Vaccination With Detoxified Leukocidin AB Reduces Bacterial Load in a Staphylococcus aureus Minipig Deep Surgical Wound Infection Model.

Vaccines against Staphylococcus aureus have eluded researchers for >3 decades while the burden of staphylococcal diseases has increased. Early vaccine attempts mainly used rodents to characterize preclinical efficacy, and all subsequently failed in human clinical efficacy trials. More recently, leukocidin AB (LukAB) has gained interest as a vaccine antigen. We developed a minipig deep surgical wound infection model offering 3 independent efficacy readouts: bacterial load at the superficial and at the deep-seated surgical site, and dissemination of bacteria. Due to similarities with humans, minipigs are an attractive option to study novel vaccine candidates. With this model, we characterized the efficacy of a LukAB toxoid as vaccine candidate. Compared to control animals, a 3-log reduction of bacteria at the deep-seated surgical site was observed in LukAB-treated minipigs and dissemination of bacteria was dramatically reduced. Therefore, LukAB toxoids may be a useful addition to S. aureus vaccines and warrant further study.

Mineralized Trichobezoars in a Rhesus Macaque (Macaca mulatta).

A five-year-old female rhesus macaque (Macaca mulatta) presented with a thin body condition and multiple palpable mid-abdominal masses. Mineralized cecal trichobezoars were removed surgically. Thirteen months later, similar masses recurred and were confirmed with radiographs. This is the first case report of a mineralized cecal trichobezoar in a rhesus macaque.

Susceptibility to SIV Infection After Adenoviral Vaccination in a Low Dose Rhesus Macaque Challenge Model.

BACKGROUND: Vaccination with the Merck human adenovirus serotype-5 (HAdV-5) vectored HIV-1 subtype B gag/pol/nef vaccine was unexpectedly associated with enhanced susceptibility to HIV-1 infection in uncircumcised HAdV-5 seropositive men. It has been hypothesized that vaccination may have resulted in activated CD4+ T lymphocytes trafficking to mucosal sites thereby increasing targets for HIV infection. We have previously shown that AdV-vector vacci-nation in rhesus macaques resulted in an increase in the frequency of activated mucosal CD4+ T cells. However, whether this increase in activation is sufficient to increase susceptibility to HIV/SIV infection is unclear. METHODS: To examine this scenario, we developed a preliminary, proof-of-concept vaccination-challenge model in order to examine vaccine-induced SIV susceptibility in rhesus macaques. Rhesus macaques (n = 10/group) were vaccinated with a simian AdV-7 (SAdV-7)-vector encoding an irrelevant insert (SARS spike) and challenged 5 weeks post-prime in an escalating dosing regimen starting with sub-infectious doses (1:10,000 or 2TCID50) of SIVmac251. RESULTS: In contrast to our previous study, the SAdV-7 vaccine regimen did not induce detectable mucosal CD4+ T cell activation at the time points assessed in animals obtained from a different vendor and housed in a different facility. Within the power of the study, we did not observe significantly increased SIV acquisition in SAdV-7-vaccinated (5/10) versus placebo-vaccinated (3/10) macaques after repeated low-dose intra-rectal SIVmac251 challenge (P < 0.2). CONCLUSIONS: These results lay groundwork for future experiments to assess vaccine-induced SIV susceptibility in rhesus macaques. Further larger-scale studies are necessary to confirm the AdV-vector vaccination associated trend towards increased SIV/HIV acquisition and clarify associated mechanisms.

Using a Cageside Device for Testing Glycosylated Hemoglobin in Cynomolgus Macaques (Macaca fascicularis).

Nonhuman primates naturally develop type 2 diabetes mellitus and exhibit clinical features that are similar to those observed in humans, including obesity, insulin resistance, dyslipidemia, and pancreatic pathology. The glycosylated hemoglobin (HbA1C) test is the primary test used for diabetes management in humans because it reflects the average blood glucose levels over the previous 3 mo. The HbA1C results are a better predictor of potential risk of complications than are single or episodic measures of glucose levels. HbA1C levels have proven useful for the diagnosis and monitoring of blood glucose levels in NHP, but for testing by a commercial laboratory, the test requires a vial of whole blood, results are not available for several days, and the test is expensive. The cageside device requires a single drop of blood, it displays the HbA1C percentage in 5 min, and the cost per sample is less than for sending it to a commercial lab. We therefore assessed the correlation between a cageside test using a handheld unit and the commercial lab test for measuring HbA1C in cynomolgus macaques. From both normal and confirmed diabetic animals, 4 mL blood was collected from a peripheral vessel and sent to a commercial lab for HbA1C testing. At the same time, a drop of capillary blood was collected and tested immediately in the HbA1C cageside test. A comparison of the results revealed significant correlation between the cageside and commercial lab tests. Therefore, we feel that the HbA1C test using handheld device may help to rule out nondiabetics and indicate which animals require additional testing.

The HMW1 and HMW2 Adhesins Enhance the Ability of Nontypeable Haemophilus influenzae To Colonize the Upper Respiratory Tract of Rhesus Macaques.

Nontypeable Haemophilus influenzae (NTHi) initiates infection by colonizing the upper respiratory tract and is a common cause of localized respiratory tract disease. Previous work has established that the NTHi HMW1 and HMW2 proteins are potent adhesins that mediate efficient in vitro adherence to cultured human respiratory epithelial cells. In this study, we used a rhesus macaque model to assess the contributions of HMW1 and HMW2 to in vivo colonization. In experiments involving inoculation of individual isogenic derivatives of NTHi strain 12, the parent strain expressing both HMW1 and HMW2 and the mutant strains expressing either HMW1 or HMW2 were able to colonize more frequently than the double mutant strain lacking HMW1 and HMW2. In competition experiments, the parent strain efficiently outcompeted the double mutant lacking HMW1 and HMW2. Colonization with strains expressing HMW2 resulted in development of antibody against HMW2 in a number of the animals, demonstrating that colonization can stimulate an antibody response. In conclusion, we have established that the HMW1 and HMW2 adhesins play a major role in facilitating colonization of the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response.

Effect of electron radiation on vasomotor function of the left anterior descending coronary artery.

The left anterior descending (LAD, interventricular) coronary artery provides the blood supply to the mid-region of the heart and is a major site of vessel stenosis. Changes in LAD function can have major effects on heart function. In this report, we examined the effect of electron simulated solar particle event (eSPE) radiation on LAD function in a porcine animal model. Vasodilatory responses to adenosine diphosphate (ADP; 10(−9)­10(−4) M), bradykinin (BK; 10(−11)­10(−6) M), and sodium nitroprusside (SNP; 10(−10)­10(−4) M) were assessed. The LAD arteries from Control (non-irradiated) and the eSPE (irradiated) animals were isolated and exhibited a similar relaxation response following treatment with either ADP or SNP. In contrast, a significantly reduced relaxation response to BK treatment was observed in the eSPE irradiated group, compared to the control group. These data demonstrate that simulated SPE radiation exposure alters LAD function.

Ionizing radiation selectively reduces skin regulatory T cells and alters immune function.

The skin serves multiple functions that are critical for life. The protection from pathogens is achieved by a complicated interaction between aggressive effectors and controlling functions that limit damage. Inhomogeneous radiation with limited penetration is used in certain types of therapeutics and is experienced with exposure to solar particle events outside the protection of the Earth's magnetic field. This study explores the effect of ionizing radiation on skin immune function. We demonstrate that radiation, both homogeneous and inhomogeneous, induces inflammation with resultant specific loss of regulatory T cells from the skin. This results in a hyper-responsive state with increased delayed type hypersensitivity in vivo and CD4+ T cell proliferation in vitro. The effects of inhomogeneous radiation to the skin of astronauts or as part of a therapeutic approach could result in an unexpected enhancement in skin immune function. The effects of this need to be considered in the design of radiation therapy protocols and in the development of countermeasures for extended space travel.

Comparison of hindlimb unloading and partial weight suspension models for spaceflight-type condition induced effects on white blood cells.

Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems.

Acute biological effects of simulating the whole-body radiation dose distribution from a solar particle event using a porcine model.

In a solar particle event (SPE), an unshielded astronaut would receive proton radiation with an energy profile that produces a highly inhomogeneous dose distribution (skin receiving a greater dose than internal organs). The novel concept of using megavoltage electron-beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation has been described previously. Here, Yucatan minipigs were used to determine the effects of a superficial, SPE-like proton dose distribution using megavoltage electrons. In these experiments, dose-dependent increases in skin pigmentation, ulceration, keratinocyte necrosis and pigment incontinence were observed. Five of 18 animals (one each exposed to 7.5 Gy and 12.5 Gy radiation and three exposed to 25 Gy radiation) developed symptomatic, radiation-associated pneumonopathy approximately 90 days postirradiation. The three animals from the highest dose group showed evidence of mycoplasmal pneumonia along with radiation pneumonitis. Moreover, delayed-type hypersensitivity was found to be altered, suggesting that superficial irradiation of the skin with ionizing radiation might cause immune dysfunction or dysregulation. In conclusion, using total doses, patterns of dose distribution, and dose rates that are compatible with potential astronaut exposure to SPE radiation, animals experienced significant toxicities that were qualitatively different from toxicities previously reported in pigs for homogeneously delivered radiation at similar doses.

The combined effects of reduced weightbearing and ionizing radiation on splenic lymphocyte population and function.

PURPOSE: The effects of radiation +/- hypogravity on immunologic function were investigated using the Partial Weight Suspension (PWS) model ( Wagner et al. 2010 ). MATERIALS AND METHODS: Mice were exposed to 0.5, 1, or 2 Gray (Gy) dose of gamma radiation and then placed in the PWS system for 4, 24, 48 hours, or 4 days. Spleens were excised and white blood cells were prepared for flow cytometry analyses. RESULTS: The combination of PWS + radiation (1 and 2 Gy doses only) resulted in decreased cell viability at the 24 h (∼16% decrease), 48 h (∼20% decrease), and 4 day (∼20% decrease) time points, compared to the PWS (no radiation) and no treatment (non-suspended, non-irradiated) groups. The T lymphocyte (thymus-derived) population increased by ∼10% (24 h, 48 h, and 4 day time points), while the B lymphocyte (bursal or bone marrow-derived) population decreased by ∼10% (at all time points examined), when mice were exposed to PWS + radiation (2 Gy dose only), compared to the PWS or no treatment groups. T cell activation was observed in the PWS group and the 0.5 Gy +/- PWS groups at the 4 and 24 h time points, compared to the no treatment group. However, T cell activation was significantly suppressed (∼85%) at the acute time points in the 2 Gy +/- PWS groups, comparable to the no treatment group. CONCLUSIONS: Ionizing radiation in the absence and presence of simulated hypogravity results in acute lymphocyte dysfunction and compromised immune response.

Analysis of white blood cell counts in mice after gamma- or proton-radiation exposure.

In the coming decades human space exploration is expected to move beyond low-Earth orbit. This transition involves increasing mission time and therefore an increased risk of radiation exposure from solar particle event (SPE) radiation. Acute radiation effects after exposure to SPE radiation are of prime importance due to potential mission-threatening consequences. The major objective of this study was to characterize the dose-response relationship for proton and γ radiation delivered at doses up to 2 Gy at high (0.5 Gy/min) and low (0.5 Gy/h) dose rates using white blood cell (WBC) counts as a biological end point. The results demonstrate a dose-dependent decrease in WBC counts in mice exposed to high- and low-dose-rate proton and γ radiation, suggesting that astronauts exposed to SPE-like radiation may experience a significant decrease in circulating leukocytes.

Evaluation of an inhouse rapid ELISA test for detection of giardia in domestic sheep (Ovis aries).

Sheep (Ovis aries) are increasingly used at our institution as models of human disease. Within the research environment, routine husbandry and handling of sheep has potential for transmission of zoonotic agents, including Giardia. The prevalence of Giardia in sheep may approach 68%. Classic diagnostic testing involves microscopic examination for fecal cysts or trophozoites; however, limitations of microscopy include time, labor, and potential false-negative results due to intermittent shedding. We wished to determine whether a commercial rapid ELISA used for Giardia detection in dogs and cats could be used in sheep. Fecal samples collected from sheep (n = 93) were tested with a combination of 6 methods: reference laboratory fecal flotation, reference laboratory ELISA, inhouse fecal flotation, and commercially available tests (enzyme immunoassay, direct fluorescence antibody assay, and rapid ELISA). Prevalence of Giardia infection in facility sheep was 11.8% (11 of 93 animals). Of the 11 samples considered positive, 3 were confirmed by multiple testing methods, and 5 were positive by microscopy alone. Inhouse fecal flotation for 8 samples was positive on only 1 of 2 consecutive testing days. The rapid ELISA test exhibited 0% sensitivity for sheep giardiasis. Overall, the examined methods had low sensitivities and low positive predictive values. Despite limitations, microscopic analysis of repeat fecal samples remained the most accurate diagnostic method for ovine giardiasis among the methods tested.

Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio).

Despite the progressively increasing use of zebrafish (Danio rerio) in research, the most humane method of euthanasia for these fish has not been determined. Contemporary guidance documents state that hypothermia is an unacceptable method of euthanasia. The goal of this study was to compare rapid cooling and tricaine methanesulfonate (MS222) for zebrafish euthanasia. Zebrafish (n = 46) were euthanized by immersion in either an ice-water (4 degrees C or less) bath or unbuffered MS222 solution (250 mg/L; 25 to 30 degrees C). Another cohort (n = 10) was exposed to buffered MS222 to determine whether the acidity of the water alone caused distress. The times from exposure until the animals became unable to swim, right themselves, and death were measured, and signs of distress were recorded. Fish then were placed in a 'recovery tank' of system water to verify that recovery did not occur. Tissues were examined histologically. The mean time for euthanasia was longer and the number of fish exhibiting signs of distress was greater for fish exposed to MS222 than those exposed to chilled water. In addition, 4 of the 23 fish exposed to MS222 regained consciousness in the recovery tank, whereas none of 23 fish exposed to chilled water recovered. No histopathologic changes or evidence of ice crystal formation were seen in either group. In light of the faster time to death and fewer signs of distress in zebrafish euthanized by rapid cooling, we advocate this method as a humane veterinary practice.