Research Note: Curbing Salmonella Enteritidis in broiler chickens with palm-free medium-chain fatty acids.

Salmonellosis is still one of the most reported zoonoses worldwide and poultry meat is a major source, as chickens are often persistent carriers of Salmonella. Medium-chain fatty acids (MCFA) are known for their strong antimicrobial activity. MCFAs used today in the animal feed industry, however, mainly originate from the palm oil industry, which is notorious for its negative impact on the climate. We investigated the effect of a specific blend of palm-free MCFAs (ranging from C6 to C9) on Salmonella Enteritidis (SE) colonization in broiler chickens and in vitro SE characteristics. Fifty Ross 308 broiler chickens were randomly divided in 2 treatment groups. Chickens received either un-supplemented feed or feed supplemented with 300 ppm MCFAs from D0 onwards. On D7, all chickens were orally inoculated with 1600 CFU of SE. Cloacal swabs (D11) and samples of liver and caeca (D12) of all animals were collected and SE was enumerated. Percentage of SE-positive caecum samples was significantly (P = 0.044) reduced in birds receiving MCFAs compared to those receiving unsupplemented feed (36% vs. 64%). In vitro work performed with the same SE strain showed that preincubating the Salmonella bacteria with MCFAs at a sub-minimal inhibitory concentration significantly (p < 0.05) reduced bacterial adhesion to and invasion in Caco-2 cells, which may explain the observed reduction in intestinal SE colonization in the in vivo trial. Together, these results show that the tested eco-friendly MCFA blend could be a promising tool in the control of Salmonella in broilers.

Agricultural contaminants in amphibian breeding ponds: Occurrence, risk and correlation with agricultural land use.

Anthropogenic pressure such as agricultural pollution globally affects amphibian populations. In this study, a total of 178 different compounds from five agrochemical groups (i.e. antimicrobial drugs residues (ADRs), coccidiostats and anthelmintics, heavy metals, mycotoxins and pesticides) were determined monthly, from March until June 2019 in 26 amphibian breeding ponds in Flanders, Belgium. Furthermore, a possible correlation between the number and concentration of selected contaminants that were found and the percentage of arable land within a 200 m radius was studied. Within each group, the highest detected concentrations were obtained for 4-epioxytetracycline (0.422 µg L-1), levamisole (0.550 µg L-1), zinc (333.1 µg L-1), 3-acetyldeoxynivalenol (0.013 µg L-1), and terbuthylazine (38.7 µg L-1), respectively, with detection frequencies ranging from 1 (i.e. 3-acetyldeoxynivalenol) to 26 (i.e. zinc) out of 26 ponds. Based on reported acute and chronic ecotoxicological endpoints, detected concentrations of bifenthrin, cadmium, copper, cypermethrin, hexachlorobenzene, mercury, terbuthylazine, and zinc pose a substantial ecological risk to aquatic invertebrates such as Daphnia magna and Ceriodaphnia dubia, which both play a role in the food web and potentially in amphibian disease dynamics. Additionally, the detected concentrations of copper were high enough to exert chronic toxicity in the gray treefrog (Hyla versicolor). The number of detected compounds per pond ranged between 0 and 5 (ADRs), 0 - 2 (coccidiostats and anthelmintics), 1 - 7 (heavy metals), 0 - 4 (mycotoxins), and 0 - 12 (pesticides) across the four months. Furthermore, no significant correlation was demonstrated between the number of detected compounds per pond, as well as the detected concentrations of 4-epioxytetracycline, levamisole, copper, zinc, enniatin B and terbuthylazine, and the percentage of arable land within a 200 m radius. For heavy metals and pesticides, the number of compounds per pond varied significantly between months. Conclusively, amphibian breeding ponds in Flanders were frequently contaminated with agrochemicals, yielding concentrations up to the high µg per liter level, regardless of the percentage surrounding arable land, however showing temporal variation for heavy metals and pesticides. This research also identifies potential hazardous substances which may be added to the European watch list (CD 2018/408/EC) in the future.

Multi-residue analysis of 20 mycotoxins including major metabolites and emerging mycotoxins in freshwater using UHPLC-MS/MS and application to freshwater ponds in flanders, Belgium.

Mycotoxins are known for their negative impact on human and animal health as they frequently contaminate food and feed products from crop origin that are consumed by humans and animals. Furthermore, mycotoxins can leach out of plant tissue, to be transported through runoff water into nearby ponds where they can exert negative effects on aquatic organisms, such as fish, amphibians and zooplankton. The overall goal of this study was to develop a SPE-UHPLC-MS/MS method for the detection and quantification of multiple mycotoxins in amphibian breeding ponds. The method was validated and yielded acceptable within-run and between-run apparent recoveries and precision, as well as good linearity. Matrix effects (i.e. 75.7-109.6%, ≤ 17.8% RSD) were evaluated using water from 20 different ponds in Flanders, Belgium. By incorporating internal standards, overall results improved and adequate precision values (i.e. ≤ 15%) were obtained according to the EMA guideline. Additionally, extraction recovery (n = 3) was evaluated, yielding good results for all mycotoxins (i.e. 75.3-109.1%, ≤15% RSD), except for AME (i.e. 6.7 ± 0.7%), which implied the need for a matrix-matched calibration curve. Detection sensitivity was in the low nanograms per liter range. Storage stability experiments indicated that sample storage at 4 °C in amber glass bottles and analysis performed within 96 h after sampling was sufficient to avoid loss by degradation for all compounds, excluding ß-ZAL and ß-ZEL, for which analysis within 24 h is more indicated. The method was successfully applied to water samples originating from 18 amphibian breeding ponds situated across Flanders. Overall, enniatins B, B1 and A1 were most commonly detected at maximum concentrations of 6.9, 3.3 and 2.6 ng L-1, respectively, followed by detection of beauvericin (1.1 ng L-1 and < 1 ng L-1), alternariol monomethyl ether (< 10 ng L-1), HT2-toxin (< 40 ng L-1), zearalenone (< 25 ng L-1) and α-zearalanol (< 10 ng L-1). We believe that this method will boost further research into the dynamics and ecotoxicological impact of mycotoxins in aquatic environments.

Highly sensitive multi-residue analysis of veterinary drugs including coccidiostats and anthelmintics in pond water using UHPLC-MS/MS: application to freshwater ponds in Flanders, Belgium.

Veterinary drugs, such as coccidiostats and anthelmintics are routinely administered in extensive animal husbandry, finding their way into the aquatic environment through urine and/or feces of treated animals kept outdoors or by the application of contaminated liquid manure on agricultural fields and subsequent mechanisms of surface run-off, leaching and drift. Several of these compounds are known to exert acute and chronic toxicity effects on aquatic organisms, and can lead to changes in biodiversity and ecosystem functioning. The overall objective of this research was to develop, validate and apply a highly sensitive, multi-residue SPE-UHPLC-MS/MS method for the determination of 12 coccidiostats, registered as a feed supplement or veterinary medicine in Europe and three regularly used anthelmintics, in pond water, often functioning as amphibian habitat. Sample extraction was optimized using a fractional factorial resolution design. Pond water filtration efficiency (i.e. 80-118%, ≤25% RSD) and matrix effects (i.e. 72-119%, ≤39% RSD) were evaluated using water from respectively 3 and 20 different ponds in Flanders. By incorporating internal standards, overall results improved and adequate precision values (i.e.≤15%) were obtained according to the EMA guidelines. Acceptable within-run and between-run apparent recoveries, satisfactory precision as well as good linearity were demonstrated according to the CD 2002/657/EC, SANTE/12682/2019 and VICH 49 guidelines, except for robenidine for which the between-day precision was between 21.0 and 34.5%. Sample storage stability studies indicated that storage at 4 °C and analysis performed within 96 hours after sampling was sufficient to avoid loss by degradation for all compounds, excluding robenidine. Values for the limit of detection (LOD) and quantification (LOQ) were in nanograms per liter, which was essential for the environmental application of this novel method. The method was successfully applied on grab water samples from the water surface of 18 different ponds across Flanders, Belgium, detecting amprolium and levamisole at concentrations below the LOQ of 2.5 ng L-1 and at 250.0 ng L-1 or below the LOQ of 250.0 ng L-1, respectively. In conclusion, our newly developed method may provide insights about the contamination status of amphibian breeding ponds.

Evaluation of group vaccination of sows and gilts against Salmonella Typhimurium with an attenuated vaccine in subclinically infected pig herds.

Subclinical Salmonella Typhimurium infections occur frequently in pigs and constitute a major risk for human salmonellosis. With the currently available control measures, Salmonella Typhimurium infections in pigs remain difficult to control. Vaccination has been proposed to be an effective tool to control infections at farm level. In the current study, the effect of group vaccination of sows and gilts against Salmonella Typhimurium is evaluated on Salmonella prevalence in fecal and overshoe samples and ileocecal lymph nodes, and on serology in the sows and their offspring in three subclinically infected pig farms. In each farm, all sows and gilts were vaccinated twice, three weeks apart, with an attenuated histidine-adenine auxotrophic vaccine (Salmoporc®, IDT Biologika). From three months after the group vaccination onwards, all sows were given a booster dose three weeks before every farrowing. The farms were monitored bacteriologically and serologically from 12 months before until 15 months after the group vaccination. After group vaccination, no significant effect was detected in the prevalence of Salmonella Typhimurium in the fecal and overshoe samples collected in the sows (before: 2 %, after: 0 %) and their offspring at 18 weeks (before: 17 %, after: 11 %) and at 26 weeks of age (before: 15 %, after: 7 %), and when combining the results of the offspring at 18 and 26 weeks of age (before: 16 %, after: 9 %). Also, no significant effect was detected in the prevalence of Salmonella Typhimurium positive lymph nodes of sows (before and after: 0 %) and their offspring (before: 4 %, after: 7 %). Regarding serology, the mean S/P-ratios of the sows were significantly higher after the group vaccination, compared to before group vaccination (before: 1.50, after: 2.32, p < 0.001). The mean S/P-ratios of the offspring at slaughter age were significantly lower after the group vaccination, compared to before group vaccination (before: 1.71, after: 1.04, p = 0.001). In conclusion, group vaccination of sows and gilts resulted in a more beneficial serological status of the offspring, but did not significantly decrease Salmonella Typhimurium excretion and lymph node contamination.

Bacteriological evaluation of vaccination against Salmonella Typhimurium with an attenuated vaccine in subclinically infected pig herds.

Subclinical infections with Salmonella Typhimurium occur frequently in pigs. They constitute a risk for human salmonellosis and are difficult to control with currently available control measures. Vaccination against Salmonella Typhimurium in pigs can be an effective tool to control Salmonella infections at farm level. In the present study, the efficacy of an attenuated Salmonella Typhimurium vaccine (Salmoporc®, IDT Biologika) to control Salmonella infections in pigs was evaluated in three subclinically infected pig herds. The effect on Salmonella excretion and the number of pigs positive for Salmonella Typhimurium field and vaccine strains in ileocecal lymph nodes at slaughter were evaluated using five different vaccination strategies: 1. vaccination of sows, 2. vaccination of sows and piglets, 3. vaccination of sows and fattening pigs, 4. vaccination of piglets, 5. vaccination of fattening pigs, which were all compared to a non-vaccinated control group (experimental group 6). Each vaccination strategy was implemented in each farm, during two consecutive production cycles of the same sows. The prevalence of Salmonella Typhimurium field strain excretion was low; in total, 4% of the fecal and overshoe samples collected in the non-vaccinated control group were Salmonella Typhimurium field strain positive. The excretion of Salmonella Typhimurium field strain did not significantly differ between farms, production cycles and experimental groups. Applying vaccination in either sows and piglets, sows and fattening pigs, or in piglets only, resulted in a significantly reduced number of Salmonella Typhimurium field strain positive lymph nodes of slaughter pigs in the second production cycle, but not in the first production cycle. Vaccination of sows and piglets resulted in the most consistent reduction of Salmonella Typhimurium field strain positive lymph nodes at slaughter. The vaccine strain was detected in the lymph nodes of 13 pigs at slaughter, indicating the possible persistence of the vaccine strain until slaughter. Because of limitations in the study design, and the variability between farms and production cycles, the results of the current observational study should be extrapolated with care. Nevertheless, the results provide evidence that applying vaccination against Salmonella Typhimurium in sows and piglets (preferred), sows and fattening pigs, and piglets only can support the control of Salmonella Typhimurium infections by decreasing the prevalence of Salmonella Typhimurium field strain positive lymph nodes at slaughter.

Effects of attenuated vaccine protocols against Salmonella Typhimurium on Salmonella serology in subclinically infected pig herds.

Vaccination of pigs against Salmonella Typhimurium (S. Typhimurium) can be effective for the control of Salmonella infections at the farm level and reduce the risk of Salmonella contamination in the food chain. However, vaccination may interfere with herd serological status in serology-based Salmonella monitoring programs. The present study investigated the effects of an attenuated S. Typhimurium vaccine (Salmoporc, IDT Biologika) on Salmonella serology in sows, neonatal piglets and slaughter pigs from three subclinically infected herds. Within each herd, five different vaccination protocols were tested as follows: group 1, vaccination of sows; group 2, vaccination of sows and piglets; group 3, vaccination of sows and fattening pigs; group 4, vaccination of piglets; and group 5 vaccination of fattening pigs. Each group was compared to a non-vaccinated control group (group 6). Sera were analyzed by ELISA (HerdChek Swine Salmonella, IDEXX Laboratories) and sample-to-positive (S/P) ratios were calculated. At day 3 after farrowing, but not before vaccination, S/P ratios in vaccinated sows (mean: 2.21) were significantly higher than S/P ratios in non-vaccinated sows (mean: 0.87, P<0.001). S/P ratios in 3-day old piglets from vaccinated sows (mean: 2.46) were significantly higher than S/P ratios in similar piglets from non-vaccinated sows (mean: 0.73, P<0.001). At slaughter, S/P ratios in pigs from groups 2, 3, 4 and 5 were significantly higher than those in the non-vaccinated control group (P<0.001). Therefore, vaccination of piglets and fattening pigs could have implications for current serology-based Salmonella monitoring programs in slaughter pigs.

Recommendations on diagnostic tools for Batrachochytrium salamandrivorans.

Batrachochytrium salamandrivorans (Bsal) poses a major threat to amphibian, and more specifically caudata, diversity. Bsal is currently spreading through Europe, and mitigation measures aimed at stopping its spread and preventing its introduction into naïve environments are urgently needed. Screening for presence of Bsal and diagnosis of Bsal-induced disease in amphibians are essential core components of effective mitigation plans. Therefore, the aim of this study was to present an overview of all Bsal diagnostic tools together with their limitations and to suggest guidelines to allow uniform interpretation. Here, we investigate the use of different diagnostic tools in post-mortem detection of Bsal and whether competition between Bd and Bsal occurs in the species-specific Bd and Bsal duplex real-time PCR. We also investigate the diagnostic sensitivity, diagnostic specificity and reproducibility of the Bsal real-time PCR and show the use of immunohistochemistry in diagnosis of Bsal-induced chytridiomycosis in amphibian samples stored in formaldehyde. Additionally, we have drawn up guidelines for the use and interpretation of the different diagnostic tools for Bsal currently available, to facilitate standardization of execution and interpretation.

Salmonella Typhimurium DT193 and DT99 are present in great and blue tits in Flanders, Belgium.

Endemic infections with the common avian pathogen Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) may incur a significant cost on the host population. In this study, we determined the potential of endemic Salmonella infections to reduce the reproductive success of blue (Cyanistes caeruleus) and great (Parus major) tits by correlating eggshell infection with reproductive parameters. The fifth egg of each clutch was collected from nest boxes in 19 deciduous forest fragments. Out of the 101 sampled eggs, 7 Salmonella Typhimurium isolates were recovered. The low bacterial prevalence was reflected by a similarly low serological prevalence in the fledglings. In this study with a relatively small sample size, presence of Salmonella did not affect reproductive parameters (egg volume, clutch size, number of nestlings and number of fledglings), nor the health status of the fledglings. However, in order to clarify the impact on health and reproduction a larger number of samples have to be analyzed. Phage typing showed that the isolates belonged to the definitive phage types (DT) 193 and 99, and multi-locus variable number tandem repeat analysis (MLVA) demonstrated a high similarity among the tit isolates, but distinction to human isolates. These findings suggest the presence of passerine-adapted Salmonella strains in free-ranging tit populations with host pathogen co-existence.

Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry.

Bacterial Zoonoses Transmitted by Household Pets: State-of-the-Art and Future Perspectives for Targeted Research and Policy Actions.

The close contact between household pets and people offers favourable conditions for bacterial transmission. In this article, the aetiology, prevalence, transmission, impact on human health and preventative measures are summarized for selected bacterial zoonoses transmissible by household pets. Six zoonoses representing distinct transmission routes were selected arbitrarily based on the available information on incidence and severity of pet-associated disease caused by zoonotic bacteria: bite infections and cat scratch disease (physical injuries), psittacosis (inhalation), leptospirosis (contact with urine), and campylobacteriosis and salmonellosis (faecal-oral ingestion). Antimicrobial resistance was also included due to the recent emergence of multidrug-resistant bacteria of zoonotic potential in dogs and cats. There is a general lack of data on pathogen prevalence in the relevant pet population and on the incidence of human infections attributable to pets. In order to address these gaps in knowledge, and to minimize the risk of human infection, actions at several levels are recommended, including: (1) coordinated surveillance of zoonotic pathogens and antimicrobial resistance in household pets, (2) studies to estimate the burden of human disease attributable to pets and to identify risk behaviours facilitating transmission, and (3) education of those in charge of pets, animal caretakers, veterinarians and human medical healthcare practitioners on the potential zoonotic risks associated with exposure to pets. Disease-specific recommendations include incentives to undertake research aimed at the development of new diagnostic tests, veterinary-specific antimicrobial products and vaccines, as well as initiatives to promote best practices in veterinary diagnostic laboratories and prudent antimicrobial usage.

Successful treatment of Batrachochytrium salamandrivorans infections in salamanders requires synergy between voriconazole, polymyxin E and temperature.

Chytridiomycosis caused by the chytrid fungus Batrachochytrium salamandrivorans (Bsal) poses a serious threat to urodelan diversity worldwide. Antimycotic treatment of this disease using protocols developed for the related fungus Batrachochytrium dendrobatidis (Bd), results in therapeutic failure. Here, we reveal that this therapeutic failure is partly due to different minimum inhibitory concentrations (MICs) of antimycotics against Bsal and Bd. In vitro growth inhibition of Bsal occurs after exposure to voriconazole, polymyxin E, itraconazole and terbinafine but not to florfenicol. Synergistic effects between polymyxin E and voriconazole or itraconazole significantly decreased the combined MICs necessary to inhibit Bsal growth. Topical treatment of infected fire salamanders (Salamandra salamandra), with voriconazole or itraconazole alone (12.5 µg/ml and 0.6 µg/ml respectively) or in combination with polymyxin E (2000 IU/ml) at an ambient temperature of 15 °C during 10 days decreased fungal loads but did not clear Bsal infections. However, topical treatment of Bsal infected animals with a combination of polymyxin E (2000 IU/ml) and voriconazole (12.5 µg/ml) at an ambient temperature of 20 °C resulted in clearance of Bsal infections. This treatment protocol was validated in 12 fire salamanders infected with Bsal during a field outbreak and resulted in clearance of infection in all animals.

Treatment of urodelans based on temperature dependent infection dynamics of Batrachochytrium salamandrivorans.

The recently emerged chytrid fungus Batrachochytrium salamandrivorans currently causes amphibian population declines. We hypothesized that temperature dictates infection dynamics of B. salamandrivorans, and that therefore heat treatment may be applied to clear animals from infection. We examined the impact of environmental temperature on B. salamandrivorans infection and disease dynamics in fire salamanders (Salamandra salamandra). Colonization of salamanders by B. salamandrivorans occurred at 15°C and 20°C but not at 25°C, with a significantly faster buildup of infection load and associated earlier mortality at 15°C. Exposing B. salamandrivorans infected salamanders to 25°C for 10 days resulted in complete clearance of infection and clinically cured all experimentally infected animals. This treatment protocol was validated in naturally infected wild fire salamanders. In conclusion, we show that B. salamandrivorans infection and disease dynamics are significantly dictated by environmental temperature, and that heat treatment is a viable option for clearing B. salamandrivorans infections.

Wildlife disease. Recent introduction of a chytrid fungus endangers Western Palearctic salamanders.

Emerging infectious diseases are reducing biodiversity on a global scale. Recently, the emergence of the chytrid fungus Batrachochytrium salamandrivorans resulted in rapid declines in populations of European fire salamanders. Here, we screened more than 5000 amphibians from across four continents and combined experimental assessment of pathogenicity with phylogenetic methods to estimate the threat that this infection poses to amphibian diversity. Results show that B. salamandrivorans is restricted to, but highly pathogenic for, salamanders and newts (Urodela). The pathogen likely originated and remained in coexistence with a clade of salamander hosts for millions of years in Asia. As a result of globalization and lack of biosecurity, it has recently been introduced into naïve European amphibian populations, where it is currently causing biodiversity loss.

Use of a live attenuated Salmonella enterica serovar Typhimurium vaccine on farrow-to-finish pig farms.

Salmonella enterica infection in pigs is economically important and poses a zoonotic risk. In this study, the efficacy of an attenuated S. enterica serovar Typhimurium strain was evaluated in three farrow-to-finish pig herds. In each herd, 120 piglets were vaccinated orally at 3 and 24 days of age, while 120 piglets served as unvaccinated controls. Faeces, ileocaecal lymph nodes and caecal contents were examined for S. Typhimurium by isolation and serum was analysed for antibodies against S. Typhimurium by ELISA. All pigs were weighed at pre-weaning and slaughter to determine daily weight gain. In vaccinated pigs prior to slaughter, significantly fewer animals excreted S. enterica, there was a significantly lower S. enterica-specific mean antibody titre and there was a significantly higher mean daily weight gain compared to unvaccinated controls. In two herds, there were significantly lower proportions of S. enterica positive ileocaecal lymph nodes and caecal contents at slaughter between the vaccinated and control groups, but this difference was not significant across all three herds. S. enterica with the same auxotrophic characteristics and genotype as the vaccine strain was isolated from several samples of faeces, ileocaecal lymph nodes and caecal contents from vaccinated pigs. These findings indicate that vaccination with an attenuated S. Typhimurium strain reduces S. enterica shedding, but the reduction is not consistent and the vaccine strain may persist in tissues.

Microarray-based detection of Salmonella enterica serovar Enteritidis genes involved in chicken reproductive tract colonization.

Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40.

Presence of Helicobacter suis on pork carcasses.

Helicobacter (H.) suis is a world-wide spread pathogen which not only colonizes the stomach of pigs, but is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. H. suis infections are associated with gastric lesions both in pigs and in humans. Recently, the presence of viable H. suis bacteria has been demonstrated in minced pork, suggesting that manipulation or consumption of contaminated pig meat is a possible route of transmission of this zoonotic agent. The main goal of this study was to determine the extent of pork carcass contamination with H. suis at slaughter. In two consecutive studies, the occurrence of H. suis DNA was assessed in scalding water, head and mouth swabs, mesenteric lymph nodes, palatine tonsils and on the chest, shoulder and ham region of pork carcasses from three slaughterhouses using qPCR with ureA gene based H. suis-specific primers. H. suis DNA was detected on carcasses in all slaughterhouses, in 8.3% of all 1083 samples. It was found in all sampled matrices, except for the palatine tonsils and scalding water samples. Contamination levels of dressed pork samples did not exceed 184 genomic equivalents per 100cm(2) (shoulder, ham) or 300cm(2) (chest). All positive PCR products were subjected to sequence analysis of the ureA gene to confirm the identification of H. suis bacteria. Using multilocus sequence typing (MLST) on a selection of the positive samples, 5 unique sequence types (STs) could be assigned. Multiple H. suis strains were present on samples derived from one specific pig herd. Since H. suis DNA was detected in 11% (n: 90) of the mesenteric lymph nodes derived at the slaughterhouse, it was determined whether these organisms can colonize the mesenteric lymph nodes after experimental infection. Despite high-level colonization of the porcine stomachs with the H. suis strain, no H. suis DNA was detected in the mesenteric lymph nodes at four weeks after experimental infection. This might indicate that its presence in these tissues of slaughtered pigs is due to contamination during the slaughter process, but further studies are necessary to confirm this. In conclusion, we demonstrate a relatively high prevalence of H. suis on pork carcasses.

Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr-saker hybrid falcons (Falco rusticolus x Falco cherrug). Adult (n = 15) and juvenile (n = 18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin:globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and ß globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and ß globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A. fulmigatus show significant alterations, which in combination with other diagnostic procedures, assist in the early diagnosis of avian aspergillosis.