Transcriptional and Pathological Host Responses to Coinfection with Virulent or Attenuated Mycoplasma gallisepticum and Low-Pathogenic Avian Influenza A Virus in Chickens.

The avian pathogen Mycoplasma gallisepticum, the etiological agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a copathogen such as low-pathogenic avian influenza virus (LPAIV). To further investigate the intricacies of this copathogenesis, chickens were monoinfected or coinfected with either virulent M. gallisepticum strain Rlow or LPAIV H3N8 (A/duck/Ukraine/1963), with assessment of tracheal histopathology, pathogen load, and transcriptomic host responses to infection by RNA sequencing. Chickens coinfected with M. gallisepticum Rlow followed by LPAIV H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening than chickens infected with LPAIV H3N8 alone and greater viral loads than chickens infected first with H3N8 and subsequently with M. gallisepticum Rlow Recovery of live M. gallisepticum was significantly higher in chickens infected first with LPAIV H3N8 and then with M. gallisepticum Rlow, compared to chickens given a mock infection followed by M. gallisepticum Rlow The transcriptional responses to monoinfection and coinfection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as Toll-like receptor 15, Toll-like receptor 21, and matrix metallopeptidase 1. Pathway and gene ontology analyses of these differentially expressed genes suggest that coinfection with virulent M. gallisepticum and LPAIV induces decreases in the expression of genes related to ciliary activity in vivo and alters multiple immune-related signaling cascades. These data aid in the understanding of the relationship between M. gallisepticum and LPAIV during copathogenesis in the natural host and may contribute to further understanding of copathogen infections of humans and other animals.

Variable Lipoprotein Hemagglutinin A Gene (vlhA) Expression in Variant Mycoplasma gallisepticum Strains In Vivo.

Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease, is a significant poultry pathogen, causing severe inflammation and leading to economic losses worldwide. Immunodominant proteins encoded by the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role remains unknown. Previous work has demonstrated that vlhA phase variation is dynamic throughout the earliest stages of infection, with vlhA 3.03 being the predominant vlhA expressed during the initial infection, and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms. To further investigate this gene family, we assessed the vlhA profile of two well-characterized vaccine strains, GT5 and Mg7, a vlhA 3.03 mutant strain, and an M. gallisepticum population expressing an alternative immunodominant vlhA Here, we report that two M. gallisepticum vaccine strains show different vlhA profiles over the first 2 days of infection compared to that of wild-type Rlow, while the population expressing an alternative immunodominant vlhA gene reverted to a profile indistinguishable from that of wild-type Rlow Additionally, we observed a slight shift in the vlhA gene expression profile but no reduction in virulence in a vlhA 3.03 mutant. Taken together, these data further support the hypothesis that M. gallisepticum vlhA genes change in a nonstochastic temporal progression of expression and that vlhA 3.03, while preferred, is not required for virulence. Collectively, these data may be important in elucidating mechanisms of colonization and overall pathogenesis of M. gallisepticum.

Transcriptional Profiling of the Chicken Tracheal Response to Virulent Mycoplasma gallisepticum Strain Rlow.

Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease (CRD) in poultry, leads to prolonged recruitment and activation of inflammatory cells in the respiratory mucosa. This is consistent with the current model of immune dysregulation that ostensibly allows the organism to evade clearance mechanisms and establish chronic infection. To date, studies using quantitative reverse transcription-PCR (qRT-PCR) and microarrays have shown a significant transient upregulation of cytokines and chemokines from tracheal epithelial cells (TECs) in vitro and tracheal tissue ex vivo in response to virulent strain Rlow that contributes to the infiltration of inflammatory cells into the tracheal mucosa. To expand upon these experiments, RNA was isolated from tracheas of 20 chickens infected with M. gallisepticum Rlow and 20 mock-infected animals at days 1, 3, 5, and 7 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A rapid host response was observed 24 h postinfection, with over 2,500 significantly differentially expressed genes on day 3, the peak of infection. Many of these genes have immune-related functions involved in signaling pathways, including Toll-like receptor (TLR), mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways. Of interest was the increased expression of numerous cell surface receptors, including TLR4 and TLR15, which may contribute to the production of cytokines. Metabolic pathways were also activated on days 1 and 3 postinfection, ostensibly due to epithelial cell distress that occurs upon infection. Early perturbations in tissue-wide gene expression, as observed here, may underpin a profound immune dysregulation, setting the stage for disease manifestations characteristic of M. gallisepticum infection.

Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.

Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein Gene vlhA Expression during In Vivo Infection of the Natural Chicken Host.

Mycoplasma gallisepticum is the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms of M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomic vlhA gene expression directly from M. gallisepticum populations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minor vlhA gene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specific vlhA genes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominant vlhA gene expression in the colonizing bacterial population. The dominant expression of a given vlhA gene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominate vlhA gene when no longer faced with host pressures. Overall, these data indicate that vlhA phase variation is dynamic throughout the earliest stages of infection and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms.

Hydrogen peroxide production from glycerol metabolism is dispensable for virulence of Mycoplasma gallisepticum in the tracheas of chickens.

Hydrogen peroxide (H2O2) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H2O2 production on virulence in vivo have never been assessed in any Mycoplasma species. To this end, we determined that the wild-type (WT) R(low) strain of the avian pathogen Mycoplasma gallisepticum is capable of producing H2O2 when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely, glpO, glpK, and glpF, were identified. All mutants assessed were incapable of producing H2O2 and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H2O2 when grown in glycerol, while the naturally attenuated F strain does produce H2O2. Chickens were infected with one of two glpO mutants, a glpK mutant, R(low), or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all glp mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H2O2 production/cytotoxicity and virulence for this Mycoplasma species in its natural host. However, it is possible that glycerol metabolism is required by M. gallisepticum in a niche that we have yet to study.

Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

Extensive variation in surface lipoprotein gene content and genomic changes associated with virulence during evolution of a novel North American house finch epizootic strain of Mycoplasma gallisepticum.

Mycoplasma gallisepticum, a significant respiratory and reproductive pathogen of domestic poultry, has since 1994 been recognized as an emergent pathogen of the American house finch (Carpodacus mexicanus). Epizootic spread and pathognomonic characteristics of house finch-associated Mycoplasma gallisepticum (HFMG) have been studied as a model of an emergent to endemic pathogen in a novel host. Here we present comparative analysis of eight HFMG genomes, including one from an index isolate and seven isolates separated spatially and temporally (1994-2008) across the epizootic, and notably having differences in virulence. HFMG represented a monophyletic clade relative to sequenced poultry isolates, with genomic changes indicating a novel M. gallisepticum lineage and including unique deletions of coding sequence. Though most of the HFMG genome was highly conserved among isolates, genetic distances correlated with temporal-spatial distance from the index. The most dramatic genomic differences among HFMG involved phase-variable and immunodominant VlhA lipoprotein genes, including those variable in presence and genomic location. Other genomic differences included tandem copy number variation of a 5 kbp repeat, changes in and adjacent to the clustered regularly interspaced short palindromic repeats, and small-scale changes affecting coding potential and association of genes with virulence. Divergence of monophyletic isolates from similar time/space in the epizootic indicated local diversification of distinct HFMG sublineages. Overall, these data identify candidate virulence genes and reveal the importance of phase-variable lipoproteins during the evolution of M. gallisepticum during its emergence and dissemination in a novel host in nature, likely mediating an important role at the interface between pathogen virulence and host immunity.

Identification of lipoprotein MslA as a neoteric virulence factor of Mycoplasma gallisepticum.

Many lipoproteins are expressed on the surfaces of mycoplasmas, and some have been implicated as playing roles in pathogenesis. Family 2 lipoproteins of Mycoplasma pneumoniae have a conserved "mycoplasma lipoprotein X" central domain and a "mycoplasma lipoprotein 10" C-terminal domain and are differentially expressed in response to environmental conditions. Homologues of family 2 lipoproteins are Mycoplasma specific and include the lipoprotein of Mycoplasma gallisepticum, encoded by the MGA0674 gene. Comparative transcriptomic analysis of the M. gallisepticum live attenuated vaccine strain F and the virulent strain R(low), reported in this study, indicated that MGA0674 is one of several differentially expressed genes. The MGA0674-encoded lipoprotein is a proteolytically processed, immunogenic, TX-114 detergent-phase protein which appears to have antigenic divergence between field strains R(low) and S6. We examined the virulence of an R(low) Delta MGA0674 mutant (P1H9) in vivo and observed reduced recovery and attenuated virulence in the tracheas of experimentally infected chickens. The virulence of two additional R(low) Delta MGA0674 mutants, 2162 and 2204, was assessed in a second in vivo virulence experiment. These mutants exhibited partial to complete attenuation in vivo, but recovery was observed more frequently. Since only Mycoplasma species harbor homologues of MGA0674, the gene product has been renamed "Mycoplasma-specific lipoprotein A" (MslA). Collectively, these data indicate that MslA is an immunogenic lipoprotein exhibiting reduced expression in an attenuated strain and plays a role in M. gallisepticum virulence.

Comparative genomic analyses of attenuated strains of Mycoplasma gallisepticum.

Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage M. gallisepticum strain R (R(low)) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of R(low), R(high), indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R(low) isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum R(low). Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter- and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.

The influence of host tissue on M. gallisepticum vlhA gene expression.

Mycoplasma gallisepticum, a significant poultry pathogen, has evolved rapidly in its new passerine host since its first reported isolation from house finches in the US in 1994. In poultry, M. gallisepticum infects the upper respiratory tract, causing tracheal mucosal thickening and inflammation, in addition to inflammation of the reproductive tract. However, in house finches M. gallisepticum primarily causes inflammation of the conjunctiva. Given that different tissues are primarily affected by the same pathogen in different hosts, we have compared the early changes in gene expression of the phase-variable lipoproteins (vlhA) gene family of M. gallisepticum collected directly from target tissues in both hosts. Previous data have demonstrated that vlhA genes may be related to virulence, exhibiting changes in expression in a non-stochastic, temporal progression and we hypothesize that this may be influenced by differences in the target host tissue. If this is true, we would expect M. gallisepticum to display a different vlhA gene expression pattern in the chicken trachea compared to its expression pattern in house finch conjunctiva. Here we report significant differences in vlhA gene expression patterns between M. gallisepticum collected from chicken tracheas compared to those collected from house finch conjunctiva. While many of the predominant vlhA genes expressed in the input population showed an increase in expression in the chicken trachea at day one postinfection, those same vlhA genes decreased in expression in the house finch. These data suggest that discrete suites of vlhA genes may be involved in M. gallisepticum pathogenesis and tropism for unique tissues in two disparate avian hosts.

Inflammatory toxin from Mycoplasma bovis: isolation and characterization.

An inflammatory toxin was extracted from Mycoplasma bovis with 75 percent aqueous ethanol. The toxin is a complex polysaccharide composed of glucose, glucosamine or galactosamine, and a heptose, is heat-stable, devoid of protein and lipid, and has a molecular weight of 73,000. The holotoxin in the cell membrane is a glycoprotein; however, it is the polysaccharide portion that is toxic. This inflammatory toxin increases vascular permeability and is capable of activating complement. Infusion of 0.9 milligram of toxin into the bovine udder resulted in the characteristic eosinophilic mastitis produced by Mycoplasma bovine.

Rapid preclinical detection of sheeppox virus by a real-time PCR assay.

Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.

Sequence and transcriptional analysis of the genes encoding the class-II topoisomerase of Mycoplasma gallisepticum.

The gyrAB genes encoding the entire B and a portion of the A subunit of DNA gyrase (E.C. 5.99.1.3) from Mycoplasma gallisepticum (Mg), strain S6, were cloned and sequenced. These gyrAB genes are co-transcribed as a single, polycistronic mRNA transcript. The Mg gyrB appears unique among prokaryotic gyrB in its use of GUG as a start codon.

Antibody-mediated selection of a Mycoplasma gallisepticum phenotype expressing variable proteins.

A variant phenotype of Mycoplasma gallisepticum S6 was isolated from an in vitro antibody-culture system utilizing metabolism-inhibiting antibodies against the 64 kDa lipoprotein (LP64). M. gallisepticum populations grown in medium alone or medium containing normal rabbit serum maintained expression of the parental phenotype. This paper describes the identification of proteins which undergo variable expression. Several of these were integral membrane proteins, with estimated molecular masses of 91, 43, 41, 38, 37, and 18 kDa, which were expressed in the variant phenotype but not in the parental phenotype. Three proteins (LP64, p63 and p47) were expressed in the parental phenotype, but not in the variant phenotype. The data suggest that the interaction of specific immunoglobulins with target epitopes resulted in the selection of a subpopulation of organisms expressing an alternative array of membrane proteins which, lacking the target epitopes, was able to escape the metabolism-inhibiting effects of the specific antibodies.

Comparison of an infective avirulent and canine virulent Bordetella bronchiseptica.

Two Bordetella bronchiseptica isolates, S-55 and D-2, were evaluated in dogs for inducement of (i) infection, (ii) clinical bordetellosis, and (iii) histopathologic changes on tracheal and bronchiole tissues. Further, each isolate was characterized for variance in (i) toxicity for mice and (ii) intracellular proteins. Both S-55 and D-2 were detectable in test dog groups during the 26-day test period, although 545 times more D-2 was recovered than was S-55. In dogs inoculated with D-2, clinical infectious tracheobronchitis appeared in 4 days and continued for 22 days. Bordetellosis was not observed in dogs given S-55 or in noninoculated dogs. Tracheal and bronchiole tissues from dogs inoculated with the S-55 and D-2 isolates were microscopically examined for lesions. Dogs inoculated with S-55 did not have tracheal or bronchiole lesions. Lesions were not observed in noninoculated dogs. Dogs inoculated with D-2 had marked lesions in the tracheal and bronchiole tissues. The D-2 whole cells were an average 4.8 times as lethal as S-55 whole cells in mice (given intraperitoneal inoculation), whereas cell-free culture supernatants from S-55 and D-2 isolates were nontoxic. Cell-free sonicated extracts of S-55 and D-2 proved toxic to mice (intraperitoneal inoculation), but after the extracts were heated at 56 C for 30 minutes, both were nontoxic. Intracellular proteins of approximately 116,000 and 44,000 daltons were found in higher concentration in D-2 cells than in S-55 cells.

Mycoplasma sturni from blue jays and northern mockingbirds with conjunctivitis in Florida.

Northern mockingbirds (Mimus polyglottos) and blue jays (Cyanocitta cristata) in a Florida (USA) wildlife care facility developed clinical signs and gross lesions suggestive of the ongoing outbreak of Mycoplasma gallisepticum (MG) conjunctivitis in house finches (Carpodacus mexicanus) and American goldfinches (Carduelis tristis). Mycoplasmal organisms were cultured from conjunctival/corneal swabs of birds with sinusitis, conjunctivitis, and/or epiphora. All of the isolates tested were identified as Mycoplasma sturni by indirect immunofluorescence. Mycoplasma sturni as well as MG should be considered in the differential diagnosis of songbirds with conjunctivitis.

Mycoplasmal conjunctivitis in a European starling.

Bilateral conjunctivitis and episcleritis were identified in an adult European starling (Sturnus vulgaris). A novel mycoplasma species, Mycoplasma sturni, was isolated in pure culture from the conjunctiva of both eyes. The clinical presentation was similar to that of conjunctivitis in house finches (Carpodacus mexicanus) caused by Mycoplasma gallisepticum. However, the histologic lesions were distinct, by the presence of ulceration and by the absence of epithelial hyperplasia and lymphoplasmacytic infiltration [corrected]. Mycoplasma sturni ferments glucose, does not hemadsorb or hemagglutinate chicken erythrocytes, and grows rapidly at 37 C in comparison to other Mycoplasma spp. The role of M. sturni in conjunctivitis in other passerine species is presently unknown.

Comparative assessment of a metabolically attenuated Mycoplasma gallisepticum mutant as a live vaccine for the prevention of avian respiratory mycoplasmosis.

In a previous study, signature sequence mutagenesis (SSM) was used to identify a mutant with a disruption of the gene encoding the metabolic factor, dihydrolipoamide dehydrogenase, and that mutant was designated Mg 7. The current study assessed the safety, immunogenicity and efficacy of Mg 7 in comparison to two commercially available vaccines (ts-11 and F) as well as a laboratory vaccine strain, GT5. Intratracheal vaccination of chickens with all four attenuated mutants induced varying levels of protection against intratracheal challenge with virulent Mycoplasma gallisepticum strain R(low). Mg 7 vaccinated chickens rapidly cleared the challenge strain, had lower histopathologic tracheal lesion scores when compared to unvaccinated chickens, and mounted a strong humoral anti-M. gallisepticum-specific IgG response. The IgG levels increased 2- to 3-fold upon R(low) challenge. Mg 7 induced a greater level of protection against intratracheal R(low) challenge than that observed with the other three attenuated strains, as evidenced by a lower recovery of R(low) from tracheas and lower histopathologic lesion scores in tracheas and air sacs. Based on these findings, Mg 7 appears to have good potential as a safe and effective vaccine for the prevention of avian mycoplasmosis.

Sheeppox virus kelch-like gene SPPV-019 affects virus virulence.

Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, DeltaKLP, was derived from the pathogenic strain SPPV-SA. DeltaKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). DeltaKLP-infected cells exhibited a reduction in Ca(2+)-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, DeltaKLP was markedly attenuated, since all DeltaKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with DeltaKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep.