Characterization of a novel theta-type replicon of indigenous plasmid pTE15 from Lactobacillus reuteri N16.

BACKGROUND: pTE15 is a ~ 15-kb narrow-host-range indigenous plasmid from Lactobacillus reuteri N16 that does not replicate in selected Bacillus spp., Staphylococcus spp., and other Lactobacillus spp. METHODS: Combined deletion analysis the minireplicon essential of pTE15 with replicon-probe vector pUE80 (-) to confirmed sufficient for replication and from the ssDNA intermediate detection, plasmid amplification tested by chloramphenicol treatment, and replication origin sequence analysis to delineated the novel theta-type replication of pTE15. RESULTS: Single-stranded intermediate of pTE15 DNA was not detected in L. reuteri, indicating that this plasmid does not replicate via a rolling circle mechanism. The replicon of pTE15 did not display the structural organization typical of rolling-circle plasmids, nor were they similar to known rolling-circle plasmids. We further provided evidence that this plasmid applied a new mode of theta-type replication mechanism: (1) the size of this plasmid was > 10-kb; (2) the minireplicon consisted of AT-rich (directed repeat, iteron) and DnaA sequences; (3) the minireplicon did not contain double-strand origin (DSO) and essential rep genes, and it also showed no single-strand origin (SSO) structure; (4) the intermediate single-stranded DNA products were not observed for pTE15 replication; (5) the minireplicon did not contain a typical essential replication protein, Rep, (6) its copy number was decreased by chloramphenicol treatment, and (7) genes in pTE15 replication region encoded truncated RepA (TRepA), RepB and RepC, which were replication-associated proteins, but they were not essential for pTE15 replication. CONCLUSIONS: Collectively, our results strongly suggested that the indigenous plasmid pTE15 of L. reuteri N16 belongs to a new class of theta replicons.

The Anti-Cancer Effects of a Zotarolimus and 5-Fluorouracil Combination Treatment on A549 Cell-Derived Tumors in BALB/c Nude Mice.

Zotarolimus is a semi-synthetic derivative of rapamycin and a novel immunosuppressive agent used to prevent graft rejection. The pharmacological pathway of zotarolimus restricts the kinase activity of the mammalian target of rapamycin (mTOR), which potentially leads to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical influence on tumorigenesis. This study aims to examine the anti-tumor effect of zotarolimus or zotarolimus combined with 5-fluorouracil (5-FU) on A549 human lung adenocarcinoma cell line implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. We established A549 xenografts in nude mice, following which we randomly divided the mice into four groups: control, 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus combined with 5-FU. Compared the results with those for control mice, we found that mice treated with zotarolimus or zotarolimus combined with 5-FU retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; decreased inflammation cytokines levels (e.g., IL-1ß, TNF-α, and IL-6); reduced inflammation-related factors such as cyclooxygenase-2 (COX-2) protein and nuclear factor-κB (NF-κB) mRNA; enhanced anti-inflammation-related factors including IL-10 and inhibitor of NF-κB kinase α (IκBα) mRNA; and inhibited metastasis-related factors such as transforming growth factor ß (TGF-ß), CD44, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). Notably, mice treated with zotarolimus combined with 5-FU had significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with the groups of mice treated with 5-FU or zotarolimus alone. The in vivo study confirmed that zotarolimus or zotarolimus combined with 5-FU could retard lung adenocarcinoma growth and inhibit tumorigenesis. Zotarolimus and 5-FU were found to have an obvious synergistic tumor-inhibiting effect on lung adenocarcinoma. Therefore, both zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents for treatment of human lung adenocarcinoma.

Clozapine Worsens Glucose Intolerance, Nonalcoholic Fatty Liver Disease, Kidney Damage, and Retinal Injury and Increases Renal Reactive Oxygen Species Production and Chromium Loss in Obese Mice.

Clozapine is widely employed in the treatment of schizophrenia. Compared with that of atypical first-generation antipsychotics, atypical second-generation antipsychotics such as clozapine have less severe side effects and may positively affect obesity and blood glucose level. However, no systematic study of clozapine's adverse metabolic effects-such as changes in kidney and liver function, body weight, glucose and triglyceride levels, and retinopathy-was conducted. This research investigated how clozapine affects weight, the bodily distribution of chromium, liver damage, fatty liver scores, glucose homeostasis, renal impairment, and retinopathy in mice fed a high fat diet (HFD). We discovered that obese mice treated with clozapine gained more weight and had greater kidney, liver, and retroperitoneal and epididymal fat pad masses; higher daily food efficiency; higher serum or hepatic triglyceride, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine levels; and higher hepatic lipid regulation marker expression than did the HFD-fed control mice. Furthermore, the clozapine group mice exhibited insulin resistance, poorer insulin sensitivity, greater glucose intolerance, and less Akt phosphorylation; their GLUT4 expression was lower, they had renal damage, more reactive oxygen species, and IL-1 expression, and, finally, their levels of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and catalase) were lower. Moreover, clozapine reduced the thickness of retinal cell layers and increased iNOS and NF-κB expression; a net negative chromium balance occurred because more chromium was excreted through urine, and this influenced chromium mobilization, which did not help overcome the hyperglycemia. Our clozapine group had considerably higher fatty liver scores, which was supported by the findings of lowered adiponectin protein levels and increased FASN protein, PNPLA3 protein, FABP4 mRNA, and SREBP1 mRNA levels. We conclude that clozapine can worsen nonalcoholic fatty liver disease, diabetes, and kidney and retinal injury. Therefore, long-term administration of clozapine warrants higher attention.

The Ameliorative Effects of Fucoidan in Thioacetaide-Induced Liver Injury in Mice.

Liver disorders have been recognized as one major health concern. Fucoidan, a sulfated polysaccharide extracted from the brown seaweed Fucus serratus, has previously been reported as an anti-inflammatory and antioxidant. However, the discovery and validation of its hepatoprotective properties and elucidation of its mechanisms of action are still unknown. The objective of the current study was to investigate the effect and possible modes of action of a treatment of fucoidan against thioacetamide (TAA)-induced liver injury in male C57BL/6 mice by serum biochemical and histological analyses. The mouse model for liver damage was developed by the administration of TAA thrice a week for six weeks. The mice with TAA-induced liver injury were orally administered fucoidan once a day for 42 days. The treated mice showed significantly higher body weights; food intakes; hepatic antioxidative enzymes (catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD)); and a lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and C-reactive protein (CRP) levels. Additionally, a reduced hepatic IL-6 level and a decreased expression of inflammatory-related genes, such as cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA was observed. These results demonstrated that fucoidan had a hepatoprotective effect on liver injury through the suppression of the inflammatory responses and acting as an antioxidant. In addition, here, we validated the use of fucoidan against liver disorders with supporting molecular data.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum ß-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

Analysis of Pollution of Phthalates in Pork and Chicken in Taiwan Using Liquid Chromatography-Tandem Mass Spectrometry and Assessment of Health Risk.

Phthalates are widely used plasticizers that can cause endocrine disruption, mutagenicity, and carcinogenic effects and can contaminate food through various pathways. Investigations are scanty on phthalate pollution of livestock and poultry meat and their dietary exposure to humans. The present study assessed residual levels of phthalates in unpackaged pork (30 samples) and unpackaged chicken (30 samples) and their relevance to meat consumption and health risks in the Taiwanese population. Phthalate quantity was assessed by liquid chromatography-tandem mass spectrometry; the materials included diisononyl phthalate, diisodecyl phthalate, benzyl butyl phthalate, di-2-ethylhexyl phthalate (DEHP), and di-n-butyl phthalate. The Taiwan Food and Drug Administration (TFDA) has established values of tolerable daily intake (TDI) for the five phthalates. The major compound detected was DEHP, which ranged from 0.62 to 0.80 mg/kg in two pork samples, and 0.42-0.45 mg/kg in three chicken samples. Collectively, 8.33% of the phthalate-residue-containing samples tested positive for DEHP. The concentrations of DEHP were lower than the screening value of 1.0 mg/kg, as defined by the TFDA. Health risk was calculated as the estimated daily intake (DI) for any likely adverse effects; the DI of DEHP residues was <1% of the TDI value. The estimated risk was insignificant and considered to be safe, indicating that there is no risk to the health of Taiwanese population due to meat consumption. However, it is suggested that a phthalate monitoring program in meat should be instituted for any possible effects in future on human health.

Genetic Differentiation of the Bloodsucking Midge Forcipomyia taiwana (Diptera: Ceratopogonidae): Implication of the Geographic Isolation by the Central Mountain Ranges in Taiwan.

Forcipomyia (Lasiohelea) taiwana, a small bloodsucking midge, thrives in moderately moist habitats and is commonly found in grassy and bushy areas at an elevation below 250 m. This species exhibits a diurnal biting pattern and shows a marked preference for human blood. Although not known to transmit arthropod-borne diseases, the bites of F. taiwana can induce severe allergic reactions in some individuals. As a significant nuisance in Taiwan, affecting both daily life and the tourism industry, comprehensive studies on its population genetics across different geographical regions remain scarce. The central mountain ranges in Taiwan, comprising more than two hundred peaks above 3000 m in elevation, extend from the north to the south of the island, creating distinct eastern and western geographical divisions. This study utilizes microsatellite markers to explore the genetic differentiation of F. taiwana populations located in the eastern and western regions of the mountain ranges. Our findings reveal substantial genetic differentiation among populations inhabiting Taiwan's western region compared to those in the eastern region. This indicates that the topographical barriers presented by the mountain ranges significantly restrict gene flow, particularly given the species' limited active flight ability and habitat preferences. Although passive dispersal mechanisms, like wind or human activity, could contribute, this study concludes that the gene flow of F. taiwana between the western and eastern regions is primarily influenced by topographical constraints.

Characterization of a small cryptic plasmid pK50-2 isolated from Lactobacillus reuteri K50.

The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866 bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (-) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.

Molecular Characteristics and Pathogenicity of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) 1 in Taiwan during 2019-2020.

Two variants of porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV 1 and PRRSV 2, have caused abortion in pregnant sows and respiratory distress in nursery pigs worldwide. PRRSV 2 has been thoroughly researched in Taiwan since 1993; however, the first case of PRRSV 1 was not reported until late 2018. To decipher the genetic characteristics of PRRSV 1 in Taiwan, open reading frame 5 (ORF5) genes of PRRSV 1 strains collected from 11 individual pig farms in 2019-2020 were successfully sequenced. All Taiwanese ORF5 sequences were closely related to Spanish-like PRRSV strains, which are considered to share a common evolutionary origin with the strain used for the PRRSV 1 vaccine. Analyses of amino acid (aa) and non-synonymous substitutions showed that genetic variations resulted in numerously specific codon mutations scattered across the neutralizing epitopes within the ORF5 gene. The PRRSV 1 challenge experiment disclosed the pathogenetic capability of the NPUST2789 isolate in nursery pigs. These findings provide comprehensive knowledge of the molecular diversity of the PRRSV 1 variant in local Taiwanese fields and facilitate the development of suitable immunization programs against this disease.

The First Nationwide Surveillance of Severe Fever with Thrombocytopenia Syndrome in Ruminants and Wildlife in Taiwan.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September-November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

Application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection.

The spike (S) protein, containing two subunits, S1 and S2, is the major immunity-eliciting antigen of avian infectious bronchitis virus (IBV), a highly contagious disease of chickens. Several immunogenic regions, mainly located within the S1 subunit, have been identified. Nonetheless, these immune-dominant regions were defined using selected monoclonal antibodies or using a short peptide approach that involves only certain limited regions of the S protein. In addition, some immune-dominant regions are located in hypervariable regions (HVRs) which are not present in all serotypes. Hence, the aim of this study was to determine a broader range of antigenic regions that have strong antibody eliciting ability; these could then be applied for development of an IBV-diagnostic tool. Initially, the S1 and part of the S2 subunit protein (24-567 amino acids) were expressed as five fragments in prokaryotic system. The antigenicity was confirmed using IBV immunized sera. Performance of the S subfragments was evaluated by ELISA using a panel of field chicken sera with known IBV titres determined by a commercial kit. This indicated that, among the five antigenic recombinant proteins, the region S-E showed the highest specificity and sensitivity, namely 95.38 % and 96.29 %, respectively. The κ value for the in-house ELISA using the S-E fragment compared to a commercial kit was 0.9172, indicating a high agreement between these two methods. As region S-E harbors strong immunogenicity within the spike protein, it has the potential to be exploited as an antigen when developing a cost-effective ELISA-based diagnosis tool.

Evaluation of the Potential Entomopathogenic Fungi Purpureocillium lilacinum and Fusarium verticillioides for Biological Control of Forcipomyia taiwana (Shiraki).

Forcipomyia taiwana (Diptera: Ceratopogonidae) is a nuisance blood-sucking pest to humans in Taiwan. An F. taiwana bite causes itching and redness and usually causes serious harassment to human outdoor activity. In terms of F. taiwana control, chemical pesticides are ineffective. Therefore, other efforts are needed. Fungal mycosis in the larvae, pupae, and emerging F. taiwana adults was found during the rearing of F. taiwana. In this study, six fungal isolates were isolated from infected cadavers and subjected to molecular identification. In addition, their biocontrol potential was evaluated against different life stages of F. taiwana. Based on the pathogenicity screening, two fungal isolates, NCHU-NPUST-175 and -178, which caused higher mortality on the fourth instar larvae of F. taiwana, were selected for virulence tests against different life stages of F. taiwana larvae. The results of the phylogenetic analysis indicated that the NCHU-NPUST-175 and -178 belonged to Purpureocillium lilacinum and Fusarium verticillioides, respectively. Bioassay against different life stages of F. taiwana with different spore concentrations (5 × 105 to 5 × 107 conidia/mL) revealed a dose-dependent effect on larvae for both fungal isolates, while only 38% and 50% mortality was found in highest concentration (5 × 107 conidia/mL) at fourth instar larvae by Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178, respectively. Moreover, reductions in egg-hatching rate and adult emergence rate were found, when the last stage of F. taiwana was inoculated with both fungal isolates, indicating the ovicidal potential and the impact of entomopathogenic fungi on the development of F. taiwana. In conclusion, Pl-NCHU-NPUST-175 and Fv-NCHU-NPUST-178 showed larvicidal activity, ovicidal activity, and impact on adult emergence on F. taiwana.

The anticancer effects of cyanidin 3-O-glucoside combined with 5-fluorouracil on lung large-cell carcinoma in nude mice.

The haskap (Lonicera caerulea L., Caprifoliaceae) berry has been widely used in traditional medicine in Kuril Islands, Russia, Japan, and China. Cyanidin-3-O-glucoside (C3G) is the most abundant anthocyanin in haskap berries, and C3G induces antiproliferative pharmacological activity in various cancer cells. However, no study has investigated its anti-lung large-cell carcinoma (LCC) pharmacological role. Therefore, this study determined whether C3G alone or C3G combined with 5-fluorouracil (5-FU) inhibits human lung LCC. We determined the tumor growth, apoptosis, inflammation, and metastasis in the H661 lung LCC lines xenografted into BALB/c nude mice. The mice were administered saline (control), 5-FU, C3G, or both C3G and 5-FU. Relative to the control mice, those treated with C3G alone or both C3G and 5-FU exhibited impaired tumor growth; increased tumor apoptosis; decreased inflammatory cytokine levels (e.g., IL-1ß, TNF-α, C-reactive protein, and IL-6); decreased inflammation-related factors, including cyclooxygenase-2 protein and nuclear factor-κB (NF-κB) mRNA; increased inhibition of NF-κB kinase α mRNA; and downregulated metastasis-related factors, such as transforming growth factor-ß, CD44, epidermal growth factor receptor, and vascular endothelial growth factor. In addition, C3G alone or combined with 5-FU affected the expression of the tumor microenvironment-related factors Ki67, CD45, PDL1, and CD73. Compared with the mice treated with 5-FU or C3G alone, those treated with both C3G and 5-FU exhibited significantly impaired tumor growth, decreased tumor sizes, and increased tumor inhibition. This in vivo study demonstrated that C3G alone or combined with 5-FU may impair the growth of lung LCC and inhibit tumorigenesis. The findings indicate that C3G alone or C3G combined with 5-FU may be beneficial for treating human lung LCC.

Micrococcus porci sp. nov., Isolated from Feces of Black Pig (Sus scrofa).

An aerobic bacterium, designated as strain KD337-16T, was isolated from the fecal samples of a black pig. It exhibited spherical, non-motile and non−spore-forming, Gram-positive cells. KD337-16T was identified as a member of the genus Micrococcus through 16S rRNA gene sequencing, and its closest relatives were found to be Micrococcus endophyticus YIM 56238T (99.5% similarity), Micrococcus luteus NCTC 2665T (99.1%), Micrococcus yunnanensis YIM 65004T (99.1%), Micrococcus aloeverae AE-6T (99.1%), Micrococcus antarcticus T2T (98.9%), and Micrococcus flavus LW4T (98.7%). Phylogenomic trees were constructed, and strain KD337-16T was found to form its own cluster as an independent lineage of M. flavus LW4T. Between KD337-16T and its close relatives, the average nucleotide identity, average amino acid identity, and digital DNA−DNA hybridization were below the respective species delineation thresholds at 82.1−86.6%, 78.1−86.1%, and 24.4−34.9%. The major cellular fatty acids and polar lipids were anteiso-C15:0 and iso-C15:0, and DPG and PG, respectively. The predominant menaquinone was MK-8(H2). Taken together, the results indicate that strain KD337-16T is a novel species of the genus Micrococcus, for which the name Micrococcus porci sp. nov. is proposed. The type strain is KD337-16T (=BCRC 81318T = NBRC 115578T).

Effects of Para-Toluenesulfonamide on Canine Melanoma Xenotransplants in a BALB/c Nude Mouse Model.

The pharmacological pathway of para-toluenesulfonamide (PTS) restricts the kinase activity of the mammalian target of rapamycin, potentially leading to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical effect on tumorigenesis. We aimed to examine the antitumor effect of PTS or PTS combined with cisplatin on canine melanoma implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. The mice were randomly divided into four groups: control, cisplatin, PTS, and PTS combined with cisplatin. Mice treated with PTS or PTS combined with cisplatin had retarded tumor growth and increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase phosphorylation, decreased inflammatory cytokine levels, reduced inflammation-related factors, enhanced anti-inflammation-related factors, and inhibition of metastasis-related factors. Mice treated with PTS combined with cisplatin exhibited significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with those treated with cisplatin or PTS alone. PTS or PTS combined with cisplatin could retard canine melanoma growth and inhibit tumorigenesis. PTS and cisplatin were found to have an obvious synergistic tumor-inhibiting effect on canine melanoma. PTS alone and PTS combined with cisplatin may be antitumor agents for canine melanoma treatment.

Serotypes, antimicrobial susceptibility, and minimal inhibitory concentrations of Actionbacillus pleuropneumoniae isolated from slaughter pigs in Taiwan (2002-2007).

In total, 211 isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during 2002-2007. Serotypes, antimicrobial susceptibility and minimum inhibitory concentration (MIC) values were determined for each isolate of A. pleuropneumoniae to 10 antimicrobial agents. Serovar 1 of A. pleuropneumoniae was predominant in Taiwan in 138 of the 211 isolates, followed by serovars 2 and 5. More than 90% of collected isolates were sensitive to ceftiofur, cephalothin, and chloramphenical. However, lincospectin and gentamicin were relatively less susceptible with sensitivities of only 2.4 and 5.7%, respectively. Additionally, ceftiofur had the highest in vitro activity with an MIC(50) of 2.2 µg/ml, followed by cephalothin (2.7 µg/ml) and chloramphenicol (7.9 µg/ml). Lincospectin had the least activity with MIC(50) and MIC(90) values of 73.9 and 114.5 µg/ml, respectively. The data indicate that ceftiofur and cephalothin were extremely active against A. pleuropneumoniae and with minimum MIC values. These drugs are suitable for controlling and treating hemorrhagic pleuropneumonia outbreaks in swine.

Shedding pattern and serological profile of porcine circovirus type 2 infection in cesarean-derived, colostrum-deprived and farm-raised pigs.

Six 5-week-old porcine circovirus type 2 (PCV2)-free, cesarean-derived, colostrums-deprived (CDCD) pigs were inoculated intranasally with 10(6) TCID(50) of PCV2. Four CDCD pigs were untreated cohabitants. Forty farm-raised pigs from two PCV2-contaminated herds were randomly selected for PCV2 trace investigations. Blood, nasal, oropharyngeal and fecal samples were collected from all tested pigs weekly. The PCV2 DNA shed at 6-11 and 7-12 weeks of age for PCV2-inoculated pigs and cohabitants, respectively. All the CDCD pigs exhibited seroconversion after PCV2 exposure. In the farm-raised animals, PCV2 shed at 9-15 weeks of age and seroconversion started at 11 weeks of age. Collectively, the pigs had a prolonged PCV2 shedding period following viral exposure, and growing pigs were the source of horizontal PCV2 transmission in PCV2-infected herds.

Characterization of tetracycline resistance lactobacilli isolated from swine intestines at western area of Taiwan.

To investigate the frequency of tetracycline resistance (Tet-R) lactobacilli in pig intestines, a total of 256 pig colons were analyzed and found to contain typical colonies of Tet-R lactic acid bacteria in every sample, ranging from 3.2 × 10(3) to 6.6 × 10(5) CFU/cm(2). From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. Based on the EFSA (2008) breakpoints, all isolates, after MIC analysis, were qualified as Tet-R, from which the significant high Tet-R MIC(50) and MIC(90) values indicated an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0%, 22.5%, 14.4%, 8.1% and 0.9%, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % (average = 62%).

Transcriptome analysis of Lactobacillus rhamnosus GG strain treated with prebiotic - bovine lactoferrin under a cold environment.

Lactoferrin (LF) is secreted by mammals and displays extensive biological effects. We previously reported that bovine LF (BLF) can boost the cold tolerance of a well-applied probiotic strain, Lactobacillus rhamnosus GG (LGG), to grow robustly under a cold environment, but the molecular mechanism is not clear. Here, RNA-seq analysis was conducted to ascertain molecular pathways underlying cold tolerance exerted by BLF. LGG was cultured in a cold environment (22 °C) in the presence or absence of BLF. Transcriptome analysis indicated that BLF significantly elicited 1.2-3.2 fold (log2 Fold change) higher expression levels of genes related to stress, defense, cell division, and transporter in LGG, including the genes CspA, LytR, XRE, MerR, and GpsB. The KEGG pathway and GO analyses confirmed that BLF can modulate a few central pathways to boost the growth of LGG. BLF also reduced metabolic pathways involved in purine, amino acid, pyrimidine, one-carbon metabolism, and secondary metabolites in LGG. We speculate that the reduction of the above pathways may play key roles to reduce energy requirement and maintain carbon metabolism balance in LGG for surviving and growing in a cold state, and BLF can be an excellent prebiotic to LGG cultured in this cold condition (22 °C). Overall, this study uncovers the molecular effects of BLF on LGG.

Correlation of Neutralizing Antibodies (NAbs) between Sows and Piglets and Evaluation of Protectability Associated with Maternally Derived NAbs in Pigs against Circulating Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) under Field Conditions.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by a highly transmissible pathogen called porcine reproductive and respiratory syndrome virus (PRRSV), has caused severe problems, including reproductive disorders in sows and respiratory symptoms in nursery pigs worldwide, since the early 1990s. However, currently available PRRSV vaccines do not supply complete immunity to confront the viral infection. Elicitation of PRRSV-specific neutralizing antibodies (NAbs) during the preinfectious period has been deemed to be a feasible strategy to modulate this virus, especially in farms where nursery pigs are seized with PRRSVs. A total of 180 piglets in a farrow-to-finish farm that had a natural outbreak of PRRS were distributed into three groups based on the different PRRSV NAbs levels in their dams. In the present study, piglets that received superior maternal-transferred NAbs showed delayed and relatively slight viral loads in serum and, on the whole, higher survival rates against wild PRRSV infections. A positive correlation of maternal NAbs between sows and their piglets was identified; moreover, high NAbs titers in piglets can last for at least 4 weeks. These results provide updated information to develop an appropriate immune strategy for breeding and for future PRRSV control under field conditions.