Bruce S. Seal

Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle Disease Virus RNA in Clinical Samples

ABSTRACT A real-time reverse-transcription PCR (RRT-PCR) was developed to detect avian paramyxovirus 1 (APMV-1) RNA, also referred to as Newcastle disease virus (NDV), in clinical samples from birds. The assay uses a single-tube protocol with fluorogenic hydrolysis probes. Oligonucleotide primers and probes were designed to detect sequences from a conserved region of the matrix protein (M) gene that recognized a diverse set (n = 44) of APMV-1 isolates. A second primer-probe set was targeted to sequences in the fusion protein (F) gene that code for the cleavage site and detect potentially virulent NDV isolates. A third set, also directed against the M gene, was specific for the North American (N.A.) pre-1960 genotype that includes the common vaccine strains used in commercial poultry in the United States. The APMV-1 M gene, N.A. pre-1960 M gene, and F gene probe sets were capable of detecting approximately 103, 102, and 104 genome copies, respectively, with in vitro-transcribed RNA. Both M gene assays could detect approximately 101 50% egg infective doses (EID50), and the F gene assay could detect approximately 103 EID50. The RRT-PCR test was used to examine clinical samples from chickens experimentally infected with the NDV strain responsible for a recent epizootic in the southwestern United States. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.

Matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first US avian pneumovirus isolate is distinct from European strains

Avian pneumovirus (APV) is the etiological agent of turkey rhinotracheitis (TRT). Outbreaks of TRT first occurred in the US during May, 1996 and continued through June, 1997. This is the first report of these virus types in the US that was previously considered exotic to the US and Canada. The US isolate, APV/CO, was replicated in chick embryo fibroblasts (CEF) and poly-A RNA from APV/CO infected CEF cells was purified for cDNA synthesis. Degenerate oligonucleotide primers were used to amplify nucleotide sequences coding for the matrix (M) protein gene. Although the type A and B European APV M genes share 75% identity in their coding sequences, they have only 60% identity with the US APV/CO M protein gene. Predicted M proteins of European APV type A and B isolates share 89% identity in their amino acid sequence. However, the predicted M protein of APV/CO has only 78% identity with APV type A and 77% identity with APV type B protein sequences. Phylogenetically the US APV/CO isolate separates as a unique virus relative to European APV type A and B strains that cluster together. Sequence information for the APV/CO M protein gene and predicted amino acids of the M protein confirm the unique nature of this isolate compared to its European counterparts. This correlates with the inability to serologically detect the US APV/CO isolate using diagnostics based on European viruses.

Molecular Characterization of an Avian Astrovirus

ABSTRACT Astroviruses are known to cause enteric disease in several animal species, including turkeys. However, only human astroviruses have been well characterized at the nucleotide level. Herein we report the nucleotide sequence, genomic organization, and predicted amino acid sequence of a turkey astrovirus isolated from poults with an emerging enteric disease.

Phylogenetic Relationships among Virulent Newcastle Disease Virus Isolates from the 2002-2003 Outbreak in California and Other Recent Outbreaks in North America

ABSTRACT Isolates from the 2002-2003 virulent Newcastle disease virus (v-NDV) outbreak in southern California, Nevada, Arizona, and Texas in the United States were compared to each other along with recent v-NDV isolates from Mexico and Central America and reference avian paramyxovirus type 1 strains. Nucleotide sequencing and phylogenetic analyses were conducted on a 1,195-base genomic segment composing the 3′ region of the matrix (M) protein gene and a 5′ portion of the fusion (F) protein gene including the M-F intergenic region. This encompasses coding sequences for the nuclear localization signal of the M protein and the F protein cleavage activation site. A dibasic amino acid motif was present at the predicted F protein cleavage activation site in all v-NDVs, including the California 2002-2003, Arizona, Nevada, Texas, Mexico, and Central America isolates. Phylogenetic analyses demonstrated that the California 2002-2003, Arizona, Nevada, and Texas viruses were most closely related to isolates from Mexico and Central America. An isolate from Texas obtained during 2003 appeared to represent a separate introduction of v-NDV into the United States, as this virus was even more closely related to the Mexico 2000 isolates than the California, Arizona, and Nevada viruses. The close phylogenetic relationship between the recent 2002-2003 U.S. v-NDV isolates and those viruses from countries geographically close to the United States warrants continued surveillance of commercial and noncommercial poultry for early detection of highly virulent NDV.

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.

Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin.

An effective bacteriocin was identified and characterized. Lactic acid bacteria were screened against Campylobacter jejuni. One bacteriocin producer, Enterococcus faecium (NRRL B-30746), was studied. The isolate was grown, and the bacteriocin was purified to single-band homogeneity. Biochemical traits indicated that the peptide was a Class IIa bacteriocin, and it was named E 50-52. The bacteriocin had a molecular weight of 3339.7 and an isoelectric point of 8.0. The minimal inhibitory concentrations of E 50-52 against C. jejuni, Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. ranged from 0.025 to 32 microg/mL. In therapeutic broiler trials, oral treatment with E 50-52 reduced both C. jejuni and Salmonella enteritidis by more than 100,000-fold in the ceca, and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens provides a promising alternative to antibiotics.

Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates

Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolates F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.

Phylogenetic relationships of bluetongue viruses based on gene S7

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.

Analysis of capsid protein gene variation among divergent isolates of feline calicivirus

Genomic variability within the capsid protein gene of feline calicivirus (FCV) was evaluated among different isolates using hybridization analysis and enzymatic viral nucleic acid amplification. Total infected cell RNA was first hybridized with cDNA clones generated to the capsid gene of the FCV isolates CFI/68, 255, LLK, NADC and KCD. Field isolates of FCV were categorized by hybridization with capsid gene cDNA from the reference strains. Isolates that did not hybridize were positive by Western blot using a cross-reactive cat polyclonal FCV CFI/68 capsid protein antiserum. Using previously published sequence information, oligonucleotide primers were generated based on conserved sequences surrounding the hypervariable capsid protein gene regions. Analysis of the FCV capsid protein gene hypervariable regions was completed by sequencing products of FCV nucleic acid amplified by reverse transcription and polymerase chain reaction. From these data, amino acid substitutions in the hypervariable regions of the capsid protein were identified for those isolates that did not hybridize with the original cDNA clones. An association between phylogenetic relationships and serum neutralization was established among FCV isolates examined.

Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and characterization of its bacteriocin, including the antimicrobial activity spectrum.

ABSTRACT Lactobacillus salivarius 1077 (NRRL B-50053) was isolated from poultry intestinal materials, and in vitro anti-Campylobacter jejuni activity was demonstrated. The isolate was then used for bacteriocin production and its enrichment. The protein content of the cell-free supernatant from the spent medium was precipitated by ammonium sulfate and dialyzed to produce the crude antimicrobial preparation. A typical bacteriocin-like response of sensitivity to proteolytic enzymes and resistance to lysozyme, lipase, and 100°C was observed with this preparation. The polypeptide was further purified by gel filtration, ion-exchange, and hydrophobic-interaction chromatography. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Edman degradation, and isoelectrofocusing were used to characterize its 3,454-Da molecular mass, the amino acid sequence of its 37 residue components, and the isoelectric point of pI 9.1 of the bacteriocin. Bacteriocin L-1077 contained the class IIa bacteriocin signature N-terminal sequence YGNGV. MICs of bacteriocin L-1077 against 33 bacterial isolates (both Gram negative and Gram positive) ranged from 0.09 to 1.5 μg/ml. Subsequently, the therapeutic benefit of bacteriocin L-1077 was demonstrated in market-age (40- to 43-day-old) broiler chickens colonized with both C. jejuni and Salmonella enterica serovar Enteritidis. Compared with untreated control birds, both C. jejuni and S. Enteritidis counts in colonized ceca were diminished by >4 log10 and S. Enteritidis counts in both the liver and the spleen of treated birds were reduced by 6 to 8 log10/g compared with those in the nontreated control birds. Bacteriocin L-1077 appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens.