[Determination of methyl isobutyl ketone in urine by headspace coupled with gas chromatography-mass spectrometry].

Objective: To establish a method for the determination of methyl isobutyl ketone (MIBK) in urine samples by headspace-gas chromatography-mass spectrometry. Methods: Automatic headspace sampling technique was adopted to optimize the headspace conditions (headspace bottle heating temperature and equilibration time) and gas chromatographic conditions. A total of 5 ml samples were taken and added with 3.0 g ammonium sulfate into a 20 ml headspace bottle. After heated at 60 ℃ for 30 mins, gas from the upper part of headspace bottle was injected into gas chromatography with an injection volume of 100 µl. The target was separated by HP-5MS UI (30 m×0.25 mm×0.25 µm) capillary column and then detected by mass spectrometry detector. The retention time and external standard method were used for qualitative and quantitative analysis of MIBK in samples, respectively. Results: The standard curve of MIBK showed significant linearity between 20.0-1 000.0 µg/L. The standard curve was y=62.9x-652.5, and the correlation coefficient r=0.9998. The detection limit of MIBK was 5.0 µg/L and the quantification limit of MIBK was 16.0 µg/L. The average recovery rate was 95.3%~100.2% at three spiked concentrations of low (50.0 µg/L) , medium (200.0 µg/L) and high (500.0 µg/L) . The intra-day and inter-day precisions were 1.7%~3.8% (n=6) and 1.2%~4.0% (n=6) respectively. This method was stable for the determination of MIBK, and the urine could be kept 14 d at -20 ℃ without significantly loss. Conclusion: This method is proved to be simple, practical and highly sensitive. It can satisfy the request for the determination of urine samples of workers exposed to MIBK.

[Meta-analysis on the contents of trace elements in workers with occupational exposure to lead].

Objective: To quantitatively evaluate the content differences of trace elements in workers with occupational exposure to lead. Methods: In January 2021, relevant literatures on the contents of trace elements in workers with occupational exposure to lead published from 1990 to 2020 were searched through CNKI, Wanfang, VIP, PubMed, web of science and Embase. Screened and extracted the literatures, and evaluated the quality of the included literatures with Newcastle Ottawa Scale. Meta analysis was performed with Review Manager 5.3 software, and standardized mean difference (SMD) and its 95% confidence interval were used as effect indicators. Results: A total of 20 literatures were included, and the quality scores were 5-7. The results of Meta-analysis showed that compared with the control group, the contents of blood zinc (SMD=-1.01, 95%CI: -1.53, -0.49) , hair zinc (SMD=-0.17, 95%CI: -0.33, -0.01) , hair copper (SMD=-0.50, 95%CI: -1.01, 0) , hair iron (SMD=-3.91, 95%CI: -5.80, -2.03) and hair manganese (SMD=-1.09, 95%CI: -2.02, -0.15) in occupational lead exposure group were significantly lower (P<0.05) . Compared with the control group, the content of cobalt in hair of occupational lead exposure group (SMD=1.41, 95%CI: 0.72, 2.10) was higher, and the difference was statistically significant (P<0.05) . There was no significant difference in the contents of blood chromium, blood copper, blood iron, blood manganese, blood selenium and hair nickel between the two groups (P>0.05) . Conclusion: Workers with occupational exposure to lead have abnormal trace elements.

A case report of severe mycoplasma pneumonia with autoimmune haemolytic anaemia.

Mycoplasma pneumonia is a common cause of respiratory disease and more so in school going children. The spectrum of the manifestations range from haematological, dermatological, neurological, musculoskeletal, renal, cardiac and also gastrointestinal. The treatment approach has varied over time. In this report we would like to share our experience in a case of M.pneumonia with autoimmune haemolytic anaemia (AIHA).

[Determination of the butanone in urine by gas chromatography with precolumn derivation].

Objective: To establish a method for determination of the butanone in urine by gas chromatography (GC) with pre-column derivation. Methods: For detecting of butanone in urine, potassium iodide and potassium dichromate were added into urine under acidic condition, sample derivatization was undertaken in 50 ℃ water bath for 60 min and the iodine butanone was extracted with n-hexane. After the sodium thiosulfate solution was used to remove excess iodine, urine samples were centrifuged at 10000 r/min for 5 min, then the supernatant was analyzed using temperature rising programming with the Agilent Hp-5 column (30 m×0.32 mm, 0.25 µm) and electron capture detector (ECD) as the detector. The detector temperature was 300 ℃, the inlet temperature was 200 ℃, and the carrier gas was nitrogen. Results: For detecting of butanone in urine, potassium iodide and potassium dichromate were added for derivatization under the acidic condition. After extraction and centrifugation, the supernatant directly put through column and detected by ECD. In present study, the sample pretreatment condition was optimized, the relative standard deviations of intra-day and inner-day, the spiked samples and its recovery were evaluated for analyzing the accuracy of the proposed method. Conclusion: This method has proved to be simple, efficient and highly sensitivity, it can be utilized for butanone detection in occupational population.

Genetic effects of vascular endothelial growth factor and its receptor 2 on feather maturity in three chicken breeds.

1. The goal of the current study was to evaluate the genetic effects of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2) on feather maturity in the Qingyuan partridge chicken, Guangxi sanhuang chicken and Princess chicken. 2. Both SSCP-PCR and qPCR were employed to detect the polymorphism and gene expression of the VEGF and VEGFR-2 genes. 3. Four SNPs were identified in the VEGFR-2 gene. Exon10-A69G was associated with feather maturity (P < 0.01). Princess chickens with the genotype EF had higher feather maturity scores (P < 0.01). Higher expression levels of VEGF and VEGFR-2 were detected in the immature feather group of Qingyuan partridge chickens, especially in the skin. 4. The VEGF and VEGFR-2 genes play critical roles in feather maturity. In addition, exon10-A69G and genotype EF in the Princess chicken could potentially be utilised as genetic markers to improve efficiency in breeding.

[Applications of the Fe(3)O(4) nanocomposite modified by Humic Acid in determination of S-phenylmercapturic acid in urine by dispersive solid-phase extraction and HPLC].

Objective: To establish a method for determination the S-phenylmercapturic acid in urine by dispersive solid-phase extraction using Humic Acid/Fe(3)O(4) magnetic nanocomposite as adsorbent. Methods: The 5 ml of urine samples were adjusted to pH 1.0 and extracted by Fe3O4@HA. Then the analytes were separated on EC-C(18) capillary column and detected by HPLC-VWD. The S-phenylmercapturic acid was characterized by the retention time and quantified by peak area and external standard method. Results: The standard curves of SPMA showed significant linearity between 0.04~1.00 mg/L (r=0.999 7) . The average recovery was 94.2%~102.4%. The intra-day and inter-day precisions (RSD) were 2.9~6.7% (n=6) and 3.1~7.5% (n=6) respectively. The detect limit of SPMA was 0.012 g/L (S/N=3) . Conclusion: This method is simple, rapid, sensitive and accurate. It is applicable for determination of SPMA in the urine of works who were exposed to benzene.

The effects of different velogenic NDV infections on the chicken bursa of Fabricius.

BACKGROUND: Virulent Newcastle disease virus (NDV) was reported to cause rapid depletion of chicken bursa of Fabricius. Severe pathological condition of the organ is commonly associated with high levels of virus replication, intense inflammatory response and also the degree of apoptosis. In this study, the responses of chicken bursa of Fabricius infected with two different strains of velogenic NDV, namely AF2240 and IBS002, were investigated by observing cell population changes, oxidative stress, viral replication and cytokine expression in the organ. Subsequently, apoptosis of enriched bursal IgM+ cells was determined to help us elucidate possible host pathogen relationships between the chicken bursa of Fabricius and NDV infection. RESULTS: The depletion of IgM+ cells and infiltration of macrophages were observed to be higher in bursa infected with AF2240 as compared to IBS002. In line with the increment of the macrophage population, higher nitric oxide (NO) and malondialdehyde (MDA) contents which indicated higher oxidative stress were also detected in bursa infected with NDV AF2240. In addition, higher pro-inflammatory cytokines and chemokine gene expression such as chicken CXCLi2, IL-18 and IFN-γ were observed in AF2240 infected bursa. Depletion of IgM+ cells was further confirmed with increased cell death and apoptosis of the cells in AF2240 infected bursa as compared to IBS002. However, it was found that the viral load for NDV strain IBS002 was comparatively higher than AF2240 although the magnitude of the pro- inflammatory cytokines expression and cell apoptosis was lower than AF2240. CONCLUSION: The results of our study demonstrated that infection of NDV strains AF2240 and IBS002 caused apoptosis in bursa IgM+ cells and its severity was associated with increased expression of pro-inflammatory cytokines/chemokine, macrophage infiltration and oxidative stress as the infection duration was prolonged. However, of the two viruses, we observed that NDV AF2240 induced a greater magnitude of apoptosis in chicken bursa IgM+ cells in comparison to IBS002. This might be due to the high level of oxidative stress and inflammatory cytokines/chemokine as well as lower IL10 expression which subsequently led to a high rate of apoptosis in the chicken bursa of Fabricius although the detected viral load of AF2240 was lower than IBS002.

[Simultaneous determination of trichloroethylene and trichloroethanol in blood by liquid-liquid extraction-gas chromatography].

Objective: To establish a method for determing the trichloroethylene(TCE)and trichloroethanol(TCOH)in blood samples by liquid-liquid extraction-gas chromatography with electron capture detector. Methods: With this method,ether was used as extraction solvent and trichloromethane was used as an internal standard. The whole blood sample was extracted with ether, and dehydrated by anhydrous sodium sulfate. Then the analytes were separated on HP-5 capillary column(30m×0.32mm×0.15µm)and detected byECD.The retention time was for qualitative analysis and the internal standard was for quantitation. Results: The standard curves of TCE and TCOH showed significant linearity between 95.5µg/L-7640.0µg/L(r=0.9997)and 19.0µg/L-1520.0µg/L(r=0.9992). The average recovery was 95.5%-103.6%.The intra-day and inter-day precisions(RSD)were 2.5%-6.8%(n=6)and 1.6%-4.3%(n=6) respectively. The detect limit of TCE and TCOH were 2.10 µg/L and 0.56µg/L(S/N=3)respectively.The blood can be kept 7 days at-20℃ refrigerator without significantly loss. Conclusion: This method is proved to be simple,practical and highly sensitive. It can satisfy the request for the determination of blood samples of humans exposed to TCE.

In vitro characterization of chicken bone marrow-derived dendritic cells following infection with very virulent infectious bursal disease virus.

Infectious bursal disease is caused by infectious bursal disease virus (IBDV), an immunosuppressive virus that targets immune cells such as B cells and macrophages. However, the involvement of dendritic cells (DCs) during IBDV infection is not well understood. In this study the in vitro effects of live and inactivated very virulent IBDV (vvIBDV) UPM0081 on bone marrow-derived DCs (BM-DC) were characterized and compared with BM-DC treated with lipopolysaccharide (LPS). Morphologically, BM-DC treated with LPS and vvIBDV showed stellate shape when compared to immature BM-DC. In addition, LPS-treated and both live and inactivated vvIBDV-infected BM-DC expressed high levels of double positive CD86 and major histocompatibility complex class II antigens (>20%). vvIBDV-infected BM-DC showed significantly higher numbers of apoptotic cells compared to LPS. Replication of vvIBDV was detected in the infected BM-DC as evidenced by the increased expression of VP3 and VP4 IBDV antigens based on flow cytometry, real-time polymerase chain reaction and immunofluorescence tests. Levels of different immune-related genes such as interleukin-1ß (IL-1ß), CXCLi2 (IL-8), IL-18, interferon gamma (IFN-γ, IL-12α, CCR7 and Toll-like receptor-3 (TLR3) were measured after LPS and vvIBDV treatments. However, marked differences were noticed in the onset and intensity of the gene expression between these two treatment groups. LPS was far more potent than live and inactivated vvIBDV in inducing the expression of IL-1ß, IL-18 and CCR7 while expression of Th1-like cytokines, IFN-γ and IL-12α were significantly increased in the live vvIBDV treatment group. Meanwhile, the expression of TLR3 was increased in live vvIBDV-infected BM-DC as compared to control. Inactivated vvIBDV-treated BM-DC failed to stimulate IFN-γ, IL-12α and TLR3 expressions. This study suggested that BM-DC may serve as another target cells during IBDV infection which require further confirmation via in vivo studies.

[Prevalence of medicare antiviral drugs use and related factors in HIV-infected people in Ningbo].

Objective: To analyze the use of medicare antiviral drugs (ART) and related factors among HIV-infected people in Ningbo City. Methods: The retrospective data was collected related to infection and treatment of HIV-infected people in ART in Ningbo up to February 2023 through the National Infectious Disease Surveillance System. Binary logistic regression was used to analyze the factors related to medicare antiviral drug use in HIV-infected people. R 4.2.2 software was used for statistical analysis. Results: A total of 6 433 HIV-infected people with ART records were collected, among which 5 783 were in ART. The prevalence of medicare drugs use among people in ART was 24.8% (1 435/5 783, 95%CI: 23.7%-25.9%). Beilun District (8.7%, 43/497) and Fenghua District (5.7%, 14/247) had the lowest level in medicare drugs use. Among people in ART using medicare or out-of-pocket drugs, the prevalence of those who had at least one viral load test in the last year (84.9%, 1 352/1 593) was significantly lower than that of those using free drugs (91.4%, 3 829/4 190) (χ2=52.50, P<0.001). The results of multivariate logistic analysis showed that the factors influencing medicare drug use included low educational level (junior high school and below: aOR=0.24, 95%CI:0.17-0.34), farmer or worker (farmer: aOR=0.60, 95%CI: 0.39-0.91; worker: aOR=0.42, 95%CI: 0.27-0.64), low monthly income (<3 000 Yuan: aOR=0.29, 95%CI: 0.18-0.45), the longer interval time between diagnosis and treatment (≥21 days: aOR=0.47, 95%CI: 0.30-0.74). Conclusions: Significant regional differences on the prevalence of medicare antiviral drugs use in HIV-infected people exist in Ningbo City. Follow-up management program of patients should be improved to strengthen patient compliance to mobilize medicare drug promotion. Meanwhile, publicity of medicare drugs should be strengthened for farmers or workers with low education level and patients with delayed treatment.

[HIV infection and related factors of traceability efficiency among sex partners of HIV positive men who have sex with men].

Objective: To investigate the situation related to HIV infection and influencing factors of traceability efficiency among sex partners of HIV positive men who have sex with men (MSM). Methods: A cross-sectional survey was conducted to investigate the traceability among sex partners of HIV-positive MSM in Ningbo from 2018 to 2020. Limiting-antigen avidity enzyme immunoassay determined recent HIV infection. The classified data was evaluated by chi-square test, and factors of traceability efficiency were analyzed by multivariate logistic regression. Results: A total of 374 newly confirmed HIV-positive MSM were recruited to participate in the HIV test in Ningbo from 2018 to 2020.HIV positive rate of sex partner was 15.7% (75/479,95%CI:12.4%-18.9%). HIV positive rates of sex partner of recent HIV infection MSM was 31.8% (21/66,95%CI:20.3%-43.4%). The proportion of newly confirmed HIV-positive sex partners of recent HIV infection MSM (76.2%) was higher than that of long-term HIV infection MSM (48.1%). The difference was statistically significant (P=0.028). Results from the multivariate logistic regression analysis showed that HIV traceability efficiency was higher in the following subpopulations as; HIV positive MSM who were 36-45 years old (compared with 18-25 years old, OR=3.973,95%CI:1.364-11.569), HIV active detection (compared with HIV passive detection, OR=1.896, 95%CI:1.083-3.319), recent HIV infection MSM (compared with long-term HIV infection MSM, OR=3.733, 95%CI:1.844-7.556). Conclusions: HIV positive rate among partners of HIV positive MSM was very high. The traceability efficiency, which was recent HIV infection MSM and HIV active detection, was high. It is suggested to strengthen the traceability and focus on the newly confirmed HIV-positive MSM in VCT clinics.

[Characteristics and influencing factors of newly HIV infection among newly confirmed HIV/AIDS cases in Ningbo city, 2017-2020].

Objective: To identify the characteristics and influencing factors of local HIV infection among newly confirmed cases in Ningbo from 2017 to 2020 to provide evidence for traceability investigations on critical cases and facilitate the detection procedures and reduce new HIV infection. Methods: From January 1, 2017, to December 31, 2020, the newly confirmed HIV/AIDS in Ningbo were recruited. An epidemiological questionnaire was used to collect relevant information, including demography, sexual behaviors, results of HIV antibody tests, and the route of HIV transmission. According to the HIV testing, history of risk behaviors, and the level of CD4+ lymphocytes after confirmation, the HIV infection was acquired in the previous year, or the place was in Ningbo. The EpiData 3.1 and SPSS 23.0 software were used for input, sorting database and statistical analysis. Results: A total of 2 044 HIV/AIDS on-site investigations were completed. The average age of the subjects was (40.6±15.3) years old, including 1 684 males (82.4%), 758 unmarrieds (37.1%), 1 072 (52.5%) registered as permanent residents in Ningbo, 1 253 (61.3%) with junior high school education or below, 979 (47.9%) lived in Ningbo for more than five years. The proportion of local, new HIV infections was 34.34% (702/2 044). Multivariate logistic analysis showed that the proportion of local newly HIV infection was higher among those who were confirmed in 2020 (compared with the 2017 confirmed cases, OR=1.422, 95%CI:1.092-1.851), whose occupations were students/teachers/cadres/retirees (compared to commercial service/catering/public place service personnel, OR=1.682, 95%CI: 1.307-2.165), meeting sex partners via male social software locally in the last year (compared with without using related dating software, OR=1.353, 95%CI: 1.073-1.706). Conclusions: The proportion of local HIV infection of newly confirmed HIV/AIDS was relatively high in Ningbo city from 2017 to 2020. Meeting gay sex partners through local male social software appeared a risk factor for local newly HIV infection. Traceability investigations and internet intervention should be carried out for MSM. While male social software should be focused on identifying and controlling the risk of local newly HIV infection.

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia.

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan-based real-time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L⁻¹) for 10 days and challenged with WSSV by feeding WSSV-positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post-infection. The TaqMan real-time PCR assay showed a highly dynamic detection limit that spanned over 6 log10 concentrations of DNA and high reproducibility (standard deviation 0.33-1.42) and small correlation of variability (CV) (1.89-3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L⁻¹ of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L⁻¹ of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L⁻¹ (TAN) had increased susceptibility to WSSV.

Comparative Pathogenicity of Malaysian QX-like and Variant Infectious Bronchitis Virus Strains in Chickens at Different Age of Exposure to the Viruses.

Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.

Molecular Characterization of QX-Like and Variant Infectious Bronchitis Virus Strains in Malaysia Based on Partial Genomic Sequences Comprising the S-3a/3b-E-M-Intergenic Region-5a/5b-N Gene Order.

Infectious bronchitis virus (IBV) is one of the major poultry pathogens of global importance. However, the prevalence of IBV strains in Malaysia is poorly characterized. The partial genomic sequences (6.8 kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of 11 Malaysian IBVs isolated in 2014 and 2015 were sequenced using next-generation sequencing technology. Phylogenetic and pairwise sequence comparison analysis showed that the isolated IBVs are divided into two groups. Group 1 (IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015) shared 90%-95% nucleotide and deduced amino acid similarities to the QX-like strain. Among these isolates, IBS142/2015 is the first IBV detected in Sarawak state located in East Malaysia (Borneo Island). Meanwhile, IBV isolates in Group 2 (IBS037A/2015, IBS037B/2015, IBS051/2015, and IBS180/2015) were 91.62% and 89.09% identical to Malaysian variant strain MH5365/95 (EU086600) at nucleotide and amino acid levels, respectively. In addition, all studied IBVs were distinctly separate from Massachusetts (70%-72% amino acid similarity) and European strains including 793/B, Italy-02, and D274 (68%-73% amino acid similarity). Viruses in Group 1 have the insertion of three amino acids at positions 23, 121, and 122 of the S1 protein and recombinant events detected at nucleotide position 4354-5864, with major parental sequence derived from QX-like (CK-CH-IBYZ-2011) and a minor parental sequence derived from Massachusetts vaccine strain (H120). This study demonstrated coexistence of the IBV Malaysian variant strain along with the QX-like strain in Malaysia.

ß-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis.

ß-Arrestins (ß-arrs) are regulators and mediators of G protein-coupled receptor signaling, and accumulating evidence suggests that they are functionally involved in inflammation and autoimmune diseases. However, the effect of ß-arrs is unclear in inflammatory bowel disease (IBD), and the role of ß-arr2 is unknown in ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study is to investigate whether ß-arr2 encourages inflammation-induced epithelial apoptosis through endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) in colitis. In the present study, the results showed that ß-arr2 was increased in specimens from patients with UC or CD. Furthermore, a ß-arr2 deficiency significantly repressed intestinal inflammation, ameliorated colitis, and alleviated mucosal apoptosis in mice. In addition, the targeted deletion of ß-arr2 depressed ER stress, inhibited PUMA, and downregulated PUMA-mediated mitochondrial apoptotic signaling in colitis. ß-Arr2, an important modulator of G protein-coupled receptor function, binds eIF2α to activate ER stress signaling. Furthermore, the knockdown of PUMA dramatically prevented ß-arr2-induced apoptosis via alleviating ER stress in vitro. The results suggest that ß-arr2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis and that ß-arr2 is a potential therapeutic target for colitis.

Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody.

Our previous studies have shown that monoclonal antibodies (MAbs) to Blastocystis hominis react mainly with carbohydrate epitopes, while 1 MAb (1D5) reacts specifically with a protein of 30.5 kDa. In the present study, 3 monoclonal antibodies (1D5, 1E7 and 4F7) were used in immunogold localization. 1E7 and 4F7 were found to react primarily with the surface coat, while 1D5 was plasma membrane-specific. In the presence of complement, only 1D5 exhibited a cytotoxic effect on B. hominis whereas 1E7 and 4F7 did not, suggesting that the surface coat of B. hominis could serve as an immunological barrier against host antibodies. Using a recently described agar plating method, only 1D5 exhibited significant (P < 0.01) complement-independent cytotoxicity to B. hominis, inhibiting colony growth at low concentrations. Parasites that had been exposed to 1D5 were morphologically smaller than those that were not exposed to this MAb. Colonies that grew in the presence of 1D5 were isolated and grown in liquid medium containing increasing amounts of the cytotoxic MAb. Two clones that grew well in liquid medium containing 1D5 were also able to develop into colonies in soft agar. This study has shown that the 30.5 kDa protein found on the plasma membrane of B. hominis is a functionally important protein and that not all cells within a certain population would be susceptible to the cytotoxic effects of 1D5. These findings suggest that a heterogenous population exists in continuously maintained cultures of B. hominis.

Production and characterization of murine monoclonal antibodies to Blastocystis hominis.

Several hybridomas producing antibodies detected by enzyme-linked immunosorbent assay (ELISA) were established by fusions of mouse myeloma P3.X63.Ag8.U1 with spleen cells from BALB/c mice immunized against an isolate of Blastocystis hominis. Five strongly positive hybrids (6B6, 1D5, 1E7, 4F7 and 4G11) were cloned and all were found to secrete IgM monoclonal antibodies. Four MAbs (6B6, 1E7, 4F7 and 4G11) reacted in immunoblots with a number of B. hominis antigens (mol. wt ranging from 25,000 to 220,000) which were likely to be repeating oligosaccharide epitopes located on glycoproteins, as indicated by pronase and periodate treatment. Another MAb (1D5) reacted with a single antigenic band (mol. wt 30,5000). Similar results were obtained in immunoblots using 4 other B. hominis isolates. Indirect fluorescent-antibody assay (IFA) using MAbs showed 3 patterns of reactivity. 1D5 showed patchy fluorescence, 4F7 showed peripheral fluorescence and 6B6, 1E7 and 4G11 showed bright diffuse fluorescence. These patterns were observed for all 5 human Blastocystis isolates. The MAbs exhibited some cross-reactivity with 2 reptilian Blastocystis isolates but not with Giardia intestinalis, Trichomonas vaginalis or Entamoeba histolytica.

Rapid simultaneous detection of two orchid viruses using LC- and/or MALDI-mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry are capable of providing molecular mass information on biological samples with high speed, accuracy and sensitivity. With mass spectrometry, identifying a virus based on the molecular weight of its coat protein is relatively simple and accurate. The technique can be applied to all viruses with known coat protein molecular weights. Using the LC/MS and/or MALDI, this paper describes rapid simultaneous detection of the two most prevalent orchid viruses, namely cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV). The coat protein molecular weights of CymMV and ORSV were detected accurately using an extract from 1 g of virus-infected Oncidium orchid flower. Because LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps, both techniques are ideal for rapid identification of viruses from a large number of samples. This is the first report on the application of LC/MS and/or MALDI for simultaneous detection of two plant viruses from an infected plant extract.

Mechanical transport of rotavirus by the legs and wings of Musca domestica (Diptera: Muscidae).

Factors affecting the mechanical transmission of rotavirus by the legs and wings of the housefly, Musca domestica L., were examined in a laboratory study. Rotavirus was picked up when houseflies walked on thin smears of clarified rotavirus suspensions. The addition of glycerol, which increased viscosity of the virus suspension, and particulate human feces slightly increased the proportion of flies contaminated with virus. However, the addition of glycerol greatly reduced the average number of virus particles picked up per fly, whereas feces greatly increased the number of particles. The proportion of flies with virus-contaminated legs, which transferred virus to > 1 contact surface, was increased by longer contact time with the surface and when the contact surface was agar instead of glass. Most virus particles were deposited on 1st contact with the surface. Most flies dislodged virus particles inoculated on the underside of their wings soon after the start of simulated flight. Our data indicated that the nature of the virus-suspending medium has a greater effect on the level of virus contamination than on the ability to become contaminated. The importance of walking as a mode of virus transport depends on the nature of the contact surface, the risk of the contaminated fly settling first on a surface likely to come into contact with humans, and fly numbers.