Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin.
alpha toxin; antibodies; Clostridium perfringens; enterotoxaemia; perfringolysin; veal
Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.
The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.
cholesterol-dependent cytolysin; gas gangrene; myonecrosis; necrohemorrhagic enteritis; enterotoxaemia; calves
Helicobacter (H.) suis can colonize the stomach of pigs as well as humans, causing chronic gastritis and other gastric pathological changes including gastric ulceration and mucosa-associated lymphoid tissue (MALT) lymphoma. Recently, a virulence factor of H. suis, γ-glutamyl transpeptidase (GGT), has been demonstrated to play an important role in the induction of human gastric epithelial cell death and modulation of lymphocyte proliferation depending on glutamine and glutathione catabolism. In the present study, the relevance of GGT in the pathogenesis of H. suis infection was studied in mouse and Mongolian gerbil models. In addition, the relative importance of H. suis GGT was compared with that of the H. pylori GGT. A significant and different contribution of the GGT of H. suis and H. pylori was seen in terms of bacterial colonization, inflammation and the evoked immune response. In contrast to H. pyloriΔggt strains, H. suisΔggt strains were capable of colonizing the stomach at levels comparable to WT strains, although they induced significantly less overall gastric inflammation in mice. This was characterized by lower numbers of T and B cells, and a lower level of epithelial cell proliferation. In general, compared to WT strain infection, ggt mutant strains of H. suis triggered lower levels of Th1 and Th17 signature cytokine expression. A pronounced upregulation of B-lymphocyte chemoattractant CXCL13 was observed, both in animals infected with WT and ggt mutant strains of H. suis. Interestingly, H. suis GGT was shown to affect the glutamine metabolism of gastric epithelium through downregulation of the glutamine transporter ASCT2.
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Helicobacter heilmannii is a zoonotic bacterium that has been associated with gastric disease in humans. In this study, the mRNA expression of mucins in the stomach of BALB/c mice was analyzed at several time points during a 1-year infection with this bacterium, during which gastric disease progressed in severity. Markers for acid production by parietal cells and mucous metaplasia were also examined. In the first 9 weeks postinfection, the mRNA expression of Muc6 was clearly upregulated in both the antrum and fundus of the stomach of H. heilmannii-infected mice. Interestingly, Muc13 was upregulated already at 1 day postinfection in the fundus of the stomach. Its expression level remained high in the stomach over the course of the infection. This mucin is, however, not expressed in a healthy stomach, and high expression of this mucin has so far only been described in gastric cancer. In the later stages of infection, mRNA expression of H+/K+-ATPase α/β and KCNQ1 decreased, whereas the expression of Muc4, Tff2, Dmbt1, and polymeric immunoglobulin receptor (pIgR) increased starting at 16 weeks postinfection onwards, suggesting the existence of spasmolytic polypeptide-expressing metaplasia in the fundus of the stomach. Mucous metaplasia present in the mucosa surrounding low-grade mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions was also histologically confirmed. Our findings indicate that H. heilmannii infection causes severe gastric pathologies and alterations in the expression pattern of gastric mucins, such as Muc6 and Muc13, as well as disrupting gastric homeostasis by inducing the loss of parietal cells, resulting in the development of mucous metaplasia.
Devrieseasis caused by Devriesea agamarum is a highly prevalent disease in captive desert lizards, resulting in severe dermatitis and in some cases mass mortality. In this study, we assessed the contribution of autovaccination to devrieseasis control by evaluating the capacity of 5 different formalin-inactivated D. agamarum vaccines to induce a humoral immune response in bearded dragons (Pogona vitticeps). Each vaccine contained one of the following adjuvants: CpG, incomplete Freund's, Ribi, aluminium hydroxide, or curdlan. Lizards were administrated one of the vaccines through subcutaneous injection and booster vaccination was given 3 weeks after primo-vaccination. An indirect ELISA was developed and used to monitor lizard serological responses. Localized adverse effects following subcutaneous immunization were observed in all but the Ribi adjuvanted vaccine group. Following homologous experimental challenge, the incomplete Freund's as well as the Ribi vaccine were observed to confer protection in bearded dragons against the development of D. agamarum associated septicemia but not against dermatitis. Subsequently, two-dimensional gelelectrophoresis followed by immunoblotting and mass spectrometry was conducted with serum obtained from 3 lizards that showed seroconversion after immunisation with the Ribi vaccine. Fructose-bisphosphate aldolase and aldo-keto reductase of D. agamarum reacted with serum from the latter lizards. Based on the demonstrated seroconversion and partial protection against D. agamarum associated disease following the use of formalin-inactivated vaccines as well as the identification of target antigens in Ribi vaccinated bearded dragons, this study provides promising information towards the development of a vaccination strategy to control devrieseasis in captive lizard collections.
Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.
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.
deoxynivalenol; fumonisin; Fusarium mycotoxins; human; infectious diseases; mouse; pig; poultry; T-2 toxin; zearalenone
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.
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.
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.
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.
In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression.
In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1β, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1β, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05).
Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.
RT-qPCR; Equine adipose tissue; mRNA expression; Inflammation
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.
Digital Dermatitis (DD) is a common disease of dairy cows, the pathogenesis of which is still not clear. This study examined some host responses associated with the typical lesions, in an attempt to further elucidate the pathogenesis of the disease. Twenty four samples representing the 5 different clinical stages of DD (M0-M4) were collected from slaughtered cattle for histopathological and immunological analyses.
Significant increases in total epidermal thickness were found in M2, M3, and M4 when compared with M0 and M1. M3 samples, when compared with M0 and M1, were characterized by a significant increase in the thickness of the keratin layer. Counts of both eosinophils and neutrophils were at a maximum in the M2 stage and decreased in the M3 and M4 stage. A significant increase in IL8 expression was observed in the M2-M3 stages of the disease and immunohistochemical staining showed the source as keratinocytes, suggesting an important role for keratinocyte-derived IL8 in the pathogenesis of DD.
Results of the present study point to a strong stimulation of the innate immune response at the level of the keratinocytes throughout most of the clinical stages, and a delayed response of the adaptive immune reaction.
Digital dermatitis; Pathogenesis; Keratinocytes; Cytokines; Cattle
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.
Broiler chickens are rather resistant to deoxynivalenol and thus, clinical signs are rarely seen. However, effects of subclinical concentrations of deoxynivalenol on both the intestine and the liver are less frequently studied at the molecular level. During our study, we investigated the effects of three weeks of feeding deoxynivalenol on the gut wall morphology, intestinal barrier function and inflammation in broiler chickens. In addition, oxidative stress was evaluated in both the liver and intestine. Besides, the effect of a clay-based mycotoxin adsorbing agent on these different aspects was also studied. Our results show that feeding deoxynivalenol affects the gut wall morphology both in duodenum and jejenum of broiler chickens. A qRT-PCR analysis revealed that deoxynivalenol acts in a very specific way on the intestinal barrier, since only an up-regulation in mRNA expression of claudin 5 in jejunum was observed, while no effects were seen on claudin 1, zona occludens 1 and 2. Addition of an adsorbing agent resulted in an up-regulation of all the investigated genes coding for the intestinal barrier in the ileum. Up-regulation of Toll-like receptor 4 and two markers of oxidative stress (heme-oxigenase or HMOX and xanthine oxidoreductase or XOR) were mainly seen in the jejunum and to a lesser extent in the ileum in response to deoxynivalenol, while in combination with an adsorbing agent main effect was seen in the ileum. These results suggest that an adsorbing agent may lead to higher concentrations of deoxynivalenol in the more distal parts of the small intestine. In the liver, XOR was up-regulated due to DON exposure. HMOX and HIF-1α (hypoxia-inducible factor 1α) were down-regulated due to feeding DON but also due to feeding the adsorbing agent alone or in combination with DON.
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.
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.
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.
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.
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.
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.
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.
Chlamydiales; Urodelans; Candidatus Amphibiichlamydia salamandrae; Neurergus sp.; Salamandra sp.; bacteria; salamander; amphibian; chlamydia
This report describes a 1-year-old cat with acute dyspnea. Thoracic radiography revealed a pneumomediastinum and severe subcutaneous emphysema. Lower airway surgical exploration was unable to determine the cause. At postmortem examination, acute necrotizing bronchopneumonia and fibrinonecrotic tracheitis due to feline herpesvirus-1 were diagnosed.
Although aspergillosis is one of the most common diseases in captive birds, the pathogenesis of avian aspergillosis is poorly known. We studied the role of avian respiratory macrophages as a first line of defense against avian aspergillosis. The phagocytic and killing capacities of avian respiratory macrophages were evaluated using pigeon respiratory macrophages that were inoculated with Aspergillus fumigatus conidia. On average, 25% of macrophage-associated conidia were phagocytosed after one hour. Sixteen percents of these cell-associated conidia were killed after 4 h and conidial germination was inhibited in more than 95% of the conidia. A. fumigatus conidia were shown to be cytotoxic to the macrophages. Intracellularly germinating conidia were located free in the cytoplasm of necrotic cells, as shown using transmission electron microscopy. These results suggest that avian respiratory macrophages may prevent early establishment of infection, unless the number of A. fumigatus conidia exceeds the macrophage killing capacity, leading to intracellular germination and colonization of the respiratory tract.
Helicobacter (H.) suis colonizes the stomach of pigs and is the most prevalent gastric non-H. pylori Helicobacter species in humans. Limited information is available on host immune responses after infection with this agent and it is unknown if variation in virulence exists between different H. suis strains. Therefore, BALB/c and C57BL/6 mice were used to compare colonization ability and gene expression of various inflammatory cytokines, as determined by real-time PCR, after experimental infection with 9 different H. suis strains. All strains were able to persist in the stomach of mice, but the number of colonizing bacteria at 59 days post inoculation was higher in stomachs of C57BL/6 mice compared to BALB/c mice. All H. suis strains caused an upregulation of interleukin (IL)-17, which was more pronounced in BALB/c mice. This upregulation was inversely correlated with the number of colonizing bacteria. Most strains also caused an upregulation of regulatory IL-10, positively correlating with colonization in BALB/c mice. Only in C57BL/6 mice, upregulation of IL-1β was observed. Increased levels of IFN-γ mRNA were never detected, whereas most H. suis strains caused an upregulation of the Th2 signature cytokine IL-4, mainly in BALB/c mice. In conclusion, the genetic background of the murine strain has a clear impact on the colonization ability of different H. suis strains and the immune response they evoke. A predominant Th17 response was observed, accompanied by a mild Th2 response, which is different from the Th17/Th1 response evoked by H. pylori infection.