Moderate Susceptibility to Subcutaneous Plague (Yersinia pestis) Challenge in Vaccine-Treated and Untreated Sonoran Deer Mice (Peromyscus maniculatus sonoriensis) and Northern Grasshopper Mice (Onychomys leucogaster).

The variable response of wild mice to Yersinia pestis infection, the causative agent of plague, has generated much speculation concerning their role in the ecology of this potentially lethal disease. Researchers have questioned the means by which Y. pestis is maintained in nature and also sought methods for managing the disease. Here we assessed the efficacy of a new tool, the sylvatic plague vaccine (SPV), in wild-caught northern grasshopper mice (Onychomys leucogaster) and commercially acquired Sonoran deer mice (Peromyscus maniculatus sonoriensis). More than 40% of the animals survived a subcutaneous Y. pestis challenge of 175,000 colony forming units (over 30,000 times the white mouse 50% lethal dose) in both vaccine-treated and control groups. Our results indicate that SPV distribution is unlikely to protect adult mice from plague infection in field settings and corroborate the heterogeneous response to Y. pestis infection in mice reported by others.

S-Palmitoylation of junctophilin-2 is critical for its role in tethering the sarcoplasmic reticulum to the plasma membrane.

Junctophilins (JPH1-JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM). These ER/SR-PM junctions bring Ca-release channels in the ER/SR and Ca as well as Ca-activated K channels in the PM to within 10-25 nm. Such proximity is critical for excitation-contraction coupling in muscles, Ca modulation of excitability in neurons, and Ca homeostasis in nonexcitable cells. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD). Their N-terminal Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH-PM binding is not clear. We investigate whether S-palmitoylation of cysteine (Cys), a critical mechanism controlling peripheral protein binding to PM, occurs in JPHs. We focus on JPH2 that has four Cys residues: three flanking the MORN motifs and one in the TMD. Using palmitate-alkyne labeling, Cu(I)-catalyzed alkyne-azide cycloaddition reaction with azide-conjugated biotin, immunoblotting, proximity-ligation-amplification, and various imaging techniques, we show that JPH2 is S-palmitoylatable, and palmitoylation is essential for its ER/SR-PM tether function. Palmitoylated JPH2 binds to lipid-raft domains in PM, whereas palmitoylation of TMD-located Cys stabilizes JPH2's anchor in the ER/SR membrane. Binding to lipid-raft domains protects JPH2 from depalmitoylation. Unpalmitoylated JPH2 is largely excluded from lipid rafts and loses the ability to form stable ER/SR-PM junctions. In adult ventricular myocytes, native JPH2 is S-palmitoylatable, and palmitoylated JPH2 forms distinct PM puncta. Sequence alignment reveals that the palmitoylatable Cys residues in JPH2 are conserved in other JPHs, suggesting that palmitoylation may also enhance ER/SR-PM tethering by these proteins.

Age at Vaccination May Influence Response to Sylvatic Plague Vaccine (SPV) in Gunnison's Prairie Dogs (Cynomys gunnisoni).

Gunnison's prairie dogs (Cynomys gunnisoni) have been considered at greater risk from Yersinia pestis (plague) infection in the montane portion of their range compared to populations at lower elevations, possibly due to factors related to flea transmission of the bacteria or greater host susceptibility. To test the latter hypothesis and determine whether vaccination against plague with an oral sylvatic plague vaccine (SPV) improved survival, we captured prairie dogs from a C. g. gunnisoni or "montane" population and a C. g. zuniensis or "prairie" population for vaccine efficacy and challenge studies. No differences (P = 0.63) were found in plague susceptibility in non-vaccinated animals between these two populations; however, vaccinates from the prairie population survived plague challenge at significantly higher rates (P < 0.01) than those from the montane population. Upon further analysis, we determined that response to immunization was most likely associated with differences in age, as the prairie group was much younger on average than the montane group. Vaccinates that were juveniles or young adults survived plague challenge at a much higher rate than adults (P < 0.01 and P = 0.02, respectively), but no difference (P = 0.83) was detected in survival rates between control animals of different ages. These results suggest that host susceptibility is probably not related to the assumed greater risk from plague in the C. g. gunnisoni or "montane" populations of Gunnison's prairie dogs, and that SPV could be a useful plague management tool for this species, particularly if targeted at younger cohorts.

A waterborne outbreak of campylobacteriosis in the South Island of New Zealand due to a failure to implement a multi-barrier approach.

Outbreaks of waterborne gastroenteritis continue to occur in developed countries. Darfield, a rural town in the South Island of New Zealand experienced an outbreak of campylobacteriosis following a transgression of Escherichia coli on 16 August 2012. A descriptive outbreak investigation was performed. As a result, 29 cases had a laboratory-confirmed diagnosis of campylobacteriosis and 138 were identified as probable cases. Heavy rains, contamination of water with animal effluent from nearby paddocks and failures in the treatment of drinking water led to pathogens being distributed through the town's water supply. A multi-barrier approach is advocated to ensure the quality of water and many countries have legislation or programmes to address this. Although legislation for water safety plans based on a multi-barrier approach is in place in New Zealand, at the time of the outbreak it was not a requirement for the Darfield water supply. In addition, despite the awareness of the importance of a multi-barrier approach, competing interests, including those from the agricultural industry and financial restraints on water suppliers, can prevent it from being implemented. Governments need to be more willing to enforce legislation and standards to protect the public from waterborne disease.

The innate immune response may be important for surviving plague in wild Gunnison's prairie dogs.

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis, with ≥99% mortality reported from multiple studies of plague epizootics. A colony of Gunnison's prairie dogs (Cynomys gunnisoni) in the Aubrey Valley (AV) of northern Arizona appears to have survived several regional epizootics of plague, whereas nearby colonies have been severely affected by Y. pestis. To examine potential mechanisms accounting for survival in the AV colony, we conducted a laboratory Y. pestis challenge experiment on 60 wild-caught prairie dogs from AV and from a nearby, large colony with frequent past outbreaks of plague, Espee (n = 30 per colony). Test animals were challenged subcutaneously with the fully virulent Y. pestis strain CO92 at three doses: 50, 5,000, and 50,000 colony-forming units (cfu); this range is lethal in black-tailed prairie dogs (Cynomys ludovicianus). Contrary to our expectations, only 40% of the animals died. Although mortality trended higher in the Espee colony (50%) compared with AV (30%), the differences among infectious doses were not statistically significant. Only 39% of the survivors developed moderate to high antibody levels to Y. pestis, indicating that mechanisms other than humoral immunity are important in resistance to plague. The ratio of neutrophils to lymphocytes was not correlated with plague survival in this study. However, several immune proteins with roles in innate immunity (VCAM-1, CXCL-1, and vWF) were upregulated during plague infection and warrant further inquiry into their role for protection against this disease. These results suggest plague resistance exists in wild populations of the Gunnison's prairie dog and provide important directions for future studies.

Unmyelinated axons show selective rostrocaudal pathology in the corpus callosum after traumatic brain injury.

Axonal injury is consistently observed after traumatic brain injury (TBI). Prior research has extensively characterized the post-TBI response in myelinated axons. Despite evidence that unmyelinated axons comprise a numerical majority of cerebral axons, pathologic changes in unmyelinated axons after TBI have not been systematically studied. To identify morphologic correlates of functional impairment of unmyelinated fibers after TBI, we assessed ultrastructural changes in corpus callosum axons. Adult rats received moderate fluid percussion TBI, which produced diffuse injury with no contusion. Cross-sectional areas of 13,797 unmyelinated and 3,278 intact myelinated axons were stereologically measured at survival intervals from 3 hours to 15 days after injury. The mean caliber of unmyelinated axons was significantly reduced at 3 to 7 days and recovered by 15 days, but the time course of this shrinkage varied among the genu, mid callosum, and splenium. Relatively large unmyelinated axons seemed to be particularly vulnerable. Injury-induced decreases in unmyelinated fiber density were also observed, but they were more variable than caliber reductions. By contrast, no significant morphometric changes were observed in myelinated axons. The finding of a preferential vulnerability in unmyelinated axons has implications for current concepts of axonal responses after TBI and for development of specifically targeted therapies.

Resistance to plague among black-tailed prairie dog populations.

In some rodent species frequently exposed to plague outbreaks caused by Yersinia pestis, resistance to the disease has evolved as a population trait. As a first step in determining if plague resistance has developed in black-tailed prairie dogs (Cynomys ludovicianus), animals captured from colonies in a plague-free region (South Dakota) and two plague-endemic regions (Colorado and Texas) were challenged with Y. pestis at one of three doses (2.5, 250, or 2500 mouse LD50s). South Dakota prairie dogs were far more susceptible to plague than Colorado and Texas prairie dogs (p<0.001), with a mortality rate of nearly 100% over all doses. Colorado and Texas prairie dogs were quite similar in their response, with overall survival rates of 50% and 60%, respectively. Prairie dogs from these states were heterogeneous in their response, with some animals dying at the lowest dose (37% and 20%, respectively) and some surviving even at the highest dose (29% and 40%, respectively). Microsatellite analysis revealed that all three groups were distinct genetically, but further studies are needed to establish a genetic basis for the observed differences in plague resistance.

Consumption of baits containing raccoon pox-based plague vaccines protects black-tailed prairie dogs (Cynomys ludovicianus).

Baits containing recombinant raccoon poxvirus (RCN) expressing plague antigens (fraction 1 [F1] and a truncated form of the V protein-V307) were offered for voluntary consumption several times over the course of several months to a group of 16 black-tailed prairie dogs (Cynomys ludovicianus). For comparison, another group of prairie dogs (n = 12) was injected subcutaneously (SC) (prime and boost) with 40 microg of F1-V fusion protein absorbed to alum, a vaccine-adjuvant combination demonstrated to elicit immunity to plague in mice and other mammals. Control animals received baits containing RCN without the inserted antigen (n = 8) or injected diluent (n = 7), and as there was no difference in their survival rates by Kaplan-Meier analysis, all of them were combined into one group in the final analysis. Mean antibody titers to Yersinia pestis F1 and V antigen increased (p < 0.05) in the vaccinated groups compared to controls, but titers were significantly higher (p < 0.0001) in those receiving injections of F1-V fusion protein than in those orally vaccinated with RCN-based vaccine. Interestingly, upon challenge with approximately 70,000 cfu of virulent Y. pestis, oral vaccination resulted in survival rates that were significantly higher (p = 0.025) than the group vaccinated by injection with F1-V fusion protein and substantially higher (p < 0.0001) than the control group. These results demonstrate that oral vaccination of prairie dogs using RCN-based plague vaccines provides significant protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous flea bites.

Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis).

In previous studies, we demonstrated protection against plague in mice and prairie dogs using a raccoon pox (RCN) virus-vectored vaccine that expressed the F1 capsular antigen of Yersinia pestis. In order to improve vaccine efficacy, we have now constructed additional RCN-plague vaccines containing two different forms of the lcrV (V) gene, including full-length (Vfull) and a truncated form (V307). Mouse challenge studies with Y. pestis strain CO92 showed that vaccination with a combination of RCN-F1 and the truncated V construct (RCN-V307) provided the greatest improvement (P=0.01) in protection against plague over vaccination with RCN-F1 alone. This effect was mediated primarily by anti-F1 and anti-V antibodies and both contributed independently to increased survival of vaccinated mice.

Meeting the challanges in today's outpatient oncology setting: a case study.

Physicians and staff in outpatient oncology settings are feeling besieged as healthcare trends accelerate. One of these is to provide a greater number and variety of treatments on an outpatient basis. The second is the shift from indemnity insurance to a managed care environment to help control the costs. The challenge at the Center for Cancer Care at OSF Saint Anthony Medical Center in Rockford, Illinois, was to create processes that increased efficiency, dealt with expanded patient caseloads, improved the level of care provided and took into account the shortage of oncology nurses.