Biomedical subjects

B S Seal

Publications and source records attributed to B S Seal.

At least 19 recordsLinked to original sources

PubMed2006

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Adsorption↗

PubMed2006

Campylobacter spp. subtype analysis using gel-based repetitive extragenic palindromic-PCR discriminates in parallel fashion to flaA short variable region DNA sequence analysis.

AIMS: The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology. METHODS AND RESULTS: Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. CONCLUSIONS: The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.

Bacterial Typing Techniques↗

PubMed2006

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

Amino Acid Sequence↗

PubMed2006

Pathogenesis of Newcastle disease in commercial and specific pathogen-free turkeys experimentally infected with isolates of different virulence.

The pathogenesis of five different Newcastle disease virus (NDV) isolates representing all pathotypes was examined in commercial and specific pathogen-free (SPF) turkeys. Experimentally-infected birds were monitored clinically and euthanatized, with subsequent tissue collection, for examination by histopathology, by immunohistochemistry for the presence of NDV nucleoprotein, and by in situ hybridization for the presence of replicating virus. Clinically, the lentogenic pathotype did not cause overt clinical signs in either commercial or SPF turkeys. Mesogenic viruses caused depression in some birds. Turkeys infected with velogenic neurotropic and velogenic viscerotropic isolates showed severe depression, and neurologic signs. Histologic appearances for all strains had many similarities to lesions observed in chickens inoculated with the various isolates; that is, lesions were present predominantly in lymphoid, intestinal, and central nervous tissues. However, in general, disease among turkeys was less severe than in chickens, and turkeys could be considered a subclinical carrier for some of the isolates.

Animals↗

PubMed2006

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 specific-pathogen-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.

Animals↗

PubMed2002

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.

Animals↗

PubMed2001

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates.

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Amino Acid Sequence↗

PubMed2001

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus).

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.

Animals↗

PubMed2000

Development of an RT-PCR diagnostic test for an avian astrovirus.

Astroviruses are small round viruses that cause enteric disease in the young of several species. Detection and diagnosis of astrovirus infection in non-human hosts relies heavily on electron microscopy and fluorescent antibody tests. Recently, our laboratory isolated and sequenced an avian astrovirus from poult enteritis mortality syndrome affected turkeys. These studies describe the development of RT-PCR methods, which specifically detect regions of the viral capsid and polymerase genes, and demonstrate their use in detecting astrovirus infection in commercial turkey flocks.

Animals↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Newcastle disease virus phosphoprotein gene analysis and transcriptional editing in avian cells.

Nucleotide sequence was determined for the phosphoprotein (P) gene from 23 Newcastle disease virus (NDV) isolates representing all defined pathotypes with different chronological and geographic origins. Sequence variation, with synonymous substitutions dominating, occurred throughout the P gene. An exception was a conserved central region containing the transcriptional editing site. Four G nucleotide additions were detected in NDV P gene mRNA potentially creating alternative open reading frames. However, only one in-frame stop codon exists with a single G addition among all isolates that would allow for a potential V protein. A second potential stop codon does not exist in the P gene consensus sequence among all isolates with more than one G nucleotide addition at the editing site. This precludes a possible W protein in these isolates. A second potential alternative in-frame start site exists among all isolates that could encode a predicted X protein for NDV. Comparison of the P gene editing sites among the Paramyxovirinae and predicted P gene usage demonstrates that NDV more closely resembles the respiroviruses and morbilliviruses. Phylogenetic analysis of P gene sequences among NDV isolates demonstrates there are two clades of these viruses. One group includes viruses isolated in the US prior to 1970, while a second cluster includes virulent viruses circulating worldwide.

Amino Acid Sequence↗

PubMed2000

Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae.

Matrix (M) gene sequences for recent field isolates and older reference Newcastle disease viruses (NDV) were examined to determine phylogenetic relationships and population trends among these viruses. Overall, the M gene has a majority of synonymous nucleotide sequence substitutions occurring among NDV isolates. However, several predicted amino acid changes in the M protein of specific NDV isolates have occurred that correlate to phylogenetic relationships. Nucleotide substitutions in these codons have a greater number of nonsynonymous base changes. The NDV isolates arising since the 1970s belong to a population of viruses that expanded worldwide at an exponential rate. These viruses may have their origins in free-living birds, are present worldwide, and continue to circulate causing disease in poultry. A specific NDV lineage composed of virulent isolates obtained in the US prior to 1970 appears to no longer exists among free-living birds or commercial poultry. However, "vaccine-like" viruses are common in the US and continue to circulate among commercial poultry. Based on M protein amino acid sequences, NDV separates as a clade most closely related to morbilliviruses and not with their current designated category, the rubulaviruses among the Paramyxoviridae. Consequently, avian paramyxoviruses should have their own taxonomic subfamily among the Paramyxovirinae.

Animals↗

PubMed2000

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 isolate's 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.

Amino Acid Sequence↗

PubMed2000

Avian pneumoviruses and emergence of a new type in the United States of America.

Avian pneumovirus (APV) primarily causes an upper respiratory disease recognized as turkey rhinotracheitis (TRT) or swollen head syndrome (SHS) in chickens. The virus was first isolated in South Africa during the early 1970s and has subsequently been reported in Europe, Asia and South America. In February 1997, a serologically distinct APV isolate was officially reported in the USA following an outbreak of TRT during the previous year. This was the first report of these virus types in the USA; they were previously considered exotic to the USA and Canada. The predicted matrix (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 is only 78% similar to the APV type A and 77% similar to the APV type B protein sequence. The predicted amino acid sequence of the US APV isolate's fusion (F) protein has 72% sequence identity to the F protein of APV type A and 71% sequence identity to the F protein of type B. This compares with the 83% sequence identity between the predicted amino acid sequences of the F proteins of APV types A and B. The lack of sequence heterogeneity among the US APV isolates over 2 years suggests that these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the M and F proteins, together with the serological uniqueness of the US APV isolates, supports their classification as a new APV, designated type C.

Amino Acid Sequence↗

PubMed2000

Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies.

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.

Animals↗

PubMed2000

Molecular characterization of an avian astrovirus.

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.

Amino Acid Sequence↗

PubMed1999

Comparison of pathology-based techniques for detection of viscerotropic velogenic Newcastle disease virus in chickens.

Two pathology-based techniques, immunohistochemistry and riboprobe in-situ hybridization, were applied to formalin-fixed, paraffin wax-embedded tissues from chickens infected with three different isolates of velogenic viscerotropic Newcastle disease virus (VVNDV). With the immunohistochemical method, viral protein was consistently detectable in the spleen and caecum at the terminal phase of the infection. With in-situ hybridization, viral nucleic acid was consistently detected in the eyelid, spleen and caecum in both the acute and terminal phases. Hybridization with anti-sense probe to detect viral mRNA was often more intense than hybridization with sense probe to detect viral genomic RNA.

Animals↗

PubMed1999

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 anti-sense 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.

Air Sacs↗