High-throughput assay to phenotype Salmonella enterica Typhimurium association, invasion, and replication in macrophages.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

Spatial segregation of virulence gene expression during acute enteric infection with Salmonella enterica serovar Typhimurium.

UNLABELLED: To establish a replicative niche during its infectious cycle between the intestinal lumen and tissue, the enteric pathogen Salmonella enterica serovar Typhimurium requires numerous virulence genes, including genes for two type III secretion systems (T3SS) and their cognate effectors. To better understand the host-pathogen relationship, including early infection dynamics and induction kinetics of the bacterial virulence program in the context of a natural host, we monitored the subcellular localization and temporal expression of T3SS-1 and T3SS-2 using fluorescent single-cell reporters in a bovine, ligated ileal loop model of infection. We observed that the majority of bacteria at 2 h postinfection are flagellated, express T3SS-1 but not T3SS-2, and are associated with the epithelium or with extruding enterocytes. In epithelial cells, S. Typhimurium cells were surrounded by intact vacuolar membranes or present within membrane-compromised vacuoles that typically contained numerous vesicular structures. By 8 h postinfection, T3SS-2-expressing bacteria were detected in the lamina propria and in the underlying mucosa, while T3SS-1-expressing bacteria were in the lumen. Our work identifies for the first time the temporal and spatial regulation of T3SS-1 and -2 expression during an enteric infection in a natural host and provides further support for the concept of cytosolic S. Typhimurium in extruding epithelium as a mechanism for reseeding the lumen. IMPORTANCE: The pathogenic bacterium Salmonella enterica serovar Typhimurium invades and persists within host cells using distinct sets of virulence genes. Genes from Salmonella pathogenicity island 1 (SPI-1) are used to initiate contact and facilitate uptake into nonphagocytic host cells, while genes within SPI-2 allow the pathogen to colonize host cells. While many studies have identified bacterial virulence determinants in animal models of infection, very few have focused on virulence gene expression at the single-cell level during an in vivo infection. To better understand when and where bacterial virulence factors are expressed during an acute enteric infection of a natural host, we infected bovine jejunal-ileal loops with S. Typhimurium cells harboring fluorescent transcriptional reporters for SPI-1 and -2 (PinvF and PssaG, respectively). After a prescribed time of infection, tissue and luminal fluid were collected and analyzed by microscopy. During early infection (≤2 h), bacteria within both intact and compromised membrane-bound vacuoles were observed within the epithelium, with the majority expressing SPI-1. As the infection progressed, S. Typhimurium displayed differential expression of the SPI-1 and SPI-2 regulons, with the majority of tissue-associated bacteria expressing SPI-2 and the majority of lumen-associated bacteria expressing SPI-1. This underscores the finding that Salmonella virulence gene expression changes as the pathogen transitions from one anatomical location to the next.

Immunogenicity of a recombinant Rift Valley fever MP-12-NSm deletion vaccine candidate in calves.

The safety and immunogenicity of an authentic recombinant (ar) of the live, attenuated MP-12 Rift Valley fever (RVF) vaccine virus with a large deletion of the NSm gene in the pre-Gn region of the M RNA segment (arMP-12ΔNSm21/384) was tested in 4-6 month old Bos taurus calves. Phase I of this study evaluated the neutralizing antibody response, measured by 80% plaque reduction neutralization (PRNT80), and clinical response of calves to doses of 1 × 10(1) through 1 × 10(7) plaque forming units (PFU) administered subcutaneously (s.c.). Phase II evaluated the clinical and neutralizing antibody response of calves inoculated s.c. or intramuscularly (i.m.) with 1 × 10(3), 1 × 10(4) or 1 × 10(5)PFU of arMP-12ΔNSm21/384. No significant adverse clinical events were observed in the animals in these studies. Of all specimens tested, only one vaccine viral isolate was recovered and that virus retained the introduced deletion. In the Phase I study, there was no statistically significant difference in the PRNT80 response between the dosage groups though the difference in IgG response between the 1 × 10(1)PFU group and the 1 × 10(5)PFU group was statistically significant (p<0.05). The PRNT80 response of the respective dosage groups corresponded to dose of vaccine with the 1 × 10(1)PFU dose group showing the least response. The Phase II study also showed no statistically significant difference in PRNT80 response between the dosage groups though the difference in RVFV-specific IgG values was significantly increased (p<0.001) in animals inoculated i.m. with 1 × 10(4) or 1 × 10(5)PFU versus those inoculated s.c. with 1 × 10(3) or 1 × 10(5)PFU. Although the study groups were small, these data suggest that 1 × 10(4) or 1 × 10(5)PFU of arMP-12ΔNSm21/384 administered i.m. to calves will consistently stimulate a presumably protective PRNT80 response for at least 91 days post inoculation. Further studies of arMP-12ΔNSm21/384 are warranted to explore its suitability as an efficacious livestock vaccine.

Safety and immunogenicity of recombinant Rift Valley fever MP-12 vaccine candidates in sheep.

The safety and immunogenicity of two authentic recombinant (ar) Rift Valley fever (RVF) viruses, one with a deletion in the NSs region of the S RNA segment (arMP-12ΔNSs16/198) and the other with a large deletion of the NSm gene in the pre Gn region of the M RNA segment (arMP-12ΔNSm21/384) of the RVF MP-12 vaccine virus were tested in crossbred ewes at 30-50 days of gestation. First, we evaluated the neutralizing antibody response, measured by plaque reduction neutralization (PRNT(80)), and clinical response of the two viruses in groups of four ewes each. The virus dose was 1×10(5)plaque forming units (PFU). Control groups of four ewes each were also inoculated with a similar dose of RVF MP-12 or the parent recombinant virus (arMP-12). Neutralizing antibody was first detected in 3 of 4 animals inoculated with arMP-12ΔNSm21/384 on Day 5 post inoculation and all four animals had PRNT(80) titers of ≥1:20 on Day 6. Neutralizing antibody was first detected in 2 of 4 ewes inoculated with arMP-12ΔNSs16/198 on Day 7 and all had PRNT(80) titers of ≥1:20 on Day 10. We found the mean PRNT(80) response to arMP-12ΔNSs16/198 to be 16- to 25-fold lower than that of ewes inoculated with arMP-12ΔNSm21/384, arMP-12 or RVF MP-12. No abortions occurred though a single fetal death in each of the arMP-12 and RVF MP-12 groups was found at necropsy. The poor PRNT(80) response to arMP-12ΔNSs16/198 caused us to discontinue further testing of this candidate and focus on arMP-12ΔNSm21/384. A dose escalation study of arMP-12ΔNSm21/384 showed that 1×10(3)plaque forming units (PFU) stimulate a PRNT(80) response comparable to doses of up to 1×10(5)PFU of this virus. With further study, the arMP-12ΔNSm21/384 virus may prove to be a safe and efficacious candidate for a livestock vaccine. The large deletion in the NSm gene may also provide a negative marker that will allow serologic differentiation of naturally infected animals from vaccinated animals.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas.

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.

Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle.

BACKGROUND: Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle. RESULTS: Cattle were orally inoculated with either milk (control), milk with live attenuated Salmonella enterica serovar Dublin (vector), or milk with live attenuated recombinant S. Dublin expressing intimin (vaccinated) on days 0, 14 and 28. On day 98, all calves were challenged orally with E. coli O157:H7 to evaluate whether vaccination with the recombinant S. Dublin expressing intimin would reduce the level of E. coli O157:H7 fecal shedding.During the first 28 days, vaccinated calves shed both the vector strain and the intimin-expressing S. Dublin strain at a similar level. The vector strain was shed for a significantly longer period as compared to the level of recombinant vaccine strain. Calves that received the intimin-expressed vaccine ceased shedding S. Dublin from day 28 to day 63. All calves were challenged with E. coli O157:H7 on day 98 to determine the effect on fecal shedding of E. coli O157:H7. The amount of E. coli O157:H7 in feces was measured for 30 days post-challenge. We observed a transient clearance of E. coli O157:H7 from the feces in the vaccinated calves. The magnitude of fecal E. coli O157:H7 shedding did not correlate with the presence of intimin-specific fecal IgA. CONCLUSION: Oral vaccination with live attenuated recombinant S. Dublin expressing intimin reduced enteric colonization and fecal shedding of E. coli O157:H7. However, the transient clearance of E. coli O157:H7 was not associated with an enhanced IgA-mediated mucosal immune response.