The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of Ϸ20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.antifungal ͉ beauvericin ͉ ketoconazole

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathological changes, including the deposition of amyloid-beta (Aβ) peptide. Aged monkeys have proven to be invaluable in the study of AD, as their brains naturally develop amyloid plaques similar to those in AD brains. However, spontaneous development of AD-like pathologies in aged monkeys is time-consuming, often taking several years. Here, we created an experimentally induced AD model in middle-aged (16-17 years) rhesus monkeys by intracranial injection of Aβ42 and thiorphan, an inhibitor of neprilysin that is responsible for Aβ clearance. The working memory capacity of the monkeys in a delayed-response task was little affected following the delivery of Aβ42 and thiorphan. However, the administration of Aβ42 and thiorphan resulted in a significant intracellular accumulation of Aβ in the neurons of the basal ganglia, the cortex, and the hippocampus, accompanied by neuronal atrophy and loss. Moreover, immunohistochemistry revealed a degeneration of choline acetyltransferase-positive cholinergic neurons and an increase of glial fibrillary acidic protein-positive astrocytes. In conclusion, our data demonstrate a primate model of AD generated by combined infusion of Aβ42 and thiorphan, which duplicates a subset of neuropathological changes in AD brains, thereby having implications in the elucidation of this disease.

In order to meet the demands of experimental minipigs for biomedical researches, we have aimed at cultivating grazing Chinese Wuzhishan (WZS) minipigs and trying to make them useful and affordable since the 1990s. After more than ten years of captive cultivation following sound management practices and a rigorous selection program for fertility and litter size, we established an outbred WZS minipigs colony with a core group (14 males and 30 females) and an expanding group (20 males and 40 females). In 2010–2013 periods, extensive background data of this colony were recorded and analyzed. This paper was written to provide pertinent information about outbred WZS minipigs for producers, users, and others concerned with WZS minipigs. It contains physical characteristics, growth performance, productive performance, hematology and blood biochemistry, microsatellite analysis, organ coefficients, and carcass properties. Results show that WZS minipigs have characteristics of small body size, slow growth rate, long life cycle, high reproductive rate, and maintaining original genetic diversity. All data present that outbred WZS minipigs are suitable laboratory animal and model animal.

Tuberculosis (TB) in nonhuman primates is a serious menace to the welfare of the animals and human who come into contact with them, while the rapid, accurate, and robust diagnosis is challenging. In this study, we first sought to establish an appropriate primate TB model resembling natural TB in nonhuman primates. Four rhesus monkeys (Macaca mulatta) of Chinese origin were infected intratracheally with two low doses of M. tuberculosis H37Rv. Regardless of the infectious doses, all monkeys were demonstrated to be successfully infected by clinical assessments, tuberculin skin test conversions, peripheral immune responses, gross observations, histopathology analysis, and M. tuberculosis burdens. Furthermore, we extended the usefulness of this model for assessing the following immunodiagnostic antigens: CFP10, ESAT-6, CFP10-ESAT-6, and an antigen cocktail of CFP10 and ESAT-6. The data showed that CFP10 was an M. tuberculosis-specific, “early” antigen used for serodiagnosis of TB in nonhuman primates. In conclusion, we established a useful primate TB model depending on low doses of M .tuberculosis and affording new opportunities for studies of M. tuberculosis disease and diagnostics.

Numerous studies identify that IP-10 and IFN-γ are involved in leucocyte migration and activation and regarded as promising surrogate biomarkers in human and bovine tuberculosis infection, but there is lack of evidence for IP-10 in nonhuman primates. In this study, we directly determined IP-10 and IFN-γ levels in plasma from 30 healthy monkeys, 30 monkeys with naturally acquired tuberculosis, 4 monkeys experimentally infected with tuberculosis, and PPD stimulated whole blood of 14 monkeys with naturally acquired tuberculosis by ELISA. Higher plasma levels of IP-10 and IFN-γ were observed in natural tuberculosis monkeys than in healthy controls. The dynamic changes of plasma IP-10 and IFN-γ in experimental infections showed consistent representation of a transient increase during the infection period. After PPD stimulation, release of IP-10 and IFN-γ is significantly induced in natural tuberculosis monkeys, but the stimulation index of IP-10 was significantly lower than IFN-γ. Further analysis showed that positive correlation between IP-10 and IFN-γ existed in healthy and tuberculosis monkeys. Our findings support plasma IP-10 and IFN-γ as biomarkers for monitoring ongoing inflammation of nonhuman primate tuberculosis, and IFN-γ is a more valuable diagnostic biomarker.

Old tuberculin (OT) and purified protein derivative (PPD) are widely used for tuberculin skin testing (TST) in diagnosis of tuberculosis (TB) but often yield poor specificity and anergy in reaction. Therefore, it is necessary to develop new serological methods as a possible auxiliary diagnostic method for TB. In this study, we characterized the dynamic antibody responses of 10 purified recombinant antigens, PPD, and OT in rhesus monkeys experimentally infected with Mycobacterium tuberculosis and analyzed the time to antibody detection, antibody levels, and their association with the infectious doses. The antibodies were detected as early as 4 weeks after infection in response to 5 antigens (CFP10, CFP10-ESAT-6, U1, MPT64, and Ag85b). Antibodies against most of the other antigens were detected between 4 and 12 weeks after infection. The levels of antibodies were dose dependant. We further evaluated the serodiagnostic potential of these antigens by using indirect enzymelinked immunosorbent assay in 71 TST-positive and 90 TST-negative serum samples from monkeys. For all 12 antigens, the median optical density values of TST-positive monkeys were statistically significantly higher than those of TST-negative monkeys (P < 0.001). Among those antigens, Ag85b and CFP10 showed higher diagnostic potential than others. A combination of results from Ag85b, the 38-kDa antigen (Ag38kDa), and Ag14kDa reaches a sensitivity of 95.77%, indicating that these antigens may be ideal cocktails in TB diagnosis.Tuberculosis (TB) is a bacterial disease which causes serious health problems to both humans and nonhuman primates (NHPs). The zoonotic potential of TB and its potential transmission to laboratory NHPs are major concerns for researchers. Outbreaks of TB in laboratory monkey colonies are economically costly due to animal losses as well as increased expenses by disrupted research, lost time, and even delayed release of new products into the market. Though many strict control guidelines have been implemented, the absence of accurate diagnostic methods prevents effective TB control. Current TB diagnosis of NHPs largely depends on old tuberculin (OT) tuberculin skin testing (TST) and purified protein derivative (PPD) TST (OT-TST and PPD-TST, respectively), which have several serious limitations (7,18,23), including poor specificity, anergy in reaction, and intermittent positive results on repeated testing. A new method, which is based on detection of gamma interferon in whole blood (6, 22) has been developed for diagnosing TB in living NHPs. However, its sensitivity and application are still under evaluation.With the development of the cloning and expressing of M. tuberculosis-specific antigens, a serological test becomes an attractive diagnostic method for its convenience, robustness, and easy implementation, as well as for the absence of a requirement for living peripheral blood mononuclear cells (PBMCs). Several seroantigens, such as ESAT-6, CFP10, and the 38-kDa antigen (Ag38kDa), have been identified and included in many commercia...

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Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.