EHV-2 induced equine keratoconjunctivitis Evaluation of the role of immunological mechanisms as well as of viral and bacterial co-factors

Deckblatt-Impressum Widmung Inhalt Abbildungsverzeichnis Diagrammverzeichnis Tabellenverzeichnis Abkürzungen Einleitung Zielsetzung der Arbeit Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Anhang Danksagung SelbständigkeitserklärungEine Beteiligung des Equinen Herpesvirus Typ 2 (EHV-2) bei verschiedenen Formen entzündlicher Erkrankungen der Kornea und/oder Konjunktiva des Pferdes ist mehrfach belegt, die genaue Pathogenese der equinen Keratokonjunktivitis ist jedoch noch nicht vollständig geklärt. Im Mittelpunkt der hier vorgelegten Arbeit stand daher die Frage, ob EHV-2-infizierte augenkranke Pferde möglicherweise einen von der Norm abweichenden zellulären Immunstatus aufweisen. Die dabei erzielten Ergebnisse sollten Rückschlüsse auf die Pathogenese der Erkrankung möglicherweise beeinflussende immunsupprimierende Mechanismen des EHV-2 zulassen. Weiterhin sollte überprüft werden, ob andere virale und/oder bakterielle Erreger eine Rolle bei der equinen Keratokonjunktivitis spielen. Schließlich sollten die Studien zum Gewebe- und Zelltropismus des EHV-2/-5 im Auge einen weiteren Beitrag zum Verständnis der Pathogenese der Erkrankung leisten. Die Untersuchungen zum zellulären Immunstatus wurden vergleichend bei zehn augenkranken und 21 klinisch gesunden Pferden durchgeführt. Aus Blutproben isolierte PBL und Augentupferproben wurden hierfür einerseits mittels EHV-2 spezifischer nested PCR untersucht. Andererseits wurden serologische Untersuchungen durchgeführt und der zelluläre Immunstatus jedes Pferdes mittels Durchflusszytometrie und Differentialblutbildanalyse ermittelt. Bei dieser Studie wurde EHV-2 sowohl bei augenkranken als auch bei klinisch gesunden Pferden aus Blut- und Augentupferproben mittels nPCR bzw. Serologie nachgewiesen, ein signifikanter Zusammenhang zwischen EHV-2 und dem Merkmal augenkrank gelang hingegen mit keiner der genannten Methoden. Beim zellulären Immunstatus war einzig die Anzahl der B-Lymphozyten bei augengesunden EHV-2 positiven Pferden im Vergleich zu augenkranken bzw. -gesunden EHV-2 negativen Pferden signifikant häufiger erniedrigt. Dies könnte ein erstes Indiz dafür sein, dass eine EHV-2 Infektion oder Reaktivierung die BZellzahl messbar reduzieren kann. Aufgrund des geringen Stichprobenumfangs können jedoch erst weiterführende Untersuchungen Aufschluss darüber erbringen, inwieweit diese individuellen Unterschiede im Immunsystem entscheidend Einfluss nehmen auf den Ausbruch einer EHV-2-induzierten Keratokonjunktivitis. Zur Klärung der Frage, ob virale oder bakterielle Erreger als Co-Faktoren bei der EHV-2 bedingten equinen Keratokonjunktivitis beteiligt sind, wurden von einer zweiten Studienpopulation (bestehend aus 68 augenkranken und 32 klinisch gesunden Pferden) entnommene Augentupfer- und Blutproben zunächst auf EHV-2 und anschließend auch auf EHV-5 mittels nPCR untersucht. Stichprobenartig wurden Tupfer von Test- und Kontrolltieren schließlich noch mittels erregerspezifischer PCR auf EAdV-1, Chlamydien und Mykoplasmen untersucht. Die nPCR zum Nachweis von EAdV-1 war zuvor im Rahmen der hier vorgelegten Arbeit etabliert worden. Signifikante Unterschiede zwischen augenkranken und gesunden Pferden bei der Häufigkeit des Nachweises von EHV-2 aus Tupfer- bzw. Blutproben wurden auch bei dieser Studienpopulation nicht festgestellt. Auch Hinweise auf eine Beteiligung von EAdV-1 oder Chlamydien und Mykoplasmen bei entzündlichen Erkrankungen der Kornea und/oder Konjunktiva des Pferdes ergaben sich nicht. Wahrscheinlich ist hingegen, dass EHV-5 an der Pathogenese der equinen Keratokonjunktivitis beteiligt ist, da im kranken Auge EHV-5-Genom im Vergleich zu EHV-2 häufiger detektiert wurde, wenn auch wiederum nicht signifikant mit dem Merkmal augenkrank korrelierend. Die Studien zum Gewebe- und Zelltropismus wurden an verschiedenen Augengeweben (Konjunktiva, Kornea, Nervus opticus und Retina) sowie Cytobrushtupferproben durchgeführt, die post portem von 14 gesund geschlachteten Pferden entnommen wurden. Der Nachweis von EHV-2 und -5-Genom mittels nPCR gelang hierbei im Cytobrushtupfer sowie im Augengewebe ausschließlich in der Konjunktiva, wenn auch mit einer sehr niedrigen Nachweisrate (Anteil EHV-2 bzw. -5 positiver Tupfer: jeweils 11%; Anteil EHV-2 bzw. -5 positiver Konjunktiven: 7 bzw. 8%). Bei der anschließend exemplarisch am EHV-2-nPCR positiven Konjunktivalgewebe eines Pferdes und zuvor im Rahmen der hier vorgelegten Arbeit etablierten In situ-Hybridisierung konnte schließlich gezeigt werden, dass EHV-2 im Augengewebe in der Bindegewebsschicht der Conjunctiva palpebralis präsent ist und hier möglicherweise in spezifischen Immunzellen in Latenz geht.Several times, an involvement of the Equine Herpesvirus Type 2 (EHV-2) in different forms of inflammatory diseases of the cornea and/or conjunctiva in horses is documented; the exact pathogenesis of equine keratoconjunctivitis has yet not been understood completely. This project focus on the question, whether EHV-2-infected eye-diseased horses possibly show an immune status which differs from the reference value. Besides, the results of this thesis should provide conclusions, if the pathogenesis of the disease is possibly been influenced by immunosuppressive mechanisms of EHV-2. Furthermore, it should be investigated, whether other viral and/or bacterial pathogens play a role in equine keratoconjunctivitis. Finally, the investigations of the ocular tissue- and cell-tropism of EHV-2/-5 should further contribute to the understanding of the pathogenesis of this disease. Investigations of the cellular immune status had been carried out comparing ten eyediseased and 21 clinically sound horses. PBL, isolated from blood samples and swabs from the conjunctival fossa had been investigated using a EHV-2 specific nested PCR. Additionally, serological investigations had been carried out and the cellular immune status of each horse had been determined using flow cytometry and white blood cell count methods. EHV-2 was detected within this study in both, eye- diseased and clinically sound horses by serological methods respectively nPCR. However, a statistically significant correlation between EHV-2 and the characteristic eye-diseased was not observed. Referring to the cellular immune status EHV-2 positive horses without ocular diseases showed significantly more often decreased b-cell numbers than EHV-2 negative horses with and without ocular diseases, respectively. This should be seen as first indication that an EHV-2 infection or reactivation causes a result in a measurable reduction of b-cells. Due to the small sample number, only further investigations will give information about, how individual differences of the immune system may influence the outbreak of keratoconjunctivitis induced by EHV-2. To answer the question, whether other co-factors such as viral or bacterial pathogens are involved in EHV-2 induced equine keratoconjunctivitis, eye-swabs and blood samples from a second investigation group (consisting of 68 eye-diseased and 32 clinically sound horses) had been tested for EHV-2 and subsequently EHV-5, using specific nPCRs. Finally, random samples of eye-swabs had been investigated using pathogen specific PCRs for the presence of EAdV-1, chlamydiae and mycoplasmae, respectively. Within the scope of this project, the EAdV-1 specific nPCR had been established previously. A statistically significant correlation between eye-diseased and sound horses relating to the detection rate of EHV-2 in swab- and blood-samples was also not observed in this investigation group. In addition, evidence for an involvement of EAdV-1, chlamydiae and mycoplasmae, respectively in inflammatory diseases of the cornea and/or conjunctiva of horses could not been deduced. However, it is very likely that EHV-5 is involved in the pathogenesis of equine keratoconjunctivitis as EHV-5-genom was detected frequently in diseased eyes compared with EHV-2, even though there was no observation of a significant correlation between the characteristic eye-diseased . Studies about the tissue- and cell-tropism had been carried out on various ocular tissues (conjunctiva, cornea, nervus opticus and retina) as well as on cytobrush- swabs, which had all been taken post mortem from 14 healthy slaughtered horses. The genome of EHV-2 and -5 had been detected in cytobrush samples and in the conjunctiva only, even though the detection rate was very low (proportion of EHV-2 resp. -5 positive swabs: 11% in each case; proportion of EHV-2 resp. -5 positive conjunctiva: 7 resp. 8%). Finally, the exemplary investigation of a EHV-2-nPCR positive conjunctiva of one horse using the in- situ hybridisation, a method that had been established within the scope of this project revealed, that EHV-2 DNA is present in the submucosa of the conjunctiva palpebralis. Thus, specific cells of the immune system might possibly be a site of latency within this tissue

Livestock-Associated Meticillin-Resistant Staphylococcus aureus—Current Situation and Impact From a One Health Perspective

Purpose of Review In this article, we aim to provide an overview of the occurrence and characteristics of livestock-associated (LA-) meticillin-resistant Staphylococcus aureus (MRSA). We further question the role of LA-MRSA as a potential foodborne pathogen. We investigate recent findings and developments from a One Health perspective also highlighting current strategies and initiatives aiming to improve reporting, control, and prevention of LA-MRSA. Recent Findings While the overall number of invasive MRSA infections in humans is decreasing (in most European countries and the USA) or steadily increasing (in the Asia-Pacific region), the role of LA-MRSA as causative agent of invasive disease and as potential foodborne pathogen is still poorly understood. LA-MRSA prevalence in livestock remains high in many geographical regions and the acquisition of new virulence and resistance determinants constitutes a growing threat for human health. Summary The true incidence of LA-MRSA infections due to occupational exposure is unknown. Improved MRSA monitoring and tracking procedures are urgently needed. Strain typing is crucial to enable improved understanding of the impact of LA-MRSA on human and animal health

MRSA in breeding pigs in Germany in 2015

Methicillin resistant Staphylococcus aureus has been known to be prevalent in the pig production for nearly 15 years now (Meemken et al., 2010). In 2008 a survey carried out in the EU determined a high prevalence of MRSA in herds of breeding pigs also in Germany (EFSA, 2010). Likewise, MRSA were identified in Germany in herds of fattening pigs (Alt et al., 2011), pigs at slaughter (Tenhagen et al., 2009), on carcasses (Beneke et al., 2011) and in meat from pigs at retail (BVL, 2013). The current investigation was carried out to determine the current prevalence of MRSA in herds of breeding pigs, analyse patterns in the type of MRSA isolated from pigs and determine differences between the MRSA observed in units only housing sows and those housing weaned piglets. In previous studies it could be shown, that the prevalence of MRSA is higher in weaned piglets than in sows but little is known about differences in the types of MRSA that can be isolated in the different units

Longitudinal Study of the Contamination of Air and of Soil Surfaces in the Vicinity of Pig Barns by Livestock-Associated Methicillin-Resistant Staphylococcus aureus

During 1 year, samples were taken on 4 days, one sample in each season, from pigs, the floor, and the air inside pig barns and from the ambient air and soil at different distances outside six commercial livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA)-positive pig barns in the north and east of Germany. LA-MRSA was isolated from animals, floor, and air samples in the barn, showing a range of airborne LA-MRSA between 6 and 3,619 CFU/m3 (median, 151 CFU/m3). Downwind of the barns, LA-MRSA was detected in low concentrations (11 to 14 CFU/m3) at distances of 50 and 150 m; all upwind air samples were negative. In contrast, LA-MRSA was found on soil surfaces at distances of 50, 150, and 300 m downwind from all barns, but no statistical differences could be observed between the proportions of positive soil surface samples at the three different distances. Upwind of the barns, positive soil surface samples were found only sporadically. Significantly more positive LA-MRSA samples were found in summer than in the other seasons both in air and soil samples upwind and downwind of the pig barns. spa typing was used to confirm the identity of LA-MRSA types found inside and outside the barns. The results show that there is regular airborne LA-MRSA transmission and deposition, which are strongly influenced by wind direction and season, of up to at least 300 m around positive pig barns. The described boot sampling method seems suitable to characterize the contamination of the vicinity of LA- MRSA-positive pig barns by the airborne route

Factors associated with the occurrence of MRSA CC398 in herds of fattening pigs in Germany

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to investigate the prevalence of MRSA in herds of fattening pigs in different regions of Germany, and to determine factors associated with the occurrence of this pathogen. For this purpose pooled dust samples were collected, and a questionnaire covered information regarding herd characteristics and management practices. Samples were pre-enriched in high-salt medium followed by selective enrichment containing cefoxitin/aztreonam, and culturing. Presumptive colonies were confirmed by multiplex-PCR targeting <it>nuc-</it>, <it>mecA</it>- and 16S rRNA-genes. Isolates were s<it>pa- </it>and SCC<it>mec</it>-, and in selected cases, multilocus sequence-typed. Susceptibilities to 13 antimicrobials were determined by broth microdilution. Statistical analysis was carried out using backward stepwise logistic regression to calculate odds ratios with the MRSA test result as the outcome and herd characteristics as categorical covariates.</p> <p>Results</p> <p>Overall, 152 of 290 (52%) fattening pig farms tested positive for MRSA. The prevalence in the east, north- and south-west of Germany ranged from 39 to 59%.</p> <p>t011 (66%) and t034 (23%) were the most commonly identified s<it>pa</it>-types, and 85% of isolates carried SCC<it>mec </it>Type V. Identified <it>spa</it>-types were all associated with clonal complex CC398. Susceptibility testing revealed that all isolates were resistant to tetracycline. High resistance rates were also found for sulfamethoxazole/trimethoprim (40%), and quinupristin/dalfopristin (32%). In addition, 83% of strains displayed multidrug resistant (> 3 substance classes) phenotypes.</p> <p>Logistic regression revealed herd size (large farms OR: 5.4; CI: 2.7-11.2; p < 0.05), and production type (wean-to-finish OR: 4.0; CI: 1.6-10.4; p < 0.05) as risk factors associated with a positive MRSA finding in fattening pig operations.</p> <p>Conclusions</p> <p>MRSA CC398 is widely distributed among herds of fattening pigs in Germany. Farm management plays a crucial role in the dissemination of MRSA with herd size, and production type representing potential major indicators.</p

Staphylococcus aureus as a foodborne pathogen

Purpose of Review: We present recent insights on S. aureus as a foodborne pathogen, thus providing readers with an update of current findings impacting prevention and control measures. Recent Findings: Advances in disease burden assessment show the burden of S. aureus foodborne disease around the globe. In recent years, recent research has provided valuable new data improving the understanding of the pathobiology of S. aureus foodborne disease as well as proteomics and genomics of this foodborne pathogen. In particular, recent findings shed new light on the role of newly described enterotoxins and methicillin-resistant S. aureus. These new findings guide the way towards improved prevention and control strategies. Summary: S. aureus is the leading cause of foodborne intoxications worldwide. Control strategies are focused on hygiene measures to avoid food contamination and limit S. aureus growth. Outbreak investigations remain challenging and would strongly benefit from additional data on enterotoxin formation under stress conditions and novel tools allowing for detection of newly described enterotoxins

Modeling the transmission of livestock associated methicillin-resistant Staphylococcus aureus along the pig slaughter line

AbstractThe study introduces a new approach for a qualitative transmission assessment of MRSA throughout the pig slaughter process. Based on prevalence data found in literature the MRSA contamination and elimination rates of each individual slaughter step were estimated. The rates were used to set up a Monte Carlo simulation for modeling the propagation of MRSA along the process chain and to quantify the impact of a variable initial prevalence on the outcome prevalence of the carcasses. Sensitivity analyses for the model as well as three different scenarios were performed to estimate the impact of cross contamination during slaughter and to determine the process stages where hygiene interventions are most effective.Regardless of the initial extent of MRSA contamination low outcome prevalences ranging between 0.15 and 1.15% were achieved among pig carcasses indicating that the pig slaughter chain generally includes process steps with the capacity to limit carcass contamination. Especially scalding and singeing can lead to a significant reduction of superficial MRSA contamination during the first half of the slaughter process. Nevertheless, scenario analyses showed that the low MRSA outcome prevalence can only be guaranteed if recontamination during the ongoing slaughter process is obviated. In order to ensure a low MRSA load on pig carcasses at the end of slaughter the abattoir should primarily concentrate on controlling the process parameters of scalding and singeing and avoiding recontamination at subsequent process steps