Quantitative detection of streptococcosis infection in dead samples of Nile Tilapia (Oreochromis niloticus).

AIMS: The aims of the study were to evaluate whether epidemic strains of streptococcosis infected tilapia can be isolated and identified from dead fish for epidemiological investigation. METHODS AND RESULTS: Firstly, tilapias were inoculated with a lethal dose (1 × 108  CFU per fish) of Streptococcus agalactiae and brain tissues were harvested for bacteriological examination and qPCR assay 3, 12, 24 and 48 h postdeath. Streptococcus agalactiae was the only dominant bacterium cultivated on the brain heart infusion (BHI) plate and the bacterial load was about 107  CFU per mg. Secondly, tilapia were killed via ice water shock and immersed either in an aquarium containing 2·27 × 104  CFU per ml S. agalactiae or in a pond with streptococcosis outbreak. Streptococcus agalactiae failed to grow on the BHI plate but were identified (<6 × 102  CFU per mg) via qPCR assay. Finally, an epidemiological investigation of streptococcosis was conducted in the main tilapia breeding areas of South China. A total of 387 tilapia samples were collected including 24 suspected healthy, 35 moribund and 328 dead fish. The achieved detection rates were 0, 100 and 94·82% via bacteriological examination, and 0, 100 and 98·78% via qPCR assay respectively. The concentration of S. agalactiae in brain tissues ranged between 105 and 107  CFU per mg. CONCLUSIONS: Streptococcus agalactiae can survive for 48 h in the brain of dead fish. Dead tilapia can be a useful alternative for epidemiological investigation when the diagnostic analysis of moribund fish is unavailable or impractical. SIGNIFICANCE AND IMPACT OF THE STUDY: This detection method expands the sampling range, reduces the difficulty of sample collection and improves efficiency. Consequently, this method provides an alternative for epidemiological investigation of tilapia streptococcosis.

Prevalence of transmitted drug resistance among HIV-1 treatment-naive patients in Beijing.

To optimise patients' outcomes and gain insight into transmitted drug resistance (TDR) among human immunodeficiency virus (HIV)-1 treatment-naive patients in Beijing, the prevalence of TDR was assessed. Demographic and clinical data of 1241 treatment-naive patients diagnosed between April 2014 and February 2015 were collected. TDR was defined using the Stanford University HIV drug resistance mutations database. The risk factors were evaluated by multi-logistic regression analysis. Among 932 successfully amplified cases, most were male (96.78%) and infected through men having sex with men (91.74%). Genotype were CRF01_AE (56.44%), B (20.60%), CRF07_BC (19.96%), C (1.61%) and other genotypes (1.39%). The overall prevalence of TDR was 6.12%. Most frequent mutations occurred in non-nucleoside reverse transcriptase inhibitors (NNRTIs) (3.11%), followed by protease inhibitors (PIs) (2.25%) and nucleoside reverse transcriptase inhibitors (NRTIs) (1.32%). Furthermore, HIV-1 genotype was associated with high risk of resistance, in which genotype C and other genotype may have higher risk for resistance. The prevalence among treatment-naive patients in Beijing was low. Resistance to NNRTIs was higher than with PIs or NRTIs. Continuous monitoring of regional levels of HIV-1 TDRs would contribute to improve treatment outcomes and prevent failures.

[Research on risk factors of short-term outcome in AIDS patients with pneumocystis pneumonia].

Objective: To investigate the prognostic risk factors of acquired immunodeficiency syndrome (AIDS) patients with pneumocystis pneumonia (PCP), and to establish risk models for predicting early outcome. Methods: The clinical data of 418 AIDS patients with PCP admitted to Department of Infectious Diseases, Beijing You'an Hospital, Capital Medical University from January 2008 to May 2016 were retrospectively analyzed.The patients were divided into death group and survival group according to clinical outcome during hospitalization.Data of the two groups were collected including general information and laboratory test results.Multivariate Logistic regression was used to analyze risk factors affecting prognosis of patients, establish prognostic models and evaluate predictive value of the model. Results: Of the 418 AIDS patients with PCP, 388 cases were male and 30 cases were female, aged from 5 to 82 years, mean age was (40±12) years.There were 82 patients in the death group and 336 patients in the survival group.Disease course, bacterial infection and alveolar-arterial oxygen pressure difference(P(A-a)O(2)), serum lactate dehydrogenase(LDH), white blood cell (WBC), neutrophil (N), alanine aminotransferase (AST), urea nitrogen (BUN) and serum potassium (K) were significantly higher in the death group than those in the survival group (all P<0.05), and arterial oxygen pressure (PaO(2)), blood oxygen saturation (SpO(2)), CD4(+) T lymphocyte count, lymphocyte (L) , hemoglobin (Hb), platelet (PLT), albumin (ALB), prealbumin (PALB), cholinesterase (CHE), cholesterol (CHO), serum chlorine (Cl) and serum sodium (Na) were significantly lower in the death group than those in the survival group (all P<0.05). Multivariate Logistic regression analysis showed that P(A-a)O(2, )ALB, LDH, N and CD4(+) T lymphocyte count were prognostic factors of AIDS complicated with PCP.Prognostic index=9.736+ 0.112×P(A-a)O(2)-0.719×ALB+ 0.006×LDH+ 0.355×N-0.021×CD4.ROC curve of the short-term prognostic model was 0.985 (95%CI 0.977-0.994), with P value 0.000, cut-off value 0.907, sensitivity 92.0% and specificity 98.8%.The mortality rate increased with the increase of equation value. Conclusions: P(A-a)O(2, )ALB, LDH, N and CD4(+) T lymphocyte count are independent risk factors to predict short-term prognosis in these patients.The short-term prognostic model based on independent risk factors is useful in guiding clinical treatment.

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 µL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.

Orange-spotted grouper Epinephelus coioides Tak1: molecular identification, expression analysis and functional study.

In this study, the complementary (c)DNA sequence encoding orange-spotted grouper Epinephelus coioides Tak1 (ectak1) was cloned, which has an open reading frame of 1728 bp that encodes 575 amino acids (aa). Sequence analysis indicated that Ectak1 contains two characteristic conserved domains, i.e. an N-terminal serine-threonine protein kinase catalytic domain (27-275 aa) and a C-terminal coiled-coil region (499-562 aa). Ectak1 shares high sequence identity with Tak1 from other fish species, especially those of Nile tilapia Oreochromis niloticus (96%) and zebra mbuna Maylandia zebra (96%). ectak1 transcripts were expressed broadly in all of the tissues tested, but ectak1 expression was reduced mainly in the local infection sites (skin and gill) after infection with Cryptocaryon irritans. Intracellular localization analysis showed that Ectak1 was distributed mainly in the cytoplasm. A luciferase reporter assay showed that Ectak1 significantly impaired the NF-κB activity induced by E. coioides Myd88 and Traf6. Overall, these results suggest that Ectak1 functions to reduce the activity of NF-κB induced by toll-like receptor (TLR) signal molecules in HEK-293T cells, and it might have an important role in host defences against parasitic infections.

Construction of a Streptococcus iniae sortase A mutant and evaluation of its potential as an attenuated modified live vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus iniae is a major Gram-positive aquatic pathogen, which causes invasive diseases in cultured fish worldwide. The identification of potential virulence determinants of streptococcal infections will help to understand and control this disease, but only a few have been confirmed in S. iniae. Sortase A (srtA) is the key enzyme that anchors pre-mature cell wall-attached proteins to peptidoglycan and it can affect the correct positioning of surface proteins, as well as the course of Gram-positive bacterial infection, thereby making it a potential target in the study of virulence factors and disease control. In this study, the 759 bp srtA gene was cloned from pathogenic S. iniae TBY-1 strain and the mutant strain TBY-1ΔsrtA was constructed via allelic exchange mutagenesis. We found that srtA shares high similarities with sortase A from other Streptococcus spp. Direct survival rate assay and challenge experiments were performed, which showed that the mutant strain TBY-1ΔsrtA had a lower survival capacity in healthy tilapia blood and it was less virulent than the wild type strain in tilapia, thereby indicating that the deletion of sortase A affects the virulence and infectious capacity of S. iniae. The mutant strain TBY-1ΔsrtA was used as a live vaccine, which was administered via intraperitoneal injection, and it provided the relative percent survival value of 95.5% in Nile tilapia, thereby demonstrating its high potential as an effective attenuated live vaccine candidate.

A novel iron transporter in Streptococcus iniae.

Streptococcus iniae is a major pathogen that results in considerable economic loss to fish farms. Restricted availability of iron is a huge obstacle to survival for pathogenic bacteria during infection, and iron acquisition is important in bacterial virulence. In this study, S. iniae HD-1 was shown not to produce siderophores (low-molecular-weight compounds) but rather to require iron-containing proteins for growth under iron-restricted conditions. The adenosine triphosphate (ATP)-binding-cassette (ABC) transporter system (ftsABCD), which is cotranscribed by four downstream genes, namely, ftsA, ftsB, ftsC and ftsD, was identified as responsible for haem utilization of S. iniae. Analysis of the corresponding recombinant protein, FtsB, indicated that it is a putative lipoprotein which plays a role in haem utilization and is produced in vivo during infection with S. iniae HD-1, and therefore may be a potential candidate antigen for a streptococcal vaccine.

Immune-related genes expression profile in orange-spotted grouper during exposure to Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune-related genes in antiparasitic immune responses in fish, we monitored the expression change of IL-8, COX-2, C-type lectin and transferrin in local and systemic immune organs of orange-spotted grouper post-C. irritans infection. IL-8 expression was up-regulated during the course of infection in the skin, while COX-2 and transferrin expression was up-regulated in the gill. COX-2 expression was significantly down-regulated in the spleen (0·7-5% of its control) and head kidney (0·5-4% of its control) post-primary infection. Transferrin expression was also down-regulated in the spleen and head kidney from 6 h to 5 days post-primary infection. However, C-type lectin expression was up-regulated in all tested organs post-infection, with the exception of day 7 in the spleen post-primary infection where the expression level was slightly down-regulated (44% of its control). These results suggest that these four immune-related genes play an important role in grouper anti-C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

Rapid identification of Streptococcus iniae by specific PCR assay utilizing genetic markers in ITS rDNA.

The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae.

Carbapenemase-producing Enterobacterales in hospital drains in Southern Ontario, Canada.

BACKGROUND: Hospital drains may be an important reservoir for carbapenemase-producing Enterobacterales (CPE). AIM: To determine prevalence of CPE in hospital drains exposed to inpatients with CPE, relatedness of drain and patient CPE, and risk factors for drain contamination. METHODS: Sink and shower drains in patient rooms and communal shower rooms exposed to 310 inpatients with CPE colonization/infection were cultured at 10 hospitals. Using short- and long-read whole-genome sequencing, inpatient and corresponding drain CPE were compared. Risk factors for drain contamination were assessed using multi-level modelling. FINDINGS: Of 1209 exposed patient room and communal shower room drains, 53 (4%) yielded 62 CPE isolates in seven (70%) hospitals. Of 49 CPE isolates in patient room drains, four (8%) were linked to prior room occupants. Linked drain/room occupant pairs included Citrobacter freundii ST18 isolates separated by eight single nucleotide variants (SNVs), related blaKPC-containing IncN3-type plasmids (different species), related blaKPC-3-containing IncN-type plasmids (different species), and related blaOXA-48-containing IncL/M-type plasmids (different species). In one hospital, drain isolates from eight rooms on two units were Enterobacter hormaechei separated by 0-6 SNVs. Shower drains were more likely to be CPE-contaminated than hand hygiene (odds ratio: 3.45; 95% confidence interval: 1.66-7.16) or patient-use (13.0; 4.29-39.1) sink drains. Hand hygiene sink drains were more likely to be CPE-contaminated than patient-use sink drains (3.75; 1.17-12.0). CONCLUSION: Drain contamination was uncommon but widely dispersed. Drain CPE unrelated to patient exposure suggests contamination by undetected colonized patients or retrograde (drain-to-drain) contamination. Drain types had different contamination risks.

Virtual screening for finding natural inhibitor against cathepsin-L for SARS therapy.

Recently Simmons et al. reported a new mechanism for SARS virus entry into target cells, where MDL28170 was identified as an efficient inhibitor of CTSL-meditated substrate cleavage with IC(50) of 2.5 nmol/l. Based on the molecule fingerprint searching method, 11 natural molecules were found in the Traditional Chinese Medicines Database (TCMD). Molecular simulation indicates that the MOL376 (a compound derived from a Chinese medicine herb with the therapeutic efficacy on the human body such as relieving cough, removing the phlegm, and relieving asthma) has not only the highest binding energy with the receptor but also the good match in geometric conformation. It was observed through docking studies that the van der Waals interactions made substantial contributions to the affinity, and that the receptor active pocket was too large for MDL21870 but more suitable for MOL736. Accordingly, MOL736 might possibly become a promising lead compound for CTSL inhibition for SARS therapy.

Parasitic diseases of cultured marine finfishes and their surveillance in China.

More than 40 species of marine fishes are cultured in China and a wide variety of parasites are reported as lethal pathogens of these fishes in culture conditions. In the case of net cages, the culture facilities provides a good substrate for monogenean eggs to become entangled and the intensive aggregation of fishes facilitates the transmission of parasites between hosts. Relatively thorough studies on parasitic pathogens of marine fishes in China predominately concern the ciliate Cryptocaryon irritans and capsalid monogeneans (mainly Benedenia sp. and Neobenedenia sp.). Although nearly all such reports are related to treatment procedures, no single method has proved to be adequate for the effective control of these parasitic pathogens in marine cultured fishes. The National Fisheries Technology Extension Center (NFTEC) has established surveillance systems to monitor the diseases of aquaculture, including the parasitic diseases of maricultured fishes. The national monitoring stations for diseases of cultured marine fishes are distributed in the coastal counties or cities and provide remote in situ diagnoses of diseased fishes. International cooperation and effort are required for the control of parasitic diseases of marine finfish because of both the increasing international trade of eggs (seed) and larvae and commercial products in terms of live marine finfishes, which can readily result in the transmission of pathogens.

Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential.

OBJECTIVE: Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines. MATERIALS AND METHODS: MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. RESULTS: The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 µM. The lowest IC50 (0.32 µM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. CONCLUSIONS: We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.

Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia.

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Strain differences in the ontogeny of estrogen receptors in murine uterine epithelium.

The expression of estrogen receptor (ER) in the reproductive tracts of neonatal mice was examined using immunocytochemical and autoradiographic methods. Two strains of mice used in previous studies that reported contradictory results showed different rates of uterine epithelial development. In the inbred strain, BALB/c, the epithelium was devoid of receptor from birth through 5 days of age, while uterine epithelial cells of the outbred strain, CD-1, expressed ER as early as 3 days of age. Oviductal epithelium and cervical epithelium expressed ER on the day of birth in CD-1 mice. Glandular ontogeny in the uteri of CD-1 animals was also advanced by 3 days compared to that of BALB/c mice. These observations reconcile the conflicting reports of ER ontogeny in the neonatal mouse. More importantly, these results confirm our earlier observations, indicating that the cells lining uteri of 2- and 4-day-old BALB/c mice lack ER at a time when estrogen induces their proliferation.

Immunohistochemical study of HER-2/neu, epidermal growth factor receptor, and steroid receptor expression in normal and malignant endometrium.

HER-2/neu oncogene protein, epidermal growth factor receptor, progesterone receptor, and estrogen receptor were examined immunohistochemically in specimens of normal and neoplastic endometrium. Tissues obtained at the time of hysterectomy were snap-frozen at liquid nitrogen temperature and serially sectioned at 4 microns. Normal endometrial epithelial cells stained with anti-epidermal growth factor receptor and anti-HER-2/neu with intensities graded from 0 to 3+. Of the 49 endometrial malignancies studied, seven (14%) contained tissue exhibiting HER-2/neu staining in excess (4+) of any of the normal tissues or the other 42 cancer specimens. Expression of both HER-2/neu and steroid receptors was heterogeneous within these seven tumors. To examine this heterogeneity more closely, sections of these and other tumors were double-stained for HER-2/neu and progesterone receptor. It was found that the cells exhibiting 4+ HER-2/neu staining were progesterone receptor-negative. Conversely, cells that were progesterone receptor-positive within the same specimen exhibited HER-2/neu immunostaining equal to or less than 3+. All specimens containing 4+ HER-2/neu tissue were graded 1 or 2 adenocarcinomas, stage I. Thus, there is an inverse relationship between overexpression of HER-2/neu and progesterone receptor in endometrial cancer. On the other hand, overexpression of HER-2/neu in endometrial cancer does not seem to be related to loss of other differentiated characteristics. The prognostic value of these observations awaits continued study.

Some characteristics of host-parasite relationship for Cryptocaryon irritans isolated from South China.

A survey on the host range for the parasitic ciliate Cryptocaryon irritans was carried out among the major maricultured fish species in the Huizhou region of Guangdong Province in South China, and some characteristics of its host-parasite relationship were described. The survey showed that all ten investigated species of fish (representing six different families) were infected with C. irritans with similar susceptibility. In chemoattraction assays, sera and mucus collected from investigated fish strongly attracts C. irritans theronts. Sera collected from infected orange-spotted groupers and yellow spotted grunts (Plectorhynchus cinctus) could immobilize C. irritans theronts, and their immobilization titers were 1:40 and 1:6.7, respectively. The surface antigens of C. irritans were demonstrated by indirect immunofluorescence and immunostaining assays using immune orange-spotted grouper serum and a monoclonal antibody against grouper IgM.