Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

The 3' UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.

The Antimicrobial Properties of PdII - and RuII -pyta Complexes.

Infections associated with antimicrobial resistance (AMR) are poised to become the leading cause of death in the next few decades, a scenario that can be ascribed to two phenomena: antibiotic over-prescription and a lack of antibiotic drug development. The crowd-sourced initiative Community for Open Antimicrobial Drug Discovery (CO-ADD) has been testing research compounds contributed by researchers around the world to find new antimicrobials to combat AMR, and during this campaign has found that metallodrugs might be a promising, yet untapped source. To this end, we submitted 18 PdII - and RuII -pyridyl-1,2,3-triazolyl complexes that were developed as catalysts to assess their antimicrobial properties. It was found that the Pd complexes, especially Pd1, possessed potent antifungal activity with MICs between 0.06 and 0.125 µg mL-1 against Candida glabrata. The in-vitro studies were extended to in-vivo studies in Galleria mellonella larvae, where it was established that the compounds were nontoxic. Here, we effectively demonstrate the potential of PdII -pyta complexes as antifungal agents.

DksA is a conserved master regulator of stress response in Acinetobacter baumannii.

Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Cross-protection and cross-feeding between Klebsiella pneumoniae and Acinetobacter baumannii promotes their co-existence.

Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae against the cephalosporin, cefotaxime. Our study demonstrates distinct syntrophic interactions occur between A. baumannii and K. pneumoniae, helping to elucidate the basis for their co-existence in polymicrobial infections.

Identification of a Novel LysR Family Transcriptional Regulator Controlling Acquisition of Sulfur Sources in Acinetobacter baumannii.

l-cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in Escherichia coli. However, the regulation of sulfur metabolism in Acinetobacter baumannii is only partly understood, with the LysR-type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 (cbl), in a highly multidrug resistant and virulent isolate of A. baumannii. We have shown that Cbl is involved in controlling expression of the genes required for uptake and reduction of various sulfur sources in A. baumannii. Collectively, we have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in A. baumannii.

Metal Complexes as Antifungals? From a Crowd-Sourced Compound Library to the First In Vivo Experiments.

There are currently fewer than 10 antifungal drugs in clinical development, but new fungal strains that are resistant to most current antifungals are spreading rapidly across the world. To prevent a second resistance crisis, new classes of antifungal drugs are urgently needed. Metal complexes have proven to be promising candidates for novel antibiotics, but so far, few compounds have been explored for their potential application as antifungal agents. In this work, we report the evaluation of 1039 metal-containing compounds that were screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD). We show that 20.9% of all metal compounds tested have antimicrobial activity against two representative Candida and Cryptococcus strains compared with only 1.1% of the >300,000 purely organic molecules tested through CO-ADD. We identified 90 metal compounds (8.7%) that show antifungal activity while not displaying any cytotoxicity against mammalian cell lines or hemolytic properties at similar concentrations. The structures of 21 metal complexes that display high antifungal activity (MIC ≤1.25 µM) are discussed and evaluated further against a broad panel of yeasts. Most of these have not been previously tested for antifungal activity. Eleven of these metal complexes were tested for toxicity in the Galleria mellonella moth larva model, revealing that only one compound showed signs of toxicity at the highest injected concentration. Lastly, we demonstrated that the organo-Pt(II) cyclooctadiene complex Pt1 significantly reduces fungal load in an in vivo G. mellonella infection model. These findings showcase that the structural and chemical diversity of metal-based compounds can be an invaluable tool in the development of new drugs against infectious diseases.

Sugar-rich larval diet promotes lower adult pathogen load and higher survival after infection in a polyphagous fly.

Nutrition is a central factor influencing immunity and resistance to infection, but the extent to which nutrition during development affects adult responses to infections is poorly understood. Our study investigated how the nutritional composition of the larval diet affects the survival, pathogen load and food intake of adult fruit flies, Bactrocera tryoni, after septic bacterial infection. We found a sex-specific effect of larval diet composition on survival post-infection: survival rate was higher and bacterial load was lower for infected females raised on a sugar-rich larval diet than for females raised on a protein-rich larval diet, an effect that was absent in males. Both males and females were heavier when fed a balanced larval diet compared with a protein- or sugar-rich diet, while body lipid reserves were higher for those that had consumed the sugar-rich larval diet compared with other diets. Body protein reserves were lower for flies that had been raised on the sugar-rich larval diet compared with other diets in males, but not females. Both females and males shifted their nutrient intake to ingest a sugar-rich diet when infected compared with sham-infected flies without any effect of the larval diet, suggesting that sugar-rich diets can be beneficial to fight off bacterial infection as shown in previous literature. Overall, our findings show that nutrition during early life can shape individual fitness in adulthood.

Density-by-Diet Interactions during Larval Development Shape Adult Life History Trait Expression and Fitness in a Polyphagous Fly.

AbstractHabitat quality early in life determines individual fitness, with possible long-term evolutionary effects on groups and populations. In holometabolous insects, larval ecology plays a major role in determining the expression of traits in adulthood, but how ecological conditions during the larval stage interact to shape adult life history and fitness, particularly in nonmodel organisms, remains subject to scrutiny. Consequently, our knowledge of the interactive effects of ecological factors on insect development is limited. Here, using the polyphagous fly Bactrocera tryoni, we conducted a fully factorial design where we manipulated larval density and larval diet (protein rich, standard, and sugar rich) to gain insights into how these ecological factors interact to modulate adult fitness. As expected, a protein-rich diet resulted in faster larval development and heavier and leaner adults that were more fecund compared with the standard and sugar-rich diets, irrespective of larval density. Females from the protein-rich larval diet had overall higher reproductive rate (i.e., eggs per day) than females from other diets, and reproductive rate decreased linearly with density for females from the protein-rich diet but nonlinearly for females from the standard and sugar-rich diets over time. Surprisingly, adult lipid reserve increased with larval density for adults from the sugar-rich diet (as opposed to decreasing as in other diets), possibly because of a stress response to an extremely adverse condition during development (i.e., high intraspecific competition and poor nutrition). Together, our results provide insights into how ecological factors early in life interact and shape the fate of individuals through life stages in holometabolous insects.

Genomic and phenotypic analyses of diverse non-clinical Acinetobacter baumannii strains reveals strain-specific virulence and resistance capacity.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

Inhibitors of bacterial RNA polymerase transcription complex.

A series of hybrid compounds that incorporated anthranilic acid with activated 1H-indoles through a glyoxylamide linker were designed to target bacterial RNA polymerase holoenzyme formation using computational docking. Synthesis, in vitro transcription inhibition assays, and biological testing of the hybrids identified a range of potent anti-transcription inhibitors with activity against a range of pathogenic bacteria with MICs as low as 3.1 µM. A structure activity relationship study identified the key structural components necessary for inhibition of both bacterial growth and transcription. Correlation of in vitro transcription inhibition activity with in vivo mechanism of action was established using fluorescence microscopy and resistance passaging using Gram-positive bacteria showed no resistance development over 30 days. Furthermore, no toxicity was observed from the compounds in a wax moth larvae model, establishing a platform for the development of a series of new antibacterial drugs with an established mode of action.

Sex-specific effects of the microbiota on adult carbohydrate intake and body composition in a polyphagous fly.

The microbiota influences hosts' health and fitness. However, the extent to which the microbiota affects host' foraging decisions and related life history traits remains to be fully understood. Our study explored the effects of microbiota manipulation on foraging preference and phenotypic traits of larval and adult stages of the polyphagous fruit fly Bactrocera tryoni, one of the main horticultural pests in Australia. We generated three treatments: control (non-treated microbiota), axenic (removed microbiota), and reinoculation (individuals which had their microbiota removed then re-introduced). Our results confirmed that axenic larvae and immature (i.e., newly emerged 0 day-old, sexually-immature) adults were lighter than control and reinoculated individuals. Interestingly, we found a sex-specific effect of the microbiota manipulation on carbohydrate intake and body composition of 10 day-old mature adults. Axenic males ate less carbohydrate, and had lower body weight and total body fat relative to control and reinoculated males. Conversely, axenic females ate more carbohydrate than control and reinoculated ones, although body weight and lipid reserves were similar across treatments. Axenic females produced fewer eggs than control and reinoculated females. Our findings corroborate the far-reaching effects of microbiota in insects found in previous studies and show, for the first time, a sex-specific effect of microbiota on feeding behaviour in flies. Our results underscore the dynamic relationship between the microbiota and the host with the reinoculation of microbes restoring some traits that were affected in axenic individuals.

Platinum Cyclooctadiene Complexes with Activity against Gram-positive Bacteria.

Antimicrobial resistance is a looming health crisis, and it is becoming increasingly clear that organic chemistry alone is not sufficient to continue to provide the world with novel and effective antibiotics. Recently there has been an increased number of reports describing promising antimicrobial properties of metal-containing compounds. Platinum complexes are well known in the field of inorganic medicinal chemistry for their tremendous success as anticancer agents. Here we report on the promising antibacterial properties of platinum cyclooctadiene (COD) complexes. Amongst the 15 compounds studied, the simplest compounds Pt(COD)X2 (X=Cl, I, Pt1 and Pt2) showed excellent activity against a panel of Gram-positive bacteria including vancomycin and methicillin resistant Staphylococcus aureus. Additionally, the lead compounds show no toxicity against mammalian cells or haemolytic properties at the highest tested concentrations, indicating that the observed activity is specific against bacteria. Finally, these compounds showed no toxicity against Galleria mellonella at the highest measured concentrations. However, preliminary efficacy studies in the same animal model found no decrease in bacterial load upon treatment with Pt1 and Pt2. Serum exchange studies suggest that these compounds exhibit high serum binding which reduces their bioavailability in vivo, mandating alternative administration routes such as e. g. topical application.

Microbiology's next top model: Galleria in the molecular age.

Galleria mellonella has risen to fame as an invertebrate model organism given its ethical advantages, low maintenance costs, rapid reproduction time, short life cycle, high number of progeny, tolerance for human body temperatures, innate immune system and similarities to mammalian host models. It is increasingly being utilised to evaluate in vivo toxicity and efficacy of chemical compounds and antimicrobials, modelling microbial (bacterial, fungal and viral) pathogenicity and assessing host-pathogen interaction during infection. During this molecular age of genomic, transcriptomic, proteomic and genetic manipulation approaches, our understanding of microbial pathogenicity and host-pathogen interactions has deepened from high-throughput molecular studies performed in G. mellonella. In this review, we describe the use of G. mellonella in a broad range of studies involving omics, drug resistance, functional analysis and host-microbial community relationships. The future of G. mellonella in the molecular age is bright, with a multitude of new approaches and uses for this model from clinical to biotechnological on the horizon.

Nontoxic Cobalt(III) Schiff Base Complexes with Broad-Spectrum Antifungal Activity.

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug-resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it is reasonable to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad-spectrum antifungal activity. Some of these complexes show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report the first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg kg-1 are tolerated without adverse effects, paving the way for further in vivo studies of these complexes.

Parental Microbiota Modulates Offspring Development, Body Mass and Fecundity in a Polyphagous Fruit Fly.

The commensal microbiota is a key modulator of animal fitness, but little is known about the extent to which the parental microbiota influences fitness-related traits of future generations. We addressed this gap by manipulating the parental microbiota of a polyphagous fruit fly (Bactrocera tryoni) and measuring offspring developmental traits, body composition, and fecundity. We generated three parental microbiota treatments where parents had a microbiota that was non-manipulated (control), removed (axenic), or removed-and-reintroduced (reinoculation). We found that the percentage of egg hatching, of pupal production, and body weight of larvae and adult females were lower in offspring of axenic parents compared to that of non-axenic parents. The percentage of partially emerged adults was higher, and fecundity of adult females was lower in offspring of axenic parents relative to offspring of control and reinoculated parents. There was no significant effect of parental microbiota manipulation on offspring developmental time or lipid reserve. Our results reveal transgenerational effects of the parental commensal microbiota on different aspects of offspring life-history traits, thereby providing a better understanding of the long-lasting effects of host-microbe interactions.

Efficacy of gabapentin in the treatment of trigeminal neuralgia: A systematic review of randomized controlled trials.

The aim of this systematic review was to determine the efficacy of gabapentin (GBP) in the treatment of pain of  idiopathic trigeminal neuralgia (TN). A comprehensive literature search was conducted using the Cumulative Index of Nursing and Allied Health Literature (EBSCO Industries), Emcare (Ovid), Medline (Ovid), Medline (PubMed), Scopus (Elsevier) and Web of Science (Clarivate Analytics). The inclusion criteria comprised randomized controlled trials of GBP as a monotherapy in the treatment of idiopathic TN in adult participants and publications in English. All other study methodologies were excluded. The search yielded 1472 articles, and after exclusion, 11 full-text articles were eligible for full-text analysis. Only two studies met the inclusion criteria. There is insufficient evidence either to support or refute the efficacy of GBP in the management of idiopathic TN. Therefore, further well-designed placebo-controlled trials are required to confirm the efficacy of GBP in managing TN pain as a single therapy.

Macronutrients and infection in fruit flies.

Nutrition and infection are closely linked. While it is now well established that hosts can modulate their nutrition after being infected, the extent to which this change in foraging provides the host with a greater fitness remains to be fully understood. Our study explored the relationships between dietary choice, macronutrients intake [i.e., protein (P) and carbohydrate (C)], infection, survival rate and growth of pathogenic bacterial population in the true fruit fly Bactrocera tryoni. Results showed that flies injected with the bacterium Serratia marcescens decreased their macronutrient intake and shifted their diet choice to carbohydrate-biased diet compared to naïve individuals. Interestingly, flies injected with either PBS (i.e., sham-infected) or heat-killed bacteria also reduced food intake and modulated diet choice but only for a day after injection. When infected flies were restricted to the diet they selected (i.e., PC 1:8), they survived better the infection than those restricted to a protein-biased diet (i.e., PC 1:5). In addition, we did not observe any growth of pathogen load in infected flies fed PC 1:8 for the first 3 days post-infection. Finally, a decrease in lipid body reserves was found in flies injected with live bacteria and, interestingly, this loss of body lipid also occurred in flies injected with heat-killed bacteria, but in a diet-dependent manner. Our results indicated that B. tryoni flies modulated their macronutrient intake and decreased the negative effects of the infection on their survival ("nutritional self-medication") the first days following the infection.

Crowded developmental environment promotes adult sex-specific nutrient consumption in a polyphagous fly.

BACKGROUND: The fitness of holometabolous insects depends largely on resources acquired at the larval stage. Larval density is an important factor modulating larval resource-acquisition, influencing adult survival, reproduction, and population maintenance. To date, however, our understanding of how larval crowding affects adult physiology and behaviour is limited, and little is known about how larval crowding affects adult non-reproductive ecological traits. Here, larval density in the rearing environment of the polyphagous fruit fly Bactrocera tryoni ('Queensland fruit-fly') was manipulated to generate crowded and uncrowded larval treatments. The effects of larval crowding on pupal weight, adult emergence, adult body weight, energetic reserves, fecundity, feeding patterns, flight ability, as well as adult predation risk were investigated. RESULTS: Adults from the crowded larval treatment had lower adult emergence, body weight, energetic reserves, flight ability and fecundity compared to adults from the uncrowded larval treatment. Adults from the crowded larval treatment had greater total food consumption (i.e., consumption of yeast plus sucrose) relative to body weight for both sexes compared to adults from the uncrowded treatment. Furthermore, males from the crowded treatment consumed more yeast relative to their body weight than males from the uncrowded treatment, while females from the crowded treatment consumed more sucrose relative to their body weight than females from the uncrowded treatment. Importantly, an interaction between the relative consumptions of sucrose and yeast and sex revealed that the density of conspecifics in the developmental environment differentially affects feeding of adult males and females. We found no effect of larval treatment on adult predation probability. However, males were significantly more likely to be captured by ants than females. CONCLUSION: We show that larvae crowding can have important implications to ecological traits in a polyphagous fly, including traits such as adult energetic reserve, flight ability, and adult sex-specific nutrient intake. Our findings contextualise the effects of larval developmental conditions into a broad ecological framework, hence providing a better understanding of their significance to adult behaviour and fitness. Furthermore, the knowledge presented here can help us better understanding downstream density-dependent effects of mass rearing conditions of this species, with potential relevance to Sterile Insect Technique.

Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease.

Necrotic enteritis (NE) is a re-emerging disease as a result of increased restriction on the use of antibiotics in poultry. However, the molecular mechanisms underlying the pathogenesis of NE are unclear. Small RNA transcriptome analysis was performed using spleen and intestinal intraepithelial lymphocytes (IEL) from 2 inbred chicken lines selected for resistance or susceptibility to Marek's disease (MD) in an experimentally induced model of avian NE to investigate whether microRNA (miRNA) control the expression of genes associated with host response to pathogen challenge. Unique miRNA represented only 0.02 to 0.04% of the total number of sequences obtained, of which 544 were unambiguously identified. Hierarchical clustering revealed that most of miRNA in IEL were highly expressed in the MD-susceptible line 7.2 compared with MD-resistant line 6.3. Reduced CXCL14 gene expression was correlated with differential expression of several unique miRNA in MD-resistant chickens, whereas TGFßR2 gene expression was correlated with altered gga-miR-216 miRNA levels in MD-susceptible animals. In conclusion, miRNA profiling and deep sequencing of small RNA in experimental models of infectious diseases may be useful for further understanding of host-pathogen interactions, and for providing insights into genetic markers of disease resistance.