Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/µL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

A Recombinase-Aided Amplification Assay for the Detection of Chlamydia felis.

Chlamydia felis is an important zoonotic agent for humans and various animals. A recombinase-aided amplification (RAA) assay was developed for detecting C. felis. RAA can be performed in a closed tube at 39°C within 30 min. The detection limit was 10.6 copies of the C. felis plasmid DNA per reaction. No positive signals for other pathogens were detected. The coincidence rate of RAA and conventional PCR was 95.24% (20/21) and 100% (96/96) for positive and negative samples, respectively. The established RAA assay is a simple, rapid, highly sensitive, and specific method for detecting C. felis.

DON entry into the nucleus induces DNA damage, apoptosis and cycle arrest in GES-1 cells.

Deoxynivalenol (DON) is a mycotoxin produced by the genus Fusarium and belongs to the trichothecenes group B compound. At present, the mechanism of DON toxicity to mammalian cells is not fully understood. Since the stomach is the first physiological barrier against food contaminants, it is also the first target of exposure to toxins. In this research, we investigated the toxic effects of DON on human gastric mucosal epithelial cells (GES-1) as a model. We found that DON significantly inhibited cell activity, but did not induce ROS production in GES-1 cells. Although DON was unable to induce ROS production, the intracellular "redox homeostasis" was altered. Additionally, DON induced mitochondrial membrane potential decrease but ATP levels increase. DON can induce DNA damage, which in turn regulates apoptosis by regulating mitochondrial permeability by regulating p53 and in turn the Bcl-2 protein family. Furthermore, DON can activate the ATM-chk2-cdc25C and ATM-p53 signaling pathways to induce G2-phase cycle arrest in GES-1 cells. Finally, DON is able to enter the nucleus by simple diffusion, but does not directly target mitochondria. In conclusion, DON is able to enter the nucleus and cause DNA damage, apoptosis and cycle arrest in GES-1 cells. These results provide evidence for DON induced cytotoxicity and gastric disease.

Estimation of the Rural Dog Population Within a Mega-City: An Example in Jiading District, Shanghai.

Rural dog populations have long been recognized to be inadequately managed in terms of disease control and prevention. In this study we consider dog management in rural Shanghai and its implications for rabies control in the entire metropolitan area of Shanghai. The prerequisite to improve rabies vaccination coverage in rural Shanghai depends on a proper enumeration of the total rural dog population. In this study we selected one of the nine administrative districts in Shanghai (Jiading), within which there are 7 towns and 2 industrial zones (township-level division) that contain agricultural areas. A total of 9 villages (rabies model villages) were chosen from each township-level division in Jiading, and an additional 3 non-model villages were also included in the study. A household questionnaire survey was implemented in all 12 villages recruited. In 3 of the model villages and the 3 non-model villages chosen as a comparison, two methods of enumeration-a sight-resight survey and a household census survey-were implemented. Results from the household survey in these 6 villages showed that among the total 1,560 owned dogs, 80.4% were Chinese Garden Dogs, 69.1% were aged 1 to 3 years, 49.2% were homebred, and 88.3% were kept for the purpose of guarding the house. However, only 3.7% of the owned dogs were desexed. There was a higher proportion of chained or confined dogs in model compared to non-model villages. The model villages had an absolute rabies vaccination coverage of 100% among its owned dog population and a smaller number of stray dogs. It was also identified that the two enumeration methods yielded similar counts (P = 0.12), particularly within smaller villages. From the questionnaire survey implemented within all 12 villages and based on the average human-to-dog ratio, the total rural dog population of Jiading district was estimated to be 24,058. This study generated information on the general demographics of the rural dog population in Jiading, and demonstrates an approach to the study of rural dog populations within the context of a megacity. In such a context, rural dog populations need to be considered as a critical component of animal and public health.

Seroprevalence of Toxoplasma gondii infection in slaughter pigs in Shanghai, China.

Toxoplasma gondii is a pathogen that seriously threatens the health of humans and animals. However, the current infection status of T. gondii in slaughter pigs in Shanghai is still not clear. To investigate the seroprevalence of T. gondii infection and analyze the prevalence factors associated with the parasite infection, 1158 serum samples were collected from five slaughterhouses in three districts between 2015 and 2018. Serum antibodies against T. gondii were detected in 160 pigs (13.8%) by enzyme-linked immunosorbent assay (ELISA). Additionally, seroprevalence rates differed among different districts (ranging from 4.0% in JD-2 to 17.6% in JD-1), seasons (ranging from 6.7% in winter to 17.8% in autumn), and years (ranging from 8.0% in 2016 to 26.8% in 2015). Region, season, and year were the main factors affecting T. gondii infection in these pigs. There were few reports on serological monitoring of T. gondii in Shanghai slaughterhouses between 2015 and 2018, and the number of infections had steadily increased over the past several consecutive years. Therefore, our data are helpful to understand the epidemic status of T. gondii in Shanghai, which will strengthen the prevention and treatment of swine toxoplasmosis.

Epidemiology of pseudorabies in intensive pig farms in Shanghai, China: Herd-level prevalence and risk factors.

Pseudorabies (PR), also known as Aujeszky's disease, is a highly contagious disease affecting pigs and a wide range of animals. Pseudorabies is enzootic in many countries. In China, it is a priority animal disease for control and eradication, however the data on disease frequency in intensive pig farms and the information on associated risk factors is inadequate. A cross-sectional study of intensive pig farms (≥350 sows) in Shanghai was conducted to determine herd-level prevalence of PRV and associated risk factors. Following a two-stage random sampling design, a total of 1349 sow serum samples were tested by gpI-ELISA from a total of 91 intensive pig farms in Shanghai. A herd was classified as positive if at least one PRV test-positive sow was present. Information on putative risk/protective factors was collected using questionnaires to pig farm owners or veterinarians. A logistic regression model was built to identify risk/protective factors for herd positivity. The results indicated that the herd-level true prevalence was 67.6% (95% CI：57.0-77.0). In the multivariable logistic regression model using backward stepwise procedure, two risk factors were found to be significantly associated with herd positivity: 'Breeding with introduced sows in the last 12 months' (OR = 3.5, 95%CI:1.2, 10.3) and 'Presence of stray dogs or cats' (OR = 4.0, 95%CI: 1.2, 12.6). The multivariable logistic model fitted the data well. Hosmer-Lemeshow goodness of fit test showed χ2 = 10.86 (df = 8, p = 0.21 > 0.05) and the predictability (area under the ROC curve) was 0.86. This study suggested that PR was highly endemic in intensive pig farms in Shanghai. The risk and protective factors identified in this study could be useful to improve the prevention policy of PR in Shanghai and other areas of China.

Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.

[Biologic characteristics of eight Podoviridae Stx2-converting phage].

OBJECTIVE: We studied biologic characteristics of Stx2-converting phage induced from Escherichia coli O157 by mitomycin C. METHODS: Eight Stx2-converting phages were isolated from E. coli O157 and identified by PCR. The phage particles were purified and phage DNA was extracted. Random priming digoxin (DIG)-labeled stx2-specific gene probe was prepared for Southern blot. The morphology of these phages were studied by electron microscopy. Protein profiles were analyzed by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE). Restriction fragment length polymorphism (RFLP) was used to confirm the size, type, and polymorphism of the purified phage genome. RESULTS: These 8 phages were confirmed Stx2-converting phage and DIG-labeled probe was highly specific. All phages had a regular hexagonal head and a short tail, belonging to Podoviridae phage families. The Stx2 phages had genome sizes in the range of 48 to 65.3kb, consisting of double-stranded DNA. The restriction fragment length polymorphism of these phages showed different groups, although the SDS-PAGE protein profiles of these phages were similar. CONCLUSION: These 8 Stx2-converting phages with similar morphology belonged to Podoviridae phage families. The protein profiles were highly identical. We could differentiate these Stx2-converting phages according to their restriction fragment length polymorphism patterns.