Transduction of bla(CMY-2), tet(A), and tet(B) from Salmonella enterica subspecies enterica serovar Heidelberg to S. Typhimurium

Antimicrobial resistance of Salmonella spp., especially resistance mediated by extended spectrum β-lactamases (ESBL), is a growing public health concern. Understanding the mechanisms through which Salmomella spp. acquire the resistance genes can lead to the development of intervention and mitigation strategies. Thirty-one Salmonella isolates of bovine origin were analyzed by serotyping, antimicrobial susceptibility testing, phage induction and bacterial host range determination, and phage transduction of antimicrobial resistance. The Salmonella isolates consisted of 12 serovars. Resistance to 1 or more antibiotics was detected in 12 isolates. Inducible phages were recovered from 19 Salmonella (61%) by spot lysis assay. Of the 19 phage samples, 13 were able to multiply in 2 or more Salmonella of various serovars. A cross-serovar transduction of antimicrobial resistance was observed from S. Heidelberg to S. Typhimurium. Transfection of an antimicrobial-susceptible strain of S. Typhimurium with a phage propagated in a S. Heidelberg resistant to multiple β-lactam antibiotics and tetracycline resulted in independent acquisition of bla_CMY-2, tet(A), and tet(B). These data indicate that inducible phages are common in Salmonella of bovine origin, many of them demonstrate a broad host range, and wild-type phage mediated transduction may contribute to the dissemination of antimicrobial resistance, including the resistance mediated by ESBL.     

Characterization of a nonfimbrial mannose-sensitive hemagglutinin (MSH) produced by Salmonella enterica serovar enteritidis

A nonfimbrial mannose-sensitive hemagglutinin (MSH) with adhesive properties produced by Salmonella enterica serovar Enteritidis was characterized. The MSH was characterized as glycoprotein and consisted of three noncovalently bound subunits of M_r 28, 33 and 40 kDa determined by SDS-PAGE. The hemagglutinin was heat-stable and resistant to alkaline (high) or acid (low) pH, however, it was inhibited by proteolytic enzymes, by EDTA and by sodium periodate. Mouse antiserum raised against MSH reacted with the 28 kDa band in immunoblotting, and also inhibited hemagglutination and bacterial adherence to HeLa cells. Electron microscope examinations showed that MSH is not a fimbriae-like structure. MSH and anti-MSH IgG competitively inhibited bacterial adherence to HeLa cells. The immunofluorescence test, using MSH on HeLa cells and specific anti-MSH IgG, supported the view that MSH contributes to adherence of the organism. These results indicate that MSH is a nonfimbrial putative adhesive factor that may mediate the adherence of Salmonella enteritidis to eucaryotic cells.     

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.     

Molecular typing by random amplification of polymorphic DNA (RAPD) and detection of virulence genes of Salmonella enterica subspecies enterica serovar Gallinarum biovar gallinarum

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type.     

A new concept to stimulate mucosal as well as systemic immunity by parenteral vaccination as applied to the development of a live attenuated Salmonella enterica serovar Dublin vaccine

A new concept of slow "drip feeding" that enables activation of mucosal as well as systemic immunity following parenteral vaccination was demonstrated using Salmonella Dublin in a mouse model. The live vaccine candidate, N-RM25, generated from a wild S. Dublin strain utilising metabolic-drift (spontaneous chromosomal) mutations had a unique sensitivity to bile and restricted growth in the presence of a very low concentration of bile salts No. 3 (0.075% (w/v)) but also had the ability to survive in a high concentration (19.2%) of the substance. Following intraperitoneal administration with 10(7) cfu, N-RM25 colonised and survived (10(1)-10(3) cfu/g) in the liver and spleen of mice for over 24 days without causing disease. A small number of the mutant organisms also penetrated the gall bladder and gut, most likely via the enterohepatic circulation. N-RM25 induced significant levels of serum IgG, IgA and intestinal secretory IgA. A second metabolic-drift mutant (R-NM29) that was rapidly eliminated from the liver and spleen and highly unlikely to penetrate the gall bladder and gut, stimulated some systemic immunity, but induced no mucosal immunity because it did not reach the immune stimulation sites within the gut. In vaccine trials, N-RM25 was significantly more effective in eliminating the homologous challenge bacteria (S. Dublin wild strain FD436) from the internal organs and intestinal lumen when compared to R-NM29 and the negative control. N-RM25 prevented the development of systemic infection and produced 100% protection.     

Danger to pigs due to crushing can be reduced by the use of a simulated udder.

Sows that lie on their young, pig "crushing", is a significant cause of pig mortality in current production systems. Although mortality rates of pigs in farrowing crates are lower than mortality rates of pigs in pens, loss due to crushing is still estimated to be between 4.8 and 18%. During the first few days after parturition, pigs are highly attracted to the odor of their dam's udder. Thus, our research was designed to move the pigs away from the sow by competing with the sow's udder using a "simulated" udder. Fifteen Yorkshire x Landrace sows and their litters (11.4+/-.78 pigs) were assigned to either a control (C, n = 9) or an experimental group (SU, n = 6). The C pigs had access to a heat lamp, whereas the SU pigs' crate had a simulated udder. Data were collected using time-lapse photography (1 frame/.4 s) for a 3-d duration at the initiation of farrowing. When a sow stood, data were recorded by 1-min scan samples to record the number of pigs using either the heat lamp or the simulated udder. In addition, stillborn pigs, pig crushing, and death by other means also were recorded. Data were analyzed by 12-h periods using generalized estimating equations. Results indicate that from 12 to 72 h postpartum, excluding 24 to 36 h postpartum, the estimated probability that pigs were in a safe area (simulated udder or heat lamp) was .89 for SU pigs, compared with only .72 for C pigs (P = .005). During the 24- to 36-h period, it was more probable to find pigs on a simulated udder (.77) than under only a heat lamp (.61, P = .016). Stillborn pigs, pig crushing, and death by other means were not different between treatments (mean = .87, .60, 1.2; P>.20). The simulated udder drew pigs away from the sow's udder better than heat lamps alone. Considering these findings, mortality of pigs due to crushing may be decreased substantially using a simulated udder. These results are promising, but further refinement should be done, including improved udder design and investigation of the attractiveness of various stimuli.     

Application of nucleotide sequence of RNA polymerase beta-subunit gene (rpoB) to molecular differentiation of serovars of Salmonella enterica subsp. enterica.

To establish a molecular differentiation method for Salmonella enterica subsp. enterica, a hyper-variable region of RNA polymerase beta-subunit (rpoB) of S. enterica subsp. enterica (I), serotype Typhimurium, and Escherichia coli were investigated through comparison of nucleotide sequence of the region. The hyper-variable region was identified at 612-937 of the gene. After PCR amplification of the region in the 17 serotypes and two biotypes of serotype Gallinarum of S. enterica subsp. enterica (I), the nucleotide sequences of the region were determined and compared. All serotypes were distantly related to E. coli with 82.8-84.7% identities in nucleotide sequence while showing 96.6-100% identities with each other. According to the phylogenetic analysis based on the sequenced region with the neighbor-joining method, relatedness of biotype Gallinarum to serotype Enteritidis and biotype Pullorum was determined. Biotype Gallinarum was more closely related to serotype Enteritidis than biotype Pullorum. These results suggested that the 612-937 variable region of rpoB might be useful for molecular evolutionary analysis of serotypes of S. enterica subsp. enterica (I).     

Changes in the risk management of Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7) in Swedish sheep herds and sheep meat due to the results of a prevalence study 2012

Background

The prevalence of Salmonella in food producing animals is very low in Sweden due to rigorous control programmes. However, no active surveillance is in place in sheep. The authorities decided to perform a prevalence study in sheep herds because findings at slaughter indicated that sheep associated S. diarizonae (S. enterica subspecies diarizonae serovar 61:(k):1, 5, (7)) might be common in sheep. Sampling was stratified by herd size in two groups, small herds with ≤ 30 animals and large herds with > 30 animals. In each stratum, 237 herds were selected at random. Faecal samples received from 244 out of the 474 randomly selected herds were analysed.

Results

A total of 40 of 100 (40%) of large herds and 17 of 144 (12%) of small herds were positive. The overall adjusted prevalence was 17.6% (95% CI, 12.9-22.2). Sheep associated S. diarizonae was detected in all counties (n = 21). Scientific opinions and an evaluation of on-farm control measures performed concluded that the impact of sheep associated S. diarizonae on human health is very low, and that risk management measures applied in response to findings of sheep associated S. diarizonae in sheep or sheep meat can be expected to have very little impact on reducing risks to human health. As a result, Swedish authorities decided to make an exemption for sheep associated Salmonella diarizonae in sheep and sheep meat in the current Salmonella control measures.

Conclusions

Sheep associated S. diarizonae is endemic in Swedish sheep herds. It is more common in large herds and not limited to certain parts of the country. The responsible authorities concluded that current risk management actions regarding sheep associated S. diarizonae in sheep and sheep meat are not proportional to the risk. This is the first time in the history of the Swedish Salmonella control programme that an exemption from the legislation has been made for a specific serovar. If there is any future indication of an increasing risk, due to e.g. change in the pathogenicity or development of antimicrobial resistance, the risk assessment will be re-evaluated and control measures reinforced if needed.     

应用高效体积排阻色谱法测定市场抽检口蹄疫灭活疫苗中的抗原（146S）含量

应用高效体积排阻色谱法对5批市场抽检口蹄疫灭活疫苗中有效抗原(146S)含量进行检测,并使用口蹄疫O型、A型和亚洲1型抗原测试卡对5批疫苗中的抗原进行鉴别。结果显示高效体积排阻色谱法测定146S标准品浓度与峰面积线性关系良好,5批疫苗均检测到146S特征吸收峰(R2=0.9937,n=7),经计算得到灭活疫苗中有效抗原含量分别为2.05、3.73、2.03、4.87、3.41μg/m L;使用抗原测试卡鉴别了5批疫苗中的抗原类型。结果表明,高效体积排阻色谱法简便、高效,准确度较高,有望在口蹄疫灭活疫苗的质量控制中发挥重要作用。     

Reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus-Escherichia coli vaccine in the dam.

An outbreak of neonatal diarrhea occurred among beef calves (2000 animals) from one large Argentinian farm in 1985. Rotavirus was detected in 78% (106/136) and enterotoxigenic Escherichia coli in 1.5% of the samples (2/136) obtained from sick calves. In comparison rotavirus was identified in only 1.6% (1/63) of the samples from clinically healthy calves. The rotavirus strain responsible for the outbreak was characterized as serotype 6 belonging to group A. In the following three years the protective capacity of a combined rotavirus-E. coli inactivated vaccine administered to the dams during the last third of the gestation period was evaluated on this farm by comparison of morbidity due to diarrhea in calves from vaccinated vs. placebo cows within the same year. The morbidity due to diarrhea among calves from dams in the vaccinated and placebo groups was 34% and 77%, respectively in 1986; 23% and 47% in 1987, and 15% and 34%, in 1988. In 1987 morbidity of diarrhea in calves born from vaccinated heifers was 54% and 74% in calves from placebo heifers. In 1988 morbidity from diarrhea was 41% and 54%, respectively among calves in these two groups. In all experiments, calves from heifers showed significantly greater morbidity than calves from cows. Differences in diarrhea morbidity between the vaccinated and placebo groups were statistically significant (P less than 0.05). Additional studies showed that the diarrhea had a significant influence (P less than 0.05) on the average live weight of the calves at weaning (5 to 7 months) with an average weight loss of 7.8 kg per calf among the calves affected with diarrhea.     

Serologic responses of Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), wallaroos (Macropus robustus), and chimpanzees (Pan troglodytes) to an inactivated encephalomyocarditis virus vaccine.

Encephalomyocarditis virus (EMCV) is a picornavirus with a worldwide distribution, capable of infecting a wide range of species. Episodes of EMCV-associated mortality have been reported in zoos and national parks around the world, including sporadic cases at Taronga Zoo, Sydney. An inactivated EMCV vaccine was evaluated by inoculating Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), Eastern wallaroos (Macropus robustus), and chimpanzees (Pan troglodytes). A proportion of the vaccinated ungulates were administered a second vaccination 4 wk after the initial dose. Neutralizing antibody titers were monitored for a period of 12 mo. One month after vaccination, all vaccinated groups had developed significant antibody titers that persisted for at least 6 mo. Animals receiving two doses of vaccine had higher titers 3, 6, and 12 mo after the initial vaccination compared with animals receiving a single vaccine dose.     

Outbreak of subclinical mastitis due to beta hemolytic group L streptococci (S. dysgalactiae ssp. equisimilis) in an Austrian dairy herd

This study is reporting an outbreak of subclinical mastitis due to beta-hemolytic group L streptococci in an Austrian dairy herd with a history of high somatic cell count. At the first survey 16 of 33 lactating cows (28 quarters of 132) were cultured positive for beta-hemolytic, CAMP and esculin negative cocci that grew on Columbia blood agar with small grey catalase negative colonies. With the commercial API 20 Strep system (bioMerieux, F) isolates were classified as members of streptococci group L. All tested strains (eight of 28) produced acid from ribose, lactose, trehalose, amidon and glycogen; they hydrolysed hippurate and showed beta-glucuronidase, beta-galactosidase, alkaline phosphatase, leucinaminopeptidase and arginindehydrolase activity. Isolates were sensitive to bacitracin but resistant to tetracycline. Using phenotypic characterisation as well as sequence analysis of the 16S-23S intergenic spacer region of a representative strain, recovered isolates were identified as Streptococcus (S.) dysgalactiae ssp. equisimilis. Mastitis was characterized by normal milk secretions and absence of clinical abnormalities but high elevations of somatic cell count. Based on the characteristics of the strains and on the observations during the first herd survey, contagious transmission during milking as a result of poor milking hygiene was assumed. The mastitis was controlled through implementation of a strict hygiene protocol including use of single-use udder towels, post milking teat desinfection and cluster disinfection between milking cows in combination with antibiotic treatment of infected udders.     

Inability of so-called chicken anemia agent (CAA) infections to be diagnosed by anemia and hematopoietic organ atrophy alone.

In the recent past, anatomic and clinical pathologic diagnoses of so-called chicken anemia agent (CAA) infections have been based on lesions such as anemia and hematopietic organ atrophy (HOA). In the present study, significant (P less than or equal to 0.05) positive and negative correlations were seen in a lesion matrix constructed from 89 cases of anemia and HOA in Georgia broilers during 1988 and 1989. Only splenic atrophy and bursal atrophy were not significantly associated. We concluded that information regarding only HOA and anemia is not sufficient to allow pathologists to diagnose CAA in broiler chickens submitted to diagnostic laboratories such as ours.     

Detection of antibodies to S. enteritidis in broilers by means of indirect ELISA and chemiluminescent immunoassay (CLIA)

This study was conducted to develop a serological detection system for the monitoring of broiler flocks for Salmonella enteritidis infections. A specific S. enteritidis antigen (FG-Antigen) was used to compare the sensitivity and the specificity of the chemiluminescent immunoassay (CLIA) with those of the indirect ELISA. This comparison was performed using a total of 578 sera, which, depending on the microbiological and vaccination history, were categorized into groups. Most of the serum samples which were classified as positive showed higher titers in CLIA than in ELISA. Using the prevalence of positive reactors, significant differences between Groups were additionally demonstrated. The absorbance values of the passively immunized group showed the highest and those of the Salmonella-negative group the lowest correlation-coefficient. Using the mean net absorbance of the prevalence group, the ELISA system exhibited a sensitivity of 100% and a specificity of 96.2%, while CLIA had a sensitivity and a specificity of 85.7% and 96.2%, respectively. ELISA and CLIA can be used in the examination of non vaccinated flocks for S. enteritidis-infections as alternative to the bacteriological culture method. CLIA is distinguished for its fast and convenient procedure as well as for its wider measurement spectrum, while the indirect ELISA is almost as efficient as CLIA and requires less investment.     

A DNA vaccine encoding MPB83 from Mycobacterium bovis reduces M. bovis dissemination to the kidneys of mice and is expressed in primary cell cultures of the European badger (Meles meles)

Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M. bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26 kDa major antigen (MPB83) of M. bovis was evaluated in a mouse model of disseminated M. bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerin (BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression of MPB83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis.     

Assessment of serological response of young and adult sheep to conjunctival vaccination with Rev-1 vaccine by fluorescence polarization assay (FPA) and other serological tests for B. melitensis

The serological response of young and adult sheep vaccinated conjunctivally with Rev-1 vaccine was assessed by fluorescence polarization assay (FPA), Rose Bengal test (RBT), complement fixation test (CFT), modified Rose Bengal test (m-RBT), indirect ELISA (i-ELISA) and competitive ELISA (c-ELISA), at different post vaccination intervals. One hundred and thirty six adult sheep and 64 lambs were used in the study. The vaccinated animals were bled prior to vaccination (0 day) and thereafter at 21st, 42nd, 35th, 63rd, 91st, 125th, 159th, and 223rd and 330th day post vaccination. The majority of animals (young and adult) showed positive reaction by FPA, RBT, CFT, m-RBT and c-ELISA 21 days post vaccination, whereas by i-ELISA at 42 days. All tests perform equal when animals vaccinated as young are tested 125 days (4 months) post vaccination. In case of animals vaccinated at adulthood, FPA, RBT, CFT and c-ELISA perform equal if the animals are tested 223 days (approximately 8 months) post vaccination. I-ELISA and m-RBT show low specificity if ewes vaccinated at adulthood are tested 330 days (11 months) post vaccination. If control of brucellosis in sheep is based on conjunctivally vaccination of lambs with Rev-1, the vaccinated animals can be tested by any test used for diagnosis of B.melitensis infection accurately at least 4 months post vaccination. If brucellosis control is based on mass vaccination the use of m-RBT and i-ELISA is not recommended for testing adult animals at least for 330 days (11 months) post vaccination due to tests low specificity. Further research is needed so the appropriate cut-offs to be established for FPA, c-ELISA or i-ELISA to become valuable tools for the eradication of Brucella spp. infection in small ruminants in areas where vaccination is practiced.