Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Artículo regular­Proliferación de miofibroblastos perisinusoidales hepáticos y respuesta inflamatoria sistémica que precede a la hepatitis por septicemia y toxemia (sep/tox) en pollos de engorde. La septicemia-toxemia (sep/tox) se incluye en la Categoría 1 de seguridad alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA) y es la causa más común y económicamente significativa de decomisos de canales de pollos de engorde. Las lesiones hepáticas se consideran una posible consecuencia de la septicemia y de la contaminación bacteriana asociada con la canal. Por lo tanto, estas lesiones se consideran un indicador de septicemia/toxemia (hepatitis sep/tox). Este estudio se llevó a cabo para analizar las lesiones histológicas que preceden a las lesiones hepáticas muy visibles que conducen a los decomisos debido a septicemia/toxemia en la planta de procesamiento. Se utilizaron hígados de canales de pollos de engorde decomisados por los inspectores del USDA por septicemia/toxemia para establecer criterios microscópicos y generales de hepatitis en etapa terminal de la septicemia/toxemia. Después de la caracterización de la hepatitis por septicemia/toxemia, los pollos de engorde de una granja con un historial de decomisos por septicemia/toxemia se sometieron a examen post mortem e investigación bacteriológica en cuatro intervalos durante los últimos 20 días de producción. Se enviaron cinco pollos sanos y cinco clínicamente enfermos de cuatro casetas a los 18, 25, 32 y 38 días de producción (160 en total). Las lesiones microscópicas que representan la proliferación de miofibroblastos perisinusoidales hepáticos (HPMP), la granulopoyesis extramedular periportal (PEMG), la histocitosis folicular esplénica y la celularidad de la médula ósea (BMC) se clasificaron subjetivamente para cada ave, y la clasificación subjetiva se evaluó con técnicas cuantitativas digitales. La morfología de las células estrelladas hepáticas perisinusoidales y la transformación progresiva de estas células en miofibroblastos se confirmó mediante inmunohistoquímica para actina y desmina del músculo liso. Los cultivos aeróbicos de hígados y vesícula biliar de aves con septicemia/toxemia no produjeron crecimiento de bacterias asociadas con la septicemia. Se observó proliferación de miofibroblastos perisinusoidales hepáticos de leve a severa en todos los grupos de edad, lo que representa el 28% de las aves examinadas. Los aumentos en las células inflamatorias observados por granulopoyesis extramedular periportal y celularidad de la médula ósea se correlacionaron positivamente con proliferación progresiva de miofibroblastos perisinusoidales hepáticos y con hepatitis por septicemia/toxemia en etapa terminal en pollos de engorde.

Discovery of Human-Specific Immunodominant Chlamydia trachomatis B Cell Epitopes.

Chlamydia species-specific serology is compromised by cross-reactivity of the gold standard microimmunofluorescence (MIF) or commercial enzyme-linked immunosorbent assays (ELISAs). This study was conducted to discover novel C. trachomatis-specific peptide antigens that were recognized only by the antibody response of the natural human host. We evaluated a library of 271 peptide antigens from immunodominant C. trachomatis proteins by reactivity with 125 C. trachomatis antibody-positive sera from women with PCR-confirmed C. trachomatis infection and 17 C. trachomatis antibody-negative sera from low-risk women never diagnosed with C. trachomatis infection. These C. trachomatis peptide antigens had been predicted in silico to contain B cell epitopes but had been nonreactive with mouse hyperimmune sera against C. trachomatis We discovered 38 novel human host-dependent antigens from 20 immunodominant C. trachomatis proteins (PmpD, IncE, IncG, CT529, CT618, CT442, TarP, CT143, CT813, CT795, CT223, PmpC, CT875, CT579, LcrE, IncA, CT226, CT694, Hsp60, and pGP3). Using these human sera, we also confirmed 10 C. trachomatis B cell epitopes from 6 immunodominant C. trachomatis proteins (OmpA, PmpD, IncE, IncG, CT529, and CT618) as host species-independent epitopes that had been previously identified by their reactivity with mouse hyperimmune sera against C. trachomatis ELISA reactivities against these peptides correlated strongly with the C. trachomatis microimmunofluorescence (MIF) text results (Pearson's correlation coefficient [R] = 0.80; P < 10-6). These C. trachomatis peptide antigens do not cross-react with antibodies against other Chlamydia species and are therefore suitable for species-specific detection of antibodies against C. trachomatis This study identified an extended set of peptide antigens for simple C. trachomatis-specific ELISA serology.IMPORTANCE Current serological assays for species-specific detection of anti-Chlamydia species antibodies suffer from well-known shortcomings in specificity and ease of use. Due to the high prevalences of both anti-C. trachomatis and anti-C. pneumoniae antibodies in human populations, species-specific serology is unreliable. Therefore, novel specific and simple assays for chlamydial serology are urgently needed. Conventional antigens are problematic due to extensive cross-reactivity within Chlamydia spp. Using accurate B cell epitope prediction and a robust peptide ELISA methodology developed in our laboratory, we identified immunodominant C. trachomatis B cell epitopes by screening performed with sera from C. trachomatis-infected women. We discovered 38 novel human host-dependent antigens from 20 immunodominant C. trachomatis proteins, in addition to confirming 10 host-independent mouse serum peptide antigens that had been identified previously. This extended set of highly specific C. trachomatis peptide antigens can be used in simple ELISA or multiplexed microarray formats and will provide high specificity and sensitivity to human C. trachomatis serodiagnosis.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction.

X-ray crystallography has shown that an antibody paratope typically binds 15-22 amino acids (aa) of an epitope, of which 2-5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6-11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7-12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16-30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences.

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

Asymptomatic endemic Chlamydia pecorum infections reduce growth rates in calves by up to 48 percent.

Intracellular Chlamydia (C.) bacteria cause in cattle some acute but rare diseases such as abortion, sporadic bovine encephalomyelitis, kerato-conjunctivitis, pneumonia, enteritis and polyarthritis. More frequent, essentially ubiquitous worldwide, are low-level, asymptomatic chlamydial infections in cattle. We investigated the impact of these naturally acquired infections in a cohort of 51 female Holstein and Jersey calves from birth to 15 weeks of age. In biweekly sampling, we measured blood/plasma markers of health and infection and analyzed their association with clinical appearance and growth in dependence of chlamydial infection intensity as determined by mucosal chlamydial burden or contemporaneous anti-chlamydial plasma IgM. Chlamydia 23S rRNA gene PCR and ompA genotyping identified only C. pecorum (strains 1710S, Maeda, and novel strain Smith3v8) in conjunctival and vaginal swabs. All calves acquired the infection but remained clinically asymptomatic. High chlamydial infection associated with reduction of body weight gains by up to 48% and increased conjunctival reddening (P<10(-4)). Simultaneously decreased plasma albumin and increased globulin (P<10(-4)) suggested liver injury by inflammatory mediators as mechanisms for the growth inhibition. This was confirmed by the reduction of plasma insulin like growth factor-1 at high chlamydial infection intensity (P<10(-4)). High anti-C. pecorum IgM associated eight weeks later with 66% increased growth (P = 0.027), indicating a potential for immune protection from C. pecorum-mediated growth depression. The worldwide prevalence of chlamydiae in livestock and their high susceptibility to common feed-additive antibiotics suggests the possibility that suppression of chlamydial infections may be a major contributor to the growth promoting effect of feed-additive antibiotics.

Dual-emission fluorescence resonance energy transfer (FRET) real-time PCR differentiates feline immunodeficiency virus subtypes and discriminates infected from vaccinated cats.

Feline immunodeficiency virus (FIV) is among the most common infectious agents of cats. Five well-characterized FIV subtypes, A, B, C, D, and E, are recognized worldwide. As in HIV diagnosis, serum antibodies against FIV classically serve as an indicator of infection status. After the introduction of an inactivated FIV vaccine, this approach has become problematic, since antibodies generated by vaccination are indistinguishable from antibodies in response to infection. However, PCR detection of host-cell-integrated FIV DNA will differentiate infection-derived antibody from vaccination-derived positivity because presumably the RNA of inactivated vaccine virus will not integrate into the host genome. In this study, we established a gag gene-based dual-emission fluorescence resonance energy transfer (FRET) real-time PCR that amplifies single-target copies of all known FIV strains and differentiates five FIV subtypes. All blood samples from experimentally FIV-infected cats (n=5) were antibody positive and highly positive in the FIV PCR. In contrast, nine cats became antibody positive after FIV vaccination but remained negative in the FIV PCR. Of 101 FIV antibody-positive feline blood specimens submitted for FIV PCR diagnosis, 61 were positive (60%). A total of 23 of the positive PCRs identified subtype A, 11 identified subtype B1, 11 identified subtype B2/E, and 16 identified subtype C. FIV subtype D was not detected in any submitted specimens even though 13 blood specimens were from cats known to have received the FIV vaccine, which contains FIV subtype A and D inactivated virions. Therefore, this PCR quantitatively identifies FIV subtypes and unambiguously discriminates between FIV-vaccinated and FIV-infected cats.

Frequency and therapy monitoring of canine Babesia spp. infection by high-resolution melting curve quantitative FRET-PCR.

Babesia gibsoni and Babesia canis are the etiological agents of canine babesiosis, a protozoal hemolytic disease with global significance. Canine babesiosis has been diagnosed by microscopic identification of intra-erythrocytic trophozoites in blood smear, and by serological testing. Here we developed a quantitative fluorescence resonance energy transfer (FRET)-PCR that amplifies a fragment of the Babesia spp. 18S rRNA gene with high sensitivity and specificity. Melting curve analysis differentiates B. gibsoni, B. canis canis/B. canis vogeli, and B. canis rossi by the disassociation temperature of the fluorescent probes. Babesia gibsoni infection was detected in 8 of 48 canine breeds (17%) and 24 of a total of 235 specimens (10.2%) submitted from 22 states of the continental United States of America. A potential blood donor was positive for B. canis vogeli infection. In Hong Kong (China), B. gibsoni infection was detected in 30 of 64 specimens (46.9%) from 15 of the 24 breeds (63%). While the frequency of canine babesiosis did not associate with seasonal change in Hong Kong, positivity in the USA for Babesia spp. infection was higher in Spring and Summer than in Autumn and Winter. The data suggest that environmental factors associated with tick vector exposure rather than genetic susceptibility determine the incidence of canine babesiosis. Babesia spp. burdens in blood declined significantly with increasing age of the infected dogs, and therapy with atovaquone and tilmicosin eliminated B. gibsoni while doxcycline and berenil did not. This demonstrates that high-resolution real-time PCR analysis may advance diagnosis and therapy monitoring of canine babesiosis.