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1.  The Impact of Fusarium Mycotoxins on Human and Animal Host Susceptibility to Infectious Diseases 
Toxins  2014;6(2):430-452.
Contamination of food and feed with mycotoxins is a worldwide problem. At present, acute mycotoxicosis caused by high doses is rare in humans and animals. Ingestion of low to moderate amounts of Fusarium mycotoxins is common and generally does not result in obvious intoxication. However, these low amounts may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host pathogen interactions and thus a different outcome of infection. This review summarizes the current state of knowledge about the impact of Fusarium mycotoxin exposure on human and animal host susceptibility to infectious diseases. On the one hand, exposure to deoxynivalenol and other Fusarium mycotoxins generally exacerbates infections with parasites, bacteria and viruses across a wide range of animal host species. Well-known examples include coccidiosis in poultry, salmonellosis in pigs and mice, colibacillosis in pigs, necrotic enteritis in poultry, enteric septicemia of catfish, swine respiratory disease, aspergillosis in poultry and rabbits, reovirus infection in mice and Porcine Reproductive and Respiratory Syndrome Virus infection in pigs. However, on the other hand, T-2 toxin has been shown to markedly decrease the colonization capacity of Salmonella in the pig intestine. Although the impact of the exposure of humans to Fusarium toxins on infectious diseases is less well known, extrapolation from animal models suggests possible exacerbation of, for instance, colibacillosis and salmonellosis in humans, as well.
doi:10.3390/toxins6020430
PMCID: PMC3942744  PMID: 24476707
deoxynivalenol; fumonisin; Fusarium mycotoxins; human; infectious diseases; mouse; pig; poultry; T-2 toxin; zearalenone
2.  Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes 
Background
Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species.
Results
Strains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases.
Conclusions
Bovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro.
doi:10.1186/1746-6148-10-32
PMCID: PMC3913962  PMID: 24479821
4.  Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis 
Veterinary Research  2014;45(1):40.
Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.
doi:10.1186/1297-9716-45-40
PMCID: PMC3992141  PMID: 24708344
5.  Passive immunization to reduce Campylobacter jejuni colonization and transmission in broiler chickens 
Veterinary Research  2014;45(1):27.
Campylobacter jejuni is the most common cause of bacterium-mediated diarrheal disease in humans worldwide. Poultry products are considered the most important source of C. jejuni infections in humans but to date no effective strategy exists to eradicate this zoonotic pathogen from poultry production. Here, the potential use of passive immunization to reduce Campylobacter colonization in broiler chicks was examined. For this purpose, laying hens were immunized with either a whole-cell lysate or the hydrophobic protein fraction of C. jejuni and their eggs were collected. In vitro tests validated the induction of specific ImmunoglobulinY (IgY) against C. jejuni in the immunized hens’ egg yolks, in particular. In seeder experiments, preventive administration of hyperimmune egg yolk significantly (P < 0.01) reduced bacterial counts of seeder animals three days after oral inoculation with approximately 104 cfu C. jejuni, compared with control birds. Moreover, transmission to non-seeder birds was dramatically reduced (hydrophobic protein fraction) or even completely prevented (whole-cell lysate). Purified IgY promoted bacterial binding to chicken intestinal mucus, suggesting enhanced mucosal clearance in vivo. Western blot analysis in combination with mass spectrometry after two-dimensional gel-electrophoresis revealed immunodominant antigens of C. jejuni that are involved in a variety of cell functions, including chemotaxis and adhesion. Some of these (AtpA, EF-Tu, GroEL and CtpA) are highly conserved proteins and could be promising targets for the development of subunit vaccines.
doi:10.1186/1297-9716-45-27
PMCID: PMC3996517  PMID: 24589217
6.  Effects of Helicobacter suis γ- Glutamyl Transpeptidase on Lymphocytes: Modulation by Glutamine and Glutathione Supplementation and Outer Membrane Vesicles as a Putative Delivery Route of the Enzyme 
PLoS ONE  2013;8(10):e77966.
Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general.
doi:10.1371/journal.pone.0077966
PMCID: PMC3797756  PMID: 24147103
7.  Multilocus Sequence Typing of the Porcine and Human Gastric Pathogen Helicobacter suis 
Journal of Clinical Microbiology  2013;51(3):920-926.
Helicobacter suis is a Gram-negative bacterium colonizing the majority of pigs, in which it causes gastritis and decreased daily weight gain. H. suis is also the most prevalent gastric non-Helicobacter pylori Helicobacter species in humans, capable of causing gastric disorders. To gain insight into the genetic diversity of porcine and human H. suis strains, a multilocus sequence typing (MLST) method was developed. In a preliminary study, 7 housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC) of 10 H. suis isolates cultured in vitro were investigated as MLST candidates. All genes, except the ureI gene, which was replaced by part of the ureAB gene cluster of H. suis, displayed several variable nucleotide sites. Subsequently, internal gene fragments, ranging from 379 to 732 bp and comprising several variable nucleotide sites, were selected. For validation of the developed MLST technique, gastric tissue from 17 H. suis-positive pigs from 4 different herds and from 1 H. suis-infected human patient was used for direct, culture-independent strain typing of H. suis. In addition to the 10 unique sequence types (STs) among the 10 isolates grown in vitro, 15 additional STs could be assigned. Individual animals were colonized by only 1 H. suis strain, whereas multiple H. suis strains were present in all herds tested, revealing that H. suis is a genetically diverse bacterial species. The human H. suis strain showed a very close relationship to porcine strains. In conclusion, the developed MLST scheme may prove useful for direct, culture-independent typing of porcine and human H. suis strains.
doi:10.1128/JCM.02399-12
PMCID: PMC3592083  PMID: 23303499
8.  Transmission Dynamics of Methicillin-Resistant Staphylococcus aureus in Pigs 
From the mid-2000s on, numerous studies have shown that methicillin-resistant Staphylococcus aureus (MRSA), renowned as human pathogen, has a reservoir in pigs and other livestock. In Europe and North America, clonal complex (CC) 398 appears to be the predominant lineage involved. Especially worrisome is its capacity to contaminate humans in close contact with affected animals. Indeed, the typical multi-resistant phenotype of MRSA CC398 and its observed ability of easily acquiring genetic material suggests that MRSA CC398 strains with an increased virulence potential may emerge, for which few therapeutic options would remain. This questions the need to implement interventions to control the presence and spread of MRSA CC398 among pigs. MRSA CC398 shows a high but not fully understood transmission potential in the pig population and is able to persist within that population. Although direct contact is probably the main route for MRSA transmission between pigs, also environmental contamination, the presence of other livestock, the herd size, and farm management are factors that may be involved in the dissemination of MRSA CC398. The current review aims at summarizing the research that has so far been done on the transmission dynamics and risk factors for introduction and persistence of MRSA CC398 in farms.
doi:10.3389/fmicb.2013.00057
PMCID: PMC3602589  PMID: 23518663
MRSA; ST398; pigs; transmission risk factors; transmission routes; transmission pig models
9.  Genome Sequence of Helicobacter heilmannii Sensu Stricto ASB1 Isolated from the Gastric Mucosa of a Kitten with Severe Gastritis 
Genome Announcements  2013;1(1):e00033-12.
Here we report the genome sequence of Helicobacter heilmannii sensu stricto ASB1 isolated from the gastric mucosa of a kitten with severe gastritis. Helicobacter heilmannii sensu stricto has also been associated with gastric disease in humans. Availability of this genome sequence will contribute to the identification of genes involved in the pathogen’s virulence and carcinogenic properties.
doi:10.1128/genomeA.00033-12
PMCID: PMC3569310  PMID: 23405321
10.  Serological profiles in nursery piglets colonized with Staphylococcus aureus 
Veterinary Research  2013;44(1):4.
At present, the immune response of pigs in relation to Staphylococcus aureus carriage is poorly understood. This study was aimed at investigating the dynamics of the anti-staphylococcal humoral immune response in methicillin-susceptible S. aureus (MSSA)-positive piglets and at assessing the effect of the experimental introduction of a methicillin-resistant S. aureus (MRSA) Sequence Type (ST) 398 strain. Therefore, serum samples were collected at different times from 31 weaned piglets originating from four different sows. Twenty-four out of the 31 piglets were challenged with MRSA ST398. The serum samples were analyzed for IgG antibodies to 39 S. aureus antigens, using a multiplex bead-based assay (xMAP technology, Luminex Corporation). Though antibody responses showed broad inter-individual variability, serological results appeared to be clustered by litter of origin. For most antigens, an age-related response was observed with an apparent increase in antibody titers directed against staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMM), which have been shown to play a role in S. aureus colonization. In most animals, antibody titers directed against staphylococcal toxins or immune-modulating proteins decreased with age, possibly reflecting the absence of bacterial invasion. The introduction of MRSA ST398 did not elicit a significant humoral immune reaction.
This study describes, for the first time, the humoral immune response in weaned pigs colonized with S. aureus.
doi:10.1186/1297-9716-44-4
PMCID: PMC3558462  PMID: 23339425
11.  The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells 
Veterinary Research  2013;44(1):45.
Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells.
doi:10.1186/1297-9716-44-45
PMCID: PMC3722007  PMID: 23782465
12.  Diversity in bacterium-host interactions within the species Helicobacter heilmannii sensu stricto 
Veterinary Research  2013;44(1):65.
Helicobacter (H.) heilmannii sensu stricto (s.s.) is a zoonotic bacterium that naturally colonizes the stomach of dogs and cats. In humans, this microorganism has been associated with gastritis, peptic ulcer disease and mucosa associated lymphoid tissue (MALT) lymphoma. Little information is available about the pathogenesis of H. heilmannii s.s. infections in humans and it is unknown whether differences in virulence exist within this species. Therefore, a Mongolian gerbil model was used to study bacterium-host interactions of 9 H. heilmannii s.s. strains. The colonization ability of the strains, the intensity of gastritis and gene expression of various inflammatory cytokines in the stomach were determined at 9 weeks after experimental infection. The induction of an antrum-dominant chronic active gastritis with formation of lymphocytic aggregates was shown for 7 strains. High-level antral colonization was seen for 4 strains, while colonization of 4 other strains was more restricted and one strain was not detected in the stomach at 9 weeks post infection. All strains inducing a chronic active gastritis caused an up-regulation of the pro-inflammatory cytokine IL-1β in the antrum. A reduced antral expression of H+/K+ ATPase was seen in the stomach after infection with 3 highly colonizing strains and 2 highly colonizing strains caused an increased gastrin expression in the fundus. In none of the H. heilmannii s.s.-infected groups, IFN-γ expression was up-regulated. This study demonstrates diversity in bacterium-host interactions within the species H. heilmannii s.s. and that the pathogenesis of gastric infections with this microorganism is not identical to that of an H. pylori infection.
doi:10.1186/1297-9716-44-65
PMCID: PMC3750284  PMID: 23895283
13.  Porcine intestinal epithelial barrier disruption by the Fusarium mycotoxins deoxynivalenol and T-2 toxin promotes transepithelial passage of doxycycline and paromomycin 
Background
The gastrointestinal tract is the first target for the potentially harmful effects of mycotoxins after intake of mycotoxin contaminated food or feed. With deoxynivalenol (DON), T-2 toxin (T-2), fumonisin B1 (FB1) and zearalenone (ZEA) being important Fusarium toxins in the northern hemisphere, this study aimed to investigate in vitro the toxic effect of these mycotoxins on intestinal porcine epithelial cells derived from the jejunum (IPEC-J2 cells). Viability of IPEC-J2 cells as well as the proportion of apoptotic and necrotic IPEC-J2 cells was determined by flow cytometry after 72 h of exposure to the toxins. Correlatively, the integrity of the intestinal epithelial cell monolayer was studied using Transwell® inserts, in which the trans-epithelial electrical resistance (TEER) and passage of the antibiotics doxycycline and paromomycin were used as endpoints.
Results
We demonstrated that the percentage of Annexin-V-FITC and PI negative (viable) cells, Annexin-V-FITC positive and PI negative (apoptotic) cells and Annexin-V-FITC and PI positive (necrotic) IPEC-J2 cells showed a mycotoxin concentration-dependent relationship with T-2 toxin being the most toxic. Moreover, the ratio between Annexin-V-FITC positive and PI negative cells and Annexin-V-FITC and PI positive cells varied depending on the type of toxin. More Annexin-V-FITC and PI positive cells could be found after treatment with T-2 toxin, while more Annexin-V-FITC positive and PI negative cells were found after exposure to DON. Consistent with the cytotoxicity results, both DON and T-2 decreased TEER and increased cellular permeability to doxycycline and paromomycin in a time- and concentration-dependent manner.
Conclusions
It was concluded that Fusarium mycotoxins may severely disturb the intestinal epithelial barrier and promote passage of antibiotics.
doi:10.1186/1746-6148-8-245
PMCID: PMC3548726  PMID: 23244712
Mycotoxins; Cytotoxicity; Flow cytometry; Permeability; Intestinal cells
14.  The Importance of Sample Size in the Determination of a Flock-Level Antimicrobial Resistance Profile for Escherichia coli in Broilers 
Microbial Drug Resistance  2011;17(4):513-519.
Determining herd- or flock-specific antimicrobial resistance profiles is important to guide therapeutic use of antimicrobials and to assess risk factors for the development and spread of antimicrobial resistance. As such, it is of utmost importance to optimize the sampling strategy for the determination of herd-specific antimicrobial resistance profiles. However, the multitude of prevalences measured at the same time as well as the presence of variation both at the level of the animal and the bacterial population of concern make it impossible to use conventional sample size determination methods. In this article, the use of bootstrapping techniques for sample size determination was explored. In particular, one-stage and two-stage bootstrap samplings were used to determine the optimal number of animals and the optimal number of isolates within one animal. Results show that focus should be on the number of animals sampled rather than on the number of isolates tested within one animal.
doi:10.1089/mdr.2011.0048
PMCID: PMC3223426  PMID: 21875337
15.  Low MRSA prevalence in horses at farm level 
Background
In Europe, methicillin-resistant Staphylococcus aureus (MRSA) belonging to the clonal complex (CC) 398 has become an important pathogen in horses, circulating in equine clinics and causing both colonization and infection. Whether equine MRSA is bound to hospitals or can also circulate in the general horse population is currently unknown. This study, therefore, reports the nasal and perianal MRSA screening of 189 horses on 10 farms in a suspected high prevalence region (East- and West-Flanders, Belgium).
Results
Only one horse (0.53%) from one farm (10%) tested positive in the nose. It carried a spa type t011-SCCmecV isolate, resistant to β-lactams and tetracycline, which is typical for livestock-associated MRSA CC398.
Conclusion
In the region tested here, horses on horse farms seem unlikely to substantially contribute to the large animal associated ST398 MRSA reservoir present at intensive animal production units.
doi:10.1186/1746-6148-8-213
PMCID: PMC3536571  PMID: 23134703
Horse; Staphylococcus aureus; MRSA; Methicillin resistance; Horse farm
16.  Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus’s egg? 
Background
The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated.
Results
Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 μg/ml for florfenicol and 8.0 μg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 μg/ml florfenicol or 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 μg/ml but not to 10 μg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 μg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection.
Conclusions
We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.
doi:10.1186/1746-6148-8-175
PMCID: PMC3488559  PMID: 23009707
17.  Colonization and Transmission of Methicillin-Resistant Staphylococcus aureus ST398 in Nursery Piglets 
A transmission experiment was performed to evaluate the spread of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in nursery piglets. Reproduction ratios (R0) in three experimental groups were found to vary between 3.92 and 52.54, indicating that after introduction, MRSA ST398 will spread easily among weaned piglets, with a tendency to become established.
doi:10.1128/AEM.07356-11
PMCID: PMC3294494  PMID: 22194292
18.  Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent 
PLoS ONE  2012;7(7):e41481.
Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.
We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.
Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.
In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.
doi:10.1371/journal.pone.0041481
PMCID: PMC3401113  PMID: 22911798
19.  Novel Chlamydiaceae Disease in Captive Salamanders 
Emerging Infectious Diseases  2012;18(6):1020-1022.
doi:10.3201/eid1806.111137
PMCID: PMC3358148  PMID: 22608291
Chlamydiales; Urodelans; Candidatus Amphibiichlamydia salamandrae; Neurergus sp.; Salamandra sp.; bacteria; salamander; amphibian; chlamydia
20.  Waterfowl: Potential Environmental Reservoirs of the Chytrid Fungus Batrachochytrium dendrobatidis 
PLoS ONE  2012;7(4):e35038.
Infections with Batrachochytrium dendrobatidis (B. dendrobatidis), the causal agent of chytridiomycosis, have been shown to play an important role in the decline of amphibians worldwide. Spread of the fungus is poorly understood. Bird movement might possibly contribute to the spread of B. dendrobatidis in the environment. Therefore, 397 wild geese in Belgium were screened for presence of B. dendrobatidis on their toes using real-time quantitative PCR (qPCR). In addition, chemotaxis towards, adhesion, survival after desiccation and proliferation of B. dendrobatidis on keratinous toe scales from waterfowl were examined in vitro. qPCR revealed that 76 geese (15%) were positive for B. dendrobatidis. Results of the in vitro tests showed that B. dendrobatidis is attracted to the keratinous toes of aquatic birds on which they can adhere and even proliferate. However, desiccation is poorly tolerated. This suggests waterfowl are potential environmental reservoirs for B. dendrobatidis.
doi:10.1371/journal.pone.0035038
PMCID: PMC3325947  PMID: 22514705
21.  Influence of Mycotoxins and a Mycotoxin Adsorbing Agent on the Oral Bioavailability of Commonly Used Antibiotics in Pigs 
Toxins  2012;4(4):281-295.
It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health.
doi:10.3390/toxins4040281
PMCID: PMC3347004  PMID: 22606377
mycotoxins; mycotoxin binder; antibiotics; pigs; interaction; safety testing
22.  Vaccination reduces macrophage infiltration in bronchus-associated lymphoid tissue in pigs infected with a highly virulent Mycoplasma hyopneumoniae strain 
Background
Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and is responsible for significant economic losses to the pig industry. To better understand the mode of action of a commercial, adjuvanted, inactivated whole cell vaccine and the influence of diversity on the efficacy of vaccination, we investigated samples from vaccinated and non-vaccinated pigs experimentally infected with either a low (LV) or a highly virulent (HV) M. hyopneumoniae strain. Non-vaccinated and sham-infected control groups were included. Lung tissue samples collected at 4 and 8 weeks post infection (PI) were immunohistochemically tested for the presence of T-lymphocytes, B-lymphocytes and macrophages in the bronchus-associated lymphoid tissue (BALT). The number of M. hyopneumoniae organisms in bronchoalveolar lavage (BAL) fluid was determined using quantitative PCR at 4 and 8 weeks PI. Serum antibodies against M. hyopneumoniae were determined at 0, 2, 4, 6 and 8 weeks PI.
Results
The immunostaining revealed a lower density of macrophages in the BALT of the vaccinated groups compared to the non-vaccinated groups. The highest number of M. hyopneumoniae organisms in the BAL fluid was measured at 4 weeks PI for the HV strain and at 8 weeks PI for the LV strain. Vaccination reduced the number of organisms non-significantly, though for the HV strain the reduction was clinically more relevant than for the LV strain. At the level of the individual pigs, a higher lung lesion score was associated with more M. hyopneumoniae organisms in the lungs and a higher density of the investigated immune cells in the BALT.
Conclusions
In conclusion, the infiltration of macrophages after infection with M. hyopneumoniae is reduced by vaccination. The M. hyopneumoniae replication in the lungs is also reduced in vaccinated pigs, though the HV strain is inhibited more than the LV strain.
doi:10.1186/1746-6148-8-24
PMCID: PMC3349615  PMID: 22409839
23.  Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous Mycoplasma hyopneumoniae strains 
Background
Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although vaccination is an important control tool, the results observed under field conditions are variable. This may be due to antigenic differences between the strains circulating in pig herds and the vaccine strain. This study compared the protective efficacy of four bacterins against challenge infection with a highly virulent field strain of M. hyopneumoniae.
Seventy eight, one-week old piglets were randomly assigned to five treatment groups (A, B, C, D, E), 14 piglets each, and a negative control group (F) consisting of 8 piglets. All pigs were injected at 1 (D7) and 4 weeks of age (D28), with 2 ml of either a placebo or a bacterin based on selected M. hyopneumoniae strains, namely A (F7.2C), B (F20.1L), C (B2V1W20 1A-F), D (J strain), E (placebo; positive control), F (placebo; negative control). At D56, all pigs except those of group F were challenged intratracheally with 7 ml culture medium containing 107 CCU/ml of M. hyopneumoniae strain F7.2C. All pigs were euthanized and necropsied at D84. The severity of coughing and pneumonia lesions were the main parameters. Immunofluorescence (IF) testing, nested PCR testing of bronchoalveolar lavage (BAL) fluid and serology for M. hyopneumoniae were also performed.
Results
The different bacterins only slightly improved clinical symptoms (average 0.38 in vaccinated groups vs. 0.45 in group E) and histopathological lung lesions (average 3.20 in vaccinated groups vs. 3.45 in group E), but did not improve macroscopic lung lesions (score 4.30 vs. 4.03 in group E). None of the vaccines was significantly and/or consistently better or worse than the other ones. All bacterins evoked a serological response in the vaccinated animals. All pigs, except those from group F, were positive with nPCR in BAL fluid at D84.
Conclusion
The bacterins did not induce a clear overall protection against challenge infection, and there were no significant differences in protective efficacy between bacterins containing homologous and heterologous M. hyopneumoniae strains. Further research is necessary to better characterize the antigens involved in protection and to elucidate the protective immunity responses following M. hyopneumoniae vaccination and/or infection.
doi:10.1186/1746-6148-8-2
PMCID: PMC3269371  PMID: 22225838
24.  O157:H7 and O104:H4 Vero/Shiga toxin-producing Escherichia coli outbreaks: respective role of cattle and humans 
Veterinary Research  2012;43(1):13.
An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtx-producing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition.
doi:10.1186/1297-9716-43-13
PMCID: PMC3305544  PMID: 22330148
25.  T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions 
Veterinary Research  2012;43(1):22.
The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 μg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.
doi:10.1186/1297-9716-43-22
PMCID: PMC3362764  PMID: 22440148

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