Alterations in Genes rib, scpB and Pilus Island Decrease the Prevalence of Predominant Serotype V, Not III and VI, of Streptococcus agalactiae from 2008 to 2012.

Streptococcus agalactiae (GBS) can infect newborns, pregnant women and immunocompromised or elderly people. This study aimed to investigate differences in three pilus genes and virulence genes pavA, cfb, rib and scpB and changes in predominant serotypes III, V and VI from 2008 to 2012. The susceptibilities to penicillin, ceftriaxone, azithromycin, erythromycin, clindamycin, levofloxacin and moxifloxacin of 145 GBS strains of serotype III, V and VI strains from 2008 and 2012 were determined using disc diffusion method. PCR identification of ST-17, the pilus genes and virulence genes; multilocus sequence typing (MLST); and conserved domain and phylogenetic analysis of scpB-1 and scpB-2 proteins were performed. A dramatic number reduction was observed in serotype V, not III and V, from 2008 to 2012. The rate of resistance to azithromycin, clindamycin and erythromycin was the highest in serotype V. ST-17 was only found in serotype III with pilus genes PI-1+PI-2b. The major pilus genotype was PI-1+PI-2a. Serotype V without the rib gene was reduced in number between two studied years. Compared to scpB-1, scpB-2 had a 128-bp deletion in a PA C5a-like peptidase domain and putative integrin-binding motif RGD. In conclusion, reduction in serotype V may be due to presence of scpB-2 or lack of genes scpB and rib.

Evaluation of using comparative intradermal tuberculin test to diagnose bovine tuberculosis in dairy cattle in Taiwan.

The National Bovine Tuberculosis (bTB) Eradication Program for dairy cattle has been operating in Taiwan for many years and has allowed the prevalence of bTB to decrease gradually. However, 29% of intradermal tuberculin test (ITT)-positive dairy cows were later found to be TB negative based on necropsy, histopathological examination, and mycobacterial isolation results. Studies in Taiwan have indicated that Mycobacterium avium subsp. paratuberculosis (MAP) may lead to false-positive ITT. Due to the high prevalence (over 90%) of paratuberculosis (PTB) serum antibody among Taiwan's farms, comparative ITT (CITT) has been recommended to differentiate between bTB and PTB infections. In this study, we used ITT, CITT, and enzyme-linked immunosorbent assay (ELISA) to evaluate the prevalence of bTB from 2012 to 2018. We also used pathological and bacterial examination from ITT-positive dairy cows to evaluate CITT's diagnostic ability and adjust its cutoff point accordingly. After careful selection, 36 cows (including 31 cows from 11 ITT-positive farms and 5 from 2 ITT-negative farms) were examined by CITT. The cutoff point was adjusted using a receiver operating characteristic (ROC) analysis. Overall, our results identified the ITT-positive prevalence in Taiwan as 0.03-0.22%, and PTB-positive prevalence as 54.55-73.53%. The results of sensitivity, specificity, kappa, and ROC analyses have identified the optimal cutoff point for the CITT in Taiwan as ≥ 3 mm. At this cutoff point value, the sensitivity and specificity were 62.5% and 96.43%, respectively. Our findings can be used to reduce the false-positive response rate caused by PTB cross-reaction and accelerate the eradication of bTB in Taiwan.

Epidemiological survey of Toxoplasma gondii and Neospora caninum infections in dairy goats in Central-Southern Taiwan.

Toxoplasma gondii and Neospora caninum are intracellular protozoan parasites that cause reproductive disorders in ruminants and humans. Information on the risk factors of T. gondii and N. caninum infections in goats is very limited in Taiwan. The aim of the study was to investigate the epidemiology and identify the risk factors of these two infections in goats. A total of 630 caprine sera were collected from 42 dairy goat farms and the owners were interviewed by a structured questionnaire. The apparent seroprevalences of T. gondii in farm- and individual- levels were respectively 88.1% and 32.22%, while those of N. caninum were 19.05% and 2.54%, respectively. Toxoplasma gondii B1 gene was identified in 7 feed samples and 8 from the water samples whereas N. caninum was not found. Wooden flooring was the main risk factor for T. gondii infection while the frequency of visits by staff to other farms and the breed of goat were risk factors for N. caninum. The improvement of flooring materials or thorough cleaning, periodic disinfection and maintenance of dryness on the floor are highly recommended for the prevention of T. gondii infection in farmed goats. In addition, unnecessary visits to other farms should be limited to prevent the spread of N. caninum. These factors should be highlighted for the prevention of T. gondii and N. caninum in goats, particularly when raised in intensive housing system with flooring on height.

Mirtazapine Reduces Adipocyte Hypertrophy and Increases Glucose Transporter Expression in Obese Mice.

Metabolic syndrome is known to engender type 2 diabetes as well as some cardiac, cerebrovascular, and kidney diseases. Mirtazapine-an atypical second-generation antipsychotic drug with less severe side effects than atypical first-generation antipsychotics-may have positive effects on blood glucose levels and obesity. In our executed study, we treated male high-fat diet (HFD)-fed C57BL/6J mice with mirtazapine (10 mg/kg/day mirtazapine) for 4 weeks to understand its antiobesity effects. We noted these mice to exhibit lower insulin levels, daily food efficiency, body weight, serum triglyceride levels, aspartate aminotransferase levels, liver and epididymal fat pad weight, and fatty acid regulation marker expression when compared with their counterparts (i.e., HFD-fed control mice). Furthermore, we determined a considerable drop in fatty liver scores and mean fat cell size in the epididymal white adipose tissue in the treated mice, corresponding to AMP-activated protein kinase expression activation. Notably, the treated mice showed lower glucose tolerance and blood glucose levels, but higher glucose transporter 4 expression. Overall, the aforementioned findings signify that mirtazapine could reduce lipid accumulation and thus prevent HFD-induced increase in body weight. In conclusion, mirtazapine may be useful in body weight control and antihyperglycemia therapy.

Quinolone and Organophosphorus Insecticide Residues in Bivalves and Their Associated Risks in Taiwan.

Bivalves, such as freshwater clams (Corbicula fluminea) and hard clams (Meretrix lusoria), are the most extensive and widely grown shellfish in land-based ponds in Taiwan. However, few studies have examined the contamination of bivalves by quinolone and organophosphorus insecticides. Thus, we adapted an established procedure to analyze 8 quinolones and 12 organophosphorus insecticides using liquid and gas chromatography-tandem mass spectrometry. Surveys in Taiwan have not noted high residual levels of these chemicals in bivalve tissues. A total of 58 samples of freshwater or hard clams were obtained from Taiwanese aquafarms. We identified 0.03 mg/kg of enrofloxacin in one freshwater clam, 0.024 mg/kg of flumequine in one freshwater clam, 0.02 mg/kg of flumequine in one hard clam, 0.05 mg/kg of chlorpyrifos in one freshwater clam, 0.03 mg/kg of chlorpyrifos in one hard clam, and 0.02 mg/kg of trichlorfon in one hard clam. The results indicated that 5.17% of the samples had quinolone insecticide residues and 5.17% had organophosphorus residues. However, the estimated daily intake (EDI)/acceptable daily intake quotient (ADI) indicated no significant risk and no immediate health risk from the consumption of bivalves. These results provide a reference for the food-safety screening of veterinary drugs and pesticides in aquatic animals. Aquatic products should be frequently screened for residues of prohibited chemicals to safeguard human health.

Fluoroquinolone-resistant and extended-spectrum ß-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan.

BACKGROUND/PURPOSE: Escherichia coli is a common pathogen to cause clinical and subclinical mastitis in cows. A total of 57 E. coli isolates from raw milk from cows were characterized genetically and biochemically. METHODS: Extended-spectrum ß-lactamase (ESBL) genes, the mechanism for fluoroquinolone resistance, and variations in virulence genes and genomes of these E. coli isolates were investigated by the antimicrobial susceptibility test, simplex and multiplex polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE). RESULTS: All E. coli isolates were resistant to cloxacillin (100%) and to a lesser extent (50%) to tetracycline, neomycin, gentamycin, ampicillin, ceftriaxone, cefotaxime (CTX), and ceftazidime (CAZ). Nearly 70% of the isolates were resistant to at least two antimicrobials and 28.1% carried AmpA and AmpC genes simultaneously. The predominant bla gene was blaTEM, followed by blaCMY, blaCTX, blaSHV, and blaDHA. Among the six (10.5%) ESBL-producing E. coli carrying blaCTX-M15, blaCTX-M55, or blaCTX-M14, two isolates 31 of ST410 in the ST23 complex and 58 of ST167 in the ST10 complex were also resistant to ciprofloxacin, enrofloxacin, and levofloxacin, with mutations at codon 83 from serine to leucine and codon 87 from aspartic acid to asparagine in GyrA and at codon 80 from serine to isoleucine in ParC. These isolates were genetically diverse in pulsotype analysis, lacked toxin genes of human pathogenic E. coli and carried mostly the prevalent virulence genes fimH, papGII, and α-hemolysin. CONCLUSION: Lacking virulence genes examined, genetic diverse E. coli isolates are unrelated to human pathogenic E. coli. Enhancing sanitation in milk processing and transportation is needed to eliminate multidrug-resistant (MDR), fluoroquinolone-resistant, and ESBL-producing E. coli isolates.

Salmonella Serovars and Vaccination Effect on the Immune Responses of Male and Female Layers.

Salmonella Enteritidis, S. Gallinarum and S. Pullorum are common serovars to infect poultry and cause diseases differently. The antibody production and cellular immune responses of male and female layers were evaluated before and after inoculation. Before inoculation, S. Gallinarum and S. Pullorum could survive and grow in 10% sera from 6-week-old layers, and S. Enteritidis and E. coli were completely eliminated. The weights of the male and female layers were increased the lowest by inoculation with S. Gallinarum, followed by S. Pullorum, and S. Enteritidis. Inoculation with S. Enteritidis, S. Gallinarum and S. Pullorum increased the antibody titer in the males depending on the serovars and maintained same higher antibody level in females. Furthermore, an increased anti-Salmonella IgG titer was associated with bactericidal ability and the level was reduced by serovars and complemente. Despite the vaccination and serovars, the male layers expressed more IgG2a than IgG1, indicating preferential activation of the Th1 pathway. The inoculation number affected the expression level of IFN-γ and IL-12 in the blood not in the secretion of the peripheral blood mononucleated cells (PBMCs) and more inoculations increased the expression of both cytokines. Inoculation increased more reactive oxygen species (ROS) production in polymorphonuclear (PMN) cells, not the PBMCs. ROS production was greater in cells from the males than from the females and greater in the cells treated with S. Enteritidis than S. Gallinarum and S. Pullorum. These three serovars and their vaccinations differed in sera killing and immune responses.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, ß, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Mycobacterium tuberculosis and M. bovis infection in Feedlot Deer (Cervus unicolor swinhoei and C. nippon taiouanus) in Taiwan.

BACKGROUND/PURPOSE: Mycobacterium bovis frequently infects wild and farm deer species with tuberculosis. This study investigated mycobacterial infection in two native deer species Cervus unicolor swinhoei (Formosan Sambar, Sambar) and C. nippon taiouanus (Formasan Sika, Sika). METHODS: Based on different sampling sources of 19 intradermal tuberculin test (ITT) Sambar, mycobacterial infection and/or species were detected by acid-fast stain, duplex polymerase chain reaction (PCR) and multiplex nested PCR (mnPCR) methods, traditional mycobacterial culture and gross lesion. Blood samples of 167 Sambar deer and 147 Sika deer were then tested by duplex PCR and mnPCR methods to investigate the prevalence of mycobacterial infection. Sequence variations of these mycobacterial species were analyzed as well. RESULTS: Duplex PCR and mnPCR assays could differentiate between MTBC (M. bovis and M. tuberculosis) and M. avium, as well as between M. bovis and M. tuberculosis, respectively. These PCR methods showed a higher detection rate than traditional culture and matched the gross lesions examined in 19 ITT-examined Sambar. Therefore, the mycobacterial infection in blood samples of 314 deer samples was detected using these PCR methods. Duplex PCR and mnPCR showed an identical prevalence of 16.1% in Sambar and 8.2% in Sika and a significant difference in prevalence between these two deer species. M. bovis and M. tuberculosis were the species detected in feedlot Sambar and Sika. M. tuberculosis was found only and first in Sambar fed in central Taiwan. Sequence analysis revealed diverse genetic variations in M. bovis and M. tuberculosis associated with deer subspecies. CONCLUSION: Multiplex PCR methods were established, and M. bovis and M. tuberculosis were identified in feedlot deer in Taiwan. Sequence variations indicated diverse sources of both mycobacterial species.

Genetically divergent methicillin-resistant Staphylococcus aureus and sec-dependent mastitis of dairy goats in Taiwan.

BACKGROUND: Widespread in the environment, Staphylococcus spp. infect animals and humans as normal flora or pathogens. By extending our recent report of multi-drug resistant (MDR) S. aureus in dairy goats, this study investigated the staphylococcal infection and characterized the MDR-S. aureus and methicillin-resistant S. aureus (MRSA) isolates collected from goats in 2008 to elucidate the appearance of MRSA in goats and the mastitis associated staphylococcus enterotoxin (SE) types. A total of 555 samples were collected from six goat parts and three environmental sources among four dairy goat farms in southern Taiwan. Coagulase-positive and negative Staphylococcus spp. (CPS and CNS, respectively) were also identified. Furthermore, predominant SE genes of nine enterotoxin genes sea through sej along with antimicrobial resistance and genetic variations were determined. RESULTS: In total, 137 staphylococcal strains were identified and found predominantly in milk, and in the vagina, anus, and nasal cavity. The most prevalent species was S. lentus, followed by S. aureus, S. epidermidis, and S. xylosus. Enterotoxin genes were not identified in any CNS isolates, however sec and see were identified only in S. aureus associated with mastitis in goat. In compared to the isolates from 2006 to 2007, 27 S. aureus isolates from 2008 were found to be more resistant to ampicillin, cephalothin, oxacillin, oxytetracycline, penicillin G, and tetracycline. Eleven MRSA isolates were identified and belonged to SCCmec type III (nine isolates) as the major type and SCCmec type II (two isolates). These MRSA isolates revealed pulse-field gel electrophoresis (PFGE) pattern A (five isolates), C (one isolate), and D (one isolate) of human isolates. The other two isolates without pulsotypes belonged to ST59. CONCLUSION: The prevalence and infection sites of CNS differed from those of CPS. Genetic analyses indicated that genetic divergence, possible zoonotic transfer of MRSA, and the involvement of sec as important virulence factors for of S. aureus that lead to mastitis in goats.

Emergence of Salmonella enterica serovar Potsdam as a major serovar in waterfowl hatcheries and chicken eggs.

Salmonellosis is a common food-borne illness in humans caused by Salmonella-contaminated poultry and their products. In hatcheries, 110 Salmonella isolates were identified, mostly from first enrichment, and few from delayed enrichment. The Salmonella prevalence in goose and duck hatcheries was higher when measured by four multiplex PCR methods than by traditional culture (73.8% vs. 44.35%, P < 0.05); 97.3% of 110 isolates were Salmonella Potsdam of serogroup C1 and other isolates were Salmonella Montevideo of C1 and Salmonella Albany of C2. Plasmid and pulsed field gel electrophoresis genetic analysis revealed that isolates from duck hatcheries were more diverse than those from goose hatcheries. In Salmonella Potsdam, host species-specific genotypes were observed in geese for genotypes 3, 4, and 5 and in ducks for genotypes 7, 8, and 9, suggesting that Salmonella Potsdam may evolve into goose- and duck-specific isolates. An examination of 1121 eggs found that only Salmonella Potsdam was identified in 1.8% (7/591) of eggs from chickens fed on the ground, not housed in cages, and in egg content (6/7) as well as eggshell membrane (1/7). In conclusion, Salmonella Potsdam may be a major Salmonella infection in waterfowl and chicken eggs.

Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence.

Chicken coccidia are protozoan parasites of the genus Eimeria. They cause economical losses in the poultry industry globally. The various species can be distinguished on the basis of the morphology of the oocysts and parasitic site in intestine, but these criteria sometimes are unreliable. Therefore, a species-specific polymerase chain reaction (PCR) was developed. Based on variable sequence regions, specific primers were constructed for the differentiation of five Eimeria species (Eimeria acervulina, E. brunette, E. maxima, E. necatrix, and E. tenella). PCR products were amplified from coccidian vaccine (coccivac-D and coccivac-B) and E. tenella and were subsequently sequenced. Similarities of the five species sequences between the vaccines and Genbank were 94-100%. Analysis of the E. tenella internal transcribed spacer 1 (ITS-1) partial sequence from Taiwan and from Genbank indicated that the similarity was 99.6%. The PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. The five sets of primers will not amplify any non-specific bands of the chicken genome or its intestinal contents. Therefore, the five sets of specifically designed primers are guaranteed to be useful for differential diagnosis of avian coccidiosis caused by Eimeria spp.