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%).

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.

Use of the rSpaA415 antigen indicates low rates of Erysipelothrix rhusiopathiae infection in farmed cattle from the United States of America and Great Britain.

BACKGROUND: Clinical cases of Erysipelothrix rhusiopathiae, a zoonotic gram-positive bacterium, have been reported in many ruminant species, including in cattle, deer, moose and muskoxen. Fatal cases have been repeatedly reported in cattle over the years but to date there is only one Japanese study investigating the seroprevalence of this bacterium in cattle using the growth agglutination test (GAT). This technique is subjective, time-consuming, expensive and hazardous compared to modern serological tests such as enzyme-linked immunosorbent assays (ELISA) or the newly developed fluorescent microbead-based immunoassays (FMIA). RESULTS: The FMIA based on the surface protein SpaA (rSpaA415) antigen of E. rhusiopathiae developed in this study had an almost perfect agreement with the GAT (k = 0.83) and showed a sensitivity of 89.7% and a specificity of 92.9% when compared to the GAT. Overall, detection rates of E. rhusiopathiae antibody positive samples were 13.8% (51/370) in British herds and 6% (12/200) in US herds. Positive cattle were present in 34.3% (24/70) of the investigated British farms and in 34.7% (8/23) of the US farms with an on-farm prevalence of 7.1 to 100% for the British farms and 8.3-30% for the US farms. CONCLUSIONS: FMIA is a fast, safe and economic alternative to the GAT for the diagnosis of E. rhusiopathiae in cattle. This work is the first seroprevalence study of E. rhusiopathiae in healthy farmed cattle in Great Britain and the US and revealed that infection occurs at a low level. Further investigations to evaluate risks of zoonotic transmission when handling cattle are needed.

Evaluation of the efficacy of a commercial inactivated genogroup 2b-based porcine epidemic diarrhea virus (PEDV) vaccine and experimental live genogroup 1b exposure against 2b challenge.

Porcine epidemic diarrhea virus strains from the G1b cluster are considered less pathogenic compared to the G2b cluster. The aim of this study was to compare the ability of G1b-based live virus exposure against use of a commercial G2b-based inactivated vaccine to protect growing pigs against G2b challenge. Thirty-nine PEDV naïve pigs were randomly divided into five groups: EXP-IM-1b (intramuscular G1b exposure; G2b challenge), EXP-ORAL-1b (oral G1b exposure; G2b challenge), VAC-IM-2b (intramuscular commercial inactivated G2b vaccination; G2b challenge), POS-CONTROL (sham-vaccination; G2b challenge) and NEG-CONTROL (sham-vaccination; sham-challenge). Pigs were vaccinated/exposed at 3 weeks of age (day post-vaccination 0, dpv 0), VAC-IM-2b pigs were revaccinated at dpv 14, and the pigs were challenged at dpv 28. Among all groups, VAC-IM-2b pigs had significantly higher anti-PEDV IgG levels on dpv 21 and 28 while EXP-ORAL-1b pigs had significantly higher anti-PEDV IgA levels on dpv 14, 21, 28 and 35. EXP-ORAL-1b also had detectable IgA in feces. Intramuscular PEDV exposure did not result in a detectable antibody response in EXP-IM-1b pigs. The fecal PEDV RNA levels in VAC-IM-2b pigs were significantly lower 5-7 days after challenge compared to the POS-CONTROL group. Under the study conditions a commercial inactivated G2b-based vaccine protected pigs against G2b challenge, as evidenced by reduction of PEDV RNA in feces for 3-4 logs during peak shedding and a shorter viral shedding duration. The oral, but not the intramuscular, experimental G1b-based live virus exposure induced a high anti-PEDV IgA response prior to challenge, which apparently did not impact PEDV shedding compared to POS-CONTROL pigs.

First identification of porcine parvovirus 6 in Poland.

Porcine parvovirus type 1 is a major causative agent of swine reproductive failure. During the past decade, several new parvoviruses have been discovered in pigs. Porcine parvovirus type 6 (PPV6), recently identified, has been reported in pigs in China and in the USA while the PPV6 status in the European pig population remains undetermined. In the present study, PPV6 DNA was identified in serum samples collected from domestic pigs in Poland. In investigated herds, the prevalence of PPV6 was 14.9 % (15/101 samples). Sequencing was conducted, and 11 nearly complete PPV6 genomes were obtained. Phylogenetic analysis indicated that PPV6 sequences cluster into four distinct groups, and the Polish PPV6 strains from three individual farms were present in three of these four groups. In addition, the Polish PPV6 strain P15-1 was identified as a putative recombination of an ORF1 from US stains and an ORF2 from Chinese strains. This is the first identification of PPV6 in Europe, and this finding will encourage future epidemiological studies on parvoviruses in European pigs.

Increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3-10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains.

BACKGROUND: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. RESULTS: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. CONCLUSIONS: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.

Evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs.

Clinical disease associated with porcine epidemic diarrhea virus (PEDV) infection in naïve pigs is well chronicled; however, information on endemic PEDV infection is limited. To characterize chronic PEDV infection, the duration of infectious virus shedding and development of protective immunity was determined. On Day 0 (D0), a growing pig was challenged with PEDV and 13 contacts were commingled. On D7, 9 contact pigs (principal virus group (PG)), were selected, moved to a separate room and commingled with one sentinel pig (S1). This process was repeated weekly with S2, S3 and S4. The PG was PEDV-positive by PCR from D3-11, with some pigs intermittently positive to D42. Pigs S1 and S2 were PEDV-positive within 24 hours of commingling. Antibodies were detected in all PG by D21 and by 7 days post-contact in S1 and S2. Pigs S3 and S4 were PCR and antibody negative following commingling. To evaluate protective immunity, 5 naïve pigs (N) and the PG were challenged (N/C, PG/C) with homologous virus on D49. All N/C pigs were PEDV PCR-positive by D52 with detection out to D62 in 3/5 N/C pigs. All PG/C pigs were PEDV PCR-negative post-challenge. By D63, all N/C seroconverted. Although PEDV RNA was demonstrated in pigs after primary infection until D42, infectious PEDV capable of horizontal transmission to naïve pigs was only shed 14-16 days after infection to age-matched pigs. Homologous re-challenge 49 days post initial PEDV exposure did not result in re-infection of the pigs. This demonstrates potential for an effective PEDV vaccine.

Avian Hepatitis E Virus Infection in Organic Layers.

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.

Mutant USA strain of porcine circovirus type 2 (mPCV2) exhibits similar virulence to the classical PCV2a and PCV2b strains in caesarean-derived, colostrum-deprived pigs.

In 2012, a mutant porcine circovirus type 2 (mPCV2) strain was identified in cases of PCV-associated disease (PCVAD) in the USA. The mPCV2 had an additional amino acid, lysine (K), in the capsid at position 234. The objectives of this study were to compare the pathogenicity of mPCV2, PCV2a and PCV2b in pigs using biologically pure infectious virus stocks derived from respective infectious DNA clones, and to investigate the importance of genotype-specific ORF2 and the presence of lysine at position 234 of the capsid. A total of 47, 2-week-old, caesarean-derived, colostrum-deprived (CDCD) pigs were assigned to one of seven groups. At 3 weeks of age, the pigs were experimentally inoculated with saline, PCV2a, PCV2b, mPCV2, PCV2b-234-K (lysine addition in ORF2), chimeric PCV2b-ORF1/mPCV2-ORF2 or reciprocal chimeric mPCV2-ORF1/PCV2b-ORF2. All pigs were necropsied 21 days post-infection (p.i.). Gross lesions were limited to visible icterus and loss of body condition in a portion of the mPCV2 pigs. The amount of PCV2 DNA was significantly higher in pigs inoculated with mPCV2 compared with PCV2b in sera at 7 days p.i. and faecal swabs at 14 days p.i. Based on lymphoid lesions, a higher prevalence of PCVAD was seen in pigs infected with PCV2s containing the additional 234-K (64.3 %) compared with those infected with a PCV2 with the regular 233 bp ORF2 (40 %). Results indicated that all PCV2 isolates were capable of inducing severe lesions and disease in the CDCD pig model, and there was no significant difference in virulence.

High prevalence and genetic diversity of porcine bocaviruses in pigs in the USA, and identification of multiple novel porcine bocaviruses.

Viruses in the genus Bocavirus are associated with respiratory and enteric disease in dogs and cattle. In addition, novel porcine bocaviruses (PBoVs) have been identified in domestic and wild pigs in recent years, but are of unknown relevance to date. The objectives of this study were to determine the prevalence ra tes and genetic diversity of PBoVs in pigs in the USA. Using newly established multiplex real-time PCR assays, 385 lung, lymph node, serum and faecal samples from pigs with various disease conditions were investigated. A high PBoV prevalence rate ranging from 21.3 to 50.8 % was identified in the investigated samples and often two or more PBoV species were detected in the same sample. Cloning and sequencing analysis of the partial non-structural protein NS1 and the capsid proteins VP1 and VP2 of DNA samples positive for PBoV groups 1 (n = 6), 2 (n = 16) and 3 (n = 42), including subgroups 3A, 3B or 3C, revealed a high genetic diversity especially for the PBoV G3 VP2 gene, whereas the PBoV group 1 VP1 gene displayed a low nucleotide polymorphism. Using primer walking, 18 partial or nearly complete genomes of PBoVs were obtained and six of the 18 nearly complete genomes represented novel PBoV species. Recombination analysis using partial NS1, VP1 and VP2 genes and the nearly complete genomes indicated possible recombination events within and between PBoVs. Further studies will be required to reveal the possible pathogenic role of these diverse PBoVs.

Comparison of real-time reverse transcriptase PCR assays for detection of swine hepatitis E virus in fecal samples.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement (κ = 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.

Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations.

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.

Identification and characterization of novel porcine astroviruses (PAstVs) with high prevalence and frequent co-infection of individual pigs with multiple PAstV types.

Many astrovirus (AstV) species are associated with enteric disease, although extraintestinal manifestations in mammalian and avian hosts have also been described. In this study, the prevalence rates of porcine AstV types 1-5 (PAstV1-PAstV5) were investigated using faecal samples from 509 pigs of which 488 (95.9%) came from farms with a history of diarrhoea. All of the five known PAstV types were found to circulate in pigs in the USA, and co-infection of a single pig with two or more PAstV types was frequently observed. A high overall prevalence of 64.0% (326/509) of PAstV RNA-positive samples was detected, with 97.2% (317/326) of the PAstV RNA-positive pigs infected with PAstV4. Further genomic sequencing and characterization of the selected isolates revealed low sequence identities (49.2-89.0%) with known PAstV strains, indicating novel types or genotypes of PAstV2, PAstV4 and PAstV5. Some new features of the genomes of the PAstVs were also discovered. The first complete genome of a PAstV3 isolate was obtained and showed identities of 50.5-55.3% with mink AstV and the novel human AstVs compared with 38.4-42.7% with other PAstV types. Phylogenetic analysis revealed that PAstV1, PAstV2 and PAstV3 were more closely related to AstVs from humans and other animals than to each other, indicating past cross-species transmission and the zoonotic potential of these PAstVs.

Comparison of commercial real-time reverse transcription-PCR assays for reliable, early, and rapid detection of heterologous strains of porcine reproductive and respiratory syndrome virus in experimentally infected or noninfected boars by use of different sample types.

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville, MD [TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold, <30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.

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.

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.

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.

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.