Resistance against strongylid nematodes in two high prevalence Equine Recurrent Airway Obstruction families has a genetic basis

A recent study showed increased resistance against strongylid nematodes in offspring of a stallion affected by recurrent airway obstruction (RAG) compared with unrelated pasture mates. Resistance against strongylid nematodes was associated with RAG affection. Hypothesis: Resistance against strongylid nematodes has a genetic basis. The genetic variants influencing strongylid resistance also influence RAG susceptibility. Faecal samples from the half-sibling offspring of two RAG-affected Warmblood stallions 98 offspring from the first family (family 1) and 79 from the second family (family 2) were analysed using a combined sedimentation-flotation method. The phenotype was defined as a binary trait - either positive or negative for egg shedding. The influence of non-genetic factors on egg shedding was analysed using SAS, the mode of inheritance was investigated using PAP and iBay, and the association between shedding of strongyle eggs and RAG was estimated by odds ratios. Previously established genotypes for 315 microsatellite markers were used for QTL analyses using GRID QTL. The inheritance of "strongylid egg shedding" is influenced by major genes on ECA15 and ECA20. Shedding of strongylid eggs is associated with RAG in family 1 but not in family 2. Conclusions: The status of "shedding of strongyle eggs" has a genetic background. The results were inconclusive as to whether "egg shedding" and RAG share common genetic components. Our results suggest that it may be possible to select for resistance against strongylid nematodes.

Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using Ingenuity Pathway Analysis(R)

BACKGROUND: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The analysis was carried out with Ingenuity Pathway Analysis(R) bioinformatics software. RESULTS: The gene with the greatest number of indirect interactions with the set of proteins identified is Interleukin 4 Receptor (IL-4R), whose protein has also been detected in BALF. Interleukin 21 receptor and chemokine (C-C motif) ligand 24 also showed a large number of interactions with the group of detected proteins. Protein products of other genes like that of SOCS5, revealed direct interactions with the IL-4R protein. The interacting proteins NOD2, RPS6KA5 and FOXP3 found in several pathways are reported regulators of the NFkappaB pathway. CONCLUSIONS: The pathways generated with IL-4R highlight possible important intracellular signaling cascades implicating, for instance, NFkappaB. Furthermore, the proposed interaction between SOCS5 and IL-4R could explain how different genes can lead to identical clinical RAO phenotypes, as observed in two Swiss Warmblood half sibling families because these proteins interact upstream of an important cascade where they may act as a functional unit.

The interleukin 4 receptor gene and its role in recurrent airway obstruction in Swiss Warmblood horses

Recurrent airway obstruction (RAO) in horses is the result of an interaction of genetic and environmental factors and shares many characteristics with human asthma. Many studies have suggested that the interleukin-4 receptor gene (IL4R) is associated with this disease, and a QTL region on chromosome 13 containing IL4R was previously detected in one of the two Swiss Warmblood families. We sequenced the entire IL4R gene in this family and detected 93 variants including five non-synonymous protein-coding variants. The allele distribution at these SNPs supported the previously detected QTL signal. Subsequently, we investigated IL4R mRNA expression in bronchoalveolar lavage fluid cells. During exacerbation, IL4R expression was increased in RAO-affected offspring in the implicated family, but not in the other family. These findings support that IL4R plays a role in some cases of RAO.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

Increased parasite resistance and recurrent airway obstruction in horses of a high-prevalence family

BACKGROUND: Equine recurrent airway obstruction (RAO) shares many characteristics with human asthma. In humans, an inverse relationship between susceptibility to asthma and resistance to parasites is suspected. HYPOTHESIS/OBJECTIVES: Members of a high-incidence RAO half-sibling family (F) shed fewer strongylid eggs compared with RAO-unaffected pasture mates (PM) and that RAO-affected horses shed fewer eggs than RAO-unaffected half-siblings. ANIMALS: Seventy-three F and 73 unrelated, age matched PM. METHODS: Cases and controls kept under the same management and deworming regime were examined. Each individual was classified as RAO affected or RAO unaffected and fecal samples were collected before and 1-3 weeks and 3 months after deworming. Samples were analyzed by combined sedimentation-flotation and modified McMaster methods and classified into 3 categories of 0 eggs per gram of feces (EpG), 1-100 EpG, and > 100 EpG, respectively. RESULTS: PM compared with RAO-affected F had a 16.7 (95% confidence interval [CI]: 2.0-136.3) times higher risk for shedding > 100 EpG compared with 0 EpG and a 5.3 (95% CI: 1.0-27.4) times higher risk for shedding > 100 EpG compared with 0 EpG. There was no significant effect when RAO-unaffected F were compared with their PM. RAO-unaffected compared with RAO-affected offspring had a 5.8 (95% CI: 0.0-1.0) times higher risk for shedding 1-100 EpG. Age, sex, breed, and sharing pastures with other species had no significant confounding effects. CONCLUSION AND CLINICAL IMPORTANCE: RAO is associated with resistance against strongylid parasites in a high-prevalence family.

Relationship of horse owner assessed respiratory signs index to characteristics of recurrent airway obstruction in two Warmblood families

REASONS FOR PERFORMING STUDY: The horse owner assessed respiratory signs index (HOARSI-1-4, healthy, mildly, moderately and severely affected, respectively) is based on owner-reported clinical history and has been used for the investigation of recurrent airway obstruction (RAO) genetics utilising large sample sizes. Reliable phenotype identification is of paramount importance in genetic studies. Owner reports of respiratory signs have shown good repeatability, but the agreement of HOARSI with an in-depth examination of the lower respiratory tract has not been investigated. OBJECTIVES: To determine the correlation of HOARSI grades 3/4 with the characteristics of RAO and of HOARSI-2 with the characteristics of inflammatory airway disease. Further, to test whether there are phenotypic differences in the manifestation of lung disease between families. METHODS: Seventy-one direct offspring of 2 RAO-affected Warmblood stallions (33 from the first family, 38 from the second) were graded as HOARSI-1-4 and underwent a clinical examination of the respiratory system, arterial blood gas analysis, endoscopic mucus scoring, cytology of tracheobronchial secretion (TBS) and bronchoalveolar lavage fluid (BALF), and clinical assessment of airway reactivity to methacholine chloride. RESULTS: HOARSI-3/4 animals in clinical exacerbation showed signs consistent with RAO: coughing, nasal discharge, abnormal lung sounds and breathing pattern as well as increased numbers of neutrophils in TBS and BALF, excessive mucus accumulation and airway hyper-responsiveness to methacholine. HOARSI-3/4 horses in remission only had increased amounts of tracheal mucus and TBS neutrophil percentages. Clinical phenotypes were not significantly different between the 2 families. CONCLUSIONS AND CLINICAL RELEVANCE: HOARSI reliably identifies RAO-affected horses in our population.

Hibernacula Microclimate and White-nose Syndrome Susceptibility in the Little Brown Myotis (Myotis lucifugus)

The objective of this project was to determine the relationship between hibernacula microclimate and White-nose Syndrome (WNS), an emerging infectious disease in bats. Microclimate was examined on a species scale and at the level of the individual bat to determine if there was a difference in microclimate preference between healthy and WNS-affected little brown myotis (Myotis lucifugus) and to determine the role of microclimate in disease progression. There is anecdotal evidence that colder, drier hibernacula are less affected by WNS. This was tested by placing rugged temperature and humidity dataloggers in field sites throughout the eastern USA, experimentally determining the response to microclimate differences in captive bats, and testing microclimate roosting preference. This study found that microclimate significantly differed from the entrance of a hibernaculum versus where bats traditionally roost. It also found hibernaculum temperature and sex had significant impacts on survival in WNS-affected bats. Male bats with WNS had increased survivability over WNS-affected female bats and WNS bats housed below the ideal growth range of the fungus that causes WNS, Geomyces destructans, had increased survival over those housed at warmer temperatures. The results from this study are immediately applicable to (1) predict which hibernacula are more likely to be infected next winter, (2) further our understanding of WNS, and (3) determine if direct mitigation strategies, such as altering the microclimate of mines, will be effective ways to combat the spread of the fungus.

Altered Behavior in Bats Affected by White-Nose Syndrome

WNS-affected bats did so over similar time frames as WNSunaffected bats. The behaviors of bats with WNS did not change as drastically as expected. Thereseems to be little to no effect on their ability to fly/forage until much later stages of the disease when they are likely near death. WNS-affected bats are grooming more which could be altering the way they use energy reserves during hibernation possibly leading tostarvation and eventually death. The decreased likelihood of arousals in response to external cues may be the result of spending more energy during previous and increasingly frequent arousals. While it is clear that WNS does result in changes in behavior whether these changes are directly in response to fungal skin infection or to some other component of the syndrome such as decreased energy availability or loss of homeostasis is unknown. bat behavior, white-nose syndrome, behavior, video surveillance, arousal patterns White-Nose Syndrome (WNS) is a disease of hibernating bats caused by the fungal pathogen Geomyces destructans. The fungus, which was first noted in 2006, invades bats wings and other exposed membranes, eventually resulting in death. Researchers have yet to understand many aspects of this disease, including basic etiology and epidemiology. There is also a lack of information on how fungal infection may change the behavior of healthy bats during hibernation or how changes in behavior may influence disease progression. Based upon the physiological changes that are known to occur in affected bats, and upon anecdotal observations of aberrant behavior in these bats, I hypothesized that WNS would significantly change the behavior of the little brown myotis (Myotis lucifugus). My research examined the behavior of hibernating bats during arousals from torpor. I compared WNS-affected and unaffected bats, in the field and incaptivity, using motion-sensitive infrared cameras. Flight maneuverability and echolocation were also tested between WNS-affected and unaffected bats during arousalsfrom hibernation to detect changes in the bats' ability to perform basic locomotion or potentially catch insect prey. Lastly, hibernating bats were artificially disturbed and theirarousal patterns were monitored to examine changes in the response to external stimuli between WNS-affected and unaffected bats.Bats with WNS groomed for longer periods of time after arousing from torpor, both in the field and in captivity. They also engaged in longer periods of any sort of activity during these arousals. There were no changes in acoustical signaling during flight tests and changes in flight maneuverability were only found in bats were seen staging" near the entrance of the mine which is itself a unique behavior exhibited by affected bats. At this point these bats were likely near death and could barely fly at all. In response toexternal stimuli bats with WNS were less likely to arouse than unaffected bats. However when they did arouse WNS-affected bats did so over similar time frames as WNSunaffected bats. The behaviors of bats with WNS did not change as drastically as expected. Thereseems to be little to no effect on their ability to fly/forage until much later stages of the disease when they are likely near death. WNS-affected bats are grooming more which could be altering the way they use energy reserves during hibernation possibly leading tostarvation and eventually death. The decreased likelihood of arousals in response to external cues may be the result of spending more energy during previous and increasingly frequent arousals. While it is clear that WNS does result in changes in behavior whetherthese changes are directly in response to fungal skin infection or to some other component of the syndrome such as decreased energy availability or loss of homeostasis is unknown."

Physiological Consequences Of White-Nose Syndrome

White-nose syndrome (WNS) is a disease that has caused the mass mortality of hibernating bat species. Since its first discovery in the winter of 2006-2007, an estimated five million bats or more have been killed. Although infection with Pseudogymnoascus destructans (Pd, the causative agent of WNS) does not always result in death, bats that survive Pd infection may experience fitness consequences. To understand the physiological consequences of WNS, I measured reproductive rates of free-ranging hibernating bat species of the Northeastern United States. In addition, captive little brown myotis (Myotis lucifugus) bats that were infected by Pd but survived (¿WNS survivors¿) and uninfected bats were studied in order to understand the potential consequences (e.g., lower reproductive rates, decreased ability to heal wounds, degradation of wing tissue, and altered metabolic rates) of surviving WNS. No differences in reproductive rates were found between WNS-survivors and uninfected bats in either the field or in captivity. In addition, wound healing was not affected by Pd infection. However, wing tissue degradation was worse for little brown myotis 19 days post-hibernation, and mass specific metabolic rate (MSMR) was significantly higher for those infected with Pd 22 days post-hibernation. While it is clear that these consequences are a direct result of Pd infection, further research investigating the long-term consequences for both mothers and pups is necessary.

Exploration Of Survival Traits In The White-Nose Syndrome-Affected Big Brown Bat (Eptesicus Fuscus)

The widespread mortality of hibernating bats is associated with the emerging infectious disease white-nose syndrome (WNS), and has provoked a strong interest in understanding which bats will survive, and why? The ability of infected bats to resist WNS may depend upon variation in the expression of different characteristics. In a captive colony of big brown bats, I sought to characterize the phenotypic variability, repeatability, and survivability for several key ¿survival¿ traits, including: torpor patterns, microclimate preferences, and wound healing capacity. Torpor patterns were profiled using temperature sensitive dataloggers throughout the hibernation season, while microclimate preferences were quantified by using temperature-graded boxes and thermal imaging. In order to assess wound healing capacity, small wing biopsies were obtained from each bat and healing progress was tracked for one month. Individuals exhibited a wide range of phenotypes that were significantly influenced by sex and body condition. Repeatability estimates suggest that there is not a strong genetic basis for the observed variation in torpor patterns or microclimate preferences. Certain phenotypes (e.g., BMI) were associated with an increased probability of overwinter survivorship, which suggests a basis for intra-species differences in WNS susceptibility. The results from this project provide novel insight into what we know about ¿who will survive,¿ and will influence the direction and implementation of future conservation and mitigation strategies.

Robotics in ear, nose and throat (ENT) surgery

The three main classes of robotics in ear, nose and throat (ENT) surgery (telemanipulation, image-guided functional servoing, and computer numerical control) are discussed and important examples of applications are described to show both technological and clinical developments. As access to many anatomical features of the head requires very small and compact tools to accurately perform procedures, examples are give in ear surgery where manipulation of the minute ossicles requires fine, dexterous movements.

Nonlethal Screening of Bat-Wing Skin with the Use of Ultraviolet Fluorescence to Detect Lesions Indicative of White-Nose Syndrome

Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366-385 nm) elicits a distinct orange yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange yellow wing fluorescence (n=80) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n=88) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n=22) and 100% of nonfluorescent (n=40) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of long-wave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.