Characterization and complete genome analysis of a novel Escherichia phage, vB_EcoM-RPN242.

The novel Escherichia phage vB_EcoM-RPN242 was isolated using a strain of Escherichia coli originating from a diarrheic piglet as a host. The phage was able to form plaques on the E. coli lawn at 15-45 °C. Moreover, it was stable over a wide pH (4-10) and temperature (4-70 °C) range. The vB_EcoM-RPN242 genome was found to be a linear, double-stranded DNA consisting of 154,840 base pairs. There were 195 protein-encoding genes and two tRNAs detected in the genome; however, no genes associated with virulence, toxins or antimicrobial resistance were found. According to overall nucleotide sequence comparisons, vB_EcoM-RPN242 possibly represents a new species in the genus Agtrevirus.

In vivo assessment of bacteriophages specific to multidrug resistant Escherichia coli on fecal bacterial counts and microbiome in nursery pigs.

Escherichia coli is the most common cause of economic loss in swine industry. Nowadays, bacteriophages have been proven as good candidates for controlling bacterial infections. In this study, 6 phages were isolated and selected based on their high efficacy against 11 stains of E. coli isolated from diarrheal pigs. Six groups of weaned piglets were assigned (control, bacterial control (BC), two phage control (PC) and two phage treatment (PT) groups). Two titers (2 × 109 PFU/animal and 2 × 1010 PFU/animal) of phage cocktails consisting of these phages were tested in the PC and PT groups via oral gavage at 24, 48, and 72 h against an E. coli cocktail (2 × 109 CFU/animal) that was given to the piglets at 0, 12, 24, and 48 h of the trial. A significant reduction of fecal E. coli counts was observed in both PT groups from day 1 to 7 following the final phage dosage when compared to those of the BC group. Microbiomes in feces obtained 24 h after the final phage administration revealed phage therapy with both dosages could restore the gut's bacterial composition. Moreover, the given phage cocktails resulted in a significantly higher average daily gain of piglets during the first few weeks in both PC groups and the PT group receiving a higher phage dosage. These findings suggest that bacteriophages might be a potential alternative to antibiotics in the treatment of pathogens. In addition, they could also be utilized to improve pig growth performance.

PCV3 in Thailand: Molecular epidemiology and relationship with PCV2.

Porcine circovirus type 3 has been circulating throughout the world and since their first report, various clinical signs and disease developments have been documented. The virus is similar to the closely related PCV2 and is associated with several clinical signs called porcine circovirus-associated diseases (PCVAD). PCV2 or PCV3 is occasionally reported with clinical signs such as PDNS, respiratory signs and reproductive failure. Retrospective research conducted in Thailand revealed that both PCV2 and PCV3 have been circulation for decades. However, awareness about PCV3 infection has just arisen in recent years because of the similarities observed in disease circulation and clinical signs that have led to concerns. This study was conducted to find the relationship between the quantity of PCV2 and PCV3 in Thai pigs displaying the clinical signs related to PCVAD. A total of 479 serum samples with different production phases and clinical signs were sent to Kamphaeng Saen Veterinary Diagnostic Center (KVDC) for qPCR to detect the presence of PCV2 or PCV3. There was no relationship between the PCV3 and PCVAD-related clinical signs. Also, the relationship between PCV2 and PCV3 with no clinical signs suggested that both viruses might come from the same reservoir or have been circulating in Thailand for a long time, leading to common incidents in finding. The viral load of PCV2 was significantly different among the pig groups with and without clinical signs. The capsid sequence analysis of PCV3 revealed that 22 capsid sequences obtained from this study were found as clusters within PCV3a with a minor variation. Additional control measures are further needed to reduce the findings of the viruses. A future study with a control experiment may be needed to clarify the pathogenesis of PCV3.

The retrospective identification and molecular epidemiology of porcine circovirus type 3 (PCV3) in swine in Thailand from 2006 to 2017.

Porcine circovirus type 3 (PCV3) has recently been detected in pigs worldwide, with similar clinical manifestations to porcine circovirus-associated disease (PCVAD) from porcine circovirus type 2 (PCV2) infection. Here, we report the identification and molecular epidemiology of PCV3 in swine in Thailand from clinical samples retrieved from 2006 to 2017. The epidemiological data revealed co-infection with PCV2, PRRSV, and PCV2/PRRSV was common in our samples. Circulating PCV3 from this study shared a high similarity of nucleotide and deduced amino acid sequences of the partial capsid gene (96.7%-100% and 96.7%-100% respectively), indicated the genetic stability of PCV3 in Thailand. Phylogenetic analysis based on the capsid gene revealed scatter clustering with current PCV3 having no relation to the geographical origin of the virus strains. In this retrospective study, results have demonstrated that PCV3 has spread extensively within Thai swine from as early as 2006 and may also be involved in PRDC and PCVAD.

Genetic diversity of porcine circovirus type 2 (PCV2) in Thailand during 2009-2015.

Porcine circovirus type 2 (PCV2), the essential cause of porcine circovirus associated disease (PCVAD), has evolved rapidly and it has been reported worldwide. However, genetic information of PCV2 in Thailand has not been available since 2011. Herein, we studied occurrence and genetic diversity of PCV2 in Thailand and their relationships to the global PCV2 based on ORF2 sequences. The results showed that 306 samples (44.09%) from 56 farms (80%) were PCV2 positive by PCR. Phylogenetic trees constructed by both neighbor-joining and Bayesian Inference yielded similar topology of the ORF2 sequences. Thai PCV2 comprise four clusters: PCV2a (5.5%), PCV2b (29.41%), intermediate clade 1 (IM1) PCV2b (11.03%) and PCV2d (54.41%). Genetic shift of PCV2 in Thailand has occurred similarly to the global situation. The shift from PCV2b to PCV2d was clearly observed during 2013-2014. The viruses with genetically similar to the first reported PCV2 in 2004 have still circulated in Thailand. The first Thai PCV2b and PCV2d were closely related to the neighboring countries. The haplotype network analysis revealed the relationship of PCV2 in Thailand and other countries. These results indicate that genetic diversity of PCV2 in Thailand is caused by genetic drift of the local strains and intermittent introduction of new strains or genotypes from other countries. Genetic evolution of PCV2 in Thailand is similar to that occurs globally.

Characterization of lactic acid bacteria and other gut bacteria in pigs by a macroarraying method.

Lactic acid bacteria (LAB) consist of many genera, Gram-positive, and nonspore-forming micro-organisms; some members being used as probiotics while some others have negative effects on pig health. Bacterial species in the gastrointestinal tract can produce antibacterial substances, reduce serum cholesterol in their host, or can be responsible for growth reduction, diarrhea, and intestinal epithelial damage. It is therefore important for the pig industry to evaluate the impact of food and farm management on the presence of "good" or "bad" bacteria and the risk for consumers. This articles focuses on the molecular identification of gut microflora species following different diets given to pigs in UK and correlating the data on growth, health, and welfare. First of all, pig feces were individually collected from sows before and after farrowing and also from piglets before and after weaning over several months. Bacteria colonies were grown on MRS agar plates from feces and DNA was extracted (QIAamp DNA stool kit) and amplified using 16S rDNA (27f and 519r) primers. DNA sequencing and sequence alignment allowed us to identify species-specific zones, which were used as probes in a macroarray system also known as reverse line blot hybridization. Some probes were found to be species specific for the following species: Lactobacillus acidophilus, L. animalis, L. gallinarum, L. kitasanotis, L salivarius, Streptococcus alactolyticus, S. hyointestinalis, and Sarcina ventriculi. Actual studies are now focusing on the impact of diets of the microflora in different gut parts and at different stages of the animal's life.

Development of specific oligonucleotide probes to detect Vibrio species.

Many species of Vibrio are responsible for diseases in marine organisms and for economic losses to the aquaculture industry. The aim of this preliminary study was to obtain species-specific DNA zones to be used as potential probes from a phylogenetic analysis of the 23S ribosomal RNA (rRNA) gene of different Vibrio species from marine and human organisms. Species-specific probes were identified for V. parahaemolyticus, V. fortis, V. splendidus, and for two clusters of taxonomically related species, namely V. harveyi/campbelli and V. lentus/aestuarianus. A reverse line blot assay showed that the designed probes can specifically detect the different Vibrio species, thereby proving that these probes can be used to evaluate the presence of pathogenic and nonpathogenic Vibrio species in the sea and in marine organisms to assist in the investigation of environmental risks.