Yao-Chi Su

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

BackgroundWidespread 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.ResultsIn 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.ConclusionThe 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.

Comparison of the Association of Age with the Infection of Salmonella and Salmonella enterica Serovar Typhimurium in Pekin Ducks and Roman Geese

Nontyphoid Salmonella have a broad host range in poultry and mammals, and serovar Typhimurium is a threat to public health. In this study, normal and sick ducks and geese were collected from 12 farms in Taiwan to investigate the age-associated infection of Salmonella and Salmonella Typhimurium in Roman geese (Anser anser domesticus) and Pekin ducks (Anas platyrhynchos domesticus). In normal birds, the prevalence of Salmonella differed between species, and with age [e.g., 1-wk group, 37.5% (30/80) for ducks and 5.2% (6/116) for goslings (P < 0.05) vs. 4-wk group, 1% (1/96) for ducks and 12.1% (21/174) for geese]. Salmonella Typhimurium was identified from the visceral organs of moribund young geese suffering with colibacillosis and riemerellosis isolated from 2 goose farms (farm A and B, respectively). At farm B, 22.9% (27/118) of 4-wk geese with diarrhea were Salmonella Typhimurium-positive compared with 4.6% (8/174) of 4-wk normal geese. All Salmonella Typhimurium strains except one harbored a 94.7-kb virulence plasmid. Subcutaneous injection of Salmonella Typhimurium isolate 91NGL1 resulted in different clinical signs and pathogenesis between ducks and geese. In addition, the mean infectivity dose ratios of ducks to geese were 3.2 and 85.0 for 4- and 12-d birds, respectively, suggesting that goslings were more susceptible to Salmonella Typhimurium and resistance to Salmonella Typhimurium increased with age, especially for ducks. Therefore, Salmonella Typhimurium infection should be more common in goose farms than in duck farms, especially in the younger birds.

Fluoroquinolone-resistant and extended-spectrum β-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 β-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.

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.

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

Abstract 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.

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, β, 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.

Genetic and Antimicrobial Susceptibility Variations of Staphylococcus aureus from Dairy Goat Raw Milk

Eighty-six Staphylococcus aureus (S. aureus) isolates were collected from 17 dairy goat farms and were characterized as having eight biotypes, four coa genotypes and four nuc genotypes based on biochemical characteristics, coa genotyping, and nuc genotyping methods, respectively. Although the results of genotyping from these three methods differed, the most prevalent isolates detected were biotype 2, coa genotype 2, and nuc genotype A. The results of antimicrobial susceptibility tests to 16 antimicrobials showed that over 50% of isolates were resistant to ampicillin, oxytetracycline, penicillin, and tetracycline, whereas all were susceptible to methicillin, cephalothin, cefuroxime, enrofloxacin and bacitracin, and exhibited lower resistance to cloxacillin and sulfamethoxazole/trimethoprim. Nearly 80% of isolates exhibited multi-drug resistance (MDR). Gene tetM, rather then tetK, was mainly responsible for resistance to tetracycline. In conclusion, MDR S. aureus and one major genotype type circulated among dairy goat farms and no methicillin-resistant Staphylococcus aureus (MRSA) has been found.

Rapid Identification of Staphylococcal Infection in Dairy Goat Raw Milk by a Multiplex PCR Assay

To improve the diagnosis accuracy of staphylococcal infection in dairy goat with mastitis, we develops a multiplex polymerase chain reaction (PCR) assay by targeting specific regions of staphylococcal 16S rRNA for identification of staphylococcal infection, using specific nuc gene of S. aureus to distinguish S. aureus and the coagulase negative Staphylococci (CNS), and mecA gene responsible for oxacillin or methicillin resistance to determine methicillin resistant staphylococcus aureus (MRSA). The Analytical Profile Index (API) method, and PCR method were used to examine 3,427 milk samples from subclinically infected herds with traditional culture, and revealed 41.4% bacterial infection. E. coli, coagulase-negative staphylococci and S. aureus were the predominant bacteria and their prevalence differed among farms. Genotype Al S. aureus was clonally disseminated on most farms, except farms A, B, D, and E.

EFFECTS OF CHALLENGE METHODS AND FEEDING ADDITIVES ON ANTIBODY PRODUCTION AND GROWTH INHIBITION OF THE SERUM AGAINST SALMONELLA ENTERITIDIS, S. GALLINARUM AND S. PULLORUM IN CHICKEN

Salmonellosis in humans occurs by consumption of contaminated poultry and their products. To prevent the Salmonella infection in poultry, we investigated the maturity of peritoneal macrophages of chicks, the effect of feeding additives on the IgM and IgG levels and growth inhibition of Salmonella and egg contamination among four inoculation methods. The maximal yield of peritoneal macrophages was in 30- to 33-day-old chicks and intracellular Escherichia coli were eradicated in the macrophages of chicks aged >29-days old. Fungal β-glucan sources and serovars affected the IgM and IgG levels. Complement activity affected IgM measurement and the highest antibody levels were observed against S. Enteritidis compared with S. Pullorum/Gallinarum. Furthermore, β-glucans of Schizophyllum commune and Ganodermaincreased the IgM level against three serovars, while β-glucans of Ganoderma, Taiwanofungus camphoratus, and Botryosphaeria rhodina increased the IgG level against S. Enteritidis and S. Pullorum. In the growth inhibition, β-glucans from S. commune and T. camphoratus increased the inhibition against S. Gallinarum. Supplementation with β-glucan from S. commune in combination with antibiotics and/or herbal medicine or β-glucan from B. rhodina inhibited the growth of S. Pullorum. Feeding with β-glucans from S. commune and Cordyceps inhibited the growth of S. Enteritidis. Among cloacal, oral, intraperitoneal (IP) and intravenous (IV) inoculations, IP inoculation stimulated the maximum antibody level, while S. Enteritidis could not be eradicated from the eggs with the highest antibody level. In conclusion, additives with β-glucan can be used to enhance the immune responses of young and mature chicken against Salmonella infection.

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.