Genetic diversity of Chinese laboratory macaques based on 2b-RAD simplified genome sequencing.

BACKGROUND: Currently, Chinese laboratory macaques are widely used in biomedical research. Correspondingly, clarity regarding the genetic diversity of Chinese laboratory macaques is important for both vendors and users. METHODS: Genome sequences of 55 laboratory macaques (40 cynomolgus macaques and 15 rhesus macaques) bred in South China were analyzed using 2b-RAD simplified genome sequencing. RESULTS: A total of 115,681 single-nucleotide polymorphisms (SNPs) were found that were distributed in 21 chromosomes and an unplaced scaffold. Genetic diversity indices varied across populations and exhibited low values. The results of principal coordinate analysis (PCA) were consistent with those of the arithmetic mean (UPGMA) clustered tree and supported the structure analysis, demonstrating that the genetic differentiation in rhesus macaques was higher than that in cynomolgus macaques. Introgressive hybridization with the Chinese rhesus macaque was supported in more than 80% (32/40) of cynomolgus macaques. CONCLUSIONS: Chinese laboratory macaques had relatively low genetic diversity at the genomic level, and genetic differentiation in Chinese rhesus macaques was higher than in cynomolgus macaques. The genome of cynomolgus macaques has been shaped by introgression after hybridization with the Chinese rhesus macaques.

Development and evaluation of a Luminex xTAG assay for sulfonamide resistance genes in Escherichia coli and Salmonella isolates.

Clinically occurring sulfonamide resistance in gram-negative bacteria is codified by several sul genes, mostly associated with the mobilized genetic elements named integrons, and integrons are frequently found in plasmids. There are four sul genes (sul1, sul2, sul3 and sul4) that encode resistance to sulfonamides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of all four sul genes and related Class 1 integrons (int1) in Escherichia coli and Salmonella isolates. The limits of detection ranged from 10 to 1000 copies/µL of input purified plasmid DNA. Forty-one bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for sul genes than conventional PCR. It indicated that the xTAG-multiplex PCR is a convenient method for rapid identification of sul genes.

Isolation and characterization of Bordetella pseudohinzii in mice in China.

We report on the first detection and isolation of B. pseudohinzii (Bordetella pseudohinzii) in laboratory mice in China. Forty-one B. pseudohinzii strains were isolated from 3094 mice in 33 different laboratory animal facilities in southern China. The isolates were identified through culture and genome sequenceing. Phylogenetic analysis based on the sequences of 16S rRNA and OmpA genes demonstrated that these strains were on the same clade as other B. pseudohinzii strains isolated from mice. Experimental infected mice presented an asymptomatic infection. B. pseudohinzii replicated in both the respiratory tract and the digestive tract. Most importantly B. pseudohinzii shed via feces and infected a group of sentinel mice in a separate cage via cage padding contaminated with B. pseudohinzii-positive feces, indicating that B. pseudohinzii could transmit efficiently among mice and contaminate environmental facilities. Our study highlights the importance of routine monitoring of the pathogen in laboratory mice and provides vital insights into the transmission of Brodetellae in rodents and human.

In vitro responses of multiple cytokines to purified protein derivative in healthy and naturally Mycobacterium tuberculosis-infected rhesus monkeys (Macaca mulatta).

BACKGROUND: As the widely used biomarker of whole-blood stimulation assays for tuberculosis diagnosis, the release of IFN-γ might be affected by multiple factors, such as immunosuppression and some infectious agents. Here, we evaluated additional cytokines as diagnostic biomarkers. METHODS: Forty-three cytokines were measured by Luminex xMAP technologies in 30 healthy and 10 naturally Mycobacterium tuberculosis (MTB)-infected rhesus monkeys pre- and post-stimulation by purified protein derivative (PPD). RESULTS: After stimulation, production of 23 and 38 cytokines was markedly increased in healthy and MTB-infected macaques, respectively. A comparison of the stimulation index (SI) between MTB infections and healthy macaques showed that the SIs of 32 cytokines in MTB-infected macaques were significantly higher than those in healthy macaques. Pooling the results, eight cytokines were suggested as ideal biomarkers for a whole-blood stimulation assay for MTB diagnosis. CONCLUSION: PPD could induce multiple cytokine responses in either healthy or MTB-infected monkeys, and eight cytokines had reliable predictive capacity as diagnostic biomarkers of MTB infection.

Dynamics of Immune Responses during Experimental Mycobacterium kansasii Infection of Cynomolgus Monkeys (Macaca fascicularis).

To profile the dynamic changes of immune responses for M. kansasii infection, 3 cynomolgus monkeys were experimentally infected with M. kansasii by intratracheal inhalation of 1 × 106 CFU bacteria per monkey. Every 2 to 4 weeks, tuberculin skin testings (TSTs) were performed and blood samples were collected for immunoassay. Multiple cytokines in a single sample were measured by Luminex xMAP technologies. IgM and IgA were detected by double-antibody sandwich ELISA. IgG against PPD and 11 M. tuberculosis proteins were detected by using of indirect ELISA. At week 16, all animals were euthanized for necropsy and histological analysis. Positivities of TSTs emerged from week 2 to 6 postinfection. Leukocyte counts and T lymphocyte subsets experienced moderate increases. Among 44 kinds of cytokines, 36 kinds of them showed increases of different dynamic types and 8 kinds of them showed no specific changes. Total IgM and IgA showed a transient increase at an early infection stage. Positivities of M. tuberculosis specific IgM and IgA emerged as early as week 2 postinfection. All animals showed positive IgG against PPD and negative IgG responses to 38 kDa, MPT64L, TB16.3, 16 kDa, U1, and MTB81 antigens during the infection period. IgG against ESAT-6, CFP10, CFP10-ESAT-6, Ag85b, and 14 kDa antigens reached positive levels. The IgG avidities of PPD, ESAT-6, CFP10-ESAT-6, and Ag85b were all above 50 percent. In conclusion, the data indicate that M. kansasii infection in monkeys can induce positivities of TSTs, increases of multiple cytokines, and cross-reactive antibody responses to M. tuberculosis antigens.

Positive Correlation between IP-10 and IFN-γ Levels in Rhesus Monkeys (Macaca mulatta) with Either Naturally Acquired or Experimental Infection of Mycobacterium tuberculosis.

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.

Model-based evaluation of 5 Chinese medicine recipes in the treatment of H5N1 influenza virus infection in BALB/c mice.

OBJECTIVE: To investigate whether Chinese medicine (CM) recipes could ameliorate H5N1 influenza virus infection in BALB/c mice model. METHODS: BALB/c mice were orally administrated with 5 CM recipes (removing toxin, tonifying qi, cooling blood, laxation, and compound recipes), oseltamivir, or saline solution respectively for 5 consecutive days after the infection of H5N1 influenza virus. Series of indices were employed to evaluate the amelioration of the 5 CM recipes on infection, including clinical assessment, gross observation, histopathologic findings, cytokine levels and viral burden in the lungs. RESULTS: Two CM recipes (cooling blood and compound recipes) could postpone the death period of the mice infected with high-dose H5N1 influenza virus (P< 0.05). And for the mice infected with low-dose H5N1 influenza virus, CM recipes could significantly reduce the mortality and inhibit viral proliferation in the lungs as compared with the control group (P<0.05). There was no significant difference in lung coefficients between the treatment and the control groups, but histopathological findings in the lungs were improved in CM recipes groups compared to control group findings. A transient increase was observed in pro-inflammatory and anti-inflammatory cytokines during the first 6 days of infection. The levels of interleukin (IL)-12p40 and interferon-gamma of the treatment groups were significantly lower than that of the control group at day 3 post-infection (P<0.05), while only compound recipe were significantly lower in level of tumor necrosis factor alpha than the control (P<0.05). The level of IL-10 of the control was higher than others, and the differences between the control and cooling blood, removing toxin recipes were significant (P<0.05). CONCLUSIONS: Results of this study suggested the potentials of the CM recipes in ameliorating influenza virus infection by suppressing viral proliferations, improving histopathological lesions, and inhibition of over expression of inflammatory cytokines.

Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria.

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.

Systemic Candida parapsilosis Infection Model in Immunosuppressed ICR Mice and Assessing the Antifungal Efficiency of Fluconazole.

This study was to establish a systemic C. parapsilosis infection model in immunosuppressed ICR mice induced by cyclophosphamide and evaluate the antifungal efficiency of fluconazole. Three experiments were set to confirm the optimal infectious dose of C. parapsilosis, outcomes of infectious model, and antifungal efficiency of fluconazole in vivo, respectively. In the first experiment, comparisons of survival proportions between different infectious doses treated groups showed that the optimal inoculum for C. parapsilosis was 0.9 × 10(5) CFU per mouse. The following experiment was set to observe the outcomes of infection at a dose of 0.9 × 10(5) CFU C. parapsilosis. Postmortem and histopathological examinations presented fugal-specific lesions in multiorgans, especially in kidneys, characterized by inflammation, numerous microabscesses, and fungal infiltration. The CFU counts were consistent with the histopathological changes in tissues. Th1/Th2 cytokine imbalance was observed with increases of proinflammatory cytokines and no responses of anti-inflammatory cytokines in sera and kidneys. In the last experiment, model based evaluation of fluconazole indicated that there were ideal antifungal activities for fluconazole at dosages of 10-50 mg/kg/d. Data demonstrates that the research team has established a systemic C. parapsilosis infection model in immunosuppressed ICR mice, affording opportunities for increasing our understanding of fungal pathogenesis and treatment.

Establishment of a special pathogen free Chinese Wuzhishan Minipigs Colony.

To meet the increasing demands of specific pathogen free (SPF) minipigs in biomedical researches, 8 pregnant Chinese Wuzhishan minipigs (WZSP) sows with clear background were chosen to obtain SPF WZSP by hysterectomy. At 111 ± 2 days of the pregnancy, piglets were aseptically taken out from the sows and artificially suckled for 40 to 45 days in the positive isolators. Then, the piglets defined as F0 were transferred to barrier environment and fed with standard feeds. The original SPF colony was formed for breeding by selected piglets from F0 group of 6-8 months old. Biological characteristics of SPF WZSP were collected and further compared to those of conventional (CV) WZSP, including growth performance, reproductive performance, hematology and blood biochemistry, and major pathogens detection. As a result, 61 F0 piglets were obtained from 8 candidate sows, and 55 out of them survived. After strictly selection, 35 F0 piglets were used to form the original SPF colony, which produced 14 litters of SPF piglets defined as F1. Piglet survival rates, growth performance, and reproductive performance of SPF WZSP were similar to CV WZSP. Some hematology and blood biochemistry parameters showed significant differences between SPF and CV WZSP. Eighteen kinds of pathogens were identified to be free in F0 and F1 SPF colony by repeated pathogen detections. In conclusion, we established a satisfied SPF WZSP colony maintaining original characteristics, free of controlled diseases, and being proved to be a suitable laboratory animal.

Effects of repeated tuberculin skin testings on immune responses in experimental monkeys.

Though many alternative methods to tuberculin skin testing (TST) have been established and evaluated in recent years, sensitivities and specificities of most methods could not meet the requirements of golden standards. In this study, we sought to identify whether repeated TSTs could affect the immune responses in experimental monkeys. Nine natural tuberculosis (TB) monkeys receiving repeated TSTs biweekly were used to demonstrate the effect on TST responsiveness. Two healthy monkeys were administrated with repeated TSTs to analyze the immune response profiling. Intrapalpebral reactions in TB infections gradually weakened or presented intermittent positive reactions. The leukocyte counts, cytokine responses, and antibody responses to all antigens except Old tuberculin (OT) and MPT64L showed no specific changes for TB in healthy monkeys. Positive antibody responses to OT and MPT64L emerged during the first half experimental period, which may cause by their cross-reactivity with mycobacterial species. Results showed that repeated TSTs had no significant effects on immune responses in healthy monkeys but a progressive reduction in TST responsiveness in TB infections.

[Model index observations in SIVmac251-infected rhesus macaques].

In this study, five rhesus macaques were inoculated intravenously with SIVmac251 to establish a model of simian autoimmune deficiency syndrome (SAIDS). Peripheral blood samples were collected at different time points to monitor changes in the total T cell number and T lymphocyte subset. Plasma viral loads, cytokine expression levels and anti-SIV antibody levels were also assayed to acquire certain basic indexes to evaluate disease progression in the rhesus macaque SAIDS model. During the acute stage of infection, plasma viral loads reached a peak at week 1 post-inoculation and lasted for approximately 3 to 44 weeks. The CD3+ CD4+ T lymphocyte count in peripheral blood also transitorily decreased. During the same period, the level of interferon-gamma show an increasing trend, whereas IL-12 levels decreased; IL-2, IL-4, IL-10 and TNF-alpha were maintained at normal levels or could not be detected. During the asymptomatic and ARC phases, plasma viral loads persisted above 10(4) RNA copies/mL and either increased or declined during the later stages of disease; CD3+ CD4+ counts showed a steadily declining trend and the ratio of CD4 to CD8 decreased during late-stage disease. Moreover, antibodies against viral proteins were detected in the plasma and showed a significant increasing trend, while there were no apparently changes in the levels of IFN-gamma, IL-12, IL-2, IL-4, IL-10 and TNF-alpha. In conclusion, the characteristics of the SIV animal models in our study are similar to those of patients with AIDS. Therefore, the rhesus macaque SIVmac251 infection models can be applied for further studies into AIDS.

Biological Characteristics of Captive Chinese Wuzhishan Minipigs (Sus scrofa).

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.

Mycobacterium tuberculosis infection in rhesus monkeys (Macaca mulatta) and evaluation of ESAT-6 and CFP10 as immunodiagnostic antigens.

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.

Serum antibody responses to 10 Mycobacterium tuberculosis proteins, purified protein derivative, and old tuberculin in natural and experimental tuberculosis in rhesus monkeys.

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 enzyme-linked 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.

A nonhuman primate model of Alzheimer's disease generated by intracranial injection of amyloid-ß42 and thiorphan.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathological changes, including the deposition of amyloid-beta (Aß) 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 Aß42 and thiorphan, an inhibitor of neprilysin that is responsible for Aß clearance. The working memory capacity of the monkeys in a delayed-response task was little affected following the delivery of Aß42 and thiorphan. However, the administration of Aß42 and thiorphan resulted in a significant intracellular accumulation of Aß 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 Aß42 and thiorphan, which duplicates a subset of neuropathological changes in AD brains, thereby having implications in the elucidation of this disease.

[Observation on Rhesus monkeys infected second time with H5N1 avian influenza virus].

Rhesus monkeys with high specific H5N1 antibody were inoculated the second time with H5N1 virus, the result of the second time H5N1 inoculation and the effect of first time H5N1 inoculation on second inoculation was evaluated. Monkeys of NO. 3, NO. 4, NO. 5 were inoculated with H5N1 allantoic fluid and NO. 6 with noninfectious allantoic fluid by intratracheal thyrocricoid puncture. Three months later, NO. 4, NO. 5, NO. 6 monkeys were infected with 7 ml TCID50 10(4.875) H5N1 allantoic fluid and NO. 3 monkey with 7 ml noninfectious allantoic fluid at the same time by the same method. Clinical symptoms were recorded and antibody response was detected by ELISA. NO. 3, NO. 4, NO. 6 monkeys were killed after 72 h post infection and NO. 5 monkey was killed after 7 days post infection. Pathologic changes of the infected monkeys' lung were examined by HE staining,immunohistochemistry and the virus in lung was detected by RT-PCR. Results showed that NO. 3, NO. 4, NO. 5 monkeys still retained high level of specific antibody, H5N1 virus only could be detected in NO. 6 monkey's lung by immunohistochemistry and RT-PCR ,and the lung of NO. 6 monkey injured worst . It can be concluded that Rhesus monkeys inoculated with H5N1 avian influenza A virus at the first time could retain a high level of specific antibody in 90 days and the clinical symptom had almost recovered, the ability of Rhesus monkeys to resist second infection of H5N1 virus was enhanced notably at that moment.

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections.

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 approximately 20,000 microbial extracts, 12 hits were identified with broad-spectrum 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.