Growth Regulation in Amphibian Pathogenic Chytrid Fungi by the Quorum Sensing Metabolite Tryptophol.

Amphibians face many threats leading to declines and extinctions, but the chytrid fungal skin pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) have been identified as the causative factors leading to one of the greatest disease-driven losses of amphibian biodiversity worldwide. Infection may lead to different clinical outcomes, and lethal infections are commonly associated with unrestricted, exponential fungal growth in the amphibian epidermis. Mechanisms underpinning Bd and Bsal growth in the amphibian host are poorly understood. Here, we describe a quorum sensing mechanism that allows cell-to-cell communication by Bd and Bsal in order to regulate fungal densities and infection strategies. Addition of chytrid culture supernatant to chytrid cultures resulted in a concentration-dependent growth reduction and using dialysis, small metabolites were shown to be the causative factor. U-HPLC-MS/MS and in vitro growth tests identified the aromatic alcohol tryptophol as a key metabolite in regulating fungal growth. We determined tryptophol kinetics in both Bd and Bsal and confirmed the autostimulatory mode of action of this quorum sensing metabolite. Finally, we linked expression of genes that might be involved in tryptophol production, with in vitro and in vivo chytrid growth. Our results show that Bd and Bsal fungi use tryptophol to act as multicellular entities in order to regulate their growth.

Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans.

The recent arrival of Batrachochytrium salamandrivorans in Europe was followed by rapid expansion of its geographical distribution and host range, confirming the unprecedented threat that this chytrid fungus poses to western Palaearctic amphibians. Mitigating this hazard requires a thorough understanding of the pathogen's disease ecology that is driving the extinction process. Here, we monitored infection, disease and host population dynamics in a Belgian fire salamander (Salamandra salamandra) population for two years immediately after the first signs of infection. We show that arrival of this chytrid is associated with rapid population collapse without any sign of recovery, largely due to lack of increased resistance in the surviving salamanders and a demographic shift that prevents compensation for mortality. The pathogen adopts a dual transmission strategy, with environmentally resistant non-motile spores in addition to the motile spores identified in its sister species B. dendrobatidis. The fungus retains its virulence not only in water and soil, but also in anurans and less susceptible urodelan species that function as infection reservoirs. The combined characteristics of the disease ecology suggest that further expansion of this fungus will behave as a 'perfect storm' that is able to rapidly extirpate highly susceptible salamander populations across Europe.

Co-occurrence of Mycoplasma Species and Pigeon Herpesvirus-1 Infection in Racing Pigeons ( Columba livia).

Oropharyngeal swab samples were collected from 438 live racing pigeons ( Columba livia), with and without signs of respiratory disease, that were housed in 220 lofts in 3 provinces in the western part of the Netherlands. Polymerase chain reaction (PCR) was used to identify Mycoplasma species and pigeon herpesvirus-1 (PHV-1) from the samples. In 8.6% of the pigeon lofts tested, signs of respiratory disease were present in pigeons at sampling, and in 30.9% of the sampled pigeon lofts, respiratory signs were observed in pigeons during the 6-month period immediately before sampling. A total of 39.8% of tested pigeons (54.5% of tested lofts) were positive for Mycoplasma species, and 30.6% of tested pigeons (48.6% of tested lofts) were positive for PHV-1. In 15.8% of the tested pigeons (26.8% of tested pigeon lofts), coinfection by Mycoplasma species and PHV-1 was identified. The number of pigeon lofts having pigeons coinfected by Mycoplasma species and PHV-1 was higher than that where only one of the infections was identified. Neither the presence of Mycoplasma species, PHV-1, nor the co-occurrence of both infections was significantly associated with signs of respiratory disease.

Chironomidae bloodworms larvae as aquatic amphibian food.

Different species of chironomids larvae (Diptera: Chironomidae) so-called bloodworms are widely distributed in the sediments of all types of freshwater habitats and considered as an important food source for amphibians. In our study, three species of Chironomidae (Baeotendipes noctivagus, Benthalia dissidens, and Chironomus riparius) were identified in 23 samples of larvae from Belgium, Poland, Russia, and Ukraine provided by a distributor in Belgium. We evaluated the suitability of these samples as amphibian food based on four different aspects: the likelihood of amphibian pathogens spreading, risk of heavy metal accumulation in amphibians, nutritive value, and risk of spreading of zoonotic bacteria (Salmonella, Campylobacter, and ESBL producing Enterobacteriaceae). We found neither zoonotic bacteria nor the amphibian pathogens Ranavirus and Batrachochytrium dendrobatidis in these samples. Our data showed that among the five heavy metals tested (Hg, Cu, Cd, Pb, and Zn), the excess level of Pb in two samples and low content of Zn in four samples implicated potential risk of Pb accumulation and Zn inadequacy. Proximate nutritional analysis revealed that, chironomidae larvae are consistently high in protein but more variable in lipid content. Accordingly, variations in the lipid: protein ratio can affect the amount and pathway of energy supply to the amphibians. Our study indicated although environmentally-collected chironomids larvae may not be vectors of specific pathogens, they can be associated with nutritional imbalances and may also result in Pb bioaccumulation and Zn inadequacy in amphibians. Chironomidae larvae may thus not be recommended as single diet item for amphibians.

The absence of zoonotic agents in invasive bullfrogs (Lithobates catesbeianus) in Belgium and The Netherlands.

Exotic invasive bullfrogs (Lithobates catesbeianus) are considered to exert a considerable negative impact on native amphibian communities. This can be due to competition and predation, but they are also a notorious source of the infectious diseases chytridiomycosis and ranavirosis, affecting amphibian populations globally. Little is known regarding their carriage of other microbial agents that might be transferred to humans or other animals. In this study we determined the occurrence of the amphibian pathogens Ranavirus and Batrachochytrium dendrobatidis and of the zoonotic agents Coxiella burnetii, Neospora caninum, Leptospira sp., Toxoplasma gondii, Mycoplasma sp., Campylobacter sp., Salmonella sp. and extended-spectrum beta-lactamase producing Escherichia coli in 164 bullfrogs from three populations in Belgium and The Netherlands. Although B. dendrobatidis was present at a high prevalence of 63%, mean infection loads were low with an average of 10.9 genomic equivalents (SD 35.5), confirming the role of bullfrogs as B. dendrobatidis carriers, but questioning their role as primary reservoirs for B. dendrobatidis transmission to native amphibian communities. All tested samples were negative for the other infectious agents examined. These results suggest a limited role of bullfrogs as carrier of these pathogens.

The novel 'Candidatus Amphibiichlamydia ranarum' is highly prevalent in invasive exotic bullfrogs (Lithobates catesbeianus).

Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum) with a prevalence of 71% in exotic invasive bullfrog tadpoles (Lithobates catesbeianus) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well-defined clade with 'Candidatus Amphibiichlamydia salamandrae' within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.

Resistance to chytridiomycosis in European plethodontid salamanders of the genus Speleomantes.

North America and the neotropics harbor nearly all species of plethodontid salamanders. In contrast, this family of caudate amphibians is represented in Europe and Asia by two genera, Speleomantes and Karsenia, which are confined to small geographic ranges. Compared to neotropical and North American plethodontids, mortality attributed to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has not been reported for European plethodontids, despite the established presence of Bd in their geographic distribution. We determined the extent to which Bd is present in populations of all eight species of European Speleomantes and show that Bd was undetectable in 921 skin swabs. We then compared the susceptibility of one of these species, Speleomantes strinatii, to experimental infection with a highly virulent isolate of Bd (BdGPL), and compared this to the susceptible species Alytes muletensis. Whereas the inoculated A. muletensis developed increasing Bd-loads over a 4-week period, none of five exposed S. strinatii were colonized by Bd beyond 2 weeks post inoculation. Finally, we determined the extent to which skin secretions of Speleomantes species are capable of killing Bd. Skin secretions of seven Speleomantes species showed pronounced killing activity against Bd over 24 hours. In conclusion, the absence of Bd in Speleomantes combined with resistance to experimental chytridiomycosis and highly efficient skin defenses indicate that the genus Speleomantes is a taxon unlikely to decline due to Bd.

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows.

Methicillin-resistant Staphylococcus aureus (MRSA) is infrequently reported in mastitis. Yet, as in many other countries, the prevalence of methicillin resistance among S. aureus from mastitis is currently unknown in Belgium. To elucidate this, the presence of mecA was investigated in 118 S. aureus strains originating from diagnostic mastitis milk samples from 118 different farms experiencing S. aureus mastitis. MRSA strains were characterized by disk diffusion susceptibility testing, spa-typing, MLST and SCCmec-typing. In an additional study, four MRSA-positive farms were selected to assess the in-herd prevalence of MRSA, by sampling all cows in lactation. Isolated MRSA strains were similarly characterized. The mecA gene was detected in 11 (9.3%) of the 118 S. aureus isolates, indicating that nearly 10% of the Belgian farms suffering from S. aureus mastitis have an MRSA problem. The in-herd prevalence varied between 0% and 7.4%. Characterization of the MRSA strains showed that they were all resistant to tetracycline. Additional resistances to macrolides, lincosamides and aminoglycosides were frequently detected. The strains were ST398, spa-types t011 or t567 and had SCCmec-type IVa or V, proving that they belong to the emerging livestock-associated MRSA (LA-MRSA) strains of CC398. Our study shows that after detection in Belgian pigs, horses and poultry, LA-MRSA has also attained Belgian cattle. It is the first report on frequent isolation of LA-MRSA from bovine infections. As the in-herd isolation rate resembles that of regular S. aureus in farms experiencing S. aureus mastitis, the multi-resistance of LA-MRSA strains may cause future treatment problems.

A live Salmonella enterica serovar Enteritidis vaccine allows serological differentiation between vaccinated and infected animals.

Three precisely defined deletion mutants of Salmonella enterica serovar Enteritidis were constructed, a guanine auxotrophic DeltaguaB mutant, a nonflagellated DeltafliC mutant, and an auxotrophic and nonflagellated DeltaguaB DeltafliC double mutant. All three mutants were less invasive than the wild-type strain in primary chicken cecal epithelial cells and the human epithelial cell line T84 and less efficiently internalized in the chicken macrophage cell line HD11. The DeltafliC mutant was pathogenic in orally infected BALB/c mice, while the DeltaguaB mutant was attenuated and conferred protection against a challenge with the pathogenic parent strain. The DeltaguaB DeltafliC double mutant was totally asymptomatic and conferred better protection than the DeltaguaB mutant. This indicates that the major flagellar protein flagellin is not required for efficient vaccination of BALB/c mice against Salmonella infection. The DeltaguaB DeltafliC mutant was also safe for vaccination of 1-day-old chickens. After two immunizations, it induced statistically significant protection against infection of the internal organs of the birds by a virulent S. enterica serovar Enteritidis challenge strain but not against intestinal colonization. These data demonstrate that nonflagellated attenuated Salmonella mutants can be used as marker vaccines.

Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs.

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in several animal species. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections of pigs, however, is not well described. The interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants with disruptions in the SPI-1 genes hilA, sipA and sipB with porcine intestinal epithelial cells were characterized in vitro and in a ligated intestinal loop model in pigs. HilA and SipB were essential in the invasion of porcine intestinal epithelial cells in vitro. A sipA mutant was impaired for invasion using a polarized cell line, but fully invasive in a non-polarized cell line. All SPI-1 mutants induced a significant decrease in influx of neutrophils in the porcine intestinal loop model compared with the wild type strain. Pigs were orally inoculated with 10(8) colony forming units of both the wild type Salmonella Typhimurium strain and its isogenic sipB::kan mutant strain. The sipB mutant strain was significantly impaired to invade the intestinal, but not the tonsillar tissue, one day after inoculation and was unable to efficiently colonize the intestines and the GALT, but not the tonsils, 3 days after inoculation. This study shows that SPI-1 plays a crucial role in the invasion and colonization of the porcine gut and in the induction of influx of neutrophils towards the intestinal lumen, but not in the colonization of the tonsils.