Aerogene Ausbreitung von Viren: Eine Studie verschiedener Sammelgeräte und Quantifizierungsmethoden zur Virusisolierung aus der Luft

Die aerogene Übertragung von Infektionskrankheiten stellt ein sehr wichtiges Thema in der Medizin dar. Für genaue Untersuchungen dazu, sind geeignete Sammel- und Nachweismetho-den essentiell. Die Untersuchung verschiedener Sammelgeräte sowie unterschiedlicher Nach-weismethoden zur Virusquantifizierung aus der Luft war daher die zentrale Aufgabenstellung in dieser Arbeit. Als Sammelgeräte wurden der Impinger AGI 30 und der Gelatinefilter ausge-wählt. Alle grundlegenden Untersuchungen zur Ermittlung der Eignung und Effizienzen der Geräte bezüglich der Virusisolierung aus Luftproben wurden an experimentell erzeugten Virus-aerosolen durchgeführt. Dabei wurden zwei Geflügelviren verwendet, das Newcastle-Disease-Virus Stamm LaSota (NDV) und das Infektiöse-Bursitis-Virus Stamm Cu-1M (IBDV). Die quan-titative Bestimmung der Viren aus den Luftproben erfolgte durch Titration im Zellkultursystem bzw. in embryonierten Hühnereiern. Parallel dazu wurde eine Titerbestimmung mittels einer in dieser Arbeit etablierten quantitativen Real-Time-PCR durchgeführt. Zusätzlich wurden die Sammelgeräte unter praktischen Bedingungen getestet und verglichen. Dazu erfolgten lufthy-gienische Messungen nach Vakzinierung von Geflügel mit Lebendimpfstoffen (NDV LaSota und IBDV Cu-1M). Es wurden umfangreiche Untersuchungen unter experimentellen Beding¬ungen und später exemplarische Untersuchungen in konventionellen Geflügelhaltungen durch-geführt. Die Evaluierung der Sammelgeräte mit Hilfe der experimentell erzeugten Virusaerosole er-gab, dass mit beiden Geräten sowohl infektionsfähiges Virus als auch Virusgenom nachgewie-sen und quantifiziert werden kann. Die Effizienzen unterschieden sich jedoch z.T. deutlich. So stellte sich der Gelatinefilter zur Sammlung in Kombination mit dem quantitativen Virusnach-weis mittels Real-Time-PCR als die Methode mit der höchsten Virusnachweisrate (angegeben als geometrischer Mittelwert mit geometrischer Standardabweichung) von 22,3 % ×/ 3,1 für NDV und 36,1 % ×/ 3,4 für IBDV heraus. Der Nachweis von infektionsfähigen Viren jedoch, war für beide Testkeime aus den Proben des Impingers erfolgreicher (NDV 4,0 % ×/ 1,7 und IBDV 31,8 % ×/ 1,8). Die signifikant niedrigere Nachweisrate des Newcastle-Disease-Virus ist auf die höhere Empfindlichkeit dieses behüllten Virus beim Sammelprozess und daraus folgen-der Inaktivierung zurückzuführen. Die Quantifizierung mit Hilfe der Real-Time-PCR erfolgte mit Normalisierung aller Proben. Diese bisher zur Analyse von Luftproben noch nicht ange-wandte Methode erwies sich als sehr gut. Durch die Normalisierung werden nicht nur die ab-weichenden Effizienzen der Nukleinsäureisolierung sowie der reversen Transkription ausgegli-chen, sondern auch die unterschiedliche Inhibition der PCR der Proben verschiedener Luftkeimsammler. Damit sind die Proben untereinander besser vergleichbar und die Quantifi-zierung exakter. Erstmals wurden auch systematische Untersuchungen zu Geräteeffizienzen in Kombination mit verschiedenen Virusnachweismethoden durchgeführt. Bei den lufthygienischen Untersuchungen nach Vakzinierung von ca. 50 Hühnern gegen Newcastle Disease (ND) bzw. Infektiöse Bursitis (IBD) unter experimentellen Bedingungen, waren drei Luftproben nach der Impfung gegen ND viruspositiv, jedoch keine nach der gegen IBD. Diese positiven Nachweise gelangen mit dem Gelatinefilter und nachfolgender Analyse mittels quantitativer Real-Time-PCR. Schlussfolgernd ist zumindest bei dem NDV eine aeroge-ne Ausscheidung des Impfvirus anzunehmen. Wahrscheinlich liegt die Viruskonzentration in der Luft jedoch meist unter der Nachweisgrenze der eingesetzten Geräte. Auch die Auswertung von Luftproben nach Vakzinierung gegen oben genannte Krankheiten in konventionellen Tierhal-tungen mit mehreren Tausend Tieren Besatz pro Stall führte zu keinen viruspositiven Ergebnis-sen. Anscheinend wurden die Viren auch trotz der großen Tierzahl in der Luft so stark verdünnt oder in so geringem Maße ausgeschieden, dass sie mit den in dieser Arbeit entnommenen Luft-probenvolumina von 1000 l nicht detektiert werden konnten. Für weiterführende Untersuchun-gen müsste daher ein Sammelgerät verwandt werden, mit welchem schnell große Probenvolu-mina entnommen werden können und das Endvolumen der Probenflüssigkeit dennoch gering ist, um die luftgetragenen Viren optimal zu konzentrieren. Schlussfolgernd erwies sich die Filtration in Kombination mit der normalisierten quantitati-ven Real-Time-PCR dennoch insgesamt als eine sehr valide und praktikable Methode zum Nachweis luftgetragener Viren aus Tierhaltungen

Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398

Colonized vertebrates including humans and pigs are to date the main reservoirs of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Currently, the mechanisms underlying colonization of pigs are not fully understood. We investigated the influence of piglet pre-immune status on airborne MRSA colonization. Three groups of MRSA-negative piglets were primed and exposed to airborne LA-MRSA (104 colony forming units (cfu)/m3) in an aerosol chamber for 24 h. One group was treated intramuscularly with dexamethasone (1 mg/kg body weight) to imitate weaning stress. The second group was exposed to bacterial endotoxin containing MRSA aerosol. Both conditions play a role in the development of multifactorial diseases and may promote MRSA colonization success. The third group served as control. The piglets' MRSA status was monitored for 21 days via swab samples. At necropsy, specific tissues and organs were analyzed. Blood was collected to examine specific immunological parameters. The duration of MRSA colonization was not extended in both treated groups compared to the control group, indicating the two immune-status influencing factors do not promote MRSA colonization. Blood sample analysis confirmed a mild dexamethasone-induced immune suppression and typical endotoxin-related changes in peripheral blood. Of note, the low-dose dexamethasone treatment showed a trend of increased MRSA clearance

Detection of Viable but Non-Culturable (VBNC)-Campylobacter in the Environment of Broiler Farms: Innovative Insights Delivered by Propidium Monoazide (PMA)-v-qPCR Analysis

Campylobacteriosis cases in humans are of global concern, with high prevalence rates in the poultry reservoir considered the most important source of infection. Research findings show Campylobacters’ ability to enter a viable but non-culturable (VBNC) state, remaining “viable” but unable to grow on culture media. We explored the persistence of VBNC states in specific environments, particularly at broiler farms, as this state may lead to an underestimation of the present Campylobacter prevalence. For VBNC detection, a propidium monoazide PMA-dye viability qPCR (v-qPCR) was used in combination with cultivation methods. We examined samples collected from broiler farm barns and their surroundings, as well as chicken manure from experimental pens. In addition, the tenacity of culturable and VBNC-Campylobacter was studied in vitro in soil and water. In a total of three visits, Campylobacter was not detected either culturally or by v-qPCR (no Campylobacter DNA) in the environment of the broiler farms. In four visits, however, VBNC-Campylobacter were detected both inside and outside the barns. The overall prevalence in environmental samples was 15.9% for VBNC-Campylobacter, 62.2% for Campylobacter DNA, and 1.2% for culturable C. jejuni. In the experimental pens, no cultivable C. jejuni was detected in chicken manure after 24 h. Strikingly, “VBNC-Campylobacter” persisted even after 72 h. “VBNC-Campylobacter” were confirmed in barn surroundings and naturally contaminated chicken manure. Laboratory studies revealed that VBNC-Campylobacter can remain intact in soil for up to 28 days and in water for at least 63 days, depending on environmental conditions

Stability of Feline Coronavirus in aerosols and dried in organic matrices on surfaces at various environmental conditions

Enveloped respiratory viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be transmitted through aerosols and contact with contaminated surfaces. The stability of these viruses outside the host significantly impacts their transmission dynamics and the spread of diseases. In this study, we investigated the tenacity of Feline Coronavirus (FCoV) in aerosols and on surfaces under varying environmental conditions. We found that airborne FCoV showed different stability depending on relative humidity (RH), with higher stability observed at low and high RH. Medium RH conditions (50–60%) were associated with increased loss of infectivity. Furthermore, FCoV remained infectious in the airborne state over 7 h. On stainless-steel surfaces, FCoV remained infectious for several months, with stability influenced by organic material and temperature. The presence of yeast extract and a temperature of 4 °C resulted in the longest maintenance of infectivity, with a 5 log10 reduction of the initial concentration after 167 days. At 20 °C, this reduction was achieved after 19 days. These findings highlight the potential risk of aerosol and contact transmission of respiratory viruses, especially in enclosed environments, over extended periods. Studying surrogate viruses like FCoV provides important insights into the behavior of zoonotic viruses like SARS-CoV-2 in the environment

Low dose colonization of broiler chickens with ESBL-/AmpC- producing escherichia coli in a seeder-bird model independent of antimicrobial selection pressure

Extended-spectrum beta-lactamase- (ESBL-) and AmpC beta-lactamase- (AmpC-) producing Enterobacteriaceae pose a risk for both human and animal health. For livestock, highest prevalences have been reported in broiler chickens, which are therefore considered as a reservoir of multidrug-resistant bacteria. The possibility of transfer to humans either by a close contact to colonized broiler flocks or through contaminated retail meat results in the necessity to develop intervention measures for the entire broiler production chain. In this regard, a basic understanding of the colonization process is mandatory including the determination of the minimal bacterial load leading to a persistent colonization of broiler chickens. Therefore, we conducted a bivalent broiler colonization study close to real farming conditions without applying any antimicrobial selection pressure. ESBL- and AmpC- negative broiler chickens (Ross 308) were co- colonized on their third day of life with two strains: one CTX-M-15-producing Escherichia coli-ST410 and one CMY-2/mcr-1-positive E. coli-ST10. Colonization was assessed by cloacal swabs over the period of the trial, starting 24 h post inoculation. During the final necropsy, the contents of crop, jejunum, cecum, and colon were quantified for the occurrence of both bacterial strains. To define the minimal oral colonization dosage 104 to 101 colony forming units (cfu) were orally inoculated to four separately housed broiler groups (each n = 19, all animals inoculated) and a dosage of already 101 cfu E. coli led to a persistent colonization of all animals of the group after 3 days. To assure stable colonization, however, a dosage of 102 cfu E. coli was chosen for the subsequent seeder-bird trial. In the seeder-bird trial one fifth of the animals (seeder, n = 4) were orally inoculated and kept together with the non-inoculated animals (sentinel, n = 16) to mimic the route of natural infection. After 35 days of trial, all animals were colonized with both E. coli strains. Given the low colonization dosage and the low seeder/sentinel ratio, the rapid spread of ESBL- and AmpC- producing Enterobacteriaceae in conventional broiler farms currently seems inevitably resulting in an urgent need for the development of intervention strategies to reduce colonization of broilers during production

Impact of different management measures on the colonization of broiler chickens with ESBL- and pAmpC- producing Escherichia coli in an experimental seeder-bird model

The colonization of broilers with extended-spectrum beta-lactamase- (ESBL-) and plasmid-mediated AmpC beta-lactamase- (pAmpC-) producing Enterobacteriaceae has been extensively studied. However, only limited data on intervention strategies to reduce the colonization throughout the fattening period are available. To investigate practically relevant management measures for their potential to reduce colonization, a recently published seeder-bird colonization model was used. Groups of 90 broilers (breed Ross 308) were housed in pens under conventional conditions (stocking of 39 kg/m(2), no enrichment, water and feed ad libitum). Tested measures were investigated in separate trials and included (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/m(2), and (III) the use of an alternative broiler breed (Rowan x Ranger). One-fifth of ESBL- and pAmpC- negative broilers (n = 18) per group were orally co-inoculated with two E. coli strains on the third day of the trial (seeder). One CTX-M-15-positive E. coli strain (ST410) and one CMY-2 and mcr-1-positive E. coli strain (ST10) were simultaneously administered in a dosage of 10(2) cfu. Colonization of all seeders and 28 non-inoculated broilers (sentinel) was assessed via cloacal swabs during the trials and a final necropsy at a target weight of two kilograms (= d 36 (control, I-II), d 47 (III)). None of the applied intervention measures reduced the colonization of the broilers with both the ESBL- and the pAmpC- producing E. coli strains. A strain-dependent reduction of colonization for the ESBL- producing E. coli strain of ST410 by 2 log units was apparent by the reduction of stocking density to 25 kg/m(2). Consequently, the tested management measures had a negligible effect on the ESBL- and pAmpC- colonization of broilers. Therefore, intervention strategies should focus on the prevention of ESBL- and pAmpC- colonization, rather than an attempt to reduce an already existing colonization

Persistent and Transient Airborne MRSA Colonization of Piglets in a Newly Established Animal Model

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was first found in 2005 and is up to date widespread in animal husbandry reservoir – focusing on pig farming. The regular detectability of MRSA in the air of pigsties as well as in exhaust air of pig farms (mean count: 102 cfu/m3) poses the question whether an airborne spread and, therefore, a MRSA colonization of animals via the airborne route exists. To answer this question, we exposed three groups of nine MRSA-negative tested piglets each to a defined airborne MRSA concentration (102, 104, and 106 cfu/m3) in our aerosol chamber for 24 h. In the following observation period of 21 days, the MRSA status of the piglets was monitored by taking different swab samples (nasal, pharyngeal, skin, conjunctival, and rectal swab). At the end of the experiment, we euthanized the piglets and investigated different tissues and organs for the spread of MRSA. The data of our study imply the presence of an airborne MRSA colonization route: the animals exposed to 106 cfu/m3 MRSA in the air were persistent colonized. The piglets exposed to an airborne MRSA concentration of 104 cfu/m3 were transient, and the piglets exposed to an airborne MRSA concentration of 102 cfu/m3 were not colonized. Consequently, a colonization via the airborne route was proven

Transmission pathways of campylobacter spp. at broiler farms and their environment in Brandenburg, Germany

Broiler meat is widely known as an important source of foodborne Campylobacter jejuni and Campylobacter coli infections in humans. In this study, we thoroughly investigated transmission pathways that may contribute to possible Campylobacter contamination inside and outside broiler houses. For this purpose we carried out a comprehensive longitudinal sampling approach, using a semi-quantitative cultivation method to identify and quantify transmissions and reservoirs of Campylobacter spp.. Three german broiler farms in Brandenburg and their surrounding areas were intensively sampled, from April 2018 until September 2020. Consecutive fattening cycles and intervening downtimes after cleaning and disinfection were systematically sampled in summer and winter. To display the potential phylogeny of barn and environmental isolates, whole genome sequencing (WGS) and bioinformatic analyses were performed. Results obtained in this study showed very high Campylobacter prevalence in 51/76 pooled feces (67.1%) and 49/76 boot swabs (64.5%). Average counts between 6.4 to 8.36 log10MPN/g were detected in pooled feces. In addition, levels of 4.7 and 4.1 log10MPN/g were detected in boot swabs and litter, respectively. Samples from the barn interior showed mean Campyloacter values in swabs from drinkers 2.6 log10MPN/g, walls 2.0 log10MPN/g, troughs 1.7 log10MPN/g, boards 1.6 log10MPN/g, ventilations 0.9 log10MPN/g and 0.7 log10MPN/g for air samples. However, Campylobacter was detected only in 7/456 (1.5%) of the environmental samples (water bodies, puddles or water-filled wheel tracks; average of 0.6 log10MPN/g). Furthermore, WGS showed recurring Campylobacter genotypes over several consecutive fattening periods, indicating that Campylobacter genotypes persist in the environment during downtime periods. However, after cleaning and disinfection of the barns, we were unable to identify potential sources in the broiler houses. Interestingly, alternating Campylobacter genotypes were observed after each fattening period, also indicating sources of contamination from the wider environment outside the farm. Therefore, the results of this study suggest that a potential risk of Campylobacter transmission may originate from present environmental sources (litter and water reservoirs). However, the sources of Campylobacter transmission may vary depending on the operation and farm environmental conditions

Antimicrobial effect of a drinking water additive comprising four organic acids on Campylobacter load in broilers and monitoring of bacterial susceptibility

Application of organic acids via feed or drinking water is under discussion as a possible intervention strategy to reduce Campylobacter (C.) load in primary poultry production. A previous in vitro study showed that reduced concentrations of sorbic acid, benzoic acid, propionic acid, and acetic acid were required for antibacterial activity against Campylobacter when using a mixture of these 4 acids compared to when using the single acids. The present study aimed at determining the antibacterial efficiency of this combination in vivo as a drinking water additive for reducing shedding and intestinal C. jejuni colonization in broilers. Furthermore, we assessed whether the inoculated C. jejuni strain BfR-CA-14430 adapted in vivo to the applied organic acids. Results of this study showed that adding the organic acids consistently reduced Campylobacter loads in cloacal swabs. While significant reductions were observed within the entire study period, a maximum 2 log reduction occurred at an age of 18 d. However, after dissection at the end of the trial, no significant differences were detected in Campylobacter loads of cecal and colon contents compared to the control group. Susceptibility testing of re-isolates from cloacal swabs and cecal content revealed equal minimum inhibitory concentration (MIC) values compared to the inoculated test strain, suggesting that C. jejuni remained susceptible throughout the trial

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