猕猴结核分枝杆菌PCR检测方法的建立与初步应用

目的建立猕猴结核分枝杆菌PCR快速检测方法。方法根据GenBank中报道的人型结核分枝杆菌标准株H37RV全基因序列,针对其中致病性结核分枝杆菌所特有的保守区域ESAT-6设计一对引物进行TouchdownPCR反应,利用此方法对100份野生来源猕猴全血标本进行检测,并与传统的PPD实验和咽拭子抗酸染色实验结果作比对。结果抽取33份野生来源猕猴的外周血,提取DNA进行扩增,扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的序列基本相同,检测标本中有4份（4／33）为阳性,其中3份（3／33）标本与结核菌素试验和咽拭子抗酸染色试验结果相符合。结论建立了从猴全血中直接检测猴结核分枝杆菌DNA的TouchdownPCR方法,具有敏感性和特异性高,且简便的优点。     

Intestinal microbial patterns of the common marmoset and rhesus macaque

The intestinal microflora of common marmosets and rhesus monkeys were compared by enumerating bacteria from the small and large intestines. Rhesus monkeys had a consistent microflora pattern manifest by higher concentrations of total and Gram-negative aerobic and facultatively anaerobic bacteria, as well as aerobic and anaerobic Lactobacilli, in the large intestine as compared to the small intestine. In contrast, the marmoset microflora were considerably more variable. Approximately two-thirds of the marmosets (designated group A) had an overall profile that resembled the rhesus monkeys, but they had significantly higher concentrations of Gram-negative microflora in their large intestines than the rhesus monkeys. The remaining marmosets (group B) had higher concentrations of bacteria in the small intestine as compared to the large intestine, with the large intestinal concentrations being significantly lower than in the rhesus monkeys and group A marmosets. Moreover, the marmosets did not have detectable levels of aerobic Lactobacilli, and anaerobic Lactobacilli concentrations were significantly lower than in the rhesus macaques. Although it is unknown why microflora differ across species, it is likely that evolutionary adaptations in anatomy and functioning of the gastrointestinal tract influence the concentration and types of bacteria residing as the normal intestinal microflora.     

Hepatitis A and B: serologic survey of human and nonhuman primate sera

Sera of humans and seven species of nonhuman primates were tested by radioimmunoassay and enzyme immunoassay for the presence of hepatitis A antibody, hepatitis B surface antigen and antibody to hepatitis B surface antigen. The outcome of testing a total of 276 serum or plasma specimens was as follows: with the exception of squirrel monkeys (0%) and cotton-top marmosets (0%), a considerable percentage of all other species tested had detectable antibodies to hepatitis A virus: humans 45.9%, chimpanzees 36.6%, baboons 38.2%, vervets 57.9%, cebus monkeys 40.0% and common marmosets 50.0%. Only one human and two chimpanzees were carriers of hepatitis B surface antigen. Antibodies to hepatitis B surface antigen were detected in human (11.3%), chimpanzees (29.9%), baboons (36.2%) and squirrel monkeys (5%). Chimpanzees showed an increasing prevalence of antibodies to hepatitis A virus and hepatitis B surface antigen with age.     

Hepatic expression of cytochrome P450 enzymes in non‐human primate species

Cytochromes P450 (P450) largely remain to be characterized in great apes. Comparative immunochemical detection of drug metabolizing forms of P450s 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4A, and 4F in liver microsomes from chimpanzees, gorillas, orangutans, gibbons, cynomolgus and rhesus macaques, and common marmosets were carried out.     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

非人灵长类动物种质冷冻保存研究进展和展望

非人灵长类动物是生物多样性的重要组成部分,也是生物医学研究的珍贵实验动物,然而,由于人类活动、栖息地破坏、狩猎和遗传隔离等原因,许多非人灵长类动物的野生种群数量急剧下降,甚至处于灭绝的边缘。种质冷冻保存对拯救非人灵长类动物和保存遗传物质资源具有重要意义。本文综述了新大陆猴、旧大陆猴和巨猿等类群动物精子、卵子、胚胎和性腺组织等种质冷冻保存的研究进展,介绍了狨猴、松鼠猴、恒河猴、食蟹猴和黑猩猩等种质冷冻保存的主要方法,并对未来种质冷冻保存的研究方向进行了讨论。     

The importance of surface-based cues for face discrimination in non-human primates

Understanding how individual identity is processed from faces remains a complex problem. Contrast reversal, showing faces in photographic negative, impairs face recognition in humans and demonstrates the importance of surface-based information (shading and pigmentation) in face recognition. We tested the importance of contrast information for face encoding in chimpanzees and rhesus monkeys using a computerized face-matching task. Results showed that contrast reversal (positive to negative) selectively impaired face processing in these two species, although the impairment was greater for chimpanzees. Unlike chimpanzees, however, monkeys performed just as well matching negative to positive faces, suggesting that they retained some ability to extract identity information from negative faces. A control task showed that chimpanzees, but not rhesus monkeys, performed significantly better matching face parts compared with whole faces after a contrast reversal, suggesting that contrast reversal acts selectively on face processing, rather than general visual-processing mechanisms. These results confirm the importance of surface-based cues for face processing in chimpanzees and humans, while the results were less salient for rhesus monkeys. These findings make a significant contribution to understanding the evolution of cognitive specializations for face processing among primates, and suggest potential differences between monkeys and apes.     

Apolipoprotein A-I of primates

Monkey apoA-I was isolated by ultracentrifugation or immunoprecipitation and analyzed by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. The plasma apoA-I of 26 Old World monkeys (12 cynomolgus and 14 rhesus), 40 New World monkeys (8 cebus, 8 squirrel, 8 spider, 8 owl, and 8 marmosets), 6 prosimians (lemurs) and 10 apes (5 gibbons and 5 chimpanzees) were compared with each other as well as with human apoA-I. These analyses showed that monkey apoA-I contained one major and one to three minor (two basic and one acidic) isoproteins. The basic and acidic minor isoproteins differed by +2, +1, and -1 charges from the major apoA-I isoprotein designated apoA-I2. We have observed profound differences among the apoA-I electrophoretic patterns of the various primate species studied. The apparent isoelectric points of the major isoproteins of apoA-I of prosimians, Old World monkeys, New World monkeys, chimpanzees, gibbons, and humans were 5.70, 5.80, 5.35, 5.64, 5.42, and 5.64, respectively. The entire apoA-I isoprotein pattern of prosimians, Old World monkeys, chimpanzees, gibbons, and New World monkeys with respect to humans was shifted by approximately +1.5, +0.5, 0, -2.0, and -2.5 charges, respectively. The apoA-I synthesized by organ cultures of cynomolgus monkey intestine and liver overlaps on the two-dimensional system with the corresponding most basic minor plasma apoA-I isoprotein designated apoA-I2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nasal,Oral and Ear Swabs for Canine Visceral Leishmaniasis Diagnosis: New Practical Approaches for Detection of Leishmania infantum DNA

Background

The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil.

Methodology/Principal Findings

Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05).

Conclusions

Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.     

Video-task acquisition in rhesus monkeys (Macaca mulatta) and chimpanzees (Pan troglodytes): A comparative analysis

This study describes video-task acquisition in two nonhuman primate species. The subjects were seven rhesus monkeys (Macaca mulatta) and seven chimpanzees (Pan troglodytes). All subjects were trained to manipulate a joystick which controlled a cursor displayed on a computer monitor. Two criterion levels were used: one based on conceptual knowledge of the task and one based on motor performance. Chimpanzees and rhesus monkeys attained criterion in a comparable number of trials using a conceptually based criterion. However, using a criterion based on motor performance, chimpanzees reached criterion significantly faster than rhesus monkeys. Analysis of error patterns and latency indicated that the rhesus monkeys had a larger asymmetry in response bias and were significantly slower in responding than the chimpanzees. The results are discussed in terms of the relation between object manipulation skills and video-task acquisition.     

The mechanism of end-organ resistance to 1 alpha,25-dihydroxycholecalciferol in the common marmoset.

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Fetal and infant head circumference sexual dimorphism in primates

Studies have shown that after controlling for the effects of body size on brain size, the brains of adult humans, rhesus monkeys, and chimpanzees differ in relative size, where males have a greater volume of cerebral tissue than females. We assess whether head circumference sexual dimorphism is present during early development by evaluating sex differences in relative head circumference in living fetuses and infants within the first year of life. Head circumference is used as a proxy for brain size in the fetus and infant. Femur length is used as a proxy for body length in the fetus. Ultrasonography was used to obtain fetal measures, and anthropometry was used to obtain postnatal measures in humans, rhesus monkeys, baboons, and common marmosets. We show that statistically significant but low levels of head circumference sexual dimorphism are present in humans, rhesus monkeys, and baboons in early life. On average, males have head circumferences about 2% larger than females of comparable femur/body length in humans, rhesus monkeys, and baboons. No evidence for head circumference sexual dimorphism in the common marmoset was found. Dimorphism was present across all body size ranges. We suggest that head circumference sexual dimorphism emerges largely postnatally and increases throughout maturation, particularly in humans who reach adult dimorphism values greater than the monkeys. We suggest that brain dimorphism is not likely to impose an additional energetic burden to the gestating or lactating mother. Finally, some of the problems with ascribing functional significance to brain size sexual dimorphism are discussed, and the energetic implications for brain size sexual dimorphism in infancy are assessed.     

Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum

Non-human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non-invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20-32 days of gestation (term 165 +/- 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real-time PCR analysis of the rhesus Y-chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false-positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non-invasive method for routine gender screening.     

Characterization of a quinol peroxidase mutant in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.     

Adaptation of a strain of Plasmodium vivax from India to New World monkeys, chimpanzees, and anopheline mosquitoes.

A strain of Plasmodium vivax from India was adapted to develop in splenectomized Saimiri boliviensis, Aotus lemurinus griseimembra, A vociferans, A. nancymai, A. azarae boliviensis, hybrid Aotus monkeys, and splenectomized chimpanzees. Infections were induced via the inoculation of sporozoites dissected from the salivary glands of Anopheles stephensi and An. dirus mosquitoes to 12 Aotus and 8 Saimiri monkeys; transmission via the bites of infected An. stephensi was made to 1 Aotus monkey and 1 chimpanzee. The intravenous passage of infected erythrocytes was made to 9 Aotus monkeys and 4 chimpanzees. Gametocytes in 13 Aotus monkeys and 4 chimpanzees were infectious to mosquitoes. Infection rates were markedly higher in mosquitoes fed on chimpanzees. PCR studies on 10 monkeys injected with sporozoites revealed the presence of parasites before their detection by microscopic examination. The India VII strain of P. vivax develops in Aotus and Saimiri monkeys and chimpanzees following the injection of parasitized erythrocytes, or sporozoites, or both. The transmission rate via sporozoites to New World monkeys of approximately 50% may be too low for the testing of sporozoite vaccines or drugs directed against the exoerythrocytic stages. However, the strain is highly infectious to commonly available laboratory-maintained anopheline mosquitoes. Mosquito infection is especially high when feedings are made with gametocytes from splenectomized chimpanzees.     

Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A gamma-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in gamma-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.     

实验动物CAR杆菌16SrRNA基因PCR监测方法的建立

目的建立CAR杆菌的PCR监测方法 ,筛查国内部分实验动物样本中CAR杆菌携带状况。方法利用CAR杆菌的特有16SrRNA基因序列片段267bp设计引物,通过从日本实验动物中央研究所获取的CAR标准株DNA,建立实验动物CAR杆菌16SrRNA基因PCR监测方法。结果利用建立的CAR杆菌16SrRNA基因PCR监测方法对国内455份实验动物样本进行筛查,未检出CAR杆菌感染。结论建立了敏感性好,特异性高的实验动物CAR杆菌PCR监测方法 ,未见动物携带CAR杆菌。     

Rapid detection and quantification of five periodontopathic bacteria by real-time PCR

The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LightCycler system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from 10(3) to more than 10(8) cells (r2 = 0.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.     

Quantification in Soil and the Rhizosphere of the Nematophagous Fungus Verticillium chlamydosporium by Competitive PCR and Comparison with Selective Plating

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A γ-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in γ-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.