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Surveillance of antimicrobial administration in livestock production is an important factor in global policies to reduce spreading of antimicrobial resistance. In recent years, many studies have been carried out concerning the usage of antimicrobials in animal production and in some countries recording of antimicrobial quantities dispensed to famers is mandatory. On cattle farms, antimicrobial treatments are recorded for fattening calves under 8 months of age and for fattening cattle older than 8 months in Germany and treatment frequencies are then calculated. However, with the entry into force of Regulation (EU) 2019/6 on 01/28/2022, antimicrobial monitoring will gradually be extended to all animal species and age groups. Therefore, an effective, fast and accurate transfer of data on the use of antimicrobials, especially in the field of livestock farming, into corresponding databases is required to determine the treatment frequencies for the individual animal species or types of use. For this purpose, an electronic interface was programmed to transfer the data on antimicrobial use in dairy cattle farms from a herd management software program directly into a database. To test the practicability and effectiveness of this interface, 10 dairy cattle farms from Saxony were initially selected. Based on an in-depth analysis of the treatment frequencies of antimicrobial administration of 7 different age groups of animals after a two-year observation period, the functionality of the electronic interface could be established. The greatest potential for reduction of antimicrobials is in newborn calves, as they represent the age group with the highest treatment frequency.

We studied the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in dairy calves as part of a routine health check protocol. In addition, data regarding antimicrobial use (AMU), farm hygiene, and farm management were collected in order to identify possible risks for ESBL occurrence. Ten farms participated in the study with a median of 781 milking cows (319-1701). All calves investigated were younger than two weeks with an average age of 6.8 (±3.9) days. The farms were visited and samples were collected twice at an interval of 7-11 months. Faecal samples diluted 1:10, were plated onto BrillianceTM ESBL agar in duplicates. After 24 hours at 37°C, colonies were counted and total colony forming units (cfu)/ml calculated. Bacteria species were identified biochemically. ESBL-production was phenotypically confirmed using the MICRONAUT-S β-Lactamases system. Additionally, antimicrobial susceptibility was tested using VITEK® 2 technology. Phylotyping of E. coli isolates and screening for bla genes was performed by PCR. ESBL-producing enterobacteria were detected on all farms and 96.5% of calves investigated shed ESBL-positive bacteria. Of all ESBL-producing isolates, the majority were E. coli (92.9%), followed by Enterobacter cloacae (5.1%) and Klebsiella pneumoniae subsp. pneumoniae (2.0%). The majority of E. coli isolates was clearly assigned to phylogroup C (25.0%), followed by phylogroups A (15.2%) and E (14.1%). CTX-M group 1 was most frequently detected (80.4%). E. cloacae contained blaCTX-M and blaTEM or blaSHV. K. pneumoniae harboured blaSHV only. Besides resistance to penicillins and cephalosporins, the majority of isolates was also resistant to one or more antibiotic classes, with a high proportion being resistant against fluoroqinolones. 52.5% of isolates were further characterised as threefold multidrug resistant gram-negative bacteria (3MDR-GNB) according to the German Commission for Hospital Hygiene and Infection Prevention. None of the isolates were 4MDR-GNB, i.e. none revealed carbapenem-resistance. Penicillins were the most frequently administered antibiotics to calves on most farms and were the predominant substance class at herd level on all farms. Overall, the number of calves treated prior to sampling was rather low (11.7%). Analyses of data regarding the farm management identified weaknesses in biosecurity and cleaning and disinfection. Besides beta-lactam antibiotics being the most commonly used antibiotics no other risk factors could be identified. In summary, the prevalence of ESBL-carriers in dairy calves was exceptionally high and should be motivation to develop strategies for the reduction of multidrug-resistant bacteria in farm animals.

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.

Presurgical hand asepsis is part of the daily routine in veterinary medicine. Nevertheless, basic knowledge seems to be low, even among specialised veterinary surgeons. The major objectives of our study were to assess current habits for presurgical hand preparation (phase 1) among personnel in a veterinary hospital and their effectiveness in reducing bacteria from hands in comparison to a standardised protocol (phase 2). Assessment of individual habits focused on time for hand washing and disinfection, the amount of disinfectant used, and the usage of brushes. The standardised protocol defined hand washing for 1 min with liquid neutral soap without brushing and disinfection for 3 min. All participants (2 surgeons, 8 clinic members, 32 students) used Sterillium®. Total bacterial counts were determined before and after hand washing, after disinfection, and after surgery. Hands were immersed in 100 ml sterile sampling fluid for 1 min and samples were inoculated onto Columbia sheep blood agar using the spread-plate method. Bacterial colonies were manually counted. Glove perforation test was carried out at the end of the surgical procedure. Differences in the reduction of relative bacterial numbers between current habits and the standardised protocol were investigated using Mann-Whitney-Test. The relative increase in bacterial numbers as a function of operation time (≤60 min, >60 min) and glove perforation as well as the interaction of both was investigated by using ANOVA. Forty-six and 41 preparations were carried out during phase 1 and phase 2, respectively. Individual habits differed distinctly with regard to time (up to 8 min) and amount of disinfectant (up to 48 ml) used both between participants and between various applications of a respective participant. Comparison of current habits and the standardised protocol revealed that the duration of hand washing had no significant effect on reducing bacteria. Contrary, the reduction in bacterial numbers after disinfection by the standardised protocol was significantly higher (p

High amounts of aerial pollutants like dust and microorganisms can pose serious health hazards to animals and humans. The aim of the current study therefore was, to assess the efficiency of UVC irradiation combined to air filtration in reducing airborne microorganisms at laboratory scale. In a second part, a UVC-combined recirculating air filtration module (UVC module) was implemented in a small animal facility in order to assess its improvement of air quality with regard to airborne bacteria and dust. Tests at laboratory scale were performed using aerosols of Staphylococcus (S.) aureus, Actinobacillus pleuropneumoniae, porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus. We varied relative humidity (RH) to evaluate its effect on UVC irradiation efficiency. In addition, viability of pathogens inside the filter material was determined over up to six months. UVC-combined air filtration resulted in a more than 99% reduction of viral and bacterial particles. RH had no influence on UVC efficiency. Viability in the filter matter varied depending on the pathogen used and RH with S. aureus and PPV being most resistant. In our small pig facility consisting of two separated barns, weekly air measurements were conducted over a period of 13 weeks (10 piglets) and 16 weeks (11 piglets), respectively. Airborne bacterial numbers were significantly lower in the barn equipped with the UVC module compared to the reference barn. On average a reduction to 37% of reference values could be achieved for bacteria, whereas the amount of total dust was reduced to a much lesser extent (i.e. to 78% of reference values). Measures taken in front of and behind the UVC module revealed a reduction of 99.4% for airborne bacteria and 95.0% for total dust. To conclude, recirculating air filtration combined to UVC provided efficient reduction of pathogens at laboratory and experimental scale. The implementation of such devices might improve the overall environmental quality in animal facilities.

Biosecurity is defined as the implementation of measures that reduce the risk of disease agents being introduced and/or spread. For pig production, several of these measures are routinely implemented (e.g. cleaning, disinfection, segregation). However, air as a potential vector of pathogens has long been disregarded. Filters for incoming and recirculating air were installed into an already existing ventilation plant at a fattening piggery (3,840 pigs at maximum) in Saxony, Germany. Over a period of three consecutive fattening periods, we evaluated various parameters including air quality indices, environmental and operating parameters, and pig performance. Animal data regarding respiratory diseases, presence of antibodies against influenza A viruses, PRRSV, and Actinobacillus pleuropneumoniae and lung health score at slaughter were recorded, additionally. There were no significant differences (p = 0.824) in total bacterial counts between barns with and without air filtration. Recirculating air filtration resulted in the lowest total dust concentration (0.12 mg/m3) and lung health was best in animals from the barn equipped with recirculating air filtration modules. However, there was no difference in animal performance. Antibodies against all above mentioned pathogens were detected but mostly animals were already antibody-positive at re-stocking. We demonstrated that supply air filtration as well as recirculating air filtration technique can easily be implemented in an already existing ventilation system and that recirculating air filtration resulted in enhanced lung health compared to supply air-filtered and non-filtered barns. A more prominent effect might have been obtained in a breeding facility because of the longer life span of sows and a higher biosecurity level with air filtration as an add-on measure.

Air filtration has been shown to be efficient in reducing pathogen burden in circulating air. We determined at laboratory scale the retention efficiency of different air filter types either composed of a prefilter (EU class G4) and a secondary fiberglass filter (EU class F9) or consisting of a filter mat (EU class M6 and F8-9). Four filter prototypes were tested for their capability to remove aerosol containing equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), bovine enterovirus 1 (BEV), Actinobacillus pleuropneumoniae (APP), and Staphylococcus (S.) aureus from air. Depending on the filter prototype and utilisation, the airflow was set at 1,800 m3/h (combination of upstream prefilter and fiberglass filter) or 80 m3/h (filter mat). The pathogens were aerosolized and their concentration was determined in front of and behind the filter by culture or quantitative real-time RT-PCR. Furthermore, survival of the pathogens over time in the filter material was determined. Bacteria were most efficiently filtered with a reduction rate of up to 99.9% depending on the filter used. An approximately 98% reduction was achieved for the viruses tested. Viability or infectivity of APP or PRRSV in the filter material decreased below the detection limit after 4 h and 24 h, respectively, whereas S. aureus was still culturable after 4 weeks. Our results demonstrate that pathogens can efficiently be reduced by air filtration. Consequently, air filtration combined with other strict biosecurity measures markedly reduces the risk of introducing airborne transmitted pathogens to animal facilities. In addition, air filtration might be useful in reducing bioaerosols within a pig barn, hence improving respiratory health of pigs.

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.

Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16 °C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation.

BackgroundBats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. This is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. Nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. But the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date.Methodology/principal findingsBetween 2002 and 2009, 486 deceased bats of 19 European species (family Vespertilionidae) were collected in different geographic regions in Germany. Most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. The bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. Trauma and disease represented the most important causes of death in these bats. Comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least 12% of the bats investigated.Conclusions/significanceOur data demonstrate the importance of diseases and infectious agents as cause of death in European bat species. The clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats.