Chicken Flocks in Australia.

Currently, there is no available vaccine against hemorrhagic enteritis virus (HEV) in Australia. Although it is assumed that subclinical HEV infections occur and may be associated with an increase in colibacillosis in Australian commercial turkey flocks, the prevalence of infection with this virus in the country is largely unknown. The aims of this study were to determine the extent of HEV infection in commercial flocks in Australia and to investigate the diversity of Australian HEV strains. Serum and spleen samples were collected from breeder and grower turkeys and serum was collected from breeder and grower chickens by the two major poultry integrator companies in Australia. Of the turkey samples, 727/849 (86%) sera were positive for anti-HEV antibodies by ELISA. HEV DNA was detected in 215/278 (77%) spleen samples positive by PCR. Of the meat chicken sera, 115/144 (80%) samples were seropositive. Sequencing the whole genome of three HEV field isolates showed that the Australian strains are highly similar and cluster separately from strains from other geographic regions although several point mutations were shared with HEV strains considered to be virulent. In conclusion, HEV infection is ubiquitous in Australian commercial poultry flocks. The impact of the many genomic point mutations detected in Australian HEV strains on virus pathogenicity is unclear.

Circulación y caracterización molecular del virus de la enteritis hemorrágica en parvadas comerciales de pavo y pollos de engorde en Australia. Actualmente, no existe una vacuna disponible contra el virus de la enteritis hemorrágica (HEV) en Australia. Aunque se supone que se producen infecciones subclínicas por el virus de la enteritis hemorrágica y pueden estar asociadas con un aumento de la colibacilosis en las parvadas comerciales de pavos australianos, se desconoce en gran medida la prevalencia de la infección por este virus en el país. Los objetivos de este estudio fueron determinar la diseminación de la infección por el virus de la enteritis hemorrágica en parvadas comerciales en Australia e investigar la diversidad de cepas del virus de la enteritis hemorrágica australianas. Se recolectaron muestras de suero y bazo de pavos reproductores y de engorda y las dos principales empresas integradoras avícolas de Australia recolectaron suero de pollos reproductores y de engorde. De las muestras de pavo, 727/849 (86%) sueros fueron positivos para anticuerpos contra la enteritis hemorrágica por ELISA. Se detectó ADN del virus de la enteritis hemorrágica en 215/278 (77%) muestras de bazo positivas por PCR. De los sueros de carne de pollo, 115/144 (80%) muestras fueron seropositivas. La secuenciación del genoma completo de tres aislados de campo del virus de la enteritis hemorrágica mostró que las cepas australianas son muy similares y se agrupan por separado de las cepas de otras regiones geográficas, aunque se compartieron varias mutaciones puntuales con las cepas del virus de la enteritis hemorrágica consideradas virulentas. En conclusión, la infección por el virus de la enteritis hemorrágica es ubicua en las parvadas avícola comerciales australianas. No está claro el impacto de las diferentes mutaciones puntuales genómicas detectadas en las cepas australianas del virus de la enteritis hemorrágica sobre la patogenicidad del virus.

Molecular-based monitoring of live vaccines in dust samples from experimental and commercial chicken flocks and its potential use as a screening test.

Despite the high cost of vaccination programmes, conventional methods to evaluate vaccine uptake are often impractical and costly. More recently, molecular-based testing of poultry dust has been used to monitor the "take" of Marek's disease virus and infectious laryngotracheitis virus (ILTV) live vaccines. This study aimed to provide proof-of-concept for detecting other poultry pathogens by using molecular detection of vaccine microorganisms in poultry dust of vaccinated flocks. Dust and choanal cleft and cloacal swabs were collected from chickens vaccinated against avian encephalomyelitis virus (AEV), fowlpox virus (FPV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using live vaccines in an experimental flock. Dust samples were collected weekly from 5 commercial breeder or layer flocks from day-old up to 25 weeks of age. These flocks were vaccinated against Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), ILTV, fowl adenovirus (FAdV), MG and MS. Samples were tested for nucleic acids of these microorganisms by PCR or reverse transcriptase PCR. Genomes of all targeted vaccines were detected in dust samples from the experimental and commercial flocks except for FPV, which was detected only in the experimental flock. FAdV was detected in unvaccinated commercial flocks. These findings suggest that PCR detection of target organisms in dust samples has potential as a relatively simple and inexpensive population-level test to monitor vaccine take and/or pathogen status in chicken flocks. Further studies comparing the detection of each of these microorganisms in poultry dust with individual birds samples are required to validate this approach.

A preliminary study of the localization of infectious laryngotracheitis virus glycoprotein E within specific peripheral blood lymphocytes.

Infectious laryngotracheitis virus (ILTV) DNA has been detected in blood fractions, but the cell phenotype with which the virus is associated is unknown. This study investigated the presence of ILTV antigen in peripheral blood cells of six acutely ILTV-infected chickens (5 or 9 days post ocular inoculation with a virulent isolate) and three sham-inoculated chickens using immunofluorescent staining. Blood fractions were separated by Ficoll-Paque density gradient centrifugation, and smears were prepared from erythrocyte and leukocyte fractions. The smears were stained for ILTV glycoprotein E and the leukocyte markers CD4, CD8, Bu-1 (B cell), KUL01 (monocyte/macrophage), TCRγδ, and TCRαß/Vß2 and examined under a confocal microscope. In samples from infected birds, ILTV gE-specific fluorescence was localized in B cells and all evaluated T cell types, but not in monocytes and erythrocytes. The percentage of CD4, CD8, TCRγδ, TCRαß/Vß1, TCRαß/Vß2 and B cells positive for ILTV antigen ranged from 13.3% to 22.3%. None of the samples from the sham-inoculated chickens exhibited fluorescence for ILTV gE. The results of this pilot study suggest that ILTV has a tropism for peripheral blood T and B cells. Further research is required to investigate whether these cells support ILTV productive replication. RESEARCH HIGHLIGHTSSelective tropism of ILTV for peripheral blood cells was demonstrated in acutely infected birds.The ILTV antigen gE was detected in blood CD4, CD8, TCRγδ, TCRαß and B cells but not in monocytes and erythrocytes.The highest percentage of ILTV antigen was observed in CD4 cells (22.3%) followed by TCRαß/Vß1 (20.6%), CD8 (15.4%), TCRαß/Vß2 or B cells (14.4%) and TCRγδ cells (13.3%).

Centennial Review: Factors affecting the chicken gastrointestinal microbial composition and their association with gut health and productive performance.

Maintenance of "gut health" is considered a priority in commercial chicken farms, although a precise definition of what constitutes gut health and how to evaluate it is still lacking. In research settings, monitoring of gut microbiota has gained great attention as shifts in microbial community composition have been associated with gut health and productive performance. However, microbial signatures associated with productivity remain elusive because of the high variability of the microbiota of individual birds resulting in multiple and sometimes contradictory profiles associated with poor or high performance. The high costs associated with the testing and the need for the terminal sampling of a large number of birds for the collection of gut contents also make this tool of limited use in commercial settings. This review highlights the existing literature on the chicken digestive system and associated microbiota; factors affecting the gut microbiota and emergence of the major chicken enteric diseases coccidiosis and necrotic enteritis; methods to evaluate gut health and their association with performance; main issues in investigating chicken microbial populations; and the relationship of microbial profiles and production outcomes. Emphasis is given to emerging noninvasive and easy-to-collect sampling methods that could be used to monitor gut health and microbiological changes in commercial flocks.

Microbial taxa in dust and excreta associated with the productive performance of commercial meat chicken flocks.

BACKGROUND: A major focus of research on the gut microbiota of poultry has been to define signatures of a healthy gut and identify microbiota components that correlate with feed conversion. However, there is a high variation in individual gut microbiota profiles and their association with performance. Population level samples such as dust and pooled excreta could be useful to investigate bacterial signatures associated with productivity at the flock-level. This study was designed to investigate the bacterial signatures of high and low-performing commercial meat chicken farms in dust and pooled excreta samples. Poultry house dust and fresh pooled excreta were collected at days 7, 14, 21, 28 and 35 of age from 8 farms of two Australian integrator companies and 389 samples assessed by 16S ribosomal RNA gene amplicon sequencing. The farms were ranked as low (n = 4) or high performers (n = 4) based on feed conversion rate corrected by body weight. RESULTS: Permutational analysis of variance based on Bray-Curtis dissimilarities using abundance data for bacterial community structure results showed that company explained the highest variation in the bacterial community structure in excreta (R2 = 0.21, p = 0.001) while age explained the highest variation in the bacterial community structure in dust (R2 = 0.13, p = 0.001). Farm performance explained the least variation in the bacterial community structure in both dust (R2 = 0.03, p = 0.001) and excreta (R2 = 0.01, p = 0.001) samples. However, specific bacterial taxa were found to be associated with high and low performance in both dust and excreta. The bacteria taxa associated with high-performing farms in dust or excreta found in this study were Enterococcus and Candidatus Arthromitus whereas bacterial taxa associated with low-performing farms included Nocardia, Lapillococcus, Brachybacterium, Ruania, Dietzia, Brevibacterium, Jeotgalicoccus, Corynebacterium and Aerococcus. CONCLUSIONS: Dust and excreta could be useful for investigating bacterial signatures associated with high and low performance in commercial poultry farms. Further studies on a larger number of farms are needed to determine if the bacterial signatures found in this study are reproducible.

A molecular based method for rapid detection of Salmonella spp. in poultry dust samples.

Salmonellosis, caused by Salmonella spp., is a widely reported foodborne zoonosis frequently associated with ingestion of poultry products. Salmonella vaccination of chickens can be used to reduce bacterial shedding and risk of human infection. To determine Salmonella burden in chicken farms, culture methods of environmental samples that require a turn-around time of 5-7 days are usually used. Rapid screening using molecular assays such as PCR of pre-enriched broth has been reported for Salmonella spp. detection in feed, floor dust, and drag swabs within 2-3 days. Here we report an adaptation of the method for detection of Salmonella in poultry dust samples collected using a settle plate method under experimental conditions. Key features:•Passive dust sample collection using dry settle plates without media suspended from dropper lines of drinkers.•Small amount of sample required for the pre-enrichment process.•Quantification of Salmonella DNA with high sensitivity using an inexpensive extraction protocol.

Microbial communities of poultry house dust, excreta and litter are partially representative of microbiota of chicken caecum and ileum.

Traditional sampling methods for the study of poultry gut microbiota preclude longitudinal studies as they require euthanasia of birds for the collection of caecal and ileal contents. Some recent research has investigated alternative sampling methods to overcome this issue. The main goal of this study was to assess to what extent the microbial composition of non-invasive samples (excreta, litter and poultry dust) are representative of invasive samples (caecal and ileal contents). The microbiota of excreta, dust, litter, caecal and ileal contents (n = 110) was assessed using 16S ribosomal RNA gene amplicon sequencing. Of the operational taxonomic units (OTUs) detected in caecal contents, 99.7% were also detected in dust, 98.6% in litter and 100% in excreta. Of the OTUs detected in ileal contents, 99.8% were detected in dust, 99.3% in litter and 95.3% in excreta. Although the majority of the OTUs found in invasive samples were detected in non-invasive samples, the relative abundance of members of the microbial communities of these groups were different, as shown by beta diversity measures. Under the conditions of this study, correlation analysis showed that dust could be used as a proxy for ileal and caecal contents to detect the abundance of the phylum Firmicutes, and excreta as a proxy of caecal contents for the detection of Tenericutes. Similarly, litter could be used as a proxy for caecal contents to detect the abundance of Firmicutes and Tenericutes. However, none of the non-invasive samples could be used to infer the overall abundance of OTUs observed in invasive samples. In conclusion, non-invasive samples could be used to detect the presence and absence of the majority of the OTUs found in invasive samples, but could not accurately reflect the microbial community structure of invasive samples.

Airborne Transmission of Vaccinal and Wild Type Infectious Laryngotracheitis Virus and Noninfectivity of Extracts of Excreta from Infected Chickens.

Infectious laryngotracheitis virus (ILTV) is thought to exit the host in respiratory aerosols and enter by inhalation of these. High levels of ILTV DNA have been detected in excreta, raising the possibility of alternative routes of shedding from the host. However, it is not known whether or not the ILTV DNA in excreta represents infective virus. This study investigated transmission of wild type and vaccinal ILTV from infected to susceptible commercial meat chickens. Airborne- and excreta-mediated transmission of two field isolates of ILTV (Classes 9 and 10) and three vaccine strains (SA2, A20, and Serva) were tested. To test airborne transmission, air from isolators containing infected birds was ducted through a paired isolator containing uninfected chickens. To test excreta transmission, aliquots were prepared from excreta containing a high level of ILTV DNA within the first week after infection. Chicks were infected bilaterally by eye drop. Clinical signs were monitored daily and choanal cleft swab samples for ILTV detection by quantitative PCR were collected at 4, 8, 15, 22, and 28 days postinfection (DPI) in the airborne transmission study and at 7 and 14 DPI from the excreta transmission studies. There was no transmission of ILTV from excreta, suggesting that ILTV is inactivated during passage through the gut. All strains of ILTV were transmitted by the airborne route but only to a limited extent for the vaccine viruses. The field viruses induced clinical signs, pathology, and greatly elevated ILTV genome copies in swabs. In summary, these findings confirm the suspected airborne transmission of ILTV, demonstrate differential transmission potential between wild type and vaccine strains by this route, and indicate that excreta is unlikely to be important in the transmission of ILTV and the epidemiology of ILT.

Artículo regular­Transmisión aérea del virus de la laringotraqueítis infecciosa de tipo vacunal y silvestre y ausencia de infecciosidad de los extractos de excrementos de pollos infectados. Se cree que el virus de la laringotraqueítis infecciosa (ILTV) se elimina del huésped en forma de aerosoles respiratorios y entra por la inhalación de los mismos. Se han detectado altos niveles de ADN del virus de la laringotraqueítis en las excretas, lo que aumenta la posibilidad de rutas alternas de eliminación por el hospedador. Sin embargo, no se sabe si el ADN del virus de la laringotraqueítis presente en las excretas representa virus infeccioso. Este estudio investigó la transmisión del virus de la laringotraqueítis de tipo silvestre y vacunal de pollos de carne comerciales infectados a pollos susceptibles. Se evaluó la transmisión por vía aérea y mediada por excretas de dos cepas de campo del virus de la laringotraqueítis (clases 9 y 10) y tres cepas vacunales (SA2, A20 y Serva). Para evaluar la transmisión aérea, el aire de los aisladores que contienen aves infectadas se canalizó a través de un aislador emparejado que contenía pollos no infectados. Para probar la transmisión de excretas, se prepararon alícuotas a partir de excretas que contenían un alto nivel de ADN del virus de la laringotraqueítis durante la primera semana después de la infección. Los pollos se infectaron mediante aplicación de gota ocular de forma bilateral. Los signos clínicos se monitorearon diariamente y se recolectaron muestras de hisopado de la hendidura coanal para la detección del virus de la laringotraqueítis mediante PCR cuantitativa a los 4, 8, 15, 22 y 28 días después de la infección (DPI) en el estudio de transmisión aérea y a los 7 y 14 después de la inoculación en los estudios de transmisión de excretas. No se observó transmisión del virus de la laringotraqueítis de las excretas, lo que sugiere que este virus se inactiva durante el paso a través del intestino. Todas las cepas del virus de la laringotraqueítis se transmitieron por vía aérea, pero sólo de forma limitada con los virus vacunales. Los virus de campo indujeron signos clínicos, patología y números muy altos de copias del genoma del virus de la laringotraqueítis en muestras hisopos. En resumen, estos hallazgos confirman la sospecha de transmisión aérea del virus de laringotraqueítis, demuestran el diferente potencial de transmisión entre las cepas de tipo silvestre y vacunales por esta vía, e indican que es poco probable que las excretas sean importantes en la transmisión del virus de la laringotraqueítis y en la epidemiología del virus de la laringotraqueítis infecciosa.Key words: infectious laryngotracheitis virus, airborne transmission, meat chicken, excreta, epidemiology.

Detection and Quantification of Clostridium perfringens and Eimeria spp. in Poultry Dust Using Real-Time PCR Under Experimental and Field Conditions.

Infection of poultry with Eimeria spp., the causative agent of coccidiosis, can predispose birds to necrotic enteritis (NE) caused by netB gene-positive strains of Clostridium perfringens. The detection of Eimeria spp., C. perfringens, and netB were examined in settled dust from broiler flocks under experimental and field conditions. Dust samples were collected from settle plates twice weekly from two experimental flocks inoculated with three species of pathogenic Eimeria in 9-day-old chicks, followed by netB gene-positive C. perfringens 5 days later to produce subclinical and clinical NE. A noninoculated flock was sampled weekly from day 0 and served as a control flock. An additional 227 dust samples from commercial broiler flocks were collected at the end-of-batch (6-7 wk of age; one scraped dust sample per flock). In the NE-subclinical and NE-clinical flocks, high levels of Eimeria spp. and C. perfringens were detected after inoculation followed by a gradual decline over time. In the control flock, C. perfringens and netB were detected at low levels. No significant effect of sampling location was evident on Eimeria spp., C. perfringens, and netB load within poultry houses. These results provide evidence that Eimeria spp., C. perfringens, and netB gene copies can be readily measured in poultry dust samples collected in settle plates and may provide an alternative sampling method for monitoring flock coccidiosis and NE status. Further studies are required to assess the utility for such a test in commercial flocks.

Artículo regular­Detección y cuantificación de Clostridium perfringens y Eimeria spp. en polvo de instalaciones avícolas mediante PCR en tiempo real bajo condiciones experimentales y de campo. La infección de aves comerciales con Eimeria spp., el agente causante de la coccidiosis, puede predisponer a las aves a enteritis necrótica (NE) causada por cepas de Clostridium perfringens positivas a la presencia del gene netB. La detección de Eimeria spp., C. perfringens y del gene netB se examinó en el polvo sedimentado de parvadas de pollos de engorde bajo condiciones experimentales y de campo. Se recolectaron muestras de polvo por sedimentación en placa dos veces por semana de dos parvadas experimentales inoculadas con tres especies de Eimeria patógena en pollitos de nueve días, seguidas de C. perfringens positiva al gene netB cinco días después para producir enteritis necrótica subclínica y clínica. Una parvada no inoculada se muestreó semanalmente desde el día cero y sirvió como parvada control. Se recolectaron 227 muestras adicionales de polvo de parvadas de pollos de engorde comerciales al final del lote (6 a 7 semanas de edad; una muestra de polvo por raspado por parvada). En las parvadas con enteritis necrótica subclínica y clínica, se detectaron niveles altos de Eimeria spp. y de C. perfringens después de la inoculación seguida de una disminución gradual con el tiempo. En la parvada control, C. perfringens y el gene netB se detectaron en niveles bajos. No fue evidente ningún efecto significativo de la ubicación del muestreo sobre la carga de Eimeria spp., C. perfringens y del gene netB dentro de las casetas. Estos resultados proporcionan evidencia de que las copias genéticas de Eimeria spp., C. perfringens y del gene y netB se pueden medir fácilmente en muestras de polvo de instalaciones avícolas recolectadas mediante sedimentación en placa y pueden proporcionar un método de muestreo alternativo para monitorear coccidiosis y el estado de enteritis necrótica en la parvada. Se requieren más estudios para evaluar la utilidad de tal prueba en parvadas comerciales.

Evaluation of autofluorescence quenching techniques on formalin- fixed chicken tissues.

Autofluorescence (AF) in formalin-fixed and paraffin-embedded tissues limit their use in immunofluorescence staining techniques. Various methods have been used to reduce AF in human and animal tissues but no protocol has been optimized for avian tissues. The present study was undertaken to evaluate different treatment methods including ammonium chloride, glycine, Trypan blue, sodium borohydride, Sudan Black B, potassium permanganate, LED light, cupric sulphate combined with glycine, ammonium chloride and cupric sulphate in reducing AF in FFPE chicken tissues for the detection of FITC labelled antibodies against immune cell markers. Chicken tissues including conjunctiva, trachea and Harderian gland presented intense non-homogenous AF in cells resembling erythrocytes, connective cells and melanocytes. Only Sudan Black B effectively reduced AF in FFPE tissues; however, no specific fluorescent signal was observed for six FITC labelled antibodies against immune cell markers. Specific fluorescent signal from the FITC-labelled antibodies was observed in frozen chicken tissue sections with minimal AF, suggesting that the AF in FFPE tissues is related to the use of formaldehyde fixatives. In conclusion, this study demonstrates for the first time that AF quenching methods commonly used for other animal species are not appropriate for use in avian tissues and that frozen tissue sections are recommended for immunofluorescence staining techniques in poultry.

Characterization of poultry house dust using chemometrics and scanning electron microscopy imaging.

Poultry house dust is composed of fine particles which likely originate from a diverse range of materials such as feed, litter, excreta, and feathers. Little is known about the contribution of these sources to broiler house airborne dust so the present study was designed to identify the relative contributions of these sources. Samples of feed, excreta, feather, and bedding, known mixtures of these and settled dust from 28 broiler chicken flocks were tested for the concentration of 18 chemical elements. A chemometrics approach (the application of multivariate statistical techniques to chemical analysis data) was used to identify the primary source material in broiler chicken house dust samples. Scanning electron microscopy (SEM) was also used to analyze dust sample particulates based on examination of source materials. Excreta was found to be the main component of broiler chicken house dust, both by SEM and chemometric analysis. SEM of experimental flock dust between 7 and 35 days of age (d) revealed that the contribution of excreta to dust increased with age from 60% at 7 d to 95% at 28 d (P < 0.001). The proportion of bedding and feed in dust declined with age while the contribution of feather material remained low throughout. This study demonstrates that excreta provides the bulk of the material in poultry dust samples with bedding material, feed and feather material providing lower proportions. The relative contributions of these materials to dust varies with age of birds at dust collection. Additional research is required to determine the health and diagnostic implications of this variation.

Transmission of infectious laryngotracheitis virus vaccine and field strains: the role of degree of contact and transmission by whole blood, plasma and poultry dust.

Understanding the mechanisms of transmission of infectious laryngotracheitis virus (ILTV) is critical to proper control as both vaccine and wild-type strains circulate within chicken flocks with potential adverse consequences. The relative efficiency of transmission by direct contact between chickens and airborne transmission has not been investigated. Furthermore, relatively high levels of ILTV DNA have been detected in poultry dust and blood but the infectivity of these is unknown. In this study, comparison of in-contact and airborne transmission of two vaccine and one field strain of ILTV revealed that all transmitted to 100% of in-contact birds by 6 days post-exposure (dpe). Airborne transmission without contact resulted in 100% transmission by 14 and 17 dpe for the wild-type and Serva vaccine virus but only 27% transmission by 21 dpe for the A20 vaccine virus. The infectivity of dust or extracts of dust and blood or plasma from infected chickens at various stages of infection was assessed by inoculation into susceptible chickens. There was no transmission by any of these materials. In conclusion, direct contact facilitated efficient ILTV transmission but the virus was unable to be transmitted by dust from infected chickens suggestive of a limited role in the epidemiology of ILTV.

Comparison of tracheal and choanal cleft swabs and poultry dust samples for detection of Newcastle disease virus and infectious bronchitis virus genome in vaccinated meat chicken flocks.

This study assessed different methods (tracheal and choanal cleft swabs from individual birds, and poultry dust as a population level measure) to evaluate the shedding kinetics of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) genome in meat chicken flocks after spray vaccination at hatchery. Dust samples and tracheal and choanal cleft swabs were collected from four meat chicken flocks at 10, 14, 21 and 31 days post vaccination (dpv) and tested for IBV and NDV genome copies (GC) by reverse transcriptase (RT)-PCR. IBV and NDV GC were detected in all sample types throughout the study period. Detection rates for choanal cleft and tracheal swabs were comparable, with moderate and fair agreement between sample types for IBV (McNemar's = 0.27, kappa = 0.44) and NDV (McNemar's = 0.09; kappa = 0.31) GC respectively. There was no significant association for IBV GC in swabs and dust samples (R2 = 0.15, P = 0.13) but NDV detection rates and viral load in swabs were strongly associated with NDV GC in dust samples (R2 = 0.86 and R2 = 0.90, P<0.001). There was no difference in IBV and NDV GC in dust samples collected from different locations within a poultry house. In conclusion, dust samples collected from any location within poultry house show promise for monitoring IBV and NDV GC in meat chickens at a population level and choanal cleft swabs can be used for detection of IBV and NDV GC instead of tracheal swabs in individual birds.

Molecular detection of Eimeria species and Clostridium perfringens in poultry dust and pooled excreta of commercial broiler chicken flocks differing in productive performance.

Necrotic enteritis and coccidiosis are the most economically detrimental enteric diseases of broiler chickens. This study aimed to investigate the association of DNA load of Clostridium perfringens, netB, and five Eimeria species (E. brunetti, E. maxima, E. necatrix, E. acervulina and E. tenella) in poultry house dust and pooled excreta with flock productive performance. The dust and pooled excreta from the floor were collected weekly at days 7, 14, 21, 28 and 35 of chicken age from 16 flocks of eight farms from two Australian integrator companies. The farms were ranked as high or low performers by each integrator according to the production performance of studied flocks. Eimeria tenella and necatrix were not detected in any farm while E. brunetti was detected in a low-performance farm and netB was detected in a high-performance farm. C. perfringens, E. acervulina and E. maxima DNA were detected on all farms with no significant differences in DNA load between high and low-performance farms or companies. The lack of association of pathogen DNA load and farm performance is possibly due to overall low to moderate pathogen DNA load detected in this study. Further studies on a larger number of farms are needed to determine whether these population level measurements of key pathogens based on PCR detection of nucleic acids are correlated with performance variables.

Detection of infectious laryngotracheitis virus (ILTV) in tissues and blood fractions from experimentally infected chickens using PCR and immunostaining analyses.

The ability of infectious laryngotracheitis virus (ILTV) to replicate in organs outside of the upper respiratory tract and conjunctiva associated-lymphoid tissues is still not well understood. This study investigated the tissue distribution of an Australian field strain of ILTV (class 9) on birds experimentally inoculated via eye-drop at 7 days of age by using quantitative PCR (qPCR) and immunohistochemistry. Tissues including conjunctiva, caecal tonsil, kidney, liver, lung, spleen, thymus, trachea and blood were collected from sham-inoculated (control group; n = 2) and ILTV-inoculated (n = 8) birds at 7 days post-inoculation (dpi). Blood was collected from 13 infected birds at 14 dpi and fractionated using ficoll-paque. At 7 dpi, the highest detection rate and genomic copies (GC) were in conjunctiva (8/8; 8.08 ± 0.48 log10 GC/mg) followed by trachea (8/8; 4.64 ± 0.48) and thymus (8/8; 4.52 ± 0.48), kidney (8/8; 3.97 ± 0.48), lung (8/8; 3.65 ± 0.48), spleen (8/8; 3.55 ± 0.48), liver (8/8; 3.51 ± 0.48), caecal tonsil (7/8; 3.76 ± 0.48) and plasma (4/8; 2.40 ± 0.48 log10 GC/ml). ILTV antigen was only detected in conjunctiva (7/8), trachea (6/8) and lung (4/8) samples. At 14 dpi, ILTV detection rate and genomic copies in buffy coat cells were 12/13 and 2.86 ± 0.39 log10 GC/mg, respectively while those of plasma were 11/13 and 4.29 ± 0.39 log10 GC/ml and red blood cell were 3/13 and 0.36 ± 0.39 log10 GC/mg. In conclusion, ILTV DNA was detected in a wide range of tissues and blood fractions but ILTV antigen was only detected in respiratory organs and conjunctiva.