Corrie C. Brown

An approach to the control of disease transmission in pig-to-human xenotransplantation

Abstract: Although several major immunologic hurdles need to be overcome, the pig is currently considered the most likely source animal of cells, tissues and organs for transplantation into humans. Concerns have been raised with regard to the potential for the transfer of infectious agents with the transplanted organ to the human recipient. This risk is perceived to be increased as it is likely that the patient will be iatrogenically immunocompromised and the organ‐source pig may be genetically engineered in such a way to render its organs particularly susceptible to infection with human viruses. Furthermore, the risk may not be restricted to the recipient, but may have consequences for the health of others in the community. The identification of porcine endogenous retroviruses and of hitherto unknown viruses have given rise to the most concern. We document here the agents we believe should be excluded from the organ‐source pigs. We discuss the likelihood of achieving this aim and outline the potential means by which it may best be achieved.

Pathogenesis of Newcastle Disease in Chickens Experimentally Infected with Viruses of Different Virulence

Groups of 4-week-old White Rock chickens were inoculated intraconjunctivally with nine isolates of Newcastle disease virus representing all pathotypes. Birds were monitored clinically and euthanatized sequentially, with collection of tissues for histopathologic examination and in situ hybridization using an antisense digoxigenin-labeled riboprobe corresponding to the sequence of the gene coding for the matrix protein. Disease was most severe with velogenic viscerotropic pathotypes and was characterized by acute systemic illness with extensive necrosis of lymphoid areas in the spleen and intestine. Viral nucleic acid was detected in multiple tissues but most prominently in macrophages associated with lymphoid tissue. Velogenic neurotropic isolates caused central nervous system disease despite minimal amounts of viral nucleic acid detected in neural tissue. Mesogenic and lentogenic pathotypes caused no overt disease; however, viral nucleic acid was present in myocardium and air sac epithelium following infection with these isolates. Compromise of air sac and myocardium may predispose mesogen- and lentogen-infected chickens to secondary infection and/or decreased meat and egg production.

Newcastle disease a review of field recognition and current methods of laboratory detection

Newcastle disease (ND) remains a constant threat to the poultry industry and is a limiting disease for poultry producers worldwide. The variety of clinical presentations and the emergence and spread of new genetic variants make recognition and diagnosis challenging. The current review details the pertinent features of the clinicopathologic disease in the main susceptible species, including chicken, turkey, duck, goose, pigeon, and other birds such as cormorants, psittacines, and canaries. Furthermore, the available and emerging laboratory diagnostic methodologies for the detection and typing of the virus are reviewed, including traditional techniques such as virus isolation and immunohistochemistry as well as rapid procedures based on molecular tools, such as real-time polymerase chain reaction, gene sequencing, and microarrays. The relevant genetic variability of ND viruses probably represents the major limitation in the validation and application of the current, advanced diagnostic molecular techniques. This underscores the importance of a multidisciplinary and comprehensive diagnostic approach, which should include, next to the new generation assays of the genomic era, the more traditional techniques such as histopathology, immunohistochemistry, and virus isolation.

Characterization of "Candidatus Piscichlamydia salmonis" (Order Chlamydiales), a Chlamydia-Like Bacterium Associated With Epitheliocystis in Farmed Atlantic Salmon (Salmo salar)

ABSTRACT To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon (Salmo salar), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales. “Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

Virulence of pigeon-origin Newcastle disease virus isolates for domestic chickens

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.

Experimental Pathogenesis for Chickens, Turkeys, and Pigeons of Exotic Newcastle Disease Virus from an Outbreak in California during 2002-2003:

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specificpathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.

Virulent Newcastle disease virus elicits a strong innate immune response in chickens

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100% mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1β and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.

Pathogenesis of Six Pigeon-Origin Isolates of Newcastle Disease Virus for Domestic Chickens

The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus 1 (PPMV-1), and two isolates were classified as avian paramyxovirus 1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI. Tissues were examined by histopathology, immunohistochemistry (IHC) for the presence of NDV nucleoprotein, and in situ hybridization (ISH) for the presence of viral mRNA for the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1-infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP. Among all the isolates, the IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC.

Clinicopathological Characterization in Poultry of Three Strains of Newcastle Disease Virus Isolated From Recent Outbreaks

Newcastle disease is a severe threat to the poultry industry and is caused by Newcastle disease virus, a member of the genus Avulavirus, family Paramyxoviridae. The virus is rapidly evolving, and several new genotypes have been discovered in the past few years. Characterization of these strains is important to evaluate field changes, anticipate new outbreaks, and develop adequate control measures. Three Newcastle disease isolates (APMV-1/duck/Vietnam, Long Bien/78/2002, APMV-1/chicken/Australia/9809-19-1107/1998, and APMV-1/double-crested cormorant/USA, Nevada/19529-04/2005) from recent outbreaks were investigated via clinicopathological assessment, immunohistochemistry (IHC), in situ hybridization, virus isolation, and serology in experimentally infected 4-week-old chickens. Phylogenetic studies showed that Australia isolate belongs to class II genotype I, Long Bien to class II genotype VIId, and Nevada cormorant to class II genotype V. Even though all 3 viruses had a virulent fusion protein cleavage site and ICPI values greater than 1.5, they all differed in their ability to cause clinical signs, in their lesions, and in their viral distribution in body tissues. The Long Bien isolate showed the most severe clinicopathological picture and the most widespread viral distribution. The Australia and Nevada cormorant isolates had a milder pathological phenotype, with viral replication restricted to only a few organs. The variability in clinicopathological characteristics despite the similarity in ICPI suggests that full clinicopathological assessment is necessary to fully characterize new isolates and that there are differences in pathogenesis among viruses of different genotypes.

Highly Divergent Virulent Isolates of Newcastle Disease Virus from the Dominican Republic Are Members of a New Genotype That May Have Evolved Unnoticed for Over 2 Decades

ABSTRACT A Newcastle disease virus (NDV) outbreak in chickens was reported in the Dominican Republic in 2008. The complete genome of this isolate, chicken/DominicanRepublic(JuanLopez)/499-31/2008 (NDV-DR499-31/08), and the fusion proteins of three other related viruses from the Dominican Republic and Mexico were sequenced and phylogenetically analyzed. Genetically, these four isolates were highly distinct from all other currently known isolates of NDV, and together, they fulfill the newly established criteria for inclusion as a novel genotype of NDV (genotype XVI). The lack of any reported isolation of viruses related to this group since 1986 suggests that virulent viruses of this genotype may have evolved unnoticed for 22 years. The NDV-DR499-31/08 isolate had an intracerebral pathogenicity index (ICPI) score of 1.88, and sequencing of the fusion cleavage site identified multiple basic amino acids and a phenylalanine at position 117, indicating this isolate to be virulent. These results were further confirmed by a clinicopathological assessment in vivo. In 4-week-old chickens, NDV-DR499-31/08 behaved as a velogenic viscerotropic strain with systemic virus distribution and severe necrohemorrhagic lesions targeting mainly the intestine and the lymphoid organs. The clear phylogenetic relationship between the 2008, 1986, and 1947 ancestral viruses suggests that virulent NDV strains may have evolved in unknown reservoirs in the Caribbean and surrounding regions and underlines the importance of continued and improved epidemiological surveillance strategies to detect NDV in wild-bird species and commercial poultry.