Detection of Enterococcus cecorum to identify persistently contaminated locations using faecal and environmental samples in broiler houses of clinically healthy flocks.

RESEARCH HIGHLIGHTS: Methodology is suitable to detect EC during production and after C&D.Locations were detected that may serve as a reservoir for EC.Cycles with fewer positive samples were observed.Cleaning and disinfection had a major impact on the detection of EC.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Serological monitoring of Enterococcus cecorum specific antibodies in chickens.

Pathogenic Enterococcus cecorum (EC) has gained increasing importance as the cause of skeletal infections in meat-type chicken production. Since effective intervention strategies are scarce, it must be focused on preventive measures. Vaccination of meat-type breeder chicken flocks is common practice to protect the progeny against infection with EC. However, no data are available on seroconversion after infection or vaccination. The aim of the present study was the serological monitoring of chickens for EC-specific immunoglobulin Y (IgY) using a newly established EC-specific, indirect ELISA for chickens. Sera from previous infection studies were used for the establishment of the assay. Serum samples from confirmed EC-positive meat-type chicken flocks, vaccinated, and non-vaccinated meat-type chicken breeder flocks were analyzed for EC-specific IgY. Comparison of ELISA results with results from real-time PCR and/or bacteriological examination via culture revealed fair to substantial agreement. In infected chickens, more samples were classified as positive via ELISA than via real-time PCR and/or bacteriological examination via culture. Focusing on chickens experimentally infected at 1 day post-hatch (dph), the highest proportion of positive results and highest S/P ratios were found at 42 dph (p < 0.05). A similar trend was observed for the samples from naturally infected chickens (p < 0.05). Adjustment of the secondary antibody against immunoglobulin M (IgM) may open possibilities to use the assay during the early phase of the growing period, when there is still a chance to treat the infection. The examination of samples from vaccinated and non-vaccinated meat-type breeder chickens revealed no significant differences of S/P ratios independent of farm and autogenous vaccine used. In addition to that, monitoring of a non-vaccinated meat-type breeder chicken flock at 4, 10, 15, and 19 weeks post-hatch showed a continuous increase of ELISA-positive serum samples associated with an increase of S/P ratios. This may be explained by cross reactivity with antibodies to Enterococcus hirae or natural antibodies. The usage of EC-specific, recombinant proteins for coating of the plates may help to reduce unspecific background and increase the assay's specificity in future applications. In conclusion, the newly developed ELISA provides a suitable tool for serological monitoring of meat-type chickens during experimental studies with EC under standardized conditions. Remarkably, the assay is able to detect a higher proportion of EC-positive chickens than other methods, which are currently available. However, the assay is not yet suitable for the monitoring of breeder flocks due to high background.

Research Note: Preliminary results, first detection of Enterococcus cecorum from environmental samples by streaking on X-Gluc containing selective media.

The isolation of cultivable E. cecorum from the environment of poultry houses remains a challenge. Environmental samples (dust wipes, equipment swabs, pooled feces) and samples from culled bird vertebras were collected from an infected broiler flock on d 37 posthatching. To isolate the bacterium from the cultivable microbiota, suspensions from the environmental samples were streaked onto a blood agar base medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexylammonium salt (X-Gluc), colistin sulfate, and nalidixin. The chromogenic reaction facilitated the isolation of E. cecorum from contaminated surfaces and pooled feces. Isolates from both the environment and vertebras were confirmed using MALDI-TOF and PCR analysis. Colony appearance and antimicrobial susceptibility tests revealed no phenotypic differences among the isolates. It remained unclear whether the isolates originated from the same clone. However, the principle of isolating the pathogen by streaking on a chromogenic agar may motivate researchers to investigate the transmission routes of infectious isolates, potentially leading to the optimization of biosecurity measures.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Characterization of Temperate LPS-Binding Bordetella avium Phages That Lack Superinfection Immunity.

Bordetella avium causes a highly infectious upper respiratory tract disease in turkeys and other poultry with high economic losses. Considering the antimicrobial resistance crisis, bacteriophages (phages) may be an alternative approach for treating bacterial infections such as bordetellosis. Here, we describe seven B. avium phages, isolated from drinking water and feces from chicken and turkey farms. They showed strong bacteriolytic activity with a broad host range and used lipopolysaccharides (LPS) as a host receptor for their adsorption. All phages are myoviruses based on their structure observed by transmission electron microscopy. Genome sequence analyses revealed genome assembly sizes ranging from 39,087 to 43,144 bp. Their permutated genomes were organized colinearly, with a conserved module order, and were packed according to a predicted headful packing strategy. Notably, they contained genes encoding putative markers of lysogeny, indicative of temperate phages, despite their lytic phenotype. Further investigation revealed that the phages could indeed undergo a lysogenic life cycle with varying frequency. However, the lysogenic bacteria were still susceptible to superinfection with the same phages. This lack of stable superinfection immunity after lysogenization appears to be a characteristic feature of B. avium phages, which is favorable in terms of a potential therapeutic use of phages for the treatment of avian bordetellosis. IMPORTANCE To maintain the effectiveness of antibiotics over the long term, alternatives to treat infectious diseases are urgently needed. Therefore, phages have recently come back into focus as they can specifically infect and lyse bacteria and are naturally occurring. However, there is little information on phages that can infect pathogenic bacteria from animals, such as the causative agent of bordetellosis of poultry, B. avium. Therefore, in this study, B. avium phages were isolated and comprehensively characterized, including whole-genome analysis. Although phenotypically the phages were thought to undergo a lytic cycle, we demonstrated that they undergo a lysogenic phase, but that infection does not confer stable host superinfection immunity. These findings provide important information that could be relevant for potential biocontrol of avian bordetellosis by using phage therapy.

Influence of heat stress on intestinal integrity and the caecal microbiota during Enterococcus cecorum infection in broilers.

Enterococcus cecorum (EC) is one of the most relevant bacterial pathogens in modern broiler chicken production from an economic and animal welfare perspective. Although EC pathogenesis is generally well described, predisposing factors are still unknown. This study aimed to understand the effect of heat stress on the caecal microbiota, intestinal integrity, and EC pathogenesis. A total of 373 1-day-old commercial broiler chicks were randomly assigned to four groups: (1) noninoculated, thermoneutral conditions (TN); (2) noninoculated, heat stress conditions (HS); (3) EC-inoculated, thermoneutral conditions (TN + EC); and (4) EC-inoculated, heat stress conditions (HS + EC). Birds were monitored daily for clinical signs. Necropsy of 20 broilers per group was performed at 7, 14, 21, and 42 days post-hatch (dph). A trend towards enhanced and more pronounced clinical disease was observed in the EC-inoculated, heat-stressed group. EC detection rates in extraintestinal tissues via culture were higher in the HS + EC group (~19%) than in the TN + EC group (~11%). Significantly more birds were colonized by EC at 7 dph in the HS + EC group (100%) than in the TN + EC group (65%, p < 0.05). The caecal microbiota in the two EC-inoculated groups was significantly more diverse than that in the TN group (p < 0.05) at 14 dph, which may indicate an effect of EC infection. An influence of heat stress on mRNA expression of tight junction proteins in the caecum was detected at 7 dph, where all six investigated tight junction proteins were expressed at significantly lower levels in the heat stressed groups compared to the thermoneutral groups. These observations suggest that heat stress may predispose broilers to EC-associated disease and increase the severity thereof. Furthermore, heat stress may impair intestinal integrity and promote EC translocation.

Health Status of Bycaught Common Eiders (Somateria mollissima) from the Western Baltic Sea.

The Common Eider (Somateria mollissima) inhabits the entire northern hemisphere. In northern Europe, the flyway population reaches from the southern Wadden Sea to the northern Baltic coast. The European population is classified as endangered due to declines in Common Eider numbers across Europe since 1990. In this study, we assessed 121 carcasses of Common Eiders, captured incidentally in gillnets in the Western Baltic between 2017 and 2019. The most common findings were parasitic infections of the intestine by acanthocephalans in 95 animals, which correlated with enteritis in 50% of the cases. Parasites were identified as Profilicollis botulus in 25 selected animals. Additionally, oesophageal pustules, erosions, and ulcerations, presumably of traumatic origin, were frequently observed. Nephritis and hepatitis were frequent, but could not be attributed to specific causes. Lung oedema, fractures and subcutaneous haemorrhages likely resulted from entangling and drowning. Two Common Eiders had mycobacterial infections and in one of these, Mycobacterium avium subspecies (ssp.) avium was identified. This study gives an overview of morphological changes and infectious diseases from one location of the European flyway population. It contributes to future health studies on Common Eiders in the Baltic and Wadden Seas by providing baseline information to compare with other areas or circumstances.

Proposal of a Method for Harmonized Broth Microdilution Antimicrobial Susceptibility Testing of Avibacterium gallinarum.

Avibacterium (Av.) gallinarum is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial susceptibility testing of this pathogen, so this study aimed at developing a harmonized broth microdilution method for Av. gallinarum that is suitable for diagnostic laboratories. For this, the Av. gallinarum CCUG 12391T type strain and 42 field isolates were collected and their species was confirmed by using a species-specific PCR assay and biochemical reactions. To select epidemiologically unrelated isolates, ApaI macrorestriction analysis was performed. Preliminary growth experiments were conducted with six culture media, and based on the results, four media were selected to compile growth curves with four isolates. Independent repetitions of MIC determinations were then performed to evaluate the reproducibility of the values. Cation-adjusted Mueller-Hinton broth (CAMHB) was initially selected as broth medium, but did not show sufficient homogeneity of MICs. Therefore, CAMHB plus 1% chicken serum and 0.0025% NADH was selected and showed a good homogeneity of MICs after 20 h and 24 h of incubation at 35 ± 2°C. This was reflected in essential MIC agreements ranging between 96% and 100%. Testing of a larger Av. gallinarum collection (n = 43) revealed that easily readable MICs could be obtained for the type strain and all isolates. Some Av. gallinarum showed elevated MICs of enrofloxacin (n = 35), nalidixic acid (n = 35), penicillin (n = 2), tetracycline (n = 19), and/or trimethoprim-sulfamethoxazole (n = 1). By using PCR analyses, the following antimicrobial resistance genes were detected: blaTEM, dfrA14, sul2, tet(B), tet(H). The study demonstrated that the proposed medium is suitable for a harmonized broth microdilution susceptibility testing of Av. gallinarum with a recommended incubation time of 20 to 24 h.

Influence of lincomycin-spectinomycin treatment on the outcome of Enterococcus cecorum infection and on the cecal microbiota in broilers.

BACKGROUND: Enterococcus cecorum (EC) is one of the main reasons for skeletal disease in meat type chickens. Intervention strategies are still rare and focus mainly on early antibiotic treatment of the disease, although there are no data available concerning the effectivity of this procedure. The present study aimed to investigate the effectivity of early lincomycin-spectinomycin treatment during the first week of life after EC-infection. Furthermore, the impact of lincomycin-spectinomycin treatment and EC infection on the development of cecal microbiota was investigated. METHODS: A total of 383 day-old broiler chicks were randomly assigned to four groups (non-infected and non-treated, non-infected and treated, EC-infected and non-treated, and EC-infected and treated). The EC-infected groups were inoculated orally with an EC suspension at the day of arrival and at study day 3. The treatment groups were treated with lincomycin-spectinomycin via the drinking water for six consecutive days, starting two hours after the first inoculation. Necropsy of 20 chickens per group was performed at study days 7, 14, 21, and 42. Bacteriological examination via culture and real-time PCR was performed to detect EC in different extraintestinal organs. Cecal samples of nine chickens per group and necropsy day were analyzed to characterize the composition of the cecal microbiota. RESULTS: No clinical signs or pathologic lesions were found at necropsy, and EC was not detected in extraintestinal organs of the EC-infected and treated birds. Lincomycin-spectinomycin promoted the growth of the bacterial genus Escherichia/Shigella and reduced the amount of potentially beneficial Lactobacillus spp. in the ceca regardless of EC-infection. Unexpectedly, the highest abundances of the genus Enterococcus were found directly after ending antibiotic treatment in both treatment groups, suggesting the growth of resistant enterococcal species. EC was not detected among the most abundant members of the genus Enterococcus. Oral EC-infection at the first day of life did not influence the development of cecal microbiota in the present study. CONCLUSIONS: Lincomycin-spectinomycin treatment during the first week of life can prevent the EC-associated disease in broiler type chickens and has a direct impact on the development of the cecal microbiota. The low abundance of EC in the ceca of infected chickens underlines the pathogenic nature of the disease-causing EC strains. Further research on alternative prevention and intervention strategies is needed with regard to current efforts on reducing the use of antibiotics in livestock animals.

Development of a harmonized method for antimicrobial susceptibility testing of Bordetella avium using broth microdilution and detection of resistance genes.

AIMS: In response to a request from the Clinical and Laboratory Standards Institute (CLSI), the objective of this study was to develop a harmonized method for broth microdilution susceptibility testing of Bordetella (B.) avium, the major causative agent of infectious coryza in poultry. METHODS AND RESULTS: To find a suitable test medium, growth curves with four epidemiologically unrelated B. avium isolates were created in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB + 2.5% lysed horse blood and veterinary fastidious medium. All isolates showed good growth in CAMHB, therefore MIC values were determined using this medium and the homogeneity of the values was determined. An essential MIC agreement of 99.7% was calculated. Testing of a larger strain collection (n = 49) for their susceptibility to 24 antimicrobials confirmed the suitability of the tested method and revealed some isolates with elevated MICs of florfenicol (n = 1), streptomycin (n = 2), tetracyclines (n = 5), and trimethoprim/sulfamethoxazole (n = 6). PCR assays detected the resistance genes aadA1, dfrB1, floR, sul1, sul2 and tet(A). CONCLUSIONS: The method used enables easy reading and a good reproducibility of MIC values for B. avium. SIGNIFICANCE AND IMPACT OF STUDY: Application of the tested method allows harmonized resistance testing of B. avium and identification of isolates with elevated MIC values.

Different virulence levels of Enterococcus cecorum strains in experimentally infected meat-type chickens.

In recent years, pathogenic strains of Enterococcus cecorum (EC) have emerged as a causing agent of septicemia and skeletal infection in broiler chickens with a high economic impact worldwide. Although research has been conducted, many aspects of the pathogenesis of the EC-associated disease are still unknown. In the present study, an experimental infection model was established in broiler chickens. Two different EC strains (EC14 and EC15) were compared in two different concentrations of each strain (2 × 106 and 2 × 108 colony-forming units per milliliter (CFU/mL)) after oral infection of one-day-old chicks. Clinical signs and gross lesions of the EC-associated disease were monitored in the following seven weeks. Although both EC strains were originally isolated from clinical disease outbreaks and had a high embryonic lethality, only EC14 successfully induced the typical course of the EC-associated disease with characteristic clinical signs and gross lesions. In total, 23% of the birds in the two EC14-groups were EC-positive in extraintestinal organs on culture, and no differences were found between the two infectious doses. EC14 was frequently detected via real-time PCR in the free thoracic vertebra (FTV) and femoral heads without any detectable gross lesions. The number of EC positive spleens from infected broilers was comparable using bacterial isolation and a specific real-time PCR. Interestingly, EC15 was not detected in extraintestinal organs, although birds in the EC15 groups were colonized by EC in the ceca after experimental infection. The present study represents first proof that virulence differs among EC strains in experimentally infected chickens, and emphasizes the need to further characterize virulence factors and pathogenic mechanisms of EC. The strain EC14 at a dose of 106 CFU is suitable for reproduction of the EC-associated disease. The experimental infection model reported here provides the basis for further research on the EC pathogenesis and possible prevention and intervention strategies.

Examination of common swifts (Apus apus) for salmonella shedding in the area of Hannover, Lower Saxony, Germany.

OBJECTIVE: The available literature indicates a high prevalence of the zoonotic pathogen Salmonella (S.) enterica serovar Infantis in the common swift (Apus apus). This long-distance migrant, which only consumes aerial plankton, can reach high population densities in places with suitable breeding sites. Dedicated competent private persons take part in the hand rearing of juvenile common swifts in wildlife rescue centres, which unavoidably results in close contact with these avian patients. For this reason, we examined common swifts for shedding of Salmonella spp. MATERIAL AND METHODS: In the years 2014 and 2019, intestinal swabs or fresh faeces of common swifts (2014: n = 54; 2019: n = 62) were examined microbiologically (DIN EN ISO 6579; Annex D) in the area of Hannover, Lower Saxony, Germany. RESULTS: Salmonella spp. could not be detected in any of the examined common swifts within the investigation period and the studied area in 2014 and 2019. CONCLUSION AND CLINICAL RELEVANCE: The results illustrate that the common swift is unlikely to be a natural reservoir of Salmonella spp. For the transmission of salmonella by swifts the local conditions with the corresponding environmental impact seem to play a significant role, and the risk of transmission should be assessed according to the region to be examined.

Tenacity of Enterococcus cecorum at different environmental conditions.

AIMS: Our aim was to analyse the survival of Enterococcus cecorum (EC) at various temperatures, relative air humidities and on different substrates commonly existing in broiler houses. METHODS AND RESULTS: A pathogenic EC isolate (EC14) was used to inoculate sterile litter, polyvinyl chloride (PVC) and dust samples. Incubation at 37, 25 or 15°C with either 32% relative humidity (RH) or 78% RH followed. At defined time points (0-4272 h post-inoculation), samples were examined in triplicate for the total viable count. Selected combinations were repeated for a non-pathogenic and two additional pathogenic EC strains. For EC14, the measured survival time ranged from 48 to 4272 h (178 days) depending on the substrate-humidity-temperature combination. The longevity was the highest on litter, followed by dust and then PVC. Lower temperatures facilitated its survival, lower relative air humidity favoured the survival only in combination with 25 or 15°C. All three pathogenic strains showed longer survival times (up to 432 h, 18 days) compared to the non-pathogenic EC strain (168 h, 7 days) under the same conditions. CONCLUSIONS: Enterococcus cecorum demonstrates a high persistence in the environment especially at 15°C and 32% RH. SIGNIFICANCE AND IMPACT OF THE STUDY: Hygiene management plans should consider the durability of EC and the risk of a carry-over to control consecutive EC outbreaks.

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety.

Tropical shrimp, like Litopenaeus vannamei, in land-based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.

Development of an in-house ELISA for detection of antibodies against Enterococcus cecorum in Pekin ducks.

Enterococcus cecorum (EC) is known to cause skeletal lesions in broiler chickens and also systemic infections in Pekin ducks. Despite the importance of the pathogen, there is still a lack of serological diagnostic tools for the detection of EC infections. Here we describe the development of an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the detection of EC-specific antibodies and its application by examination of 67 sera from experimentally infected Pekin ducks, 710 field samples from four Pekin duck breeder flocks previously vaccinated with inactivated vaccines, and 80 samples from commercial Pekin ducks coming from vaccinated parent flocks. All groups that had been experimentally inoculated via the air sac route were positive in the new ELISA, with significantly (P ≤ 0.05) increased mean sample/positive (S/P) ratios of 0.71-2.70 at days 7, 14 and 21 post-infection, while orally inoculated ducks and the EC-free control group remained negative with mean S/P ratios of 0.0-0.15. Antibodies were also detected in each of four vaccinated Pekin duck breeder flocks; 67.8% of the samples were antibody positive. The highest S/P ratios were found between 16 and 26 weeks (median S/P ratios from 0.15 to 1.03), but antibodies were still detected in some serum samples in weeks 61-67 post-hatch. No antibodies were detected in the commercial Pekin ducks. Antibody development in the ducks may be influenced by the composition of the inactivated vaccine. The new ELISA provides a useful tool for investigations of response to EC infections and vaccinations.

Impact of Bacteriophage-Supplemented Drinking Water on the E. coli Population in the Chicken Gut.

Among intestinal coliform microbes in the broiler gut, there are potentially pathogenic Escherichia (E.) coli that can cause avian colibacillosis. The treatment with antibiotics favors the selection of multidrug-resistant bacteria and an alternative to this treatment is urgently required. A chicken model of intestinal colonization with an apathogenic model strain of E. coli was used to test if oral phage application can prevent or reduce the gut colonization of extraintestinal pathogenic E. coli variants in two individual experiments. The E. coli strain E28 was used as a model strain, which could be differentiated from other E. coli strains colonizing the broiler gut, and was susceptible to all cocktail phages applied. In the first trial, a mixture of six phages was continuously applied via drinking water. No reduction of the model E. coli strain E28 occurred, but phage replication could be demonstrated. In the second trial, the applied mixture was limited to the four phages, which showed highest efficacy in vitro. E. coli colonization was reduced in this trial, but again, no reduction of the E. coli strain E28 was observed. The results of the trials presented here can improve the understanding of the effect of phages on single strains in the multi-strain microbiota of the chicken gut.

In vitro characterization and genetic diversity of Bordetella avium field strains.

Bordetella avium (BA) is a respiratory pathogen of particular importance for turkeys. Specific adherence and damage to the respiratory epithelia are crucial steps of the pathogenesis, but knowledge about the mechanisms and the variety of virulence in field strains is limited. We analysed 17 BA field strains regarding their in vitro virulence-associated properties in tracheal organ cultures (TOC) of turkey embryos, and their genetic diversity. The TOC adherence assay indicated that BA field strains differ considerably in their ability to adhere to the tracheal mucosa, while the TOC ciliostasis assay illustrated a high degree of diversity in ciliostatic effects. These two virulence-associated properties were associated with each other in the investigated strains. Three of the investigated strains displayed significantly (P > 0.05) lower in vitro virulence in comparison to other strains. Genetic diversity of BA strains was analysed by core genome multilocus sequence typing (cgMLST). We applied a cgMLST scheme comprising 2667 targets of the reference genome (77.3% of complete genome, BA strain 197N). The results showed a broad genetic diversity in BA field strains but did not demonstrate a correlation between sequence type and virulence-associated properties. The cgMLST analysis revealed that strains with less marked virulence-associated properties had a variety of mutations in the putative filamentous haemagglutinin gene. Likewise, amino acid sequence alignment indicated variations in the protein. The results from our study showed that both adherence and ciliostasis assay can be used for virulence characterization of BA. Variations in the filamentous haemagglutinin protein may be responsible for reduced virulence of BA field strains.

Diagnostic methods for identifying different Aeromonas species and examining their pathogenicity factors, their correlation to cytotoxicity and adherence to fish mucus.

Aeromonas spp. are ubiquitous in the aquatic environment, acting as facultative or obligate pathogens for fish. Identifying Aeromonas spp. is important for pathogenesis and prognosis in diagnostic cases but can be difficult because of their close relationship. Forty-four already characterized isolates of Aeromonas spp. were analysed by 16S rRNA gene sequencing, by gyrase B sequencing, by analysing their fatty acid profiles, by biochemical reactions and by MALDI-TOF MS. To determine their pathogenicity, cytotoxicity, adhesion to mucus and the expression of 12 virulence factors were tested. The susceptibility of the isolates towards 13 different antibiotics was determined. MALDI-TOF MS was found to be an acceptable identification method for Aeromonas spp. Although the method does not detect all species correctly, it is time-effective and entails relatively low costs and no other methods achieved better results. A high prevalence of virulence-related gene fragments was detected in almost all examined Aeromonas spp., especially in A. hydrophila and A. salmonicida, and most isolates exhibited a cytotoxic effect. Single isolates of A. hydrophila and A. salmonicida showed multiple resistance to antibiotics. These results might indicate the potentially pathogenic capacity of Aeromonas spp., suggesting a risk for aquatic animals and even humans, given their ubiquitous nature.

Recommendations for identifying pathogenic Vibrio spp. as part of disease surveillance programmes in recirculating aquaculture systems for Pacific white shrimps (Litopenaeus vannamei).

Due to their pathogenic potential, identifying Vibrio species from recirculating aquaculture systems (RAS) for Pacific white shrimp (Litopenaeus vannamei) is of great importance to determine the risk for animal's as well as for the consumer's health. The present study compared identification results for a total of 93 Vibrio isolates, including type strains and isolates from shrimp aquaculture. Results from biochemical identifications, 16S rRNA sequencing, sequencing of the uridylate kinase encoding gene pyrH and analysis of the protein spectra assessed by MALDI-TOF MS were compared. The results achieved by these different methods were highly divergent for many of the analysed isolates and for several Vibrio spp difficulties in reliably identifying occurred. These difficulties mainly resulted from missing entries in digital databases, a low number of comparable isolates analysed so far, and high interspecific similarities of biochemical traits and nucleotide sequences between the closely related Vibrio species. Due to the presented data, it can be concluded that for identifying Vibrio spp. from samples in routine diagnostics, it is recommended to use MALDI-TOF MS analysis for a quick and reliable identification of pathogenic Vibrio sp. Nevertheless, editing the database, containing the main spectra of Vibrio is recommended to achieve reliable identification results.