A Palearctic view of a bat fungal disease.

The fungal infection causing white-nose disease in hibernating bats in North America has resulted in dramatic population declines of affected species, since the introduction of the causative agent Pseudogymnoascus destructans. The fungus is native to the Palearctic, where it also infects several bat species, yet rarely causes severe pathology or the death of the host. Pseudogymnoascus destructans infects bats during hibernation by invading and digesting the skin tissue, resulting in the disruption of torpor patterns and consequent emaciation. Relations among pathogen, host, and environment are complex, and individuals, populations, and species respond to the fungal pathogen in different ways. For example, the Nearctic Myotis lucifugus responds to infection by mounting a robust immune response, leading to immunopathology often contributing to mortality. In contrast, the Palearctic M. myotis shows no significant immunological response to infection. This lack of a strong response, resulting from the long coevolution between the hosts and the pathogen in the pathogen's native range, likely contributes to survival in tolerant species. After more than 15 years since the initial introduction of the fungus to North America, some of the affected populations are showing signs of recovery, suggesting that the fungus, hosts, or both are undergoing processes that may eventually lead to coexistence. The suggested or implemented management methods of the disease in North America have encompassed, for example, the use of probiotics and fungicides, vaccinations, and modifying the environmental conditions of the hibernation sites to limit the growth of the pathogen, intensity of infection, or the hosts' responses to it. Based on current knowledge from Eurasia, policy makers and conservation managers should refrain from disrupting the ongoing evolutionary processes and adopt a holistic approach to managing the epizootic.

Vista paleártica de una enfermedad fúngica de murciélagos Resumen La enfermedad fúngica que produce el síndrome de nariz blanca en murciélagos en hibernación en Norte América ha resultado en declinaciones poblacionales dramáticas en las especies afectadas desde la introducción del agente causante, Pseudogymnoascus destructans. El hongo es nativo del Paleártico, donde también infecta a varias especies de murciélagos; sin embargo, raramente causa patología severa o la muerte del hospedero. Pseudogymnoascus destructans infecta a los murciélagos durante la hibernación invadiendo y digiriendo el tejido de la piel, lo que resulta en la disrupción de los patrones de torpor y la consecuente emaciación. Las relaciones entre el patógeno, el huésped y el ambiente son complejas, y los individuos, las especies y poblaciones responden al patógeno fúngico de distintas maneras. Por ejemplo, Myotis lucifugus, especie del Neártico, responde a la infección montando una respuesta inmune robusta, produciendo una inmunopatología que a menudo contribuye a la mortalidad. En contraste, M. myotis del Paleártico no presenta respuesta inmunológica significativa a la infección. La falta de una fuerte respuesta, resultado de la larga coevolución entre hospederos y el patógeno en el rango nativo de distribución del patógeno, probablemente contribuye a la supervivencia en especies tolerantes. Después de más de 15 años desde la introducción del hongo en Norte América, algunas de las poblaciones afectadas están mostrando señales recuperación, lo que sugiere que el hongo, hospederos, o ambos, están pasando por procesos que eventualmente pueden conducir a la coexistencia. Los métodos de manejo de la enfermedad sugeridos o implementados en Norte América han abarcado, por ejemplo, el uso de probióticos y fungicidas, vacunaciones y modificación de las condiciones ambientales de los sitios de hibernación para limitar el crecimiento del patógeno, la intensidad de la infección o las respuestas de los hospederos. Con base en conocimiento actual de Eurasia, los formuladores de políticas y los manejadores de la conservación deberían abstenerse de alterar los procesos evolutivos en curso y adoptar un enfoque holístico para gestionar la epizootia.

When the host's away, the pathogen will play: the protective role of the skin microbiome during hibernation.

The skin of animals is enveloped by a symbiotic microscopic ecosystem known as the microbiome. The host and microbiome exhibit a mutualistic relationship, collectively forming a single evolutionary unit sometimes referred to as a holobiont. Although the holobiome theory highlights the importance of the microbiome, little is known about how the skin microbiome contributes to protecting the host. Existing studies focus on humans or captive animals, but research in wild animals is in its infancy. Specifically, the protective role of the skin microbiome in hibernating animals remains almost entirely overlooked. This is surprising, considering the massive population declines in hibernating North American bats caused by the fungal pathogen Pseudogymnoascus destructans, which causes white-nose syndrome. Hibernation offers a unique setting in which to study the function of the microbiome because, during torpor, the host's immune system becomes suppressed, making it susceptible to infection. We conducted a systematic review of peer-reviewed literature on the protective role of the skin microbiome in non-human animals. We selected 230 publications that mentioned pathogen inhibition by microbes residing on the skin of the host animal. We found that the majority of studies were conducted in North America and focused on the bacterial microbiome of amphibians infected by the chytrid fungus. Despite mentioning pathogen inhibition by the skin microbiome, only 30.4% of studies experimentally tested the actual antimicrobial activity of symbionts. Additionally, only 7.8% of all publications studied defensive cutaneous symbionts during hibernation. With this review, we want to highlight the knowledge gap surrounding skin microbiome research in hibernating animals. For instance, research looking to mitigate the effects of white-nose syndrome in bats should focus on the antifungal microbiome of Palearctic bats, as they survive exposure to the Pseudogymnoascus destructans -pathogen during hibernation. We also recommend future studies prioritize lesser-known microbial symbionts, such as fungi, and investigate the effects of a combination of anti-pathogen microbes, as both areas of research show promise as probiotic treatments. By incorporating the protective skin microbiome into disease mitigation strategies, conservation efforts can be made more effective.

Immune responses in hibernating little brown myotis (Myotis lucifugus) with white-nose syndrome.

White-nose syndrome (WNS) is a fungal disease responsible for decimating many bat populations in North America. Pseudogymnoascus destructans (Pd), the psychrophilic fungus responsible for WNS, prospers in the winter habitat of many hibernating bat species. The immune response that Pd elicits in bats is not yet fully understood; antibodies are produced in response to infection by Pd, but they may not be protective and indeed may be harmful. To understand how bats respond to infection during hibernation, we studied the effect of Pd inoculation on the survival and gene expression of captive hibernating Myotis lucifugus with varying pre-hibernation antifungal antibody titres. We investigated gene expression through the transcription of selected cytokine genes (Il6, Il17a, Il1b, Il4 and Ifng) associated with inflammatory, Th1, Th2 and Th17 immune responses in wing tissue and lymph nodes. We found no difference in survival between bats with low and high anti-Pd titres, although anti-Pd antibody production during hibernation differed significantly between infected and uninfected bats. Transcription of Il6 and Il17a was higher in the lymph nodes of infected bats compared with uninfected bats. Increased transcription of these cytokines in the lymph node suggests that a pro-inflammatory immune response to WNS is not restricted to infected tissues and occurs during hibernation. The resulting Th17 response may be protective in euthermic bats, but because it may disrupt torpor, it could be detrimental during hibernation.

Molecular Evidence of Chlamydia-Like Organisms in the Feces of Myotis daubentonii Bats.

Chlamydia-like organisms (CLOs) are recently identified members of the Chlamydiales order. CLOs share intracellular lifestyles and biphasic developmental cycles, and they have been detected in environmental samples as well as in various hosts such as amoebae and arthropods. In this study, we screened bat feces for the presence of CLOs by molecular analysis. Using pan-Chlamydiales PCR targeting the 16S rRNA gene, Chlamydiales DNA was detected in 54% of the specimens. PCR amplification, sequencing, and phylogenetic analysis of the 16S rRNA and 23S rRNA genes were used to classify positive specimens and infer their phylogenetic relationships. Most sequences matched best with Rhabdochlamydia species or uncultured Chlamydia sequences identified in ticks. Another set of sequences matched best with sequences of the Chlamydia genus or uncultured Chlamydiales from snakes. To gain evidence of whether CLOs in bat feces are merely diet borne, we analyzed insects trapped from the same location where the bats foraged. Interestingly, the CLO sequences resembling Rhabdochlamydia spp. were detected in insect material as well, but the other set of CLO sequences was not, suggesting that this set might not originate from prey. Thus, bats represent another potential host for Chlamydiales and could harbor novel, previously unidentified members of this order. IMPORTANCE: Several pathogenic viruses are known to colonize bats, and recent analyses indicate that bats are also reservoir hosts for bacterial genera. Chlamydia-like organisms (CLOs) have been detected in several animal species. CLOs have high 16S rRNA sequence similarity to Chlamydiaceae and exhibit similar intracellular lifestyles and biphasic developmental cycles. Our study describes the frequent occurrence of CLO DNA in bat feces, suggesting an expanding host species spectrum for the Chlamydiales As bats can acquire various infectious agents through their diet, prey insects were also studied. We identified CLO sequences in bats that matched best with sequences in prey insects but also CLO sequences not detected in prey insects. This suggests that a portion of CLO DNA present in bat feces is not prey borne. Furthermore, some sequences from bat droppings not originating from their diet might well represent novel, previously unidentified members of the Chlamydiales order.

Interspecific variation in redox status regulation and immune defence in five bat species: the role of ectoparasites.

Harmful reactive oxygen species (ROS) produced during metabolism and immune responses are neutralized in part by a powerful enzymatic antioxidant system. Inter-species variability in the baseline activity of antioxidant enzymes may be explained by a variety of life history traits. For instance, ectoparasites can elicit repeated immune responses, thus increasing the production of reactive oxygen species. The bat species studied so far have been acknowledged to have effective antioxidant defences. However, interspecific comparisons within the clade do not exist. The present study compares the antioxidant defence and immune function activities in five northern boreal bat species relative to their ectoparasite prevalence and intensity (wing mites and louse flies) to reveal inter-species differences. Antioxidant enzyme and immune defense activities, which differ between species, are positively associated, with total ectoparasite (mites and bat flies) frequencies, total ROS, and protein carbonylation in Daubenton's bats, but enzyme activities are also independently influenced by sampling date with activities increasing towards the autumn. Antioxidant activities are also positively associated with total reactive oxygen species and oxidative damage (protein carbonylation) in the Daubenton's bat. Our results suggest that antioxidant activities are associated with ecological factors such as parasite load and season, and we consider it likely that these may partly explain the observed interspecific variation.

Resistance to oxidative damage but not immunosuppression by organic tin compounds in natural populations of Daubenton's bats (Myotis daubentonii).

The acute toxicity of organic tin compounds (OTCs) has been studied in detail. However, due to their complex nature, very little is known about species-specific methods of accumulation and consequences for food-webs. Chironomids, on which e.g. Daubenton's bats feed, may act as vectors for the transport of organic tin compounds from aquatic to terrestrial ecosystems. Bats are prone to environmental toxins because of their longevity and their ecological role as top predators. Organic tin compounds are associated with increased formation of reactive oxygen species and associated oxidative damage as well as suppression of immune function. The present paper investigates whether the OTC, tributyltin (TBT) and its metabolite, dibutyltin (DBT), accumulate in natural populations of Daubenton's bats and whether TBT-associated effects are seen in general body condition, redox balance, redox enzyme activities, associated oxidative damage of red blood cells and complement function. We discovered the concentration of bat fur DBT correlated with local marine sediment TBT concentrations. However, we did not find a correlation between the explanatory factors, bat fur DBT and marine sediment TBT concentrations, and several physiological and physical response variables apart from complement activity. Higher DBT concentrations resulted in weaker complement activity and thus a weaker immune response. Although the observed physiological effects in the present study were not strongly correlated to butyltin concentrations in fur or sediment, the result is unique for natural populations so far and raises interesting questions for future ecotoxicological studies.

Sediment organic tin contamination promotes impoverishment of non-biting midge species communities in the Archipelago Sea, S-W Finland.

Chironomid species are a vital component in many benthic and terrestrial food webs; they have an important role in the detritus cycle, and are an important source of food for many species. We studied how tributyltin (TBT) in brackish water sediments affect the composition of chironomid species communities. Emergence traps were used at selected sites on a TBT gradient in the Archipelago Sea, S-W Finland. Increased sediment TBT concentration was associated with significant chironomid species turnover, which in turn was related to decreased species diversity (number of species and genera). However, the overall number of individuals did not decrease markedly with increasing TBT contamination. This suggests that the ecological role of chironomids in the food web may be preserved even under severe impoverishment of the chironomid community due to organic tin contamination. The increased prevalence of more TBT tolerant species can potentially lead to a transport of organic tin compounds between aquatic and terrestrial food webs. Furthermore, the reduced diversity of an ecologically influential group might lower the resistance of the entire food web to other environmental hazards and perturbations.

First encounter of European bat lyssavirus type 2 (EBLV-2) in a bat in Finland.

In Finland, rabies in bats was suspected for the first time in 1985 when a bat researcher, who had multiple bat bites, died in Helsinki. The virus isolated from the researcher proved to be antigenically related to rabies viruses previously detected in German bats. Later, the virus was typed as EBLV-2b. Despite an epidemiological study in bats 1986 and subsequent rabies surveillance, rabies in bats was not detected in Finland until the first case in a Daubenton's bat (Myotis daubentonii) was confirmed in August 2009. The bat was paralysed, occasionally crying, and biting when approached; it subsequently tested positive for rabies. The virus was genetically typed as EBLV-2. This is the northernmost case of bat rabies ever detected in Europe. Phylogenetic analyses showed that the EBLV-2b isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related, demonstrating that EBLV-2 may have been circulating in Finland for many years.

A digital image analysis system for the assessment of tooth whiteness compared to visual shade matching.

OBJECTIVE: To assess tooth whiteness clinically using an image analysis system and a whiteness algorithm to compare with visual shade matching. METHODOLOGY: Images of the maxillary anterior teeth of 20 subjects were obtained using an image analysis system adapted for tooth whiteness measurement. Red, green, and blue values from each image, and Commision Internationale de l'Eclairage whiteness index (CIE WI) values were calculated. A visual shade match was also carried out using a Vitapan 3D Master 26-tab shade guide. RESULTS: The range of CIE WI values for image analysis and visual shade matching was 32.86-85.91 and 32.68-79.84, respectively. Limits of agreement between methods were +17.71 and -14.60 CIE WI units. The 95% confidence interval of the difference was calculated as -0.176 to +3.083. A two-tailed Student t-test with a 95% confidence level showed significant differences (p = 0.028) between the paired CIE WI values of each tooth for the two measurement techniques. As the accuracy and reliability of the image analysis system was found to be high in previous in vitro studies, the differences between the two methods were probably related to the inherent subjectivity of visual shade matching. CONCLUSION: The adapted digital image analysis system could be used as an alternative to, or in conjunction with visual shade matching.

Visual whiteness ranking of a Vitapan 3D Master shade guide by untrained assessors.

OBJECTIVE: This study was conducted to obtain the ranking order of a 29-tab (including three "bleaching tabs") Vita Shade Guide for perceived "whiteness" by untrained assessors, and to compare this ranking with both the shade guide manufacturer's rank order and a colorimetrically derived rank order. METHODOLOGY: A total of 85 people, not trained in color assessment by shade guides, were asked to rank order the tabs of a Vitapan 3D Master shade guide for perceived "whiteness" under standardized lighting conditions. A whiteness ranking was also obtained colorimetrically using a Minolta CM-2600d spectrophotometer calibrated to give CIE whiteness values. The data were analysed using means, standard deviations, and standard errors. RESULTS: The assessors varied more in their rankings for darker shades than the lighter ones. The order derived by the assessors and the colorimetry order did not match well with the manufacturer's order, especially toward the darker shades. CONCLUSION: For use in studies of whitening products, a new order of the Vitapan 3D Master shade guide tabs has been developed in relation to whiteness.

The measurement of tooth whiteness by image analysis and spectrophotometry: a comparison.

Digital image capturing and analysis techniques have been used to measure the colour of teeth and to compare with spectrophotometric results and visual observations. A non-linear image analysis approach was developed and, for the colour range of human teeth, allows device-dependant digital camera colour data to be quantitatively transformed to Commission Internationale de l'Eclairage (CIE) colorimetric values. With reference to a CIE standard illuminant, two different lighting arrays have been used. For flat and non-translucent white and yellow surfaces, spectrophotometric results showed that this transformation achieves required accuracy. It was found, in all of the present studies, which included measurements on the VITA Lumin Vacuum shade guide and extracted teeth, that spectrophotometry invariably underestimated values of the CIE whiteness index. However, the results from these two types of measurement correlated well. There was also a reasonably good correlation between earlier data obtained by visual assessment and the present data by the two instrumental methods. For extracted teeth, both instrumental methods used in this work did not confirm a whitening effect for 2-min brushing with toothpaste, but did show significant whitening results for bleaching with 15% hydrogen peroxide.

Synergy of binary poly(oxypropylene-oxyethylene) copolymers in reducing retention of Streptococcus sanguis to hydroxyapatite.

Binary poly(oxypropylene-oxyethylene) block copolymer systems have been investigated as a non-bactericidal approach to reduce the retention of an oral bacterium, Streptococcus sanguis. Using a previously validated hydroxyapatite-coated microtitre model to simulate the tooth, the performance of copolymer pairs was measured experimentally. A synergy index and an efficacy index were defined to describe the reduction of bacterial retention by the binary systems that comprise the copolymer pairs. Relationships between the synergy and efficacy indices and their associated compositions are given. The results obtained have been rationalised using a previously developed theoretical approach in conjunction with the binding energetics of species to surfaces.

Moderation of oral bacterial adhesion on saliva-coated hydroxyapatite by polyaspartate.

AIMS: Synthetic sodium alpha,beta-polyaspartate (PA) has been investigated as a moderator of adhesion and the subsequent biofilm formation by oral bacteria. METHODS AND RESULTS: The inhibition of bacterial adhesion by PA was assessed by (i) a 30-min incubation with Streptococcus sanguis in a microtitre assay with the wells coated with hydroxyapatite (HAP) and (ii) an 18-h challenge with human salivary microflora in a HAP disc assay. In contrast to HAP-coated surfaces, clean polystyrene surfaces in the microtitre assay exhibited no anti-adhesion properties. It has been found that PA significantly and similarly adsorbs onto HAP surfaces in the presence and absence of salivary coating. The HAP disc assay also showed that PA, both in aqueous solutions and in toothpaste, reduced the level of adhered microflora and this effect was enhanced by added propylene oxide-ethylene oxide copolymers. CONCLUSION: The principal finding from this work is the potential role for PA as an inhibitor of dental plaque formation. PA may significantly modify the salivary pellicle. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates the use of PA in controlling the development of dental plaque and the formation of bacterial biofilm in general.

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization.

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.

Multiple interactions between polyphenols and a salivary proline-rich protein repeat result in complexation and precipitation.

Polyphenols (tannins) in the diet not only precipitate oral proteins, producing an astringent sensation, but also interact with dietary proteins and digestive enzymes in the gut, resulting in a variety of antinutritive and toxic effects. Salivary proline-rich proteins (PRPs), which are secreted into the oral cavity, form complexes with and precipitate dietary polyphenols, and thus, they constitute the primary mammalian defense directed against ingested tannins. In order to characterize the interaction, NMR studies were performed which involved titrating a series of polyphenols into a synthetic 19-residue PRP fragment. The results show that the predominant mode of association is a hydrophobic stacking of the polyphenol ring against the pro-S face of proline and that the first proline residue of a Pro-Pro sequence is a particularly favored binding site. Measurement of dissociation constants indicates that the larger and more complex polyphenols interact more strongly with the PRP fragment; the order of binding affinity was determined as procyanidin dimer B-2 > pentagalloylglucose > trigalloylglucose >> proanthocyanidin monomer (-)-epicatechin approximately propyl gallate. Smaller polyphenols can bind with one phenolic ring stacked against each proline residue, whereas larger polyphenols occupy two or three consecutive prolines. The more complex polyphenols interact with the PRP fragment in a multidentate fashion; moreover, they self-associate or stack when bound. Thus, a model is proposed in which multiple polyphenol/polyphenol and polyphenol/PRP interactions act cooperatively to achieve precipitation.

Tannin interactions with a full-length human salivary proline-rich protein display a stronger affinity than with single proline-rich repeats.

The protein IB5 has been purified from human parotid saliva. This protein contains several repeats of a short proline-rich sequence. Dissociation constants have been measured at several discrete binding sites using 1H-NMR for the hydrolysable tannins (polyphenols) beta-1,3,6-tri-O-galloyl-D-glucopyranose, beta-1,2,4,6-tetra-O-galloyl-D-glucopyranose and beta-1,2,3,4,6-penta-O-galloyl-D-glucopyranose and the condensed proanthocyanidin (--)-epicatechin. The dissociation constants for trigalloyl glucose and pentagalloyl glucose were 15 X 10(-5) and 1.7 X 10(-5) M, respectively, which are 115 and 1660 times stronger than those previously measured under the same conditions for a single repeat of a mouse salivary proline-rich protein. The increase in affinity is ascribed to intramolecular secondary interactions, which are strengthened by the rigidity of the interacting molecules.

Polyphenols, astringency and proline-rich proteins.

Recent, NMR and precipitation, studies of molecular recognition of proline-rich proteins and peptides by plant polyphenols are described and rationalized. The action of polysaccharides and caseins in the moderation of the astringent response, which is engendered by polyphenols present in foodstuffs and beverages, is described. The possible influence of plant cell wall glycoproteins on the process of lignification is discussed in the light of the observed affinity of phenolic substrates for prolyl residues in protein structures.

E. coli penicillin acylase isolation by selective release, aqueous two-phase partitioning and ultrafiltration.

A procedure has been developed for the purification of penicillin acylase from E. coli fermentation broths. The method described is based primarily on selective release from biomass, charge-directed partitioning in an aqueous two-phase system, and the use of ultrafiltration membranes to recycle the modified polyethylene glycol required and to further purify the enzyme. Each step has been discussed and comparisons have been made with other approaches, where possible. Suggestions have been made for optimisation in process engineering. The approach developed may be applicable to other beta-lactam antibiotic acylases or more generally to some of the other E. coli periplasmic proteins.

Study of the interaction between salivary proline-rich proteins and a polyphenol by 1H-NMR spectroscopy.

The interaction between salivary proline-rich proteins and plant polyphenols (tannins) in the oral cavity and their subsequent precipitation influences the taste, texture and nutritional value of food; it is thought to be responsible for the astringency of many foods and beverages. To investigate the interaction, two-dimensional 1H-NMR studies have been carried out on the binding of a representative polyphenol, pentagalloyl glucose, to two synthetic peptides (19 and 22 residues in length) that are typical of the repeat sequence of mouse salivary proline-rich protein MP5. Intermolecular nuclear Overhauser effects and chemical shift changes show that the main binding sites on the peptides are proline residues together with the preceding amide bond and amino acid. The interaction is principally a hydrophobic association between a galloyl ring and the pyrrolidine ring face containing the C alpha proton, but secondary hydrogen-bonding effects help to stabilise the complex. Very similar interactions are seen for both peptides. The conformation of the peptides remains extended on binding. The chemical shift changes seen for many of the peptide protons can be fitted to a simple binding curve with dissociation constant of around 40 mM, but some protons show evidence of cooperative binding involving several galloyl groups. Higher concentrations of pentagalloyl glucose lead to a reduced off-rate from the complex and eventual precipitation which implies that precipitation is caused by a kinetic competition between aggregation and dissociation of the complex.