The molecular identification of Streptococcus equi subsp. equi strains isolated within New Zealand.

AIMS: To identify Streptococcus equi subsp. equi (S. equi) by PCR analysis and obtain isolates by culture, in order to investigate the strains of S. equi infecting horses within New Zealand. METHODS: A diagnostic PCR, based on the amplification of the seeI gene for S. equi, was used on 168 samples submitted from horses with and without clinical signs of strangles. Samples were also processed and cultured on selective media for the isolation of ß-haemolytic colonies. In addition, the hypervariable region of the seM gene of S. equi was amplified and then sequenced for strain typing purposes. RESULTS: Of the 168 samples, 35 tested positive for S. equi using PCR. Thirty-two confirmed samples were from horses with a clinical diagnosis of strangles and three were from horses where clinical information was unavailable. Only 22/35 (63%) confirmed S. equi samples were successfully isolated following culture. Strain typing demonstrated that two novel seM alleles of S. equi were found in New Zealand with SeM-99 strains being restricted to the North Island while SeM-100 strains were found in both North and South Islands. CONCLUSIONS: The application of PCR for the laboratory confirmation of strangles allowed for a rapid and sensitive identification of S. equi. Moreover, seM typing revealed that within the samples examined two strains of S. equi co-circulated within the North Island of New Zealand but only one strain in the South Island. CLINICAL RELEVANCE: PCR reduces the time required to obtain laboratory confirmation of strangles compared with culture methods. It also has greater sensitivity in detecting S. equi infections, which is of particular importance in the detection of carrier animals which normally shed low numbers of bacteria. Additionally, seM molecular typing can differentiate between bacterial strains, assisting in the monitoring of local strains of S. equi subsp. equi causing disease.

The role of connexin36 gap junctions in modulating the hypnotic effects of isoflurane and propofol in mice.

Gap junction blockade is a possible mechanism by which general anaesthetic drugs cause unconsciousness. We measured the sensitivity of connexin36 knockout mice to the hypnotic effects of isoflurane and propofol. The experimental endpoint was recovery of the righting reflex of the anaesthetised animals during 0.2% step-reductions in isoflurane concentration, or following intraperitoneal injection of propofol (100 mg.kg(-1) ). Connexin36 knockout animals were more sensitive to the hypnotic effects of isoflurane than 'normal' wild-type animals. The half maximal effective concentration (EC50) for recovery of righting reflex was 0.37% for connexin36 knockout vs 0.49% for wild-type animals (p < 0.001). For propofol, connnexin36 knockout animals showed more rapid loss of righting reflex than wild-type animals (mean (SD) 2.8 (0.13) vs 3.8 (0.27) min); and young (< 60 days) connexin36 knockout animals remained anaesthetised for longer than young wild-type mice (47.2 (2.9) vs 30.5 (1.7) min; p < 0.00001). These findings suggest that the hypnotic effects of anaesthetic drugs may be moderately enhanced by gap junction blockade.

The controlled in vitro susceptibility of gastrointestinal pathogens to the antibacterial effect of manuka honey.

The susceptibility of common gastrointestinal bacteria against manuka honey with median level non-peroxide antibacterial activity (equivalent to that of 16.5% phenol) was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a standardized manuka honey with the broth microdilution method. The measured sensitivity of bacteria showed that manuka honey is significantly more effective than artificial honey (a mixture of sugars as in honey), indicating that osmolarity is not the only factor that is responsible for the antibacterial activity of the honey. Most tested gastrointestinal pathogens have MIC and MBC values in the range of 5-10% of honey, other than Enterobacter spp. which was in the range of 10-17%. The difference in efficacy between the honey with and without hydrogen peroxide removed was also studied, and it was found that both hydrogen peroxide and the non-peroxide components contribute to the bacteriostatic and bactericidal activity of the honey. It was also found that treatment against multi-antibiotic resistant microorganisms such as Salmonella typhimurium DT104 and ESBL-producing organisms with manuka honey may be promising.

Experimentally induced intramammary infection with multiple strains of Streptococcus uberis.

The effect of infusing a mixture of 5 Streptococcus uberis strains into mammary quarters of 10 lactating cows was investigated. All 5 strains, which included 2 originally isolated from the dairy environment and 3 from clinical cases of mastitis, were capable of establishing an intramammary infection when infused individually. However, when the 5 strains were infused together, a single strain predominated in 7 out of 10 quarters. One strain in particular prevailed in 4 mammary quarters and was also found to inhibit the growth of the other 4 strains with deferred antagonism on esculin blood agar. The genes required for the production of bacteriocins nisin U and uberolysin were identified in this strain, whereas the other 4 strains contained only uberolysin genes. Direct competition may have occurred between strains within the mammary gland but competition was not apparent when cultured together in UHT milk, where no strain predominated. Although the mechanism is unknown, these results imply that a selection process can occur within the mammary gland, leading to a single strain that is detected upon diagnosis of mastitis.

Somatotropic axis components and nutrient partitioning in genetically diverse dairy cows managed under different feed allowances in a pasture system.

The somatotropic axis [including growth hormone (GH), GH receptor, and insulin-like growth factor (IGF)-I] is uncoupled in high-producing cows in early lactation so that the liver fails to respond to GH and produces less IGF-I. This uncoupling was implicated in the process of nutrient partitioning, enabling high milk production. Different genetic selection goals may affect functional components of the somatotropic axis. Thus, the somatotropic axis was examined in diverse genetic strains of dairy cows [North American Holstein 1990 (NA90), New Zealand Holstein-Friesian 1990 (NZ90), and New Zealand Holstein-Friesian 1970 (NZ70)] that were managed similarly within a pasture-based system but were offered feed allowances commensurate with their genetic ability to produce milk. The NA90 cows produced more milk (26.2 +/- 0.3, 24.1 +/- 0.3, and 20.1 +/- 0.4 kg/d, for NA90, NZ90, and NZ70, respectively), but had lower milk fat percentages (4.28 +/- 0.03, 4.69 +/- 0.03, and 4.58 +/- 0.04 kg/d for NA90, NZ90, and NZ70, respectively) compared with both NZ strains. Milk protein percentages (3.38 +/- 0.02, 3.52 +/- 0.02, and 3.29 +/- 0.03 kg/d for NA90, NZ90, and NZ70, respectively) were greater for NZ90 cows. During early lactation (wk 2 to 6), the total net energy produced in milk was greater in NA90 compared with NZ90 or NZ70 cows, but total net energy in milk after wk 6 was equivalent for NA90 and NZ90 cows. The greater milk production in early lactation in NA90 cows was associated with lower body condition scores (BCS; 1 to 10 scale; 4.0 +/- 0.1) elevated blood GH concentrations (1.6 +/- 0.1 ng/mL), and low blood IGF-I concentrations (14.8 +/- 1.1 ng/mL), indicating an uncoupled somatotropic axis. In comparison, the NZ70 cows retained a coupled somatotropic axis during early lactation, maintaining greater BCS (4.6 +/- 0.1), lower blood GH (0.7 +/- 0.1 ng/mL), and greater blood IGF-I (21.9 +/- 1.2 ng/mL). The degree of uncoupling in NZ90 cows was intermediate between the other 2 strains. Additional feed allowance failed to change blood IGF-I concentrations in NA90 cows but increased IGF-I concentrations in NZ90 cows (20.9 +/- 1.4 and 13.2 +/- 1.4 ng/mL for the high and low feed allowance, respectively). Furthermore, additional feed allowance in NZ90 cows lessened BCS loss in early lactation, but did not affect BCS loss in NA90 cows. Functional components of the somatotropic axis differed for the respective strains and were consistent with strain differences in milk production, BCS, and feed allowance.

The in vitro susceptibility of Campylobacter spp. to the antibacterial effect of manuka honey.

We report the antimicrobial effect of manuka honey against Campylobacter spp. isolated by a diagnostic laboratory from specimens from a community in New Zealand. The isolates were differentiated according to species level using multiplex PCR. C. jejuni (20 strains) and C. coli (7 strains) were identified. The clinical isolates identified and type culture collection strains of these species were subjected to testing to determine the minimum inhibitory concentration (MIC) of manuka honey using a microdilution technique. The MIC of the manuka honey against all of the Campylobacter tested was found to be around 1% (v/v) honey. The low MIC values suggest that honey might still inhibit the growth of campylobacteria after dilution by fluid in the gut, but the actual concentration of honey that can be achieved in the intestine is unknown. Therefore, clinical investigation is required to establish the efficacy of honey against Campylobacter spp. in the gut environment.

Alpha1-adrenergic receptor mRNA and inflammatory mediator expression in circulating leucocytes after cardiac surgery.

Vasodilation after coronary artery bypass surgery is a common complication. Inflammatory mediators influence the expression of alpha1-adrenergic receptors. Do patients requiring high doses of postoperative inotropic support have down-regulated alpha-adrenergic receptors? Is there a characteristic pattern of preoperative inflammatory mediator expression that could predict a complicated course after the operation? Forty-four patients undergoing cardiac bypass surgery with extracorporeal circulation were prospectively investigated. Five perioperative blood samples were taken (preoperative, two hours, 12 hours, 36 hours and 72 hours postoperative). The leucocyte mRNA-expression of the three alpha1-adrenergic receptor subtypes (A, B and D) and 11 different pro-inflammatory mediators were investigated with the real-time reverse transcriptase polymerase chain reaction. The patients were divided into three groups (No-noradrenaline [No-NA]= 0 microg/min, Low-noradrenaline [Low-NA]=0.1-7 microg/min, High-noradrenaline [High-NA] >7 microg/min), according to their postoperative noradrenaline requirements. Preoperatively, alpha1(A)-receptor expression was 4.9-fold (High-NA) and 18.7-fold (Low-NA) higher than the No-NA group (P=0.005) and plasma noradrenaline levels were higher in the High-NA group (P=0.005). Across all groups at 12 hours after the operation, alpha1(A) -receptor expression decreased to approximately one-fifth of preoperative levels (P=0.01); but with greater duration and magnitude of relative decrease in the High-NA group. Patients in the No-NA group had significant postoperative increases in leucocyte inflammatory mediator expression for IL-1beta, TLR4, TREM, MPO, MMP9 and TNF genes, whereas the changes in the Low-NA and High-NA groups were not significant. Low preoperative levels of noradrenaline and low expression of alpha1(A)-adrenoreceptors in leucocytes was associated with less probability of requiring noradrenaline support after cardiac surgery.

Metabolic adaptations associated with irreversible glucose loss are different to those observed during under-nutrition.

In this study the hypothesis that irreversible glucose loss results in an 'uncoupling' of the somatotrophic axis (increasing plasma GH levels and decreasing plasma IGF-I) was tested. During periods of negative energy balance the somatotrophic axis respond by increasing plasma GH and decreasing plasma IGF-I levels. In turn, elevated GH repartitions nutrient by increasing lipolysis and protein synthesis, and decreases protein degradation. Irreversible glucose loss was induced using sub-cutaneous injections of phloridizin. Seven non-lactating cows were treated with 8g/day phloridizin (PHZ) and seven control animals (CTRL, 0g/day), while being restricted to a diet of 80% maintenance. PHZ treatment increased urinary glucose excretion (P<0.001), resulting in hypoglycemia (P<0.001). As a response to this glucose loss, the PHZ treated animals had elevated plasma NEFA (P<0.005) and BHBA (P<0.001) levels. Average plasma insulin concentrations were not altered with PHZ treatment (P=0.059). Plasma GH was not different between the two groups (P>0.1), whereas plasma IGF-I levels decreased significantly (P<0.001) with PHZ treatment. The decline in plasma IGF-I concentrations was mirrored by a decrease in the abundance of hepatic IGF-I mRNA (P=0.005), in addition the abundance of hepatic mRNA for both growth hormone receptors (GHR(tot) and GHR(1A)) was also decreased (P<0.05). Therefore, the irreversible glucose loss resulted in a partial 'uncoupling' of the somatotrophic axis, as no increase in plasma GH levels occurred although plasma IGF-I levels, hepatic IGF-I mRNA declined, and the abundance of liver GH receptor mRNA declined.

Field observations on the variation of Streptococcus uberis populations in a pasture-based dairy farm.

Microbiological and molecular tools were used to monitor Streptococcus uberis populations on farm tracks and paddocks on a dairy farm during different seasons of a year to identify and profile potential environmental niches of Strep. uberis in a pasture-based dairying system. Farm tracks of high or low cow traffic were sampled every 2 wk for an entire year and Strep. uberis numbers were enumerated from a selective medium. During each season of the year, paddocks were sampled for the presence of Strep. uberis before and after grazing by dairy cows. Farm tracks of high cow traffic generally had greater concentrations of Strep. uberis isolated compared with tracks with less cow traffic, but there was also significant variation in the concentrations of Strep. uberis contamination among seasons, being highest in winter and lowest in summer. The bacterium was detected in paddocks only after cow grazing had occurred, but the bacteria could still be detected in soil for up to 2 wk following grazing in winter. Multilocus sequence typing showed great heterogeneity, with some commonality between farm track and milk isolates, which may help explain cow-to-environment or environment-to-cow transmission of the bacterium in the dairy setting.

Parenteral treatment of clinical mastitis with tylosin base or penethamate hydriodide in dairy cattle.

The objective of this study was to compare the clinical and bacteriological cure rates of cows with clinical mastitis following treatment with either tylosin base (5 g injected 3 times at 24-h intervals; n = 306) or penethamate hydriodide (5 g injected 3 times at 24-h intervals; n = 289). Duplicate milk samples were collected before treatment and again 14 +/- 3 and 21 +/- 3 d later for microbiological analysis. Only those quarters from which gram-positive mastitis pathogens were isolated before treatment were included in the analyses. Streptococcus uberis was the most prevalent isolate. The number of cows with clinical failure (i.e., retreated within 21 d of enrollment) did not differ between treatments (64 vs. 63, respectively). At the quarter level, there was no difference in the proportion of bacteriological cure between treatments (81.2 vs. 83.8% for penethamate hydriodide or tylosin, respectively). The proportions of clinical and bacteriological cure were influenced by age, herd, severity of mastitis, number of glands within the cow with clinical mastitis, bacterial species, and days postpartum at enrollment. There was no difference between treatment groups for SCC (4.46 vs. 4.44 +/- 0.08, mean +/- standard error of the difference in ln SCC for cows treated with penethamate hydriodide or tylosin, respectively) or production of milk solids (1.45 vs. 1.48 +/- 0.02 kg/d of milk fat + protein, for the penethamate hydriodide or tylosin treatment, respectively). Overall, there was no difference in the proportions of clinical failure (17.3 vs. 16.5% of cows treated with penethamate hydriodide or tylosin, respectively) or bacteriological cure (79.8 vs. 82.0% of cows treated with penethamate hydriodide or tylosin, respectively), or in SCC or milk production between dairy cows with clinical mastitis and those treated for clinical mastitis with 1 of 2 parenteral antibiotic therapies.

Virulence genes of Escherichia coli strains isolated from mastitic milk.

Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis. Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E. coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e. Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA. The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%). The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E. coli isolates and the systemic clinical condition of the respective infected cows.

The use of real time rtPCR to quantify inflammatory mediator expression in leukocytes from patients with severe sepsis.

Real-time reverse transcriptase polymerase chain reaction (RT rtPCR) was used to quantify the pattern of inflammatory mediator mRNA expression in circulating leukocytes from adult patients diagnosed with severe sepsis. We analysed 29 blood samples from 26 severely septic patients with different septic sources and eight samples from eight healthy adult volunteers. RT rtPCR was used to quantify mRNA expression of 21 different inflammatory mediators in peripheral leukocytes. The median variability in gene expression in the sepsis patients was 10.5 times greater than the variability of the healthy comparison group. We found a significant change in the regulation for the following genes: C5aR (20-fold, P < 0.001), IL-8 (29-fold, P < 0.001), MMP9 (72-fold, P < 0.001), HSP70 (2.4-fold, P = 0.02), and RIP2 (1.8-fold, P < 0.04) were up-regulated. Conversely the median expression of IFNgamma, and IL-6 were zero (P < 0.001), and mtHSP (0.4-fold, P = 0.02) was significantly down-regulated. Using linear discriminant analysis, IFNgamma, IL-12, and TLR4 were correlated to a negative outcome. Different septic sources (peritonitis, burn, pneumonia and musculo-skeletal infections) resulted in significantly different mRNA patterns. The RT rtPCR is a useful tool to monitor the immune response in septic patients. We found a very high variability in inflammatory mediator expression among septic patients compared to healthy volunteers. This suggests that any future immune-modulatory therapy may need to be individualized to the patient's requirements as monitored by RT rtPCR. Different sources of sepsis may result in markedly different activation patterns.

Messenger RNA expression of multiple immune mediators in leukocytes from elective orthopaedic surgical patients.

The aim of this longitudinal observational study was to investigate and describe the spectrum of messenger ribonucleic acid (mRNA) expression of multiple inflammatory markers in circulating leukocytes after major orthopaedic surgery. We studied ten elective arthroplasty patients perioperatively on the orthopaedic ward, and eight healthy volunteers for a comparison group. Venous blood specimens were collected preoperatively, and 6, and 24 hours postoperatively, together with 6- and 24-hour postoperative wound drain specimens. The mRNA of 21 different inflammatory mediators was measured by real-time reverse transcriptase Polymerase Chain Reaction. Comparisons were made with the venous blood of eight healthy comparison subjects. There were significant differences (P<0.01) between preoperative specimens and normal comparisons (i.e. higher MPO, PDGF, TREM and IRAKM; lower mtHSP) reflecting the effects of chronic inflammation associated with osteoarthritis. There were significant increases (P<0.01) in expression of IL-8, MPO, IL-1beta, TREM, MMP9, and C5aR in circulating blood at 24 hours postoperatively, but not at six hours. There was no significant decrease in expression of any inflammatory mediator. There was no statistical difference in inflammatory mediator expression between drain specimens and venous specimens taken at the same time. We conclude that, in uncomplicated orthopaedic surgical patients, there was up-regulation of some cytokine mRNAs at both the local and systemic levels during the first day after surgery. We observed no evidence of immune compartmentalization, and found no evidence for innate immune paresis within the first day after surgery.

Molecular typing of Streptococcus uberis strains isolated from cases of bovine mastitis.

The discriminatory power of two polymerase chain reaction-based DNA fingerprinting methods, random amplified polymorphic DNA and repetitive extragenic palindrome were compared by subtyping 128 isolates of Streptococcus uberis cultured from cows in six different dairy herds in New Zealand. The typing results demonstrated that the majority of isolates possessed unique fingerprint profiles except on occasions where multiple isolates were obtained from individual cows. On these occasions, individual quarters of the mammary gland were generally, but not exclusively, infected by the same strain of bacteria. Both random amplified polymorphic DNA and repetitive extragenic palindromic typing assays were simple to perform, relatively inexpensive ($11.00 per reaction), and provided reliable and reproducible results. Furthermore, when these assays were used in conjunction with each other, they provided a means of confirmation of the specific DNA fingerprint patterns obtained.

Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing.

OBJECTIVE: To confirm the sensitivity of the polymerase chain reaction (PCR) technique (versus blood cultures) and to gain a better understanding of the incidence of true- and false-positive results when using this technique. DESIGN: Observational study. SETTING: Fourteen-bed, level 3 intensive care unit. PATIENTS: Hundred twenty-six critically ill adult patients. Hundred ninety-seven blood culture and PCR samples taken as clinically indicated for suspected sepsis, according to routine ICU protocol. MEASUREMENTS AND RESULTS: The PCR product (16SrDNA: 341F-1195R) was sequenced and compared with a database of known species (Genebank) to identify the bacterial nucleic acid. The PCR or blood culture result was classified as a true-positive if there was other microbiological or clinical supporting evidence.

Generic polymerase chain reaction followed by DNA sequencing as a means of diagnosing bacteraemia.

There is increasing use of polymerase chain reaction techniques to diagnose infection. We report the use of polymerase chain reaction using a generic section of the bacterial 16S rDNA gene--followed by nucleotide sequencing--to determine the species of the infecting bacteria. In the first case, the clinical and microbiological diagnosis of meningococcal septicaemia was in agreement with the results from polymerase chain reaction technique. In the second case, a Yersinia enterocolitica bacteremia was detected by the polymerase chain reaction technique, but missed with conventional blood culture techniques.

Single-strand conformational polymorphism (SSCP)-detected p53 gene mutations are a less sensitive marker of malignancy in pleural fluids than p53 immunostaining.

p53 immunostaining has been advocated as a marker of malignancy in pleural biopsies and serous fluids. The object of this study was to compare the sensitivity and specificity of p53 immunostaining for the detection of malignant cells in pleural fluids with a technique designed to detect p53 gene mutations in exons 5, 6, 7 and 8 by SSCP and nucleotide sequencing. Five out of eight pleural fluids containing adenocarcinoma showed p53 immunostaining and two of these also showed polymorphisms on SSCP and a mutation on sequencing. None of the 10 benign pleural fluids showed immunostaining for p53 or polymorphisms on SSCP. We believe that the poor sensitivity of p53 gene mutation by SSCP is mainly due to DNA from the background reactive cells 'swamping' the mutant DNA. We do not advocate its use as a diagnostic aid.

The use of polymerase chain reaction to detect septicemia in critically ill patients.

OBJECTIVE: To describe the use of bacterial DNA amplification of conserved bacterial 16S ribosomal DNA nucleotide sequences by polymerase chain reaction (PCR) to detect the presence of septicemia in critically ill septic patients. DESIGN: Case series of blood samples from septic patients comparing the PCR results with conventional blood culture results. SETTING: A general intensive care unit in a tertiary referral hospital. PATIENTS: Two sets of samples (n = 101 and n = 55) from patients diagnosed as clinically septic and requiring blood cultures. They were classified by internationally accepted criteria into systemic inflammatory response syndrome, severe sepsis, and septic shock groups. INTERVENTIONS: Blood samples taken in a sterile fashion concurrently for blood culture, and PCR of the bacterial 16S ribosomal RNA gene in leukocytes and plasma. Two different DNA extraction techniques for PCR were tried sequentially. MEASUREMENTS AND MAIN RESULTS: Blood culture and PCR positivity were measured in relation to the clinical classification of severity of sepsis. Using the initial extraction method (n = 101), ten patients were positive by both PCR and blood culture, eight patients were PCR positive and blood culture negative, and seven patients were blood culture positive and PCR negative. From the clinical criteria, PCR detected at least six true positives that had been missed on blood culture and missed four true Gram-positive bacteremias. When the initial code was broken, this deficiency was rectified using the improved extraction technique (n = 55), in which ten patients were positive by PCR and blood culture, 29 patients were PCR positive and blood culture negative, and two patients were PCR negative and PCR positive. CONCLUSIONS: We conclude that the use of PCR (for the 16S ribosomal DNA in the plasma) was significantly more sensitive than the use of conventional blood culturing techniques for the detection of bacteremia in seriously ill patients. This could prove to be a valuable adjunct to conventional blood cultures.