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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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest. Matthew Hilliar, who was a PhD candidate at University of New England at the time in which the study was carried out, is currently employed by Turosi. The funder provided support in the form of salaries for author MH during the write up of this study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the ‘author contributions’ section. MH employment by Turosi after the current study was conducted does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

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.

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 log 10 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 log 10 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 log 10 GC/mg, respectively while those of plasma were 11/13 and 4.29 ± 0.39 log 10 GC/ml and red blood cell were 3/13 and 0.36 ± 0.39 log 10 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The objectives of this study were to compare the virulence of contemporary infectious laryngotracheitis virus (ILTV) field isolates of classes 9, 10, and 14 in meat and layer chickens, and to evaluate cloacal and oropharyngeal swabs and dust as sample types for ILTV detection. A total of 211 chickens were divided into groups and inoculated with ILTV class 9, 10, or 14, or sham-inoculated via eye drop at 15 or 22 days of age. Chickens were euthanized at 5 and 9 days post-infection. Virulence was assessed by scoring of clinical signs (conjunctivitis, dyspnoea, and demeanour), ILTV genomic copies (GC) in oropharyngeal and cloacal swabs, mortality and microscopic lesions in conjunctiva and trachea. Class 14 caused subclinical infection, while inoculation with class 9 or class 10 resulted in severe clinical signs and microscopic lesions. Compared to class 14 (2.25 ± 0.36 log 10 GC), higher viral load was observed in oropharyngeal swabs of classes 9 (7.86 ± 0.48) and 10 (7.53 ± 0.36), with a higher proportion of positive oropharyngeal and cloacal swabs in the latter groups ( P  < 0.0001). Viral detection in cloacal swabs was delayed at early stages of infection compared to oropharyngeal swabs. Dust samples from class 9- and class 10-inoculated groups showed a trend towards higher GC than that of class 14. Overall, clinical scores, mortality, viral load, and microscopic lesions were similar for classes 9 and 10, but class 9 caused more severe disease in layer chickens than meat chickens. In summary, ILTV classes 9 and 10 exhibited severe virulence, while class 14 exhibited very mild virulence. RESEARCH HIGHLIGHTS Wide variation in the virulence of three field Australian field ILTV strains. Class 9 and class 10 strains were highly virulent, while class 14 was mildly virulent. The highly virulent strains were associated with significantly higher viral genome copies in various sample types than the mildly virulent strain.

Bedding material or litter is an important requirement of meat chicken production which can influence bird welfare, health, and food safety. A substantial increase in demand and cost of chicken bedding has stimulated interest in alternative bedding sources worldwide. However, risks arising from the use of alternative bedding materials for raising meat chickens are currently unknown. Organic chemicals, elemental, and biological contaminants, as well as physical and management hazards need to be managed in litter to protect the health of chickens and consequently that of human consumers. This requires access to information on the transfer of contaminants from litter to food to inform risk profiles and assessments to guide litter risk management. In this review, contaminants and hazards of known and potential concern in alternative bedding are described and compared with existing standards for feed. The contaminants considered in this review include organic chemical contaminants (e.g., pesticides), elemental contaminants (e.g., arsenic, cadmium, and lead), biological contaminants (phytotoxins, mycotoxins, and microorganisms), physical hazards, and management hazards. Reference is made to scientific literature for acceptable levels of the above contaminants in chicken feed that can be used for guidance by those involved in selecting and using bedding materials., (Copyright © 2020. Published by Elsevier Inc.)

Infectious laryngotracheitis is an important disease of chickens caused by infectious laryngotracheitis virus (ILTV). Outbreaks commonly occur in meat chicken flocks and mass vaccination with live attenuated vaccines, usually in water, is used to control the disease in these populations. Vaccination with live virus via water and nipple drinkers requires stringent adherence to protocols to ensure success, but vaccine administration monitoring is not currently assessed due to a lack of economically viable methods. Vaccinal ILTV has been shown to be detectable in dust in experimental studies and has potential as a method of assessing vaccination success. However, the pattern of vaccinal ILTV detection in dust following vaccination under commercial conditions has not been defined. We report the longitudinal profile of ILTV genome copies (GC) in poultry house dust collected on settle plates following vaccination of 8 flocks of commercial meat chickens on four farms. ILTV GC was enumerated using quantitative real-time polymerase chain reaction (qPCR). There was considerable variation between flocks in the levels of ILTV GC detected post vaccination and this variation was significantly associated with vaccine take measured in individual birds in a companion study. There was no effect of sampling location on ILTV GC in dust but the amount of dust collected was greater in locations closer to the exhaust fans in artificially ventilated houses. Results indicate that measurement of ILTV GC in single or pooled dust samples at 7-8 days post vaccination enables detection of poor vaccine takes and provides a practical means of monitoring ILT vaccination., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Monitoring of Marek's disease virus (MDV) and infectious laryngotracheitis virus (ILTV) genome using poultry dust can be useful to monitor on-farm vaccination protocols but there are no set guidelines for collection of this sample type. This study assessed different dust collection methods for MDV and ILTV detection in a vaccinated layer flock (n = 1700) from day-old to 50 weeks of age. Birds were vaccinated against MDV at day-old, and ILTV by drinking water at week 6 and eye drop at week 12. Dust samples were collected weekly by settle plates (1-3 plates/15 m 2 ) or by scraping surfaces in the poultry shed and tested for ILTV and MDV genomic copies (GC) by PCR. ILTV GC were detected 4 weeks post water vaccination, peaked at weeks 12-14 and became mostly undetectable after week 18. MDV was detected in dust on week 1, peaked at weeks 3-6, declined 3 logs by week 26 and remained detectable at this level until week 50. There was no difference in the detection rates of ILTV and MDV collected from settle plates in different locations of the shed (P > 0.10). There was no difference between settle plate and scraped samples in ILTV GC load but higher MDV GC were found in scraped samples. The settle plate method appears to reflect the current level of vaccine virus in the flock while the scrape method likely represents a cumulative record of shedding. Assessment of viral GC in dust samples is a good candidate for a practical method of estimating successful vaccine administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A series of studies were undertaken to optimize the propagation of hemorrhagic enteritis virus (HEV) in specific-pathogen-free (SPF) chickens. A total of 562 SPF chickens were orally inoculated with an Australian avirulent HEV isolate of turkey origin at 9, 14, 21, or 28 days of age with 5, 6, 7, or 8 log  10 genomic copies (GC), while 102 chickens served as uninfected controls. No clinical signs were observed in infected chickens. There was an inoculum-dose-dependent increase in the relative spleen and liver weight ( P < 0.01). Relative spleen weight 7 days post-HEV inoculation was up to 85% higher in chickens that were inoculated with 6 to 7 GC compared with controls, with no further increase at higher doses. Relative liver weight increased up to 14% in chickens inoculated with 6 GC, with no further increase. Birds inoculated with a 7 GC dose had a higher frequency of HEV DNA-positive birds (77% to 86%) than birds inoculated with lower doses (33% to 59%; P < 0.01). The most efficient dose for live passage propagation was 7 GC inoculated in 9-to-14-day-old birds, yielding an infection rate of 81%. Livers and spleens from infected birds at all doses were processed to produce a putative vaccine with a final GC recovery in the vaccine material of 8.6 GC/bird. In summary, HEV of turkey origin can be readily propagated in SPF chickens, and conditions to maximize viral retrieval were established.

The objective of this study was to determine patterns of avian HEV infection in naturally infected chicken farms. A total of 310 serum samples and 62 pooled fecal samples were collected from 62 chicken flocks on seven commercial in-line egg farms in the Midwestern United States and tested for avian HEV circulation. Serum samples were tested for the presence of anti-avian HEV IgY antibodies by a fluorescent microbead immunoassay (FMIA) which was developed for this study. The FMIA was validated using archived samples of chickens with known exposure (n = 96) and compared to the results obtained with an enzyme-linked immunosorbent assay (ELISA) based on the same capture antigen. There was an overall substantial agreement between the two assays (κ = 0.63) with earlier detection of positive chickens by the FMIA (P = 0.04). On the seven farms investigated, the overall prevalence of anti-avian HEV IgY antibodies in serum samples from commercial chickens was 44.8% (20-82% per farm). Fecal samples were tested for avian HEV RNA by a nested reverse-transcriptase PCR. The overall detection rate of avian HEV RNA in fecal samples was 62.9% (0-100% per farm). Sequencing analyses of partial helicase and capsid genes showed that different avian HEV genotype 2 strains were circulating within a farm. However, no correlation was found between avian HEV RNA detection and egg production, egg weight or mortality. In conclusion, avian HEV infection is widespread among clinically healthy laying hens in the United States., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Between 2012 and 2014, 141 chickens from 10 organic layer flocks with a history of severe drop in egg production (up to 40%) and slight increased mortality (up to 1% per week) were submitted to the Avian Health and Food Safety Laboratory (Puyallup, WA). At necropsy, the most common finding was pinpoint white foci on the liver and regressed ova without any other remarkable lesions. Histologically, there was multifocal mild-to-severe acute necrotizing hepatitis present. No significant bacteria were recovered from liver samples, and tests for mycotoxins were negative. Twenty-six serum samples from four affected flocks tested were positive for avian hepatitis E virus (HEV) immunoglobulin Y antibodies. Avian HEV RNA was detected in 10 livers of chickens from two different affected flocks. The avian HEV was characterized by sequencing and determined to belong to genotype 2. The diagnosis of a clinical manifest HEV was based solely on the demonstration of specific viral RNA and the absence of other causative agents in samples from flocks, as the clinical sings and pathologic lesions were atypical.

Two commercial Midwestern egg-type chicken flocks experienced significant increases in mortality rates in April 2013 with clinical signs appearing in 17-week-old pullets on Farm A and in 46-week-old hens on Farm B. Average weekly mortality was 0.44% over a 4-week period on Farm A and 0.17% over an 8-week period on Farm B. On Farm A, flocks in the affected house had a 45% decrease in daily egg production from weeks 19 to 27 when compared with standard egg production curves (P < 0.01) while no decrease in egg production was noticed on Farm B. Post-mortem examination revealed changes consistent with hepatitis-splenomegaly syndrome, including hepatomegaly with serosanguineous fluid in the coelomic cavity and hepatic subcapsular haemorrhages. Microscopic lesions were characterized by multifocal necrotizing hepatitis and intrahepatic haemorrhage. No significant bacteria were recovered from liver samples, but 72 to 100% of the liver samples from affected chickens on Farm A (8/11) and Farm B (7/7) contained detectable amounts of avian hepatitis E virus (aHEV) RNA as determined by polymerase chain reaction. Sequencing and phylogenetic analysis of a 361-base-pair fragment of the helicase gene demonstrated 98.6 to 100% nucleotide identity between the aHEV genomes from Farm A and Farm B, whereas identities ranged from 74.6 to 90.5% when compared with other representative sequences. Sequences from this study clustered within aHEV genotype 2 previously recognized in the USA. In contrast to other reported aHEV outbreaks that occurred in 30-week-old to 80-week-old chickens, in the present investigation clinical aHEV was identified in 17-week-old chickens on one of the farms.