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Salmonella enterica infections in pigeons are generally associated with pigeon-adapted strains of serovar Typhimurium that are of little public health concern. Here, we isolated Salmonella Enteritidis phage type 4 (PT4), an important human pathogen, from a population of feral pigeons in Brussels, which was further characterized by Pulsed-Field Gel Electrophoresis. All pigeon isolates belonged to the same pulsotype, which has been present in Belgian pigeons at least since 2001 and is associated with poultry and disease in humans. A high prevalence of 33% of Salmonella Enteritidis in Brussels combined with dense pigeon populations suggest that feral pigeons may constitute a significant, but unrevealed reservoir for contracting salmonellosis in the urban environment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A number of Helicobacter species cause gastrointestinal or hepatic disease in humans, including H. pylori, gastric non-H. pylori helicobacters from animal origin and enterohepatic Helicobacter species. Little is known on the presence of Helicobacter species in great apes, our closest living relatives and potential reservoirs of microorganisms that might emerge in humans. The aim of the present study was to investigate the presence of gastric and enterohepatic Helicobacter species in African chimpanzees and gorillas. Fresh fecal samples were collected from wild endangered chimpanzees and critically endangered western lowland gorillas from different African National Parks, as well as wild-born captive animals from primate sanctuaries. Intact Helicobacter bacteria were demonstrated in feces by fluorescence in situ hybridization. Screening using a Helicobacter genus-specific PCR revealed the presence of Helicobacter DNA in the majority of animals in all groups. Cloning and sequencing of 16S rRNA gene fragments revealed a high homology to sequences from various zoonotic enterohepatic Helicobacter species, including H. cinaedi and H. canadensis. A number of gorillas and chimpanzees also tested positive using PCR assays designed to amplify part of the ureAB gene cluster and the hsp60 gene of gastric helicobacters. Phylogenetic analysis revealed the presence of a putative novel zoonotic gastric Helicobacter taxon/species. For this species, we propose the name 'Candidatus Helicobacter homininae', pending isolation and further genetic characterization. The presence of several Helicobacter species not only implies a possible health threat for these endangered great apes, but also a possible zoonotic transmission of gastric and enterohepatic helicobacters from these primate reservoirs to humans., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Helicobacter suis is a zoonotically important bacterium, that has been associated with gastritis and ulcerative lesions of the pars oesophagea of the stomach in pigs. Its exact role in these pathologies, however, still remains controversial. Therefore, a total of 29 medicated early weaned piglets were inoculated intragastrically or orally, with a total of 2 × 10(9) viable H. suis bacteria and the effect on gastric pathology and weight gain was determined. Twenty-three medicated early weaned piglets were inoculated with a sterile culture medium and used as sham-inoculated controls. The animals were euthanized between 28 and 42 days after inoculation. Infected animals showed a more severe gastritis compared to the control group. There was also a significant reduction of approximately 60 g per day (10%) in weight gain in H. suis inoculated animals compared to the sham-inoculated control animals. In conclusion, this study demonstrates for the first time that a pure in vitro culture of H. suis not only causes gastritis but also a marked decrease of the daily weight gain in experimentally infected pigs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A longitudinal study was carried out to investigate the diversity and persistence of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains in four infected pig herds. In each herd, 20 pigs were randomly selected and blood and/or bronchoalveolar lavage (BAL) fluid was collected at 6, 10, 14 and 26 weeks of age. In the BAL fluid, quantitative PCR and MLVA (multiple-locus variable number of tandem repeats (VNTR) analysis) testing were performed for detection and typing of M. hyopneumoniae strains, respectively. At 26 weeks of age, the prevalence and severity of lung lesions were recorded at slaughter (minimum 50 pigs belonging to the same batch as the investigated pigs). The percentage of pigs testing positive on qPCR increased from 35% at 6 weeks to 96% at 26 weeks of age. With MLVA testing, positive pigs were found from 14 weeks onwards. Within each herd, only one distinct strain was detected, although clonal variants were identified in two herds. In three of the herds, the strain remained present until slaughter age. The percentage of pigs with Mycoplasma-like lesions ranged from 38% to 98%, and the average pneumonia score ranged from 1.7 to 11.9, respectively. The present field study documented that within a herd, mainly one distinct M. hyopneumoniae strain was present that persisted in the same animals for at least 12 weeks. This implies that the immune response of the animals following infection is not able to rapidly clear the infection from the respiratory tract., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Over the past decade, an increasing awareness has arisen of the role of neuroendocrine hormones in the susceptibility of mammalian hosts to a bacterial infection. During a stress response, glucocorticoids, catecholamines and neuroendocrine factors are released into the circulation of the host. For a long time the effects of stress on the course of an infection have been exclusively ascribed to the direct effect of stress-related hormones on the immune system and the intestinal barrier function. Chronic stress is known to cause a shift from T helper 1-mediated cellular immunity toward T helper 2-mediated humoral immunity, which can influence the course of an infection and/or the susceptibility to a microorganism. Bacteria can however also respond directly to stress-related host signals. Catecholamines can alter growth, motility, biofilm formation and/or virulence of pathogens and commensal bacteria, and as a consequence influence the outcome of infections by these bacteria in many hosts. For some bacteria, such as Salmonella, Escherichia coli and Pseudomonas aeruginosa it was shown that this influence is regulated by quorum sensing mechanisms. In this manuscript an overview of how and when stress influences the outcome of bacterial infections in animals is provided., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Helicobacter suis is a very fastidious porcine gastric pathogen, which is also considered to be of zoonotic importance. In vitro antimicrobial susceptibility cannot be determined using standard assays, as this agent only grows in a biphasic medium with an acidic pH. Therefore, a combined agar and broth dilution method was used to analyse the activity of nine antimicrobial agents against nine H. suis isolates. After 48 h microaerobic incubation, minimal inhibitory concentrations (MICs) were determined by software-assisted calculation of bacterial growth. Only for enrofloxacin a bimodal distribution of MICs was demonstrated, indicating acquired resistance in one strain, which showed an AGT→AGG (Ser→Arg) substitution at codon 99 of gyrA. In conclusion, the assay developed here is suitable for determination of the antimicrobial susceptibility of H. suis isolates, although activity of acid sensitive antimicrobial agents may be higher than predicted from MIC endpoints., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Campylobacter-contaminated poultry meat is an important source of foodborne gastroenteritis and poses a serious health burden in industrialized countries. Broiler chickens are commonly regarded as a natural host for this pathogen and infected birds carry a very high Campylobacter load in their gastrointestinal tract, especially the ceca. This results in contaminated carcasses during processing. While hygienic measures at the farm and control measures during carcass processing can have some effect on the reduction of Campylobacter numbers on the retail product, intervention at the farm level by reducing colonization of the ceca should be taken into account in the overall control policy. This review gives an up-to-date overview of suggested on-farm control measures to reduce the prevalence and colonization of Campylobacter in poultry., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Persistent Salmonella Typhimurium infections in pigs are a major concern for food safety and human health. Tonsils play a key role in the persistence of Salmonella Typhimurium in pigs. Previous studies indicated that Salmonella virulence genes involved in invasion and intracellular survival are of little importance for the colonization of porcine tonsils, suggesting a predominantly extracellular location of the Salmonella bacteria. Biofilm formation might promote extracellular persistence of Salmonella Typhimurium. The aim of this study was to determine whether the bacterium resides predominantly intra- or extracellularly in tonsils of pigs and to examine the contribution of biofilm-associated genes csgA, csgD and adrA in Salmonella persistence in porcine tonsils. Single cell suspensions were prepared from tonsils of orally inoculated pigs (2 x 10(7)colony forming units (CFU) wild type Salmonella Typhimurium) to determine the ratio of extracellular versus intracellular bacteria. Both at 5 and 28 days post-inoculation (pi), the majority of Salmonella bacteria was found extracellularly in porcine tonsils. To determine the contribution of biofilm formation in extracellular persistence, pigs were orally inoculated with a mixture of 2 x 10(7)CFU of the Salmonella Typhimurium wild type strain and 2 x 10(7)CFU of one of the Salmonella Typhimurium csgA, csgD or adrA mutants. At 10 days pi, equal numbers of both wild type and mutant Salmonella bacteria were found not only in tonsils, but also in ileum, ileum contents, ileocecal lymph nodes and faeces. In conclusion, we showed that Salmonella Typhimurium resides extracellularly in porcine tonsils, using a biofilm independent mechanism., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

To investigate the efficacy of voriconazole for the treatment of aspergillosis, three groups of six racing pigeons (Columba livia domestica) were inoculated in the apical part of the right lung with 2x10(7) conidia of an avian derived Aspergillus fumigatus strain. The minimal inhibitory concentration of voriconazole for this strain was 0.25 microg/ml. In two groups, voriconazole treatment was started upon appearance of the first clinical signs and continued for fourteen days. The third group was sham treated. The voriconazole-treated pigeons received voriconazole orally at a dose of 10 mg/kg body weight (BW) q12h (group 1) or 20 mg/kg BW q24h (group 2). Sixteen days post-inoculation all surviving pigeons were euthanized. Weight loss, clinical scores, daily mortality, lesions at necropsy and isolation of A. fumigatus were compared between all groups. In both voriconazole-treated groups, a significant reduction in clinical signs and lesions was observed. Administering voriconazole at 10 mg/kg BW q12h eliminated A. fumigatus and administering voriconazole at 20 mg/kg BW q24h reduced A. fumigatus isolation rates. Mild histological liver abnormalities were found in group 1 (10 mg/kg BW q12h), while mild histological as well as macroscopic liver abnormalities were found in group 2 (20 mg/kg BW q24h). In conclusion, voriconazole at 10 mg/kg BW q12h in pigeons reduces clinical signs and eliminates A. fumigatus in racing pigeons experimentally infected with A. fumigatus.

Dermatitis caused by Devriesea agamarum poses a major health problem for the captive maintenance of several desert lizard species. This study was conducted to determine the optimal antimicrobial treatment to eliminate D. agamarum infections from lizards. First, the in vitro susceptibility of 42 D. agamarum isolates was determined for 10 different antimicrobial agents using an agar dilution method. In none of the isolates acquired antimicrobial resistance was demonstrated. Then, two intramuscular treatment protocols using either enrofloxacin or ceftiofur were tested in bearded dragons (Pogona vitticeps) experimentally infected with a D. agamarum strain showing a MIC of 2 microg/ml for enrofloxacin and 0.12 microg/ml for ceftiofur. While D. agamarum could no longer be isolated after 17-18 days of ceftiofur administration, enrofloxacin administration and sham treatment failed in clearing the infection after 27 days of treatment. Based on these results, intramuscular injection of ceftiofur at 5 mg/kg BW q24h was used to treat naturally and clinically infected Uromastyx lizards. This resulted in marked clinical improvement and clearance of infection after 12 days on average. In conclusion, intramuscular administration of ceftiofur at 5 mg/kg BW q24h eliminates D. agamarum in lizards, resulting in clinical cure.

Necrotic enteritis in broiler chickens is associated with Clostridium perfringens type A, carrying the NetB toxin. C. perfringens type A is also a member of the normal intestinal microbiota of broilers. Clinically healthy chickens carry several different C. perfringens clones in their intestine. In flocks suffering from necrotic enteritis, however, mostly only one single clone is isolated from the gut of all the diseased animals. Selective proliferation of these clinical outbreak strains in the gut and spread within the flock seems likely, but an explanation has not yet been given. The hypothesis that necrotic enteritis associated C. perfringens strains might suppress the growth of normal microbiota C. perfringens strains, was therefore tested. Twenty-six C. perfringens strains isolated from healthy broilers and 24 clinical outbreak isolates were evaluated for their ability to induce intra-species growth-inhibition in an in vitro setup. A significantly higher proportion of the C. perfringens clinical outbreak strains inhibited the growth of other C. perfringens strains compared to C. perfringens strains isolated from the gut of healthy chickens. It is proposed that, in addition to toxin production, intra-species growth-inhibition may be a virulence trait that contributes to the ability of certain C. perfringens strains to cause necrotic enteritis in broilers.

Although poultry meat is now recognized as the main source of Campylobacter jejuni gastroenteritis, little is known about the strategy used by the bacterium to colonize the chicken intestinal tract. In this study, the mechanism of C. jejuni colonization in chickens was studied using four human and four poultry isolates of C. jejuni. The C. jejuni strains were able to invade chicken primary cecal epithelial crypt cells in a predominantly microtubule-dependent way (five out of eight strains). Invasion of cecal epithelial cells was not accompanied by necrosis or apoptosis in the cell cultures, nor by intestinal inflammation in a cecal loop model. C. jejuni from human origin displayed a similar invasive profile compared to the poultry isolates. Invasiveness of the strains in vitro correlated with the magnitude of spleen colonization in C. jejuni inoculated chicks. The C. jejuni bacteria that invaded the epithelial cells were not able to proliferate intracellularly, but quickly evaded from the cells. In contrast, the C. jejuni strains were capable of replication in chicken intestinal mucus. These findings suggest a novel colonization mechanism by escaping rapid mucosal clearance through short-term epithelial invasion and evasion, combined with fast replication in the mucus.

Virulence genes regulated by the SsrA/B system are indispensable for systemic disease in BALB/c mice. The role of this regulating system in the pathogenesis of Salmonella Typhimurium infections in pigs is not documented. In the present study, the interactions of Salmonella Typhimurium and an ssrA/B mutant were compared in vitro and in vivo. The ssrA/B mutant strain displayed decreased Salmonella Pathogenicity Island 2 (SPI-2) expression levels, showed a replication defect in mouse macrophages and was attenuated in a mouse model after oral inoculation. Using real time qRT-PCR and a porcine ileal loop model, it was shown that the ssrA/B mutant strain was not significantly attenuated in overall virulence and SPI-1 expression in specific. Flowcytometric analysis demonstrated that the ssrA/B mutant strain was defective in intracellular replication in porcine macrophages. After oral inoculation of piglets with the wild type strain or the ssrA/B mutant strain, the animals of both groups excreted Salmonella and were colonized by Salmonella to the same extent. In an intravenous mixed infection model, the ssrA/B mutant strain was defective in the colonization of several internal organs. These results suggest that the ssrA/B gene of Salmonella Typhimurium plays a limited role in the persistent intestinal colonization of pigs.

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.

Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.

Reptile-associated salmonellosis in humans is an increasing public health issue. This study aimed at characterizing Salmonella isolates from captive lizards and to compare them to human isolates. Salmonella was isolated from 25 of 33 cloacal and 47 of 79 faecal samples from captive lizards (75.8 and 59.5%, respectively). The strains belonged to 44 serotypes of subspecies I (27 serotypes), II (9), IIIb (3) and IV (5). Two strains, one of serotype Enteritidis and one of serotype Amsterdam, were resistant to nitrofurantoin. Invasion assays in Caco-2 cells were performed with 40 saurian isolates of subspecies I, 15 isolates of subspecies II, 4 strains of subspecies IIIb, 6 subspecies IV isolates and 17 human isolates of corresponding serotypes of subspecies I. Saurian isolates belonging to subspecies I invaded the Caco-2 cells to a higher extent than those from the other subspecies. The human isolates invaded the Caco-2 cells to a lesser degree compared to their saurian counterparts. In the same strains, the presence of virulence genes agfA, shdA, spvR, pefA and sopE was determined using PCR. Whereas agfA was detected in all strains, pefA was only detected in one saurian and in the human serotype Enteritidis strains. The spvR gene was detected in the same serotype Enteritidis strains and in 33% of the subspecies IV strains. The shdA gene was present in all the human isolates and in 86% of subspecies I saurian isolates. SopE was found in 17% of the human isolates, in 24% of the saurian subspecies I strains and in all of the subspecies IV strains.

This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen-host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed.

The pathogenesis of Salmonella enterica subsp. enterica serovar Muenchen infections in the aquatic turtle Trachemys scripta scripta was studied. After oral infection with 5x10(5)cfu of serovar Muenchen of 10-14-month-old turtles, kept at 26 degrees C, the intestine and especially the ileum, caecum and colon was colonized. Invasion of the intestinal wall, causing histopathological lesions, and colonization of internal organs were not observed. Serovar Muenchen was only isolated from turtles for 8 days after exposure. Keeping the turtles at 37 degrees C caused colonization of liver and spleen in two of six orally infected turtles and augmented the numbers of bacteria in the intestinal tract. In contrast to oral infections, intraperitoneal infections of turtles with serovar Muenchen enabled the bacterium to persist inside the host for at least 5 weeks. Clearance of serovar Muenchen from the liver and blood was more pronounced at 26 degrees C than at 37 degrees C. ELISA antibodies were demonstrated in intraperitoneally but not in orally infected turtles kept at 26 degrees C. In conclusion, the lack of persistence and invasiveness of serovar Muenchen in T. s. scripta after oral exposure might be due to the turtle's relatively low body temperature and/or the absence of well-organized gut-associated lymphoid tissue.