Genetic diversity and relatedness of feline parvovirus in Vietnam and its potential implications for canine-feline transmission.

Feline panleukopenia, caused by feline parvovirus (FPV), has been studied worldwide, but there have been very few studies conducted in Vietnam. In this study, 19 rectal swab samples were collected from northern Vietnam in 2018-2019 and screened for the presence of FPV using PCR. Through sequence analysis of the full-length VP2 gene, it was found that the FPV strains detected in Vietnam were closely related to those obtained from dogs in Vietnam, Asia, Europe, and America. Moreover, the FPV strains found in Vietnam may constitute a distinct group, related to viruses sampled in China. Interestingly, most of the nucleotide changes identified were T-C substitutions.

Endolysin LysSTG2: Characterization and application to control Salmonella Typhimurium biofilm alone and in combination with slightly acidic hypochlorous water.

The gene encoding LysSTG2, an endolysin from Salmonella-lytic bacteriophage STG2, was cloned, overexpressed, and characterized. LysSTG2 consists of a single domain belonging to the Peptidase_M15 superfamily. LysSTG2 showed strong lytic activity against chloroform-treated S. Typhimurium cells after incubation at 4-50 °C for 30 min, at pH ranging from 7.0 to 11.0, and in the presence of NaCl from 0 to 300 mmol/L. It also showed lytic activity against all the 14 tested Gram-negative strains treated with chloroform, including Salmonella, E. coli, and Pseudomonas aeruginosa, but not against the Gram-positive bacteria tested. In addition, LysSTG2 (100 µg/mL) reduced the viability of S. Typhimurium NBRC 12529 planktonic cells by 1.2 log and that of the biofilm cells after 1-h treatment. Sequential treatment of slightly acidic hypochlorous water (SAHW) containing 40 mg/L available chlorine and LysSTG2 (100 µg/mL) was effective on S. Typhimurium NBRC 12529 biofilm cells, removing more than 99% of biofilm cells. These results demonstrate that LysSTG2 alone can effectively kill S. Typhimurium cells after permeabilization treatment and successfully control S. Typhimurium in biofilms in combination with SAHW, suggesting that the combined use of LysSTG2 and SAHW might be a novel and promising method for combating S. Typhimurium in food industries.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Isolation and application of bacteriophages alone or in combination with nisin against planktonic and biofilm cells of Staphylococcus aureus.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. • Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. • SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. • Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6 log CFU/mL in broth after 6 h of incubation at 25 and 8 °C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9 log CFU/piece after 24 h of storage at 25 and 8 °C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0 log CFU/piece after 24 h of incubation at 25 and 8 °C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.

Identification of the newly identified subtilase cytotoxin-encoding gene (subAB2-2) among clinical Shiga toxin-producing Escherichia coli isolates.

Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99% and 100% identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.

Prevalence and Antibiotic Resistance Profile of Clostridium perfringens Isolated from Pork and Chicken Meat in Vietnam.

Clostridium perfringens is one of the most important zoonotic pathogens as it can cause food poisoning in humans and necrotic enteritis in both animals and humans. Meat, especially pork and chicken meat, is considered the main vehicle for the transmission of C. perfringens from animals to humans. The purpose of this study was to determine the prevalence, toxinotype, and antimicrobial resistance profile of C. perfringens isolated from pork and chicken meat sold in Vietnam. The isolation results showed that 15/50 (30%) of pork samples and 8/50 (16%) of chicken meat samples were contaminated with C. perfringens. The isolates exhibited their highest resistance rate to tetracycline (21/23; 91.30%) and clindamycin (10/23; 43.48%). On the contrary, their lowest resistance rates were observed in response to imipenem (2/23; 8.70%) and cefoxitin (1/23; 4.35%). In particular, 34.78% (8/23) of C. perfringens isolates were identified to be multidrug-resistant strains. The results of toxin genotyping indicated that all isolates were positive for the cpa gene and belonged to type A.

Antibiotic Resistance Profile and Bio-Control of Multidrug-Resistant Escherichia coli Isolated from Raw Milk in Vietnam Using Bacteriophages.

E. coli is an important zoonotic pathogen capable of causing foodborne illness and bovine mastitis. Bacteriophages have been increasingly considered a promising tool to control unwanted bacteria. The aim of this study is to determine the antibiotic resistance profile of E. coli isolated from raw milk and the efficacy of phage in controlling multidrug-resistant E. coli in raw milk. Antibiotic susceptibility testing showed the highest resistance rates of E. coli isolates to co-trime (27.34%) and ampicillin (27.34%), followed by streptomycin (25.18%), tetracycline (23.02%), and the lowest resistance rates to ciprofloxacin, gentamycin, and ceftazidime, all at a rate of 2.16%. All isolates were susceptible to meropenem. Of the 139 E. coli isolates, 57 (41.01%) were resistant to at least one antibiotic, and 35 (25.18%) were classified as MDR strains. Molecular characterization indicated that 5 (3.6%) out of the 139 isolates were STEC strains carrying stx1 gene. Seven (5.04%) isolates were phenotypically identified as ESBLEC, and four isolates (2.88%) were resistant to colistin. The results of the genotypic test revealed that four out of seven ESBLEC strains carried both blaTEM and blaCTX-M-1, two harbored blaTEM, and one possessed blaCTX-M-1, while mcr-1 was detected in all four colistin-resistant E. coli isolates. In particular, one isolated E. coli strain (EM148) was determined to be a multidrug-resistant strain simultaneously carrying blaTEM, blaCTX-M-1, and mcr-1. A total of eight phages were successfully recovered from raw milk. The application of phage PEM3 significantly reduced viable counts of multidrug-resistant host EM148 in raw milk by at least 2.31 log CFU/mL at both 24 °C and 4 °C.

An approach for differentiating echovirus 30 and Japanese encephalitis virus infections in acute meningitis/encephalitis: a retrospective study of 103 cases in Vietnam.

BACKGROUND: In recent decades, Echovirus 30 (E30) and Japanese encephalitis virus (JEV) have been reported to be the common causative agents of acute meningitis among patients in South East Asia. An E30 outbreak in Vietnam in 2001-2002 gained our interest because the initial clinical diagnosis of infected patients was due to JEV infection. There are few clinical insights regarding E30 cases, and there are no reports comparing E30 and JEV acute meningitis/encephalitis cases based on clinical symptoms and case histories. We therefore aimed to identify reliable clinical methods to differentiate E30 and JEV acute meningitis/encephalitis. METHODS: A retrospective, cross-sectional study was conducted to compare E30 and JEV acute meningitis/encephalitis cases. We collected and analyzed the clinical records of 43 E30 confirmed cases (E30 group) and 60 JEV confirmed cases (JEV group). Clinical data were compared between the E30 and the JEV groups. Differences of clinical parameters were analyzed by certain statistical tests. RESULTS: Fever, headache, and vomiting were the most common symptoms in both the E30 and the JEV groups. Combined symptoms of headache and vomiting and the triad of symptoms of fever, headache, and vomiting were observed in more patients in the E30 group (E30 vs. JEV: 19% vs. 0%, p < 0.001; 74% vs. 27%, p < 0.001, respectively). On the other hand, strong neurological symptoms such as seizure (5% vs. 73%, p < 0.001) and altered consciousness (12% vs. 97%, p < 0.001) were manifested primarily in the JEV group. CSF leukocytosis was observed predominantly in the E30 group (80 vs. 18 cells/µL, p = 0.003), whereas decreasing CSF sugar level was observed predominantly in the JEV group (58.7 vs. 46.9 mg/dL, p < 0.001). CONCLUSION: Fever, headache, vomiting, absence of neurological symptoms (seizure, altered consciousness), and presence of CSF leukocytosis are important parameters to consider in differentiating E30 from JEV cases during early infection. Then, proper measures can be adopted immediately to prevent the spread of the disease in the affected areas.

The Use of Phage Cocktail and Various Antibacterial Agents in Combination to Prevent the Emergence of Phage Resistance.

Bacterial food poisoning cases due to Salmonella and E. coli O157:H7 have been linked with the consumption of a variety of food products, threatening public health around the world. This study describes the combined effects of a phage cocktail (STG2, SEG5, and PS5), EDTA, nisin, and polylysine against the bacterial cocktail consisting of S. typhimurium, S. enteritidis, and E. coli O157:H7. Overall, phage cocktail (alone or in combination with nisin or/and polylysine) not only showed great antibacterial effects against bacterial cocktail at different temperatures (4 °C, 24 °C, and 37 °C), but also totally inhibited the emergence of phage resistance during the incubation period. These results suggest that the combination of phages with nisin or/and polylysine has great potential to simultaneously control S. typhimurium, S. enteritidis, and E. coli O157:H7.

Genomic characterization and application of a novel bacteriophage STG2 capable of reducing planktonic and biofilm cells of Salmonella.

As one major foodborne pathogen, Salmonella can cause serious food poisoning outbreaks worldwide. Bacteriophage therapy is increasingly considered as one of the promising antibacterial agents for the biocontrol of foodborne pathogens. In the current study, a lytic phage STG2 capable of infecting S. enteritidis and S. typhimurium was characterized, and its efficacy in reducing these foodborne pathogens in both planktonic and biofilm forms was evaluated on cabbage and various surfaces. Genomic characterization revealed that phage STG2 was Siphoviridae phage (Epseptimavirus genus) with a dsDNA genome comprising of 114,275 bp and its genome does not contain any genes associated to antibiotic resistance, toxins, lysogeny, or virulence factors. Additionally, phage STG2 exhibited great efficacy in reducing (>2 Log) planktonic cells on cabbage as well as the biofilms formed on cabbage, polystyrene, and stainless steel, suggesting that phage STG2 is capable of simultaneously controlling both S. enteritidis and S. typhimurium contaminations on food and food-related surfaces.

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range.

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0-11.0) and thermal tolerance (4-60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30-4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Static Aerated Composting of African Swine Fever Virus-Infected Swine Carcasses with Rice Hulls and Sawdust.

Identifying and ensuring the inactivation of the African Swine Fever virus in deadstock is a gap in the swine industry's knowledge and response capabilities. The results of our study demonstrate that ASFv in deadstock was inactivated using static aerated composting as the carcass disposal method. Replicated compost piles with whole market hogs and two different carbon sources were constructed. In-situ bags containing ASFv-infected spleen tissue were placed alongside each of the carcasses and throughout the pile. The bags were extracted at days 0, 1, 3, 7, 14, 28, 56, and 144 for ASFv detection and isolation. Real-time PCR results showed that DNA of ASFv was detected in all samples tested on day 28. The virus concentration identified through virus isolation was found to be below the detection limit by day 3 in rice hulls and by day 7 in sawdust. Given the slope of the decay, near-zero concentration with 99.9% confidence occurred at 5.0 days in rice hulls and at 6.4 days in sawdust. Additionally, the result of virus isolation also showed that the virus in bone marrow samples collected at 28 days was inactivated.

Viability of African Swine Fever Virus with the Shallow Burial with Carbon Carcass Disposal Method.

African swine fever (ASF) is a highly contagious swine disease with high mortality. In many countries, culling pigs infected and exposed to the ASF virus is mandatory to control the disease, which poses a real challenge in the disposal of large numbers of carcasses during ASF outbreaks. Shallow burial with carbon (SBC) Thanks ew mortality disposal method developed from deep burial and composting. The present study investigates the effectiveness of SBC in disposing of ASF virus-infected pigs. The real-time PCR results showed that DNA of the ASF virus was still detected in bone marrow samples on day 56, while the virus isolation test revealed that the infectious ASF virus was destroyed in both spleen and bone marrow samples on day 5. Interestingly, decomposition was found to occur rapidly in these shallow burial pits. On day 144, only large bones were found in the burial pit. In general, the results of this study indicated that SBC is a potential method for the disposal of ASF-infected carcasses; however, further studies are needed to provide more scientific evidence for the efficacy of SBC in different environment conditions.

Isolation and genetic characterization of canine distemper virus in domestic dogs from central and northern provinces in Vietnam.

Canine distemper virus (CDV) is a pathogen causing fatal disease in a wide range of carnivores. Sequence analysis of CDV strains has been classified into several geographically-related lineages, and the evolution and emergence of these strains are not fully yet investigated. In this study, the complete H gene sequences of 15 CDV strains isolated on Vero DST cell culture from clinical samples of vaccinated domestic dogs in Vietnam were investigated. Fifteen CDV isolates belonging to Asia-1 CDV variants were predominant antigenic type circulated in Central and Northern Vietnam with notable differences regarding the region and some genetic variation, and the most closely related Asia-1 variants lineage reported in Vietnam, China, Taiwan, and Japan. All identified CDV isolates clustered into 2 novel clades Asia-1-C1 and Asia-1-C2. The major amino acid mutation variants of Vietnamese Asia-1 CDV strains were found at sites 51, 157, 159, 160, 171, 178, 186, 235, 245, 277, 288, 313, 324, 330, 337, 345, 358, 359, 365, 383, 446, 475, 517, 530, 584, 598 which include N-glycosylation sites and neutralizing epitope regions in H gene. The results of the virus neutralization titer (VNT) assay showed that the dogs vaccinated with commercial vaccines had significantly low VNT (4.89 and 12.8) against field CDV isolate strains (VNUA NA04, HN18, and NB05) isolated in northern and central Vietnam, respectively. These data may suggest the need for further research in CDV monitoring and development of preventative measures against CDV in Vietnam.

The use of composting for the disposal of African swine fever virus-infected swine carcasses.

African swine fever (ASF) has been considered as one of the most important and devastating swine diseases with high mortality rates. Since effective vaccines and treatment are not available, mass euthanasia of infected and exposed pigs has been known to be the best measure to control ASF. Although composting has been proved to be a safe method for the rapid disposal of animal carcasses during outbreaks, there is no information about the effect of composting on the viability of ASF virus in swine carcasses. This study investigates the survival of the ASF virus in swine carcasses during composting. The findings suggested that the DNA of the ASF virus was detected in all samples tested. On the contrary, infectious ASF virus particles were rapidly destroyed at day 3.

Complete Genome Sequence of Campylobacter coli Bacteriophage CAM-P21.

Bacteriophage CAM-P21, isolated from a beef mince sample in Japan using Campylobacter coli, has a 12,587-bp genome encoding 18 putative coding sequences with an average GC content of 31.19%.

Isolation, characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157:H7 in different food matrices.

Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli O157:H7 are the most important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages are increasingly considered as novel antibacterial agents to control foodborne pathogens. In this study, 8 Salmonella phages and 10 E. coli O157:H7 phages were isolated from chicken products. A polyvalent phage PS5 capable of infecting S. Enteritidis, S. Typhimurium, and E. coli O157:H7 was further characterized and its efficacy in reducing these foodborne pathogens was evaluated in in vitro and in foods. Morphology, one-step growth, and stability assay showed that phage PS5 was a myovirus, with relatively short latent periods, large burst sizes, and high stability. Genome sequencing analysis revealed that the genome of PS5 does not contain any genes associated to antibiotic resistance, toxins, lysogeny, and virulence factors. In broth, phage PS5 significantly decreased the viable counts of all the three bacterial hosts by more than 1.3 log CFU/mL compared to controls after 2 h of incubation at 4 °C and 24 °C. In foods, treatment with PS5 also resulted in significant reductions of viable counts of all the three bacterial hosts compared to controls at temperatures tested. This is the first report on single phage capable of simultaneously controlling S. Enteritidis, S. Typhimurium and E. coli O157:H7 in both in vitro and in foods.

Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli.

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.

Improved Bowel Function in Patients with Spina Bifida After Bone Marrow-Derived Mononuclear Cell Transplantation: A Report of 2 Cases.

BACKGROUND Bowel dysfunction is observed in 42.2-71.2% of patients with spina bifida. Traditional treatments yield limited results. The objective of this paper is to report on improvement in bowel function in 2 children with spina bifida following bone marrow-derived mononuclear cells transplantation. CASE REPORT Two patients - 14 years old and 11 years old - with bowel dysfunction after myelomeningocele repair underwent 2 BMMNC transplantations without complications. Those patients had normal defecation, assessed through follow-ups of 21 months and 16 months, respectively. CONCLUSIONS BMMNC transplantation can improve bowel function, as demonstrated in 2 patients with spina bifida.