The Salmonella enterica serotype typhimurium effector proteins SipA,SopA, SopB,SopD, and SopE2 act in concert to induce diarrhea in calves

Salmonella enterica serotype Typhimurium requires a functional type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves. Salmonella serotype Typhimurium strains having mutations in sptP, avrA, sspH1, or slrP induced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wild type. In contrast, mutations in sipA, sopA, sopB, sopD, or sopE2 significantly reduced fluid accumulation in bovine ligated ileal loops at 8 h postinfection. A strain carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (sipA sopABDE2 mutant) caused the same level of fluid accumulation in bovine ligated ileal loops as a strain carrying a mutation in sipB, a SPI1 gene required for the translocation of effector proteins into host cells. A positive correlation was observed between the severity of histopathological lesions detected in the ileal mucosa and the levels of fluid accumulation induced by the different mutants. After oral infection of calves, the Salmonella serotype Typhimurium sipAsopABDE2 mutant caused only mild diarrhea and was more strongly attenuated than strains having only single mutations. These data demonstrate that SipA, SopA, SopB, SopD, and SopE2 are major virulence factors responsible for diarrhea during Salmonella serotype Typhimurium infection of calves.     

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.     

Analysis of the contribution of Salmonella pathogenicity islands 1 and 2 to enteric disease progression using a novel bovine ileal loop model and a murine model of infectious enterocolitis

We have developed a novel ileal loop model for use in calves to analyze the contribution of Salmonella enterica serovar Typhimurium type III secretion systems to disease processes in vivo. Our model involves constructing ileal loops with end-to-end anastamoses to restore the patency of the small intestine, thereby allowing experimental animals to convalesce following surgery for the desired number of days. This model overcomes the time constraint imposed by ligated ileal loop models that have precluded investigation of Salmonella virulence factors during later stages of the infection process. Here, we have used this model to examine the enteric disease process at 24 h and 5 days following infection with wild-type Salmonella and mutants lacking the virulence-associated Salmonella pathogenicity island 1 (SPI-1) or SPI-2 type III secretion systems. We show that SPI-2 mutants are dramatically attenuated at 5 days following infection and report a new phenotype for SPI-1 mutants, which induce intestinal pathology in calves similar to wild-type Salmonella in the 5-day ileal loop model. Both of these temporal phenotypes for SPI-1 and SPI-2 mutants were corroborated in a second animal model of enteric disease using streptomycin-pretreated mice. These data delineate novel phenotypes for SPI-1 and SPI-2 mutants in the intestinal phase of bovine and murine salmonellosis and provide working models to further investigate the effector contribution to these pathologies.     

Analysis of Salmonella enterica serotype-host specificity in calves: avirulence of S. enterica serotype gallinarum correlates with bacterial dissemination from mesenteric lymph nodes and persistence in vivo

Host and bacterial factors that determine whether Salmonella serotypes remain restricted to the gastrointestinal tract or penetrate beyond the mucosa and cause systemic disease remain largely undefined. Here, factors influencing Salmonella host specificity in calves were assessed by characterizing the pathogenesis of different serotypes. Salmonella enterica serotype Dublin was highly virulent intravenously, whereas S. enterica serotype Choleraesuis was moderately virulent. Both serotypes were virulent in calves infected orally. In contrast, S. enterica serotypes Gallinarum and Abortusovis were avirulent by either route. Serotypes Dublin, Gallinarum, and Abortusovis colonized the intestinal tract 24 h after oral inoculation, yet only serotype Dublin was consistently recovered from systemic tissues. Serotypes Dublin and Gallinarum invaded bovine intestines in greater numbers and induced greater enteropathogenic responses than serotypes Choleraesuis and Abortusovis. However, only serotype Dublin was able to persist within the intestinal mucosa, and use of a novel cannulation model demonstrated that serotype Dublin was able to pass through the mesenteric lymph nodes in greater numbers than serotype Gallinarum. Together, these results suggest that initial interactions with the intestinal mucosa do not correlate with host specificity, although persistence within tissues and translocation via efferent lymphatics appear to be crucial for the induction of bovine salmonellosis.     

Salmonella typhimurium Virulence Genes Are Induced upon Bacterial Invasion into Phagocytic and Nonphagocytic Cells

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.     

Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.     

Characterization of Salmonella-induced cell death in human macrophage-like THP-1 cells

Salmonella strains are facultative intracellular pathogens that produce marked cytopathology during infection of host cells. Different forms of cytopathic effects have been associated with the virulence systems encoded by the two Salmonella pathogenicity islands (SPI-1 and SPI-2) and the spv locus. We used Salmonella enterica serovar Dublin to investigate the induction of cytopathology during infection of the human macrophage-like cell line THP-1. Analysis of host cells by flow cytometry using a fluorescent terminal deoxynucleotidyltransferase dUTP-biotin nick end labeling (TUNEL) assay revealed that 70% of THP-1 cells showed DNA fragmentation after 4 h of infection, increasing to greater than 90% by 5.5 h. Moreover, the results showed that gentamicin-killed or chloramphenicol-treated bacteria did not induce DNA fragmentation. Serovar Dublin strains with mutations in SPI-1, SPI-2, or spvB induced these cytopathic effects similar to wild-type bacteria. In contrast, a mutation in the phoP regulatory gene abolished DNA fragmentation in the TUNEL assay. Caspase-3 activation was detected during Salmonella infection of THP-1 cells, but caspase-8 and caspase-9 activities were not found. However, inhibition of caspase-3 did not block Salmonella-induced DNA fragmentation. These results identify a previously undetected apoptotic effect in Salmonella-infected cells that is dependent on phoP gene function.     

Contribution of Salmonella typhimurium Virulence Factors to Diarrheal Disease in Calves

Limited knowledge is available about the virulence mechanisms responsible for diarrheal disease caused by Salmonella typhimurium. To assess the contribution to diarrheal disease of virulence determinants identified in models of infection, we tested a collection of S. typhimurium mutants for their ability to cause enteritis in calves. S. typhimurium strains carrying mutations in the virulence plasmid (spvR), Salmonella pathogenicity island 2 (SPI-2) (spiB), or SPI-5 (sopB) caused mortality and acute diarrhea in calves. An S. typhimurium rfaJ mutant, which is defective for lipopolysaccharide outer core biosynthesis, was of intermediate virulence. Mutations in SPI-1 (hilA and prgH) or aroA markedly reduced virulence and the severity of diarrhea. Furthermore, histopathological examination of calves infected with SPI-1 or aroA mutants revealed a marked reduction or absence of intestinal lesions. These data suggest that virulence factors, such as SPI-1, which are required during intestinal colonization are more important for pathogenicity in calves than are genes required during the systemic phase of S. typhimurium infection, including SPI-2 or the spv operon. This is in contrast to the degree of attenuation caused by these mutations in the mouse.     

Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle

Salmonella enterica serovar Dublin is a host-restricted serovar associated with typhoidal disease in cattle. In contrast, the fowl-associated serovar S. enterica serovar Gallinarum is avirulent in calves, yet it invades ileal mucosa and induces enteritis at levels comparable to those induced by S. enterica serovar Dublin. Suppression subtractive hybridization was employed to identify S. enterica serovar Dublin strain SD3246 genes absent from S. enterica serovar Gallinarum strain SG9. Forty-one S. enterica serovar Dublin fragments were cloned and sequenced. Among these, 24 mobile-element-associated genes were identified, and 12 clones exhibited similarity with sequences of known or predicted function in other serovars. Three S. enterica serovar Dublin-specific regions were homologous to regions from the genome of Enterobacter sp. strain 638. Sequencing of fragments adjacent to these three sequences revealed the presence of a 21-kb genomic island, designated S. enterica serovar Dublin island 1 (SDI-1). PCR analysis and Southern blotting showed that SDI-1 is highly conserved within S. enterica serovar Dublin isolates but rarely found in other serovars. To probe the role of genes identified by subtractive hybridization in vivo, 24 signature-tagged S. enterica serovar Dublin SD3246 mutants lacking loci not present in Salmonella serovar Gallinarum SG9 were created and screened by oral challenge of cattle. Though attenuation of tagged SG9 and SD3246 Salmonella pathogenicity island-1 (SPI-1) and SPI-2 mutant strains was detected, no obvious defects of these 24 mutants were detected. Subsequently, a ΔSDI-1 mutant was found to exhibit weak but significant attenuation compared with the parent strain in coinfection of calves. SDI-1 mutation did not impair invasion, intramacrophage survival, or virulence in mice, implying that SDI-1 does not influence fitness per se and may act in a host-specific manner.     

Flagella-mediated bacterial motility accelerates but is not required for Salmonella serotype Enteritidis invasion of differentiated Caco-2 cells

The relative contributions of the flagellum and the flagella-associated bacterial motility in the invasion of Caco-2 cells by Salmonella serotype Enteritidis were investigated using an fliC mutant defective in flagellin production and a motA mutant that carries flagella but is non-motile. Infection assays demonstrated that, at 1 h of infection, both the fliC and the motA mutants were severely impaired in bacterial invasion compared to the parental strain. Infection assays at 3 h infection demonstrated virtually equal invasion levels for both non-motile mutants and the parental strain. Together these data suggest that flagella-mediated bacterial motility accelerates the invasion of Salmonella but is not required for the invasion event per se.     

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.     

Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.     

Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens

Salmonella enterica serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between Salmonella serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by Salmonella serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of Salmonella serovar Gallinarum in the host.     

The Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 type III secretion systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken.

Salmonella enterica serovar Typhimurium infection of chickens is a major public and animal health problem. In young chicks, S. Typhimurium infection results in severe systemic infection; in older chicks, infection results in prolonged gastrointestinal tract colonization. Here we determined the role of the Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) type III secretion systems in systemic infection and gastrointestinal tract colonization of the chicken though experimental infection of chicks with a S. Typhimurium strain with mutations in the genes encoding the secretion system machinery of SPI-1 (spaS) and SPI-2 (ssaU) that prevent secretion of effector proteins. In 1-day-old chicks, mutation of SPI-2 lead to a decrease in both systemic bacterial numbers and pathology, although no difference in gastrointestinal numbers was observed. Mutation of SPI-1 had little effect in 1-day old chicks. In 1-week-old animals the SPI-2 mutants could not be detected systemically and colonized the gastrointestinal tract only in low numbers in comparison with the parent strain, and was cleared in 1 week. The SPI-1 mutant showed greatly reduced levels of systemic infection, and colonized the gastrointestinal tract at a lower level than the parent strain. The findings show that the SPI-2 type III secretion system is required for systemic S. Typhimurium infection in both infection models, and that it plays a significant role in gastrointestinal colonization. The SPI-1 system is involved in both systemic infection and gastrointestinal colonization, but does not appear absolutely essential for either infection process.     

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.     

In-vitro and in-vivo characteristics of TnphoA mutant strains of Salmonella serotype Gallinarum not invasive for tissue culture cells.

Insertion mutants of a strain of Salmonella serotype Gallinarum, the cause of fowl typhoid, were produced with transposon TnphoA. Eight mutants were identified as being less invasive in cell culture than the parent strain. Neither the parent strain nor the mutants showed mannose-sensitive haemagglutination of various red blood cell species. Although two mutants gave mannose-resistant (MR) haemagglutination of red cells of different animal species these MR activities could not be correlated with other characteristics in vitro or in vivo. The mutant strains were divisible into three classes by their patterns of invasiveness and adhesiveness in vitro and by changes in membrane proteins. Strains of classes 1 and 2 had single transposon insertions, detected by Southern hybridisation, of 8.9 and 2.4 kb EcoRV-digested chromosomal-fragments, respectively, and were slightly less invasive than the parent strain. They were no less virulent for chickens by the oral route than a mutant strain shown to be as invasive in vitro as the parent strain. The class-2 mutant was also less adhesive than the other strains. The single mutant strain of class 3 which contained insertions in several chromosomal fragments, was non-invasive in Vero cells (less than log10 1.0 cfu/ml recovered compared with log10 2.88 for the parent strain) and showed reduced virulence by both oral and intramuscular routes. The mutant strains of all classes were taken up equally rapidly from the blood by the spleen. Intramuscular immunisation of chickens with the class-3 mutant strain gave complete protection against oral challenge 3 weeks later with 1000 oral LD50 doses of the virulent parent strain.     

The Salmonella enterica serovar typhimurium translocated effectors SseJ and SifB are targeted to the Salmonella-containing vacuole

The Salmonella enterica serovar Typhimurium type III secretion system (TTSS) encoded in Salmonella pathogenicity island 2 (SPI-2) promotes replication within host cells and systemic infection of mice. The SPI-2 TTSS is expressed following Salmonella internalization into host cells and translocates effectors across the membrane of the Salmonella-containing vacuole (SCV). Two effectors with similar amino-terminal domains, SseJ and SifB, localize to the SCV membrane in infected HEp-2 cells and subsequently traffic away from the SCV along Salmonella-induced-filaments (Sifs). Following infection of RAW cells, SseJ and SifB localize to the SCV as well as LAMP-1-positive, vesicular-appearing structures distant from the SCV. Trafficking of SseJ and SifB away from the SCV requires the SPI-2 effector SifA. Deletion of sseJ, but not sifB, leads to attenuation of Salmonella replication in mice following intraperitoneal inoculation. The contribution of SseJ to in vivo replication is identical in wild-type and sifA deletion backgrounds, suggesting that SseJ trafficking away from the SCV along Sifs is unnecessary for its virulence function.     

The pathogenesis of wild type and drug resistant mutant strains of bovine herpesvirus-1 (BHV-1) in the natural host

Summary An experimental infection with bovine herpesvirus-1 was established in calves by means of intranasal inoculation. Three calves were infected with the parental strain BHV-1 w/t, three with the TK-defective strain, B 1 and four with the HPMPA-resistant strain, 3 A. Inoculation with w/t virus resulted in a reproducible clinical disease characterised by respiratory distress, fever and the presence of virus in nasal mucus. Following the acute infection, w/t-inoculated animals became seropositive for BHV-1 specific antibody. The TK-defective mutant (BHV-1 B 1) produced an acute infection similar to the parental virus in all three calves inoculated. The HPMPA-resistant mutant (BHV-1 3 A), however, showed a reduced pattern of infection and virus of lower titre was isolated from three of four calves; the antibody responses were generally lower, and one calf remained seronegative until reactivation. Following stimulation with dexamethasone 72 days after the primary inoculation, virus was re-isolated from all wild type-inoculated calves. In contrast, no evidence of reactivation was obtained from the three B 1-inoculated animals. However, all four animals inoculated with the mutant 3 A showed virus reactivation including the calf which had remained seronegative following primary virus inoculation. Previous studies have suggested that drug-resistance mutations in herpesviruses frequently are associated with reduced pathogenicity on the basis of experiments in laboratory models. The importance of the present study is the demonstration that two different drug-resistant variants of an alpha herpesvirus both have altered pathogenicity in the natural host for that infection. These results also have implications for the design and use of attenuated vaccine strains.     

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.