Genetic diversity, population structure and origin of the native goats in Central Laos.

Maintaining genetic diversity and variation in livestock populations is critical for natural and artificial selection promoting genetic improvement while avoiding problems due to inbreeding. In Laos, there are concerns that there has been a decline in genetic diversity and a rise in inbreeding among native goats in their village-based smallholder system. In this study, we investigated the genetic diversity of Lao native goats in Phin, Songkhone and Sepon districts in Central Laos for the first time using Illumina's Goat SNP50 BeadChip. We also explored the genetic relationships between Lao goats with 163 global goat populations from 36 countries. Our results revealled a close genetic relationship between Lao native goats and Chinese, Mongolian and Pakistani goats, sharing ancestries with Guangfen, Jining Grey and Luoping Yellow breeds (China) and Teddi goats (Pakistan). The observed (Ho) and expected (He) heterozygosity were 0.292 and 0.303 (Laos), 0.288 and 0.288 (Sepon), 0.299 and 0.308 (Phin) and 0.289 and 0.305 (Songkhone), respectively. There was low to moderate genetic differentiation (FST: 0.011-0.043) and negligible inbreeding coefficients (FIS: -0.001 to 0.052) between goat districts. The runs of homozygosity (ROH) had an average length of 5.92-6.85 Mb, with short ROH segments (1-5 Mb length) being the most prevalent (66.34%). Longer ROH segments (20-40 and >40 Mb length categories) were less common, comprising only 4.81% and 1.01%, respectively. Lao goats exhibit moderate genetic diversity, low-inbreeding levels and adequate effective population size. Some genetic distinctions between Lao goats may be explained by geographic and cultural features.

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%).

Evaluation of in vitro methods of anthelmintic efficacy testing against Ascaridia galli.

To investigate methods for in vitro assessment of anthelmintic efficacy against the chicken nematode Ascaridia galli this study firstly evaluated sample preparation methods including recovery of eggs from excreta using different flotation fluids and induced larval hatching by the deshelling-centrifugation method and the glass-bead method with or without bile. It then evaluated two in vitro assays, the in-ovo larval development assay (LDA) and larval migration inhibition assay (LMIA), for anthelmintic efficacy testing against A. galli using fresh eggs and artificially hatched larvae, respectively. Four anthelmintics, thiabendazole (TBZ), fenbendazole (FBZ), levamisole (LEV) and piperazine (PIP) were employed using an A. galli isolate of known susceptibility. The results suggested that the LDA and LMIA could successfully be used to generate concentration response curves for the tested drugs. The LDA provided EC50 values for inhibition of egg embryonation of 0.084 and 0.071 µg/ml for TBZ and FBZ, respectively. In the LMIA, the values of effective concentration (EC50) of TBZ, FBZ, LEV and PIP were 105.9, 6.32, 349.9 and 6.78 × 107 nM, respectively. For such in vitro studies, a saturated sugar solution showed high egg recovery efficiency (67.8%) and yielded eggs of the highest morphological quality (98.1%) and subsequent developmental ability (93.3%). The larval hatching assays evaluated did not differ in hatching efficiency but the deshelling-centrifugation method yielded larvae that had slightly better survival rates. For final standardization of these tests and establishment of EC50 reference values, tests using isolates of A. galli of defined resistance status need to be performed.

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.

Marked differences in virulence of three Australian field isolates of infectious laryngotracheitis virus in meat and layer chickens.

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 log10 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.

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens.

Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.

Protection provided by Rispens CVI988 vaccine against Marek's disease virus isolates of different pathotypes and early prediction of vaccine take and MD outcome.

We tested the level of protection provided by the Rispens CVI988 (Rispens) vaccine against challenge with a virulent Marek's disease virus (MDV) pathotype (vMDV) and a very virulent pathotype (vvMDV) and the accuracy of a range of predictive measures of Marek's disease (MD) incidence and vaccine take. Commercial layer chicks (n = 236) were vaccinated (or not) with 4000 plaque-forming units (pfu) of Rispens vaccine at hatch and challenged (or not) with 500 pfu of each challenge virus five days post vaccination. The vvMDV pathotype FT158 induced higher MD incidence (65%) and mortality (33%) when compared with the vMDV pathotype MPF57 (39% and 8%, respectively). The protective index provided by the Rispens vaccine against FT158 (61%) did not differ significantly from that against MPF57 (66%). This provides additional evidence that protection provided by the Rispens vaccine is not influenced by pathotype determined in studies using vaccines of other Mardivirus species. The challenge viruses did not differ in MDV or Rispens viral load in spleen at 14 dpc (days post challenge) determined by specific quantitative polymerase chain reaction test. MDV load in peripheral blood leucocytes at 7 and 14 dpc, splenocytes at 14 dpc, feather cells at 14 and 21 dpc and isolator dust at 21 dpc were significant early indicators of subsequent MD incidence to 56 dpc. These are potentially useful as the sampling can be carried out well before the onset of MD and some measures are non-invasive. The Rispens viral load in both invasive and non-invasive samples was more useful as a measure of vaccine take.

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

Vaccination-challenge interval markedly influences protection provided by Rispens CVI988 vaccine against very virulent Marek's disease virus challenge.

The Rispens (CVI988) vaccine is widely used to vaccinate chickens worldwide. We tested the protective effects of the Rispens vaccine against challenge with very virulent Marek's disease virus (vvMDV) at various intervals at, before or after vaccination. The experiment used commercial ISA Brown layers and vvMDV isolate 02LAR. The protective index (PI) was measured for vaccination challenge intervals (VCI) of -10, -5, 0, 5 and 10 days, with the negative values indicating challenge prior to vaccination. Chickens were challenged by injection with 400 plaque-forming units (PFU) of 02LAR and/or vaccinated with 3200 PFU of the Rispens vaccine virus at days 0, 5 and 10 of age, with appropriate negative controls injected with diluent only. The presence of visible Marek's disease tumours was assessed up to 56 days post challenge. MDV challenge in unvaccinated chickens resulted in tumours in 52% of chickens. The Rispens vaccine provided no significant protection when challenge preceded vaccination, with PIs of -4 and 21% for VCI of -5 and -10 days respectively. On the other hand, it provided PIs of 60, 85 and 100% at VCI of 0, 5 and 10 days respectively. The study also revealed that the vvMDV load in peripheral blood lymphocytes or feather tips at 14 and 21 days post infection as determined by quantitative real-time polymerase chain reaction, which can distinguish pathogenic MDV from the Rispens vaccine strain, was an accurate early predictor of Marek's disease incidence at 56 days post challenge. The load of Rispens virus in peripheral blood lymphocytes or feathers at the same times post vaccination did not offer similar predictive power.

Viral kinetics, shedding profile, and transmission of serotype 1 Marek's disease vaccine Rispens/CVI988 in maternal antibody-free chickens.

Probably the most effective current vaccine against Marek's disease is the live Rispens (CVI988) attenuated serotype 1 Marek's disease virus (MDV). It is unknown whether the currently available Rispens vaccines transmit effectively between chickens. To investigate the kinetics and shedding of three commercially available strains of this virus and the extent of lateral transmission, we measured the shedding rate in dander and the viral load in peripheral blood lymphocytes (PBLs) and feather tips over time. Four identical climate-controlled rooms were stocked with a total of 70 specific-pathogen-free chickens for 56 days. In each of three rooms, 10 chickens were vaccinated with one of the commercial vaccines at day old and left in contact with 10 unvaccinated chickens. The fourth room contained 10 unvaccinated control chickens. As determined by MDV-specific quantitative real-time polymerase chain reaction of weekly room dust and individual PBLs and feather tip samples, the vaccine virus was shed from the vaccinated chickens in dander from day 7 postvaccination and transmitted effectively from vaccinated to in-contact chickens with a lag period of 2-3 wk. Viral load in PBLs and feather tips peaked at days 7 and 14, respectively, and declined thereafter, whereas viral load in dust increased rapidly to day 21 and then increased gradually thereafter. Antibody titer at day 56 was correlated with earlier measures of MDV load in PBLs but not feather tips or dust. These results show that currently available Rispens CVI988 vaccine virus is shed in significant quantities from vaccinated chickens and transmits effectively between chickens.

Prevalence of Marek's disease virus in different chicken populations in Iraq and indicative virulence based on sequence variation in the ecoRI-q (meq) gene.

A cross-sectional survey was conducted in six provinces in southern Iraq to determine the point prevalence of Marek's disease virus (MDV) in different chicken populations followed by sequencing the meq gene for phylogenetic analysis and virulence-associated polymorphisms. A total of 109 samples from unvaccinated flocks were analyzed comprising 52 dust and 30 spleen samples from commercial broiler farms and 27 spleens from local layer chickens purchased in the town markets. The overall prevalence of MDV was 49.5% with no significant differences between provinces (P = 0.08) or sample types (P = 0.89). Prevalence ranged from 36.8% in Karbala and Nasiriyah to 65% in Amarah. The percentages of positive samples were 59.1%, 46.7%, and 48.1% in broiler dust, broiler spleen, and layer spleen, respectively. The overall mean (+/- SEM) Log10 MDV viral copy number per milligram of dust or spleen as determined by quantitative PCR was 1.78 +/- 0.19, with no significant differences between provinces (P = 0.10) or sample types (P = 0.38). In positive samples only, the overall mean was 3.43 +/- 0.18. Sequencing of the meq gene from samples that showed high levels of MDV target in qPCR testing was attempted. Nine samples were sequenced. These sequences were compared with meq sequences of MDVs of different pathotype. All the Iraqi MDVs had a short meq gene of 897 base pairs because of the deletion of 123 bp relative to the reference strain Md5. The Iraqi meq sequences also contained single-nucleotide polymorphisms, resulting in differences in the amino acid sequence. All of the nine Iraqi meq genes encoded two repeats of four-proline sequences. The published negative association between four-proline repeat number and MDV virulence suggests that the Iraqi MDVs are likely to be highly virulent, but this needs to be confirmed by in vivo testing. Taken together, these results indicate that MDV is common in unvaccinated commercial and village chickens in southern Iraq, that there is limited meq gene sequence variation, that all sequenced samples had a short meq with two four-proline repeats, and that this is consistent with a high level of virulence.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.

Epidemiology and Transmission of Theileria orientalis in Australasia.

Oriental theileriosis, a disease primarily impacting cattle is caused by an apicomplexan hemoprotozoan parasite, Theileria orientalis. It has now become established in the Australasia region. The organism was long considered a benign cause of persistent infections; however, an increase in clinical outbreaks since 2006 in the eastern Australian states and New Zealand was associated with the identification of the pathogenic Ikeda (Type 2) and Chitose (Type 1) genotypes. Unlike the pathogenic T. parva and T. annulate, which target leucocytes, clinical manifestation with T. orientalis is due to its effects on erythrocytes, with the infection sometimes designated as Theileria associated bovine anemia (TABA). In Australia and New Zealand, the tick Haemaphysalis longicornis is the principal vector, though other Haemaphysalis species are also likely vectors. The endemic status of infection with pathogenic genotypes in areas with low or absent tick populations is an apparent paradox that may be attributable to alternative modes of transmission, such as mechanical transmission by hematophagous insects (lice, mosquitoes, and biting flies), vertical transmission, and transmission via iatrogenic means. This review addresses the evidence for the different modes of transmission of T. orientalis with particular focus on the reported and potential vectors in Australasia.

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.

Prevalence and magnitude of gastrointestinal helminth infections in cage-free laying chickens in Australia.

Helminth infections have been re-emerging with the growing popularity of free-range and floor-based chicken production systems. The aim of this study was to determine the prevalence and worm burdens of intestinal helminth infection in cage-free laying chickens in Australia. In an online survey about worm prevalence, a high proportion of respondents reported the detection of Ascaridia galli (77%), followed by tapeworms (69%) and caecal worms (Heterakis gallinarum) (62%), whereas fewer respondents (23%) reported the presence of hair worms (Capillaria spp.) in their flocks. Total worm recovery from 407 laying hens on four farms found that 92.1% of hens harboured one or more helminth parasite with a prevalence of 73 to 100% across farms. Mixed infections were common with 79% of hens harbouring two or more helminth species. The prevalence of nematode species H. gallinarum, A. galli and Capillaria spp. was 87, 82 and 35% respectively. Five cestode species were found with a low individual chicken prevalence (Raillietina tetragona 4.7%, Raillietina echinobothrida 3.2%, Raillietina cesticillus 5.2%, Choanotaenia infundibulum 4.4%, and Hymenolepis cantaniana 4.4%). The hens harboured an average of 71 worms with H. gallinarum having the highest mean burden (45.5 worms/hen) followed by A. galli (22.0 worms/hen), Capillaria spp. (2.7 worms/hen) and cestodes (0.8 worms/hen). The sex ratio (female:male worms) was 1.38:1 for A. galli, and 1.77:1 for H. gallinarum. There was a strong positive correlation between A. galli female worm count and excreta egg count (EECs) (rs = 0.94, P < 0.0001) and also between total nematode worm count and EEC (rs = 0.82, P < 0.0001) in individual hens. When investigating intestinal excreta (n = 10) and caecal excreta (n = 10) of 16 chicken flocks the prevalence of infection with ascarid worms in intestinal and caecal excreta was 71 and 78% respectively and 27% prevalence of Capillaria spp. in intestinal excreta with mean EECs of 407, 404, and 18 eggs/g of excreta (EPG), respectively. These results suggest that most chickens kept in free-range or floor production systems are infected with one or more helminth parasite species. Heavy worm infections would likely affect the production performance and welfare of birds with adverse economic impact. Strategic or tactical anthelmintic treatment with effective anthelmintic could reduce this impact.

Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism.

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.

Worm control practices on free-range egg farms in Australia and anthelmintic efficacy against nematodes in naturally infected layer chickens.

This study investigated worm control practices by free-range egg farmers and the efficacy of the commercial anthelmintics levamisole (LEV), piperazine (PIP), flubendazole (FLBZ) and fenbendazole (FBZ) against gastrointestinal nematodes on two free-range layer farms in Australia. An online survey comprising 36 questions was designed and implemented using SurveyMonkey. The survey contained questions about participant demographics, farm and flock characteristics, perceived intestinal worm importance, infection monitoring, deworming and other worm control practices. A link for the survey was emailed to free range egg producers from their industry body in December 2019. The anthelmintic efficacy trial was conducted in a total of 229 layers naturally infected with Ascaridia galli, Hetarakis gallinarum, Capillaria spp. and/or tape worms. Chickens received a single oral dose of LEV (28 mg/kg), PIP (100 mg/kg), FBZ (10 mg/kg) or LEV-PIP co-administered at their full individual doses, and FLBZ (Flubenol®), 30 ppm or 60 ppm) in the feed over 7 days. Anthelmintic efficacies were estimated by both worm count reduction (WCR %) and excreta egg count reduction (EECR %) tests 10 days after start of treatment. The survey with a response rate of 16/203, revealed that worm infection was of moderate concern to the producers and the majority (68%) felt that the current anthelmintics work effectively. The level of understanding of worms, monitoring and control practices did not reveal any major deficiencies of concern. The most commonly used anthelmintic was LEV (73%) followed by PIP (45%). Based on a standard cut-off value (≥90%), LEV, LEV-PIP, and FBZ attained the desired efficacy but PIP exhibited reduced efficacy against immature A. galli (61-85%), all stages of H. gallinarum (42-77%) and Capillaria spp. (25-44%). FLBZ was highly effective against all stages of roundworms and tapeworm infections. Even though there was some association between the efficacies estimated by WCR % and EECR % the latter was poorly associated in the natural infection model and hence does not provide a reasonable alternative for assessing anthelmintic efficacy when immature stages of the lifecycle are included. These results show no evidence of loss of susceptibility to the tested anthelmintics on these farms supporting the perception of producers that participated in the survey that current treatments work effectively. The reduced efficacy of PIP against some species and immature stages is related to its spectrum of activity rather than providing evidence of emerging resistance.

Method optimisation for prolonged laboratory storage of Ascaridia galli eggs.

Eggs in the infective stage of the chicken nematode Ascaridia galli are often required for in vivo and in vitro studies on this parasite. The reliability of any artificial A. galli infection depends on the viability and embryonation capacity of A. galli eggs. The aim of this study was to determine ideal storage conditions for maximising the viability of A. galli eggs and maintaining viability for the longest period. A 2 × 2 × 3 × 5 factorial experimental design was employed to investigate the effects of storage temperature (4°C or 26°C), storage condition (aerobic or anaerobic), storage medium (water, 0.1 N H2SO4 or 2% formalin) and storage period (4, 8, 12, 16 and 20 weeks). The viability of eggs was assessed after eggs in all treatment groups were held aerobically at 26°C for 2 weeks after the storage period to test embryonation capacity. Based on morphological characteristics, they were categorised as undeveloped, developing, vermiform, embryonated or dead. The maintenance of viability during storage at 4°C was optimal under anaerobic conditions while at 26°C it was optimal under aerobic conditions. Anaerobic conditions at 26°C led to a rapid loss of viability while aerobic conditions at 4°C had a less severe negative effect on maintenance of viability. Egg storage in 0.1 N H2SO4 resulted in a significantly higher viability overall (54.7%) than storage in 2% formalin (49.2%) or water (37.3%) (P < 0.0001). Untreated water was the least favourable storage medium when eggs were stored at 26°C while it was a medium of intermediate quality at 4°C. The viability of A. galli eggs decreased significantly with storage time (P < 0.0001) depending on the other factors. The lowest rate of decline was seen with storage of eggs under anaerobic conditions at 4°C or aerobic conditions at 26°C in 0.1 N H2SO4. Eggs in these treatments retained up to 72% of overall viability at 20 weeks with a decline rate of approximately 2% per week with no significant difference between the two. Therefore, this study has clearly revealed opposing aerobic conditions required for prolonged storage of A. galli eggs in the pre-embryonated state at 4°C. It has also identified that 0.1 N H2SO4 provides the best preservation against degradation during storage, particularly at 26°C under aerobic conditions. Achieving strictly anaerobic conditions can be difficult to achieve so storage aerobically at 26°C may be preferred for simplicity.

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.

Anthelmintic efficacy evaluation against different developmental stages of Ascaridia galli following individual or group administration in artificially trickle-infected chickens.

The efficacy of commercially available anthelmintics against mature and immature stages (including ovicidal effects) of two Australian field isolates of Ascaridia galli was evaluated in two separate experiments. The anthelmintics tested were levamisole (LEV), piperazine (PIP) and flubendazole (FBZ) plus LEV-PIP. A total of 192 artificially trickle-infected young cockerels (96 birds per isolate) were randomized into sixteen experimental groups of 12 cockerels each (7 treatments and 1 untreated control per isolate). Chickens received label-recommended doses of LEV (28 mg/kg), PIP (100 mg/kg) or LEV-PIP co-administered at their full individual doses as a single oral dose or in group drinking water at recommended concentrations of 0.8 mg/ml or 2.5 mg/ml over eight hours for 1 and 2 days respectively and FLBZ (30 ppm) in the feed over 7 days. Anthelmintic efficacies were assessed by worm count reduction (WCR%) and excreta egg count reduction (EECR%) estimated by two methods. Ten days post treatment, all untreated control birds harboured mixed worm population of 10.1 and 12.3/bird for each isolate respectively which was significantly higher (P < 0.0001) than counts in all treatment groups. Luminal or histotrophic larvae comprised 50-57 % of the total worm count. For LEV, PIP and LEV-PIP, individual oral administration provided a somewhat higher efficacy than group medication in drinking water. EECR% values were inconsistent with WCR% and found to be only an indicator of efficacy against adult worms. All developmental stages of the two A. galli isolates were highly susceptible to FLBZ (100 %) followed by LEV-PIP (92.4-100 %) and LEV (87.7-100 %). PIP exhibited good efficacy against adult worms (92-97 %) but reduced efficacy against luminal (79-84 %) and histotrophic (61-72 %) larvae. Embryonation capacity of eggs recovered from worms expelled after treatment with LEV (47-54 %), PIP (44-54 %) or LEV-PIP (45-48 %) did not differ from those from untreated birds (50-51 %) whereas eggs from FLBZ treated worms had a significantly lower (P < 0.05) capacity to embryonate (≤ 2 %). Put together, our results demonstrate no evidence of resistance of the test A. galli isolates to the tested anthelmintics but a significant advantage of FLBZ, followed by LEV-PIP and LEV over PIP in the control of A. galli, specifically with regard to immature stages. A. galli worms expelled after treatment with LEV, PIP or their combination, but not FLBZ contain viable eggs. This has epidemiological implications and may also provide an option for isolating eggs from mature worms for A. galli propagation experiments without having to sacrifice birds.