Comparative full genome analysis of four infectious laryngotracheitis virus (Gallid herpesvirus-1) virulent isolates from the United States.

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.

Gene expression profiles of two B-complex disparate, genetically inbred Fayoumi chicken lines that differ in susceptibility to Eimeria maxima.

This study was conducted to compare the gene expression profiles, after Eimeria maxima infection, between 2 B-complex congenic lines of Fayoumi chickens that display differences in disease resistance and innate immunity against avian coccidiosis using cDNA microarray. When compared with uninfected controls using a cutoff of >2.0-fold alteration (P < 0.05), M5.1 demonstrated altered expression of 1 (downregulated), 12 (6 up, 6 down), and 18 (5 up, 13 down) mRNA at 3, 4, and 5 d postinfection, respectively. In the M15.2 line, altered expression was observed in 6 (3 up, 3 down), 29 (11 up, 18 down), and 32 (8 up, 24 down) transcripts at the 3 time points, compared with uninfected controls. Comparison of the expression levels between M5.1 and M15.2 chickens after E. maxima infection revealed alterations in 32 (10 up, 22 down), 98 (43 up, 55 down), and 92 (33 up, 59 down) mRNA at the 3 time points. Functional analysis using gene ontology categorized the genes exhibiting the different expression patterns between 2 chicken lines into several gene ontology terms including immunity and defense. In summary, transcriptional profiles showed that more gene expression changes occurred with E. maxima infection in the M15.2 than the M5.1 line. The most gene expression differences between the 2 chicken lines were exhibited at d 4 and 5 after E. maxima infection. These results demonstrate that differential gene expression patterns associated with the host genetic difference in coccidiosis resistance provide insights into the host protective immune mechanisms and present a rational basis to target specific genes and gene products to bolster host defenses against avian coccidiosis.

Comparison of transcriptional changes associated with E. acervulina and E. maxima infections using cDNA microarray technology.

Enhanced understanding of host-pathogen interactions at local sites of infection will extend our knowledge of disease pathogenesis and will facilitate the development of novel preventive methodologies against many infectious diseases of economic importance. In the current study, a 9.6K avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) was developed to compare the local transcriptional profiles following primary and secondary infections with two major Eimeria parasites, E. acervulina (EA) and E. maxima (EM), which infect the intestinal duodenum and jejunum, respectively. Gene Ontology analyses showed that EAinfection primarily induced genes associated with lipid metabolism and intracellulartrafficking whereas EM infection upregulated the genes involved in protein biosynthesis and metabolism, and downregulated apoptosis related genes. Following primary EA infection, there was a significantly enhanced expression of genes involved in the signal pathway of T cell activation and cytoskeletal regulation. Thus, the AVIELA array provides a valuable tool for investigating host-pathogen interactions in avian coccidiosis and allows for the comparison of the transcriptional regulations induced by species of Eimeria that infect different areas of the intestine.

Transcriptional response of chicken macrophages to Salmonella enterica serovar enteritidis infection.

The transcriptional profiles of chicken macrophages (HD11) infected with Salmonella enterica serovar Enteritidis (SE) were analyzed by using an avian macrophage microarray and real time RT-PCR. Out of 4906 array elements interrogated, 269 genes exhibited a 2 fold change (P < 0.001) over a 24 h time-course. Genes coding for proinflammatory cytokines, CC and CXC chemokines, and chemokine ligand were upregulated; whereas genes associated with transcription, cell adhesion and proliferation were downregulated. Most transcriptional changes occurred at 5 hours post-inoculation (hpi), with more genes downregulated than upregulated.At 5 hpi, the levels of gallinacin 1, lymphotactin, RhoA, and MHCIB2M transcripts were significantly decreased. In contrast, the levels of Cdc42 and MHCIIBLB2 mRNA were elevated. Infection of HD11 cells with mutant SE strains carrying an inactivated type three secretion system (TTSS1 or TTSS2) induced significantly higher levels of CCL4, K203, lymphotactin, and RhoA than wild type SE. In conclusion, chicken macrophage genes belonging to diverse functional classes were transcriptionally modulated by SE and selective modulation of host innate responses involved the effectors of TTSS1/2.

A functional genomics approach to the study of avian innate immunity.

A second-generation 4,959 element cDNA microarray has been created and evaluated for its potential use in examining the avian innate immune response. The elements in this array were obtained from EST libraries of stimulated avian PMNC-derived monocytes/macrophages and supplemented by genes of interest from several specific innate immune pathways. The elements are spotted in triplicate resulting in 14,877 total spots per slide. The avian innate immunity microarray (AIIM) contains 25 avian interleukin, chemokine, and cytokine elements. The array also contains elements for several innate immune pathways, including genes involved in the Toll-like receptor (TLR) pathway (including six of the currently known avian TLR receptors), avian interferon/antiviral response pathway genes, and genes involved in apoptosis, antigen presentation and the oxidative burst. The AIIM can be used to evaluate global gene expression patterns in a number of immunologically relevant tissues and in chickens, turkeys and ducks. The array has also been evaluated for its ability to monitor the avian immune response to both bacterial (avian pathogenic Escherichia coli) and viral (avian influenza) avian pathogens.

Immunogenomic approaches to study host immunity to enteric pathogens.

With increasing consumer demands for safe poultry products, effective control of disease-causing pathogens is becoming a major challenge to the poultry industry. Many chicken pathogens enter the host through the gastrointestinal tract, and over the past few decades, in-feed antibiotics and active vaccination have been the 2 main mechanisms of disease control. However, increasing public concerns are prompting government regulations on the use of growth-promoting drugs in animal production, and the ability of current vaccines to protect against emerging hypervirulent strains of pathogens is becoming an issue. Therefore, there is a need to develop alternative control strategies against poultry pathogens of economic importance as well as to carry out basic research to enhance understanding of host-pathogen interactions at local sites of infection. Effective control strategies against pathogens can only be accomplished by comprehensive analysis of the basic immunobiology of host-pathogen interactions. Recent sequencing of the poultry genome and the availability of several tissue-specific cDNA microarrays are facilitating the rapid application of functional immunogenomic technologies to poultry disease research. Studies using functional genomic, immunology, and bioinformatic approaches have provided novel insights into disease processes and protective immunity to chicken pathogens. In this review, we summarize recent published literature concerning the host response to Eimeria and Salmonella infections with emphasis on our studies using immunogenomic tools to investigate and characterize the mechanisms of avian immunity to these mucosal pathogens. The results clearly indicate that this immunogenomic approach will lead to increased understanding of immune responses to infectious agents that will enable the development of effective prevention strategies against mucosal pathogens.

Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens.

Infectious bronchitis virus (IBV) field isolates of the Arkansas (Ark) serotype were identified by reverse transcription-polymerase chain reaction (RT-PCR) as the most common serotype isolated from 1993 to 1997. These isolates were recovered from broiler flocks with respiratory disease raised on the Delmarva peninsula in spite of Ark vaccination in the region. For the purposes of investigating this apparently paradoxical finding, five RT-PCR Ark-positive field isolates recovered in 1995 and 1996 were selected for further characterization. The isolates were compared with Ark reference strains by reciprocal virus neutralization (VN) in embryonated eggs, S-1 gene sequence analysis, and challenge of immunity studies in specific-pathogen-free (SPF) chickens. Antigenic (VN) comparisons and S-1 gene analysis confirmed that the five RT-PCR Ark-positive field isolates were of the Ark serotype but also revealed that the viruses could be readily distinguished from Ark reference strains. Four of the isolates (Ark/213/96, Ark/15C/96, Ark/1529/95, Ark/1534/95) were found to have higher antigenic relatedness percentages to each other (95%-100%) than to Ark reference strains DPI (52%-72%) and Georgia variant (Georgia var) (53%-68%) by VN. Another isolate, Ark/1535/95, was found to differ antigenically from the other four RT-PCR Ark-positive field isolates (34%-61%), Ark DPI (44%), and Georgia var (43%) strains. The trends in the S-1 gene sequencing results were similar to those observed for the VN findings. Isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 demonstrated a higher degree of predicted S-1 amino acid similarity to each other (96.5%-98.7%) than to Ark DPI (92.4%-93.7%), Ark 99 (93.2%-94.7%), and Georgia var (89.3%-90.8%). Ark/1535/95 S-1 amino acid similarity values were lower compared with those of the other four RT-PCR Ark-positive field isolates (93.4%-94.8%), Ark DPI (91.9%), Ark 99 (93.0%), and Georgia var (88.7%). Furthermore, the isolates could be distinguished from the Ark reference strains by a characteristic sequence polymorphism, a six-nucleotide deletion encoding amino acids 57 (Asp) and 58 (Asp) in hypervariable region 1 of S-1. On the basis of the VN and sequencing findings, isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 were considered to be a single subtype of the Ark serotype. The fifth isolate, Ark/1535/95, may constitute another subtype of the Ark serotype. Vaccination of SPF chickens with a high-titering commercially available live vaccine containing the Ark DPI strain provided solid protection (>90%) against challenge with the RT-PCR Ark-positive field isolates. Immunization of SPF chickens with Ark/213/96 produced 100% protection against challenge with the homologous strain, as well as isolates Ark/1535/95 and Ark 99 but lower levels of protection against Ark DPI (58%) and Georgia var (55%). Primers for RT-PCR were designed to distinguish between the Ark subtypes and the Ark reference strains on the basis of the characteristic six-nucleotide deletion identified in the S-1 gene of the Ark subtypes. Retrospective analysis of RT-PCR Ark-positive isolates found that the Ark subtypes existed as early as 1992 in Delmarva broilers and became prevalent by 1995. With RT-PCR, restriction fragment length polymorphism analysis, and DNA sequencing techniques, the presence of Ark subtype viruses was demonstrated in two commercial Ark DPI strain vaccines and in our Ark DPI laboratory stocks that were the original source of the virus used for vaccine development. The demonstration of the Ark subtype and reference strains in the Ark DPI strain is evidence of the existence of IBV quasispecies. Factors possibly influencing the emergence of the Ark subtype in commercial broilers are discussed.

Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene.

Direct automated cycle sequencing (DACS) of a reverse transcription-polymerase chain reaction (RT-PCR) product of the S-1 subunit of the spike peplomer gene was used to identify infectious bronchitis virus (IBV) serotypes. Degenerate primers CK4 and CK2, utilized previously in our laboratory, were selected for DACS because they successfully amplify a wide range of serotypes represented by various reference strains and field isolates and the resulting polymerase chain reaction (PCR) product contains diagnostically relevant S-1 sequences that can be used to identify the serotype of IBV. The S-1 nucleotide sequences generated by DACS were aligned and analyzed with commercial software to determine their relationship to the S-1 nucleotide sequences of IBV strains on deposit in the GenBank and EMBL databases. Reference strains Massachusetts (Mass) 41, Connecticut (Conn), Arkansas (Ark) DPI, JMK, and DE/072/92 were initially tested by DACS to establish the feasibility of the procedure. The DACS procedure was further evaluated with a panel of "unknowns" comprised of IBV reference strains, field isolates, and variant serotypes collected by our laboratory. The DACS procedure provided high-quality and reproducible S-1 sequence for all IBV serotypes tested, including variant serotypes that had not been sequenced previously. The S-1 nucleotide sequences for the amplified PCR products of reference strains Mass 41, Conn, Ark DPI, JMK, and DE/072/92 generated by DACS were highly homologous (>99% nucleotide identity) with their respective GenBank database sequences. In the unknown panel, the nucleotide identities of the DACS S-1 sequences of field isolates of serotypes previously identified by virus neutralization were also found to be very high (> or = 95.5%) after alignment with database sequences. In contrast, the nucleotide identities of S-1 sequences of variant serotypes 37, 3330, and PA/1220/98 and reference strain Clark 333, for which database sequences were not available, ranged from 27.7% to 73.8%, well below the identity values for a homologous serotype. With alignment software, the identities of strains in mixtures of RNAs of two different serotypes were not resolvable. DACS of IBV S-1 RT-PCR products will enable researchers to rapidly identify field strains, including new, previously unrecognized variant virus serotypes.

Acquired thermotolerance and expression of the HSP100/ClpB genes of lima bean.

Acquired thermotolerance (AT) is the ability of cells to survive a normally lethal temperature treatment as a consequence of pretreatment at an elevated but sublethal temperature. In yeast and cyanobacteria, the expression of the HSP100/ClpB protein is required for the AT response. To determine whether the HSP100/ClpB protein is associated with this response in lima bean (Phaseolus lunatus), we have cloned an HSP100/ClpB homolog and assessed expression of the two gene copies under heat stress conditions, which induce AT. Transcription of the cytoplasmically localized HSP100/ClpB protein genes is stringently controlled by heat stress in both of the laboratory and field heat stress conditions. From a heat-induced cDNA library, we identified a clone of a putative chloroplast-targeted (cp) HSP100/ClpB protein gene sequence. The cp HSP100/ClpB protein genes are constitutively expressed, but transcript levels increase post-heat stress in laboratory heat stress experiments. In field conditions the genes for the cp HSP100/ClpB are constitutively expressed. Although we were unable to correlate differences in the timing of AT response with the expression or genetic structure of the HSP100/ClpB genes in heat-tolerant or -sensitive varieties of lima bean, we clearly demonstrate the association of expression of HSP100/ClpB proteins with heat response in this species.

Infectious laryngotracheitis virus, an alpha herpesvirus that does not interact with cell surface heparan sulfate.

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4 degrees C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.

Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer (S-1) gene.

The S-1 peplomer gene sequences of 31 strains of avian coronavirus infectious bronchitis virus (IBV) from North America, Europe, and Australia were compared to identify common and unique regions for possible diagnostic applications. S-1 sequences that were conserved among serotypes and sequences that were variable between serotypes were identified. Based on conserved S-1 gene sequences, "general" degenerate oligonucleotide primers were designed that amplified IBV genomic RNA by the reverse transcriptase polymerase chain reaction (RT-PCR) procedure regardless of serotype. Primers specific for IBV serotypes Massachusetts, Connecticut, Arkansas, JMK, Delaware (DE/072/92), and California (CA/633/85) were designed from regions of the S-1 gene exhibiting extensive sequence hypervariability. The ability to identify these six serotypes of IBV by RT-PCR was demonstrated by testing the serotype-specific primers on a panel of unknown samples that included 30 reference strains and field isolates previously characterized by virus neutralization (VN). The use of serotype-specific primers in RT-PCR provides a rapid and accurate means of identifying IBV.

Characterization of MGC2, a Mycoplasma gallisepticum cytadhesin with homology to the Mycoplasma pneumoniae 30-kilodalton protein P30 and Mycoplasma genitalium P32.

A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen Mycoplasma gallisepticum. The 912-nucleotide mgc2 gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the M. pneumoniae P30 and M. genitalium P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.

Research notes: Comparison of disease susceptibility and resistance in three lines of chickens experimentally infected with infectious laryngotracheitis virus.

The susceptibility of three F1 hybrid lines of chickens to graded doses of infectious laryngotracheitis virus (ILTV) was investigated. The three F1 hybrid lines, each produced from mating two inbred lines, included the SC (B2B2) and TK (B15B21) lines and the 15I5 x 7(1) (B2B15) line. Although at 1 d of age all three lines were susceptible to ILTV, SC birds were significantly less susceptible (10%) than TK (80%) or 15I5 x 7(1) (50%) birds when exposed to 5,000 pfu of virus at 4 wk of age. The ability of each inbred F1 hybrid line to establish a protective immune response to ILTV was also determined. The SC birds required a smaller immunizing dose of virus (500 pfu) to mount a protective immune response to ILTV than the 15I5 x 7(1) line (5,000 pfu). A 5,000 pfu immunizing dose did not elicit a protective immune response in the TK line to a 10(6) pfu challenge dose of ILTV. These results also correlated with the ability to produce ILTV-specific antibodies. This study confirms and expands on observations that lines of chickens differ with respect to their susceptibility and resistance to ILTV.

Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B.

Infectious laryngotracheitis virus (ILTV) is an alpha-herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB-beta-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.

Antigenic and S-1 genomic characterization of the Delaware variant serotype of infectious bronchitis virus.

A previously unrecognized infectious bronchitis virus (IBV) serotype, referred to hereafter as the Delaware variant (DE var), was isolated from commercial broiler chickens during a severe, widespread respiratory disease epornitic in the Delmarva peninsula region of the United States in January-March 1992. The DE var serotype was found to be antigenically unrelated by virus-neutralization (VN) test to nine reference IBV serotypes from North America. Additional VN tests indicated that the DE var isolates (DE/072/92, DE/121/ 92, DE/152/92, and DE/174/92) from broilers were fully or partially neutralized by monospecific antisera prepared against themselves and against two IBV field isolates (DE/492/90 and DE/903/90) recovered from a Delmarva commercial layer flock experiencing egg production losses in 1990. Antigenic relatedness values determined by VN indicated layer isolate DE/492/90 was more closely related to the broiler DE var isolates than was layer isolate DE/903/90. Cross-challenge tests performed in specific-pathogen-free chickens also demonstrated the antigenic similarity of the broiler (DE/072/92 and DE/174/92) and the layer isolates (DE/492/90 and DE/903/90), with heterologous strain protection values ranging from 55% to 100%. Protection values of DE var isolates vs. Massachusetts 41 and Arkansas DPI were considerably lower (0-60%). The S-1 gene of the US/DE/072/92 isolate of the DE var serotype was amplified by reverse transcription polymerase chain reaction, cloned, and sequenced. The DE var S-1 gene sequence was compared with the S-1 gene sequences of IBV serotypes from North America, Europe, and Australia. A dendrogram based on this analysis supported the conclusion that the DE var serotype is highly novel among IBV. A high degree of similarity (> 88%) was observed between the S-1 genes of the DE var broiler isolates (DE/072/92 and DE/174/92) and layer isolates (DE/492/90 and DE/903/90). These data, taken with the VN and cross-challenge results, establish a genetic as well as an antigenic link between the isolates from layers and broilers and indicate the DE var serotype was responsible for both infectious bronchitis outbreaks.

Cloning and characterization of a putative cytadhesin gene (mgc1) from Mycoplasma gallisepticum.

A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S-transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas.

Identification and characterization of the infectious laryngotracheitis virus glycoprotein C gene.

The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the glycoprotein C gene of herpes simplex virus has been sequenced and identified based on its genomic location, comparative analysis to other gC proteins, and the identification of a glycosylated protein product. Located near the small subunit ribonucleotide reductase gene, the ILTV gC gene is 1242 bp in length and is predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, five potential N-linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. Antibodies raised in rabbits against a Cro-ILTV-beta-galactosidase fusion protein expressed in Escherichia coli recognize a 60-kDa ILTV-specific glycoprotein from infected cell extracts. Transcriptional analysis, using a portion of the open reading frame as a probe, identified a 1.55-kb transcript expressed with late gene kinetics. Comparison to other herpesvirus gC proteins revealed limited amino acid sequence homology and the absence of a charged extracellular region, which would normally interact with cell surface proteoglycans.

Restriction endonuclease analysis of Delmarva field isolates of infectious laryngotracheitis virus.

Restriction endonuclease (RE) digestion patterns of six Delmarva field isolates of infectious laryngotracheitis virus (ILTV) were compared with three standard reference strains. With one exception, all of the field isolates generated RE digestion patterns identical to an embryo-propagated vaccine strain of ILTV when the six-base-recognizing REs EcoRI, HindIII, PstI, and BamHI were used. In order to increase the sensitivity of the RE analysis technique, a more sensitive DNA fingerprinting approach using four-base-recognizing enzymes was developed. One field isolate could be differentiated from the embryo-propagated vaccine strain using all three enzymes, Sau3AI, MspI, and HinfI. A second isolate could be differentiated only by comparing HinfI digestion patterns. This work provides additional evidence that differentiable strains of ILTV exist in the United States. Furthermore, currently used RE analysis methods may not be sensitive enough to discriminate between field isolates and vaccine strains of ILTV, thus challenging the theory that vaccine strains of ILTV are responsible for field outbreaks of infectious laryngotracheitis.

Identification of the putative cytadhesin gene of Mycoplasma gallisepticum and its use as a DNA probe.

A portion of the putative Mycoplasma gallisepticum (MG) cytadhesin gene was identified and used as a diagnostic DNA probe. Degenerate oligonucleotide primers corresponding to conserved regions of the cytadhesin proteins from two human mycoplasmas, M. pneumoniae and M. genitalium, were synthesized for use in the polymerase chain reaction (PCR) on genomic MG DNA. A 583-base-pair MG DNA fragment was amplified and subsequently cloned and sequenced. The MG DNA fragment is predicted to encode a 193-amino-acid peptide. This peptide demonstrates significant homology to the expected portions of the two human mycoplasmal cytadhesin proteins. Used as a probe to study the distribution of this fragment in pathogenic and nonpathogenic avian mycoplasmas, the PCR product hybridized to genomic DNA from all seven MG strains tested. However, it failed to hybridize to M. synoviae, M. meleagridis, M. iowae, or M. gallinarum DNA.

Identification of the thymidine kinase gene of infectious laryngotracheitis virus.

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.