Immunohistochemical study of CD31 and α-SMA expression for age estimation of porcine skin wounds.

Age estimation of wounds in veterinary forensic investigations is based on the presence and amount of granulation tissue. However, accurate age assessment is challenging and new time-dependent markers are warranted to support and improve the current procedure. The objective of this study was to evaluate the expression of CD31-positive blood vessels and α-smooth muscle action (α-SMA)-positive myofibroblasts in granulation tissue in order to evaluate their value as markers for porcine wound age estimation in a veterinary forensic context. Immunohistochemical expression of CD31 and α-SMA in 14 experimental porcine skin wounds of different ages (4, 6, 8, 10, 18, 27 and 35 days) and 11 forensic porcine wound specimens (of unknown age) were evaluated. CD31-positive blood vessels and α-SMA-positive myofibroblasts were present in the granulation tissue in the experimental wounds at all time points. A significant decrease in the mean blood vessel counts was found in wounds aged 18, 27 and 35 days compared with wounds aged 6 days (P < 0.001), when assessing both the superficial and deep part of the wound bed. α-SMA expression was lower at 27 and 35 days post wounding compared with 6-18 days post wounding. Combined assessment of three parameters (mean blood vessel counts in the superficial and deep wound beds and α-SMA expression) could approximately specify the age of the wounds as either 6-18 days or ≥27 days. In two of the forensic cases a combination of the three parameters yielded results that were similar to the experimental wounds, indicating a wound age of 6-18 days or ≥27 days, respectively. In the remaining forensic cases a combination of the three parameters did not show the same expression pattern as in the experimental wounds. The results indicate that in some forensic cases the application of CD31 and α-SMA markers appeared to support the current procedure for porcine wound age estimation, but this must be combined with pathological characteristics.

Gross and histopathological evaluation of umbilical outpouchings in pigs.

Clinical presentations of umbilical outpouchings (UOs) in pigs cover a variety of pathological manifestations. Pigs with UOs often do not reach the abattoir as they die due to complications or are euthanized for welfare concerns. The primary objective was to characterize the gross appearance of UOs in pigs with respect to the different types of pathological manifestations. Also the association between the pathological manifestation and presence of a wound on the UO was evaluated. Pigs (in different age groups, n = 444) with an UO were sampled in Denmark from different locations (two herds and at an abattoir) and examined post mortem. Tissue samples from animals with an enterocystoma or internal umbilical proliferations were collected for histological and immunohistochemical characterization. Hernia umbilicalis was the most frequent cause (72%, n = 320) of the UOs. It was the only diagnosis in 57% (n = 254) of the pigs, and in 15% (n = 66) of the pigs the hernia appeared in combination with other manifestations. Thus, 28% (n = 124) of the pigs were diagnosed with an enterocystoma, internal umbilical proliferations, subcutaneous abscess/ fibrosis or another diagnosis, presented alone or in combination. The distribution of diagnoses varied in the different age groups. Overall, 38% (110/291) of the pigs presented a wound on the UOs post mortem. The age of the pigs confounded the relation between the pathological manifestation and the presence of a wound. The odds that an UO had a wound were lower among pigs with a subcutaneous abscess/ fibrosis compared to pigs diagnosed with an umbilical hernia or enterocystoma (OR, 0.3; 95% Cl, 0.1-0.7). The odds of wounds were higher among weaners (OR, 4.3; 95% Cl 2.3-8.3) and finishers (OR, 6.5; 95% Cl, 3.4-12.7) compared with piglets from the farrowing unit. The area of wounds ranged from 0.03 to 78.5 cm2 and increased with age (P < 0.001). Histologically and immunohistochemically the enterocystomas and internal umbilical proliferations seemed to be lined with mesothelial cells and both had a content comparable with mesenchymal embryonic connective tissue. However, only the cavities of the enterocystomas were also lined with mesothelial cells. In conclusion, UOs in pigs are caused by complex pathological conditions with hernia umbilicalis as the dominating diagnosis. Knowledge clarifying the different pathological manifestations causing an UO and the presence of wounds on the UOs is essential for future prevention strategies.

Atrial fibrillatory rate as predictor of recurrence of atrial fibrillation in horses treated medically or with electrical cardioversion.

BACKGROUND: The recurrence rate of atrial fibrillation (AF) in horses after cardioversion to sinus rhythm (SR) is relatively high. Atrial fibrillatory rate (AFR) derived from surface ECG is considered a biomarker for electrical remodelling and could potentially be used for the prediction of successful AF cardioversion and AF recurrence. OBJECTIVES: Evaluate if AFR was associated with successful treatment and could predict AF recurrence in horses. STUDY DESIGN: Retrospective multicentre study. METHODS: Electrocardiograms (ECG) from horses with persistent AF admitted for cardioversion with either medical treatment (quinidine) or transvenous electrical cardioversion (TVEC) were included. Bipolar surface ECG recordings were analysed by spatiotemporal cancellation of QRST complexes and calculation of AFR from the remaining atrial signal. Kaplan-Meier survival curve and Cox regression analyses were performed to assess the relationship between AFR and the risk of AF recurrence. RESULTS: Of the 195 horses included, 74 received quinidine treatment and 121 were treated with TVEC. Ten horses did not cardiovert to SR after quinidine treatment and AFR was higher in these, compared with the horses that successfully cardioverted to SR (median [interquartile range]), (383 [367-422] vs 351 [332-389] fibrillations per minute (fpm), P < .01). Within the first 180 days following AF cardioversion, 12% of the quinidine and 34% of TVEC horses had AF recurrence. For the horses successfully cardioverted with TVEC, AFR above 380 fpm was significantly associated with AF recurrence (hazard ratio = 2.4, 95% confidence interval 1.2-4.8, P = .01). MAIN LIMITATIONS: The treatment groups were different and not randomly allocated, therefore the two treatments cannot be compared. Medical records and the follow-up strategy varied between the centres. CONCLUSIONS: High AFR is associated with failure of quinidine cardioversion and AF recurrence after successful TVEC. As a noninvasive marker that can be retrieved from surface ECG, AFR can be clinically useful in predicting the probability of responding to quinidine treatment as well as maintaining SR after electrical cardioversion.

Dynamics of somatic cell count (SCC) and differential SCC during and following intramammary infections.

Somatic cell count is frequently used as an indicator of intramammary infections (IMI) in dairy cattle worldwide. The newly introduced differential SCC (DSCC) can potentially contribute to detection of IMI. The purpose of this study was to investigate the dynamics of SCC and DSCC after IMI. We used a data set with monthly samples from 2 Danish dairy herds through 1 yr, using bacterial culture to identify IMI. The dynamics of SCC and DSCC with regard to IMI were assessed at quarter level following new IMI with each of 3 defined pathogen groups, major, minor, or "other" pathogens, using general additive models. Both SCC and DSCC increased after IMI, with a more pronounced increase if major or other pathogens were detected compared with minor pathogens. We found that DSCC increased after IMI with other pathogens in both herds and, in herd 2, after IMI caused by major and minor pathogens. We also estimated the duration of increased SCC and DSCC when they exceeded a threshold, done separately for each pathogen group. Major pathogens had the longest-lasting effect in both herds for both SCC and DSCC. We conclude that the magnitude and duration of response of SCC and DSCC to IMI differs between herds and causative pathogens.

Half-Life Extending Modifications of Peptide YY3-36 Direct Receptor-Mediated Internalization.

Peptide YY3-36 (PYY3-36) is an endogenous ligand of the neuropeptide Y2 receptor (Y2R), on which it acts to reduce food intake. Chemically modified PYY3-36 analogues with extended half-lives are potential therapeutics for the treatment of obesity. Here we show that the common half-life extending strategies PEGylation and lipidation not only control PYY3-36's pharmacokinetics but also affect central aspects of its pharmacodynamics. PEGylation of PYY3-36 inhibited endocytosis by increasing receptor dissociation rates (koff), which reduced arrestin-3 (Arr3) activity. This is the first link between Arr3 recruitment and Y2R residence time. C16-lipidation of PYY3-36 had a negligible impact on Y2R signaling, binding, and endocytosis. In contrast, C18acid-lipidation minimized endocytosis, which indicated a decreased internalization through non-arrestin-related mechanisms. We propose a temporal model that connects the properties and position of the half-life extender with receptor Gi versus Arr3 signaling bias. We believe that this will be important for future design of peptide therapeutics.

Expert evaluation of different infection types in dairy cow quarters naturally infected with Staphylococcus aureus or Streptococcus agalactiae.

The purpose of this study was to improve the diagnostic recommendations for Staphylococcus aureus and Streptococcus agalactiae control using bacterial culture (BC), polymerase chain reaction (PCR) and somatic cell count (SCC) as diagnostic methods. The study was carried out in three steps: firstly, diagnostic test patterns for naturally infected quarters with Staph. aureus (24 quarters) and Strep. agalactiae (16 quarters) were created by sampling the quarters each day for 21 days and analysing the daily quarter milk samples using BC, PCR and SCC. Secondly, 30 mastitis experts were asked to group and describe the diagnostic test patterns and to establish a diagnosis for each group. The experts' statements regarding the groups they established were subsequently examined using qualitative content analysis to assign "infection types" to the statements. Lastly, the test performance was estimated for BC, PCR and SCC using generalised logistic regression models with the interpreted statements as a reference for infection. The experts mainly identified the Staph. aureus quarter-patterns as persistent infections, while some had more dynamic patterns. Strep. agalactiae quarter-patterns mainly involved persistent infection, yet some appeared hard to diagnose and were assigned to almost all different infection types, while experts did not agree on the interpretation. Estimates of Se for detection of Staph. aureus infection were 95.9% [93.7; 97.3] for BC, 99.5% [98.3; 99.8] for PCR, and 96.1% [94.0; 97.5] for SCC. The corresponding Sp estimates were 74.5% [65.7; 81.7], 66% [57.2; 73.8] and 43.7% [36.2; 51.5] for BC, PCR and SCC, respectively. The Se estimates of BC and PCR for Strep. agalactiae infection were 100% [83.5; 100] and 99.9% [99.6; 100], respectively, whereas the Se of SCC detecting Strep. agalactiae infection was only 34.3% [26.4; 43.3]. This indicated that Strep. agalactiae-positive BC and PCR test results were more important than SCC results to the experts when diagnosing a quarter as infected. The Sp estimates of BC, PCR and SCC for Strep. agalactiae infection were 99% [72.8; 100], 97.7% [62.1; 99.9], and 65.7% [56.7; 73.7], respectively. We conclude that PCR and BC are highly sensitive in the detection of persistent and new infections as defined by the experts, although the Se was not always 100%. An accepted lower Sp suggests that experts place less emphasis on false-positive results. We recommend that efforts are made to develop consistent terminology to characterise intramammary infections over time so that the course of infection can be taken into account at diagnosis.

Guidance on the use of the Threshold of Toxicological Concern approach in food safety assessment.

The Scientific Committee confirms that the Threshold of Toxicological Concern (TTC) is a pragmatic screening and prioritisation tool for use in food safety assessment. This Guidance provides clear step-by-step instructions for use of the TTC approach. The inclusion and exclusion criteria are defined and the use of the TTC decision tree is explained. The approach can be used when the chemical structure of the substance is known, there are limited chemical-specific toxicity data and the exposure can be estimated. The TTC approach should not be used for substances for which EU food/feed legislation requires the submission of toxicity data or when sufficient data are available for a risk assessment or if the substance under consideration falls into one of the exclusion categories. For substances that have the potential to be DNA-reactive mutagens and/or carcinogens based on the weight of evidence, the relevant TTC value is 0.0025 µg/kg body weight (bw) per day. For organophosphates or carbamates, the relevant TTC value is 0.3 µg/kg bw per day. All other substances are grouped according to the Cramer classification. The TTC values for Cramer Classes I, II and III are 30 µg/kg bw per day, 9 µg/kg bw per day and 1.5 µg/kg bw per day, respectively. For substances with exposures below the TTC values, the probability that they would cause adverse health effects is low. If the estimated exposure to a substance is higher than the relevant TTC value, a non-TTC approach is required to reach a conclusion on potential adverse health effects.

Association between teat skin colonization and intramammary infection with Staphylococcus aureus and Streptococcus agalactiae in herds with automatic milking systems.

The objective of this study was to investigate the association between teat skin colonization and intramammary infection (IMI) with Staphylococcus aureus or Streptococcus agalactiae at the quarter level in herds with automatic milking systems. Milk and teat skin samples from 1,142 quarters were collected from 300 cows with somatic cell count >200,000 cells/mL from 8 herds positive for Strep. agalactiae. All milk and teat skin samples were cultured on calf blood agar and selective media. A subset of samples from 287 quarters was further analyzed using a PCR assay (Mastit4 PCR; DNA Diagnostic A/S, Risskov, Denmark). Bacterial culture detected Staph. aureus in 93 (8.1%) of the milk samples and 75 (6.6%) of the teat skin samples. Of these, 15 (1.3%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was cultured in 84 (7.4%) of the milk samples and 4 (0.35%) of the teat skin samples. Of these, 3 (0.26%) quarters were positive in both the teat skin and milk samples. The PCR detected Staph. aureus in 29 (10%) of the milk samples and 45 (16%) of the teat skin samples. Of these, 2 (0.7%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was detected in 40 (14%) of the milk samples and 51 (18%) of the teat skin samples. Of these, 16 (5.6%) quarters were positive in both the teat skin and milk samples. Logistic regression was used to investigate the association between teat skin colonization and IMI at the quarter level. Based on bacterial culture results, teat skin colonization with Staph. aureus resulted in 7.8 (95% confidence interval: 2.9; 20.6) times higher odds of Staph. aureus IMI, whereas herd was observed as a major confounder. However, results from the PCR analyses did not support this association. Streptococcus agalactiae was isolated from the teat skin with both PCR and bacterial culture, but the number of positive teat skin samples detected by culture was too low to proceed with further analysis. Based on the PCR results, Strep. agalactiae on teat skin resulted in 3.8 (1.4; 10.1) times higher odds of Strep. agalactiae IMI. Our results suggest that Staph. aureus and Strep. agalactiae on teat skin may be a risk factor for IMI with the same pathogens. Focus on proper teat skin hygiene is therefore recommended also in AMS.

Accuracy of qPCR and bacterial culture for the diagnosis of bovine intramammary infections and teat skin colonisation with Streptococcus agalactiae and Staphylococcus aureus using Bayesian analysis.

Streptococcus agalactiae (Strep. agalactiae) and Staphylococcus aureus (Staph. aureus) are originally regarded as contagious mastitis pathogens, however, both pathogens have recently been isolated from extramammary and environmental sites, indicating that other sites than the udder might contribute to the spread of these pathogens potentially causing intramammary infections. Diagnostic tools to identify pathogens at extramammary sites are available but still needs to be validated. The objective of this cross-sectional field study was to estimate the diagnostic sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 qPCR assay and bacterial culture (BC) in identifying Strep. agalactiae and Staph. aureus from milk and teat skin samples. We randomly selected 30-40 cows with high somatic cell counts from eight Danish Strep. agalactiae-positive dairy herds with automatic milking systems. Teat skin samples and aseptic milk samples were collected from right rear quarters (n = 287) for BC and PCR analysis. Se and Sp were estimated in a Bayesian latent class analysis. For milk samples, the Se and Sp of qPCR for Strep. agalactiae were estimated to 0.97 and 0.99, respectively, whereas the Se and Sp of BC were 0.41 and 1.00, respectively. The Se and Sp of qPCR for Staph. aureus were estimated to 0.95 and 0.99, respectively, whereas the Se and Sp of BC were 0.54 and 0.77, respectively. For teat skin samples, the Se and Sp of qPCR for Strep. agalactiae were estimated to be 0.97 and 0.96, respectively, whereas the Se and Sp of BC were 0.33 and 1.00, respectively. The Se and Sp of qPCR for Staph. aureus were estimated to 0.94 and 0.98, respectively, whereas the Se and Sp of BC were 0.44 and 0.74, respectively. In conclusion, the Se for diagnosing Strep. agalactiae and Staph. aureus IMI was higher for qPCR than BC, suggesting that qPCR is a valuable method for detecting both pathogens from quarter-level milk samples. The performance of BC in the detection of Strep. agalactiae and Staph. aureus on teat skin was poor compared to qPCR, indicating that differences in the target condition of the two methods should be considered when implementing them as routine diagnostic tests for detecting teat skin colonisers. The low Se of BC may preclude the use of BC for skin testing, and qPCR is better for this task.

Determinants of antimicrobial treatment for udder health in Danish dairy cattle herds.

Societal pressure to limit the use of antibiotics in livestock production systems, including dairy cattle systems, is consistently increasing. To motivate farmers to reduce antibiotic usage, it is important to understand the factors that determine whether a cow will be treated with antibiotics or not. If farmers' usual practices regarding antibiotic treatments are taken into account, they may be motivated to adopt control measures that can facilitate prudent use of antibiotics and are at the same time cost-effective. In this study, we analyzed database recordings of milk yield and somatic cell count from the routine milk recording scheme, clinical registrations of mastitis and PCR results, and cow factors such as days in milk and parity in relation to antibiotic treatments for 518 dairy herds in Denmark. Farm-wise logistic regressions were used to predict antimicrobial treatment based on these factors. The resulting regression coefficients of 422 herds were further analyzed by principal component analysis and clustering to determine the driving predictors for treatment in different groups of farms. The results showed that determinants that were most important for predicting antibiotic treatments vary from one farm to another. Health indicators such as PCR or somatic cell count were most indicative for treatment on some farms, whereas other groups seemed to depend more on production factors (milk yield) or later culling of the cows. This shows that farmers behave differently and differences can be identified in register data. This information can be considered when developing cost-effective herd-specific control measures of mastitis to promote prudent use of antibiotics in Danish dairy cattle farms.

STARD-BLCM: Standards for the Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models.

The Standards for the Reporting of Diagnostic Accuracy (STARD) statement, which was recently updated to the STARD2015 statement, was developed to encourage complete and transparent reporting of test accuracy studies. Although STARD principles apply broadly, the checklist is limited to studies designed to evaluate the accuracy of tests when the disease status is determined from a perfect reference procedure or an imperfect one with known measures of test accuracy. However, a reference standard does not always exist, especially in the case of infectious diseases with a long latent period. In such cases, a valid alternative to classical test evaluation involves the use of latent class models that do not require a priori knowledge of disease status. Latent class models have been successfully implemented in a Bayesian framework for over 20 years. The objective of this work was to identify the STARD items that require modification and develop a modified version of STARD for studies that use Bayesian latent class analysis to estimate diagnostic test accuracy in the absence of a reference standard. Examples and elaborations for each of the modified items are provided. The new guidelines, termed STARD-BLCM (Standards for Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models), will facilitate improved quality of reporting on the design, conduct and results of diagnostic accuracy studies that use Bayesian latent class models.

Relation between tetR and tetA expression in tetracycline resistant Escherichia coli.

BACKGROUND: Tetracyclines are among the most used antibiotics in livestock worldwide. Resistance is widely disseminated in Escherichia coli, where it is generally mediated by tetracycline efflux pumps, such as TetA. Expression of tetracycline efflux pumps is tightly controlled by the repressor TetR, which has been shown to be tetracycline-responsive at sub-MIC tetracycline concentrations. The objective of this study was to investigate the effects of increasing tetracycline concentrations on the growth of TetA-producing E. coli, and to determine how expression of tetA and tetR related to each other in different growth phases in the presence of tetracycline. RESULTS: A tetracycline resistant E. coli strain containing tetA and tetR on the chromosome was constructed and cultured in the presence of increasing concentrations of tetracycline. Expression of tetR and tetA was measured at four time points in different growth phases by quantitative real-time PCR. The TetA-producing E. coli exhibited prolonged lag phase with increasing concentrations of tetracycline, while expression of tetA and tetR increased and decreased, respectively, with increasing tetracycline concentration. The levels of tetA and tetR mRNA varied depending on growth phase, resulting in a gradual decrease of the tetA/tetR ratio from approximately 4 in the lag phase to approximately 2 in the stationary phase. CONCLUSION: This study shows that the expression of tetR and tetA is tetracycline concentration- and growth phase-dependent, contributing to improved understanding of the relationships between E. coli growth, tetracycline exposure and expression of tetracycline resistance.

Immunogenic and protective efficacy of recombinant protein GtxA-N against Gallibacterium anatis challenge in chickens.

Gallibacterium anatis is a major cause of reproductive tract infections in chickens. Here, we aimed to evaluate the efficacy of the recombinant protein GtxA-N at protecting hens, by addressing three objectives; (i) evaluating the antibody response following immunization (ii) scoring and comparing lesions, following challenge with G. anatis, in immunized and non-immunized hens and (iii) investigating if the anti-GtxA-N antibody titre in individual hens correlated with the observed lesions. Two consecutive experiments were performed in hens. In the first experiment hens were immunized with GtxA-N on day 0 and day 14, infected with G. anatis on day 28 and euthanized on day 56. The GtxA-N antibody response was assessed in pooled serum samples throughout the experiment, using an indirect enzyme-linked immunosorbent assay (ELISA). In the second experiment the GtxA-N antibody titres were assessed in individual hens before and after immunization. Subsequently, the hens were inoculated with G. anatis and finally all hens where euthanized and submitted for post mortem examination 48 h after inoculation. Immunization elicited strong antibody responses that lasted at least 8 weeks (P < .0001). The individual antibody titres observed in response to immunization varied considerably among hens (range: 174,100-281,500). Lesion scores following G. anatis infection were significantly lower in immunized hens compared to non-immunized hens (P = .004). Within the immunized group, no correlation was found between the individual antibody titres and the lesion scores. This study clearly demonstrated GtxA-N as a vaccine antigen able of inducing protective immunity against G. anatis.

DENTAL LESIONS IN THE LOWLAND TAPIR (TAPIRUS TERRESTRIS).

Dental ailments, mandibular swelling, and dentoalveolar abscesses are common in tapirs, but knowledge about prevalence or etiology of these lesions in the Tapiridae family in general, and in lowland tapirs (Tapirus terrestris) in particular, is scarce. A recent study identified resorptive lesions of unknown etiology as a common problem in the Malayan tapir (Tapirus indicus). In order to investigate the type and prevalence of dental lesions occurring in lowland tapirs, and to compare these with findings with the Malayan tapir, skulls and teeth from 46 deceased lowland tapirs were visually and radiographically examined. The specimens were divided into subpopulations according to age (juveniles, young adults, adults) and origin (free-range or captive). Dental lesions were identified in 24% (11/46) of the study population. The most common pathologic findings were complicated dental fractures with associated periapical reaction (15%) and periapical reactions of various degrees without associated detectable dental pathology (13%). All these lesions likely originated from dental trauma. As in Malayan tapirs, juveniles had significantly fewer lesions than adults. This study shows that dental lesions present frequent problems for lowland tapirs, occurring both in captive and in free-ranging individuals, and indicates that increasing age should be considered a risk factor for the development of these lesions. Notably, the predominant dental problems in lowland tapirs and Malayan tapirs are not the same.

Comparison of recording of pericarditis and lung disorders at routine meat inspection with findings at systematic health monitoring in Danish finisher pigs.

BACKGROUND: The use of secondary data is widespread in a range of surveillance and monitoring applications because of the low cost and high availability associated with this form of data. However, as they are often collected for quite unrelated purposes, they are not necessarily fit for the new purpose that is required of them. Routine meat inspection data were originally collected with the purpose of safeguarding food, but have been re-tasked to also include animal welfare assessment. The objective of the present study was to compare the recording of pericarditis, pleuritis and lungs with lesions at routine meat inspection (RMI) with those performed at systematic health monitoring (SHM) in Danish pigs at slaughter, in order to assess the usefulness of RMI for monitoring the prevalence of these diseases. Data originating from 165 Danish pig herds were collected in the period September 2011 to November 2013. From each herd, a batch consisting of all pigs slaughtered on a specific day from a specific farm were included as the RMI data, while lungs and hearts sampled from the batches were used for the SHM. The RMI data and SHM data included recordings related to a) chronic pericarditis, b) chronic pleuritis and c) lung lesions. The proportion of carcases with a specific disease recording was estimated for each batch of pigs, and linear regression was used to relate the RMI-proportion to the SHM-proportion for the conditions mentioned above. RESULTS: The coefficients of determination (R(2)) were estimated as R(2),pericarditis = 0.16; R(2),pleuritis = 0.67; R(2),lungs with lesions = 0.40. R(2),pericarditis changed to 0.42 when the regression analysis included inspection type at the abattoir (with purely visual inspection of the hearts versus traditional inspection including an incision into the heart). CONCLUSIONS: Overall, the results suggest that the correlation between findings at RMI and SHM was moderate for pleuritis and lungs with lesions, but poor for pericarditis. The latter could partly be explained by the type of meat inspection conducted at the abattoir. We conclude that caution should be used whenever RMI data are used for purposes other than those for which they were originally intended.

CTX-M-1 ß-lactamase expression in Escherichia coli is dependent on cefotaxime concentration, growth phase and gene location.

OBJECTIVES: Knowledge about the regulatory mechanisms of CTX-M ß-lactamase-encoding genes in Escherichia coli is limited. The objectives of this study were to determine the growth response of CTX-M-1-producing E. coli exposed to cefotaxime and to investigate how blaCTX-M-1 expression at mRNA and protein levels is influenced by cefotaxime concentration, growth phase and gene location (chromosome versus plasmid). METHODS: Two isogenic E. coli strains, MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, containing blaCTX-M-1 on the chromosome and on a wild-type IncI1 plasmid, respectively, were constructed and the MIC of cefotaxime was determined. Growth of the two strains was studied in the presence of increasing concentrations of cefotaxime ranging from 0 to 512 mg/L. The levels of mRNA and protein in different growth phases and at different cefotaxime concentrations were studied by qPCR and selected-reaction-monitoring MS, respectively. RESULTS: The MICs of cefotaxime were 168 and 252 mg/L for MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, respectively. Both strains displayed a prolonged lag phase when exposed to cefotaxime. The mRNA of blaCTX-M-1 and CTX-M-1 protein levels increased in the presence of high cefotaxime concentrations and varied with growth phase. Higher mRNA expression levels were detected for MG1655/CTX-M-1 compared with MG1655/IncI1/CTX-M-1, but a higher protein level was found for MG1655/IncI1/CTX-M-1 compared with MG1655/CTX-M-1, the latter corresponding well with the higher MIC for this strain. CONCLUSIONS: blaCTX-M-1 mRNA expression and CTX-M-1 protein levels were dependent on cefotaxime concentration, growth phase and gene location. These results provide insight into the expression of cephalosporin resistance in CTX-M-1-producing E. coli, improving our understanding of the relationship between antimicrobial therapy and the expression of resistance mechanisms.

Assessing the influence of mechanical ventilation on blood gases and blood pressure in rattlesnakes.

OBJECTIVE: To characterize the impact of mechanical positive pressure ventilation on heart rate (HR), arterial blood pressure, blood gases, lactate, glucose, sodium, potassium and calcium concentrations in rattlesnakes during anesthesia and the subsequent recovery period. STUDY DESIGN: Prospective, randomized trial. ANIMALS: Twenty one fasted adult South American rattlesnakes (Crotalus durissus terrificus). METHODS: Snakes were anesthetized with propofol (15 mg kg(-1)) intravenously, endotracheally intubated and assigned to one of four ventilation regimens: Spontaneous ventilation, or mechanical ventilation at a tidal volume of 30 mL kg(-1) at 1 breath every 90 seconds, 5 breaths minute(-1), or 15 breaths minute(-1). Arterial blood was collected from indwelling catheters at 30, 40, and 60 minutes and 2, 6, and 24 hours following induction of anesthesia and analyzed for pH, PaO2, PaCO2, and selected variables. Mean arterial blood pressure (MAP) and HR were recorded at 30, 40, 60 minutes and 24 hours. RESULTS: Spontaneous ventilation and 1 breath every 90 seconds resulted in a mild hypercapnia (PaCO2 22.4 ± 4.3 mmHg [3.0 ± 0.6 kPa] and 24.5 ± 1.6 mmHg [3.3 ± 0.2 kPa], respectively), 5 breaths minute(-1) resulted in normocapnia (14.2 ± 2.7 mmHg [1.9 ± 0.4 kPa]), while 15 breaths minute(-1) caused marked hypocapnia (8.2 ± 2.5 mmHg [1.1 ± 0.3 kPa]). Following recovery, blood gases of the four groups were similar from 2 hours. Anesthesia, independent of ventilation was associated with significantly elevated glucose, lactate and potassium concentrations compared to values at 24 hours (p < 0.0001). MAP increased significantly with increasing ventilation frequency (p < 0.001). HR did not vary among regimens. CONCLUSIONS AND CLINICAL RELEVANCE: Mechanical ventilation had a profound impact on blood gases and blood pressure. The results support the use of mechanical ventilation with a frequency of 1-2 breaths minute(-1) at a tidal volume of 30 mL kg(-1) during anesthesia in fasted snakes.

Effect of carryover and presampling procedures on the results of real-time PCR used for diagnosis of bovine intramammary infections with Streptococcus agalactiae at routine milk recordings.

The use of PCR tests as diagnostics for intramammary infections (IMI) based on composite milk samples collected in a non-sterile manner at milk recordings is increasing. Carryover of sample material between cows and non-aseptic PCR sampling may be incriminated for misclassification of IMI with Streptococcus agalactiae (S. agalactiae) in dairy herds with conventional milking parlours. Misclassification may result in unnecessary costs for treatment and culling. The objectives of this study were to (1) determine the effect of carryover on PCR-positivity for S. agalactiae at different PCR cycle threshold (Ct) cut-offs by estimating the between-cow correlation while accounting for the milking order, and (2) evaluate the effect of aseptic presampling procedures (PSP) on PCR-positivity at the different Ct-value cut-offs. The study was conducted in four herds with conventional milking parlours at routine milk recordings. Following the farmers' routine pre-milking preparation, 411 of 794 cows were randomly selected for the PSP treatment. These procedures included removing the first streams of milk and 70% alcohol teat disinfection. Composite milk samples were then collected from all cows and tested using PCR. Data on milking order were used to estimate the correlation between consecutively milked cows in each milking unit. Factors associated with the PCR-positivity for S. agalactiae were analyzed using generalized estimating equations assuming a binomially-distributed outcome with a logit link function. Presampling procedures were only significant using cut-off 37. A first-order autoregressive correlation structure provided the best correlation between consecutively milked cows. The correlation was 13%, 11%, 9% at cut-offs <40, 37, and 34, respectively. PSP did not reduce the odds of cows being PCR-positive for S. agalactiae. In conclusion, carryover and non-aseptic sampling affected the PCR results and should therefore be considered when samples from routine milk recordings are used. In relative terms, higher cut-offs resulted in higher between-cow correlation, but the absolute amount of carryover may not be affected although this was not tested.

Spatiotemporal patterns, annual baseline and movement-related incidence of Streptococcus agalactiae infection in Danish dairy herds: 2000-2009.

Several decades after the inception of the five-point plan for the control of contagious mastitis pathogens, Streptococcus agalactiae (S. agalactiae) persists as a fundamental threat to the dairy industry in many countries. A better understanding of the relative importance of within- and between-herd sources of new herd infections coupled with the spatiotemporal distribution of the infection, may aid in effective targeting of control efforts. Thus, the objectives of this study were: (1) to describe the spatiotemporal patterns of infection with S. agalactiae in the population of Danish dairy herds from 2000 to 2009 and (2) to estimate the annual herd-level baseline and movement-related incidence risks of S. agalactiae infection over the 10-year period. The analysis involved registry data on bacteriological culture of all bulk tank milk samples collected as part of the mandatory Danish S. agalactiae surveillance scheme as well as live cattle movements into dairy herds during the specified 10-year period. The results indicated that the predicted risk of a herd becoming infected with S. agalactiae varied spatiotemporally; the risk being more homogeneous and higher in the period after 2005. Additionally, the annual baseline risks yielded significant yet distinctive patterns before and after 2005 - the risk of infection being higher in the latter phase. On the contrary, the annual movement-related risks revealed a non-significant pattern over the 10-year period. There was neither evidence for spatial clustering of cases relative to the population of herds at risk nor spatial dependency between herds. Nevertheless, the results signal a need to beef up within-herd biosecurity in order to reduce the risk of new herd infections.