Development of reverse-transcriptase quantitative PCR assays for detection of the cytokines IL-1ß, TNF-α, and IL-10 in chelonians.

In response to viral pathogens, a host releases pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) and anti-inflammatory cytokines such as interleukin-10 (IL-10). While several approaches exist to measure cytokine responses, evaluating gene transcription through reverse transcription quantitative polymerase chain reaction (RT-qPCR) provides a fast, reproducible, and sensitive method for quantifying this response. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the quantification of red-eared slider (Trachemys scripta elegans) and eastern box turtle (Terrapene carolina carolina) cytokines: IL-1ß, TNFα, IL-10 and the reference gene ß-actin. RNA was isolated from the buffy coat layer of whole blood, comprised mainly of circulating leukocytes, and complimentary DNA (cDNA) was produced. Conventional PCR was performed to obtain cytokine mRNA sequences, products were sequenced, and a hydrolysis probe-based RT-qPCR assay was designed for each cytokine. Standard curves were generated using the target gene sequences cloned within a plasmid. Efficiencies for each assay were between of 85-110%, R2 > 0.98, and limits of detection of 10-100 copies per reaction. The initial samples used to identify the novel target sequences were then used to evaluate the performance of the qPCR assays. Consistent transcription of beta actin across individuals in both species and measurable transcription of IL-1ß, TNF-α, and IL-10 transcript targets in individuals of both species were observed. The assays are a novel technique in chelonians to evaluate host innate immune response.

Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina).

Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04 × 10(7) to 1.04 × 10(1) viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease.

Development and use of a real-time polymerase chain reaction assay for the detection of Ophidiomyces ophiodiicola in snakes.

Fungal pathogens threatening the conservation of wildlife are becoming increasingly common. Since 2008, free-ranging snakes across North America have been experiencing a marked increase in the prevalence of snake fungal disease associated with Ophidiomyces ophiodiicola. Diagnosis has historically relied on histology, microbiology, and conventional polymerase chain reaction (PCR). More sensitive methods are needed to adequately characterize the epidemiology. The current study describes the development of a real-time PCR (qPCR) assay for detecting a segment of the internal transcribed spacer 1 region between the 18S and 5.8S ribosomal RNA gene. The assay was able to detect as few as 1.05 × 10(1) gene copies per reaction. An additional 4 positive cases were detected when comparing a conventional PCR (n = 3) and the qPCR (n = 7) when used on swab samples from 47 eastern massasauga rattlesnakes. The newly developed assay is a sensitive and specific tool for surveillance and monitoring in the conservation of free-ranging snakes.

Alkylation and Carbamylation Effects of Lomustine and Its Major Metabolites and MGMT Expression in Canine Cells.

DNA Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. The DNA repair enzyme O6 methylguanine DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosourea, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoid cell line 17-71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250-350 fmol/mg protein as compared to about 90 fmol/mg protein in 17-71 cells. We also show that MGMT mRNA expression in 17-71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.

Altered mRNA and protein expression of filaggrin in the skin of a canine animal model for atopic dermatitis.

BACKGROUND: Filaggrin is a structural protein that has attracted increasing interest over the past decade for its role in the pathogenesis of human atopic dermatitis (AD). Null mutations in its sequence are considered risk factors in the development of AD. HYPOTHESIS/OBJECTIVES: To investigate canine filaggrin mRNA and protein expression in the skin of atopic beagles with experimentally induced AD compared with breed-matched healthy control dogs. METHODS: All dogs were environmentally challenged for 3 days consecutively with allergens to which the atopic dogs had been sensitized. Skin biopsy specimens were taken from six healthy and seven atopic beagles before and after allergen challenge. Canine filaggrin mRNA was measured using quantitative real-time PCR. Indirect immunofluorescence was used to localize the filaggrin protein in canine skin. Analysis of variance with Tukey's multiple comparison test (over-time effect) and unpaired Student's t-test (treatment effect) were used. Values of P ≤ 0.05 were considered significant. RESULTS: Analysis of variance showed a significantly higher expression of filaggrin mRNA in atopic dogs compared with healthy control dogs (P = 0.004 on day 3 and P = 0.01 on day 10) and a decreased mRNA expression on day 3 in healthy control dogs (effect of time, P = 0.006). On blinded evaluation, filaggrin immunofluorescence was distributed homogeneously in the stratum granulosum and the stratum corneum in healthy dogs. Atopic dogs showed a patchy immunofluorescence pattern, which was exacerbated after environmental challenge. CONCLUSIONS AND CLINICAL IMPORTANCE: Altered epidermal filaggrin mRNA expression and protein distribution was detected in this experimental model.

Evaluation of canine antimicrobial peptides in infected and noninfected chronic atopic skin.

BACKGROUND: Antimicrobial peptides (AMPs) are small immunomodulatory peptides produced by epithelial and immune cells. ß-Defensins (BDs) and cathelicidins (Caths) are the most studied AMPs. Recently, increased cutaneous expression of AMPs was reported in atopic humans and in beagles with experimentally induced atopy. HYPOTHESIS/OBJECTIVES: Our goal was to analyse mRNA expression and protein levels of canine (c)BD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath in healthy and naturally affected atopic dogs, with and without active skin infection, along with their distribution in the epidermis using indirect immunofluorescence. ANIMALS: Skin biopsies were taken from 14 healthy and 11 atopic privately owned dogs. METHODS: The mRNA levels of cBD1-like, cBD2-like/122, cBD3-like, cBD103 and cCath were quantified using quantitative real-time PCR. The protein levels of cBD3-like and cCath were analysed by relative competitive inhibition enzyme-linked immunosorbent assay, while the distributions of cBD2-like/122, cBD3-like and cCath were detected by indirect immunofluorescence. RESULTS: Dogs with atopic dermatitis had significantly greater mRNA expression of cBD103 (P = 0.04) than control dogs. Furthermore, atopic skin with active infection had a higher cBD103 mRNA expression (P = 0.01) and a lower cBD1-like mRNA expression (P = 0.04) than atopic skin without infection. No significant differences in protein levels (cBD3-like and cCath) or epidermal distribution of AMPs (cBD2-like/122, cBD3-like and cCath) were seen between healthy and atopic dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Expression of cBD103 mRNA was greater, while expression of cBD1-like mRNA was lower in dogs with atopic dermatitis that had active infections. Work is needed to clarify the biological mechanisms and possible therapeutic options to maintain a healthy canine skin.

Development and validation of TaqMan quantitative PCR for detection of frog virus 3-like virus in eastern box turtles (Terrapene carolina carolina).

Ranavirus has caused disease epidemics and mass mortality events globally in free-ranging fish, amphibian, and reptile populations. Viral isolation and conventional PCR are the most common methods for diagnosis. In this study, a quantitative real-time PCR (qPCR) assay was developed using a TaqMan probe-based assay targeting a highly conserved region of the major capsid protein of frog virus 3-like virus (FV3-like) (Family Iridoviridae, genera Ranavirus). Standard curves were generated from a viral DNA segment cloned within a plasmid. The assay detected viral DNA 1000 times lower than conventional PCR. Thirty-one clinical samples (whole blood and oral swabs) from box turtles were tested using these assays and the prevalence of the virus determined. Quantitative PCR allows for a superior, rapid, sensitive, and quantitative method for detecting FV3-like virus in box turtles, and this assay will be useful for early detection and disease monitoring.

Expression and distribution of canine antimicrobial peptides in the skin of healthy and atopic beagles.

Antimicrobial peptides (AMPs) are small immuno-modulatory proteins important in defense against pathogenic organisms. Defensins and cathelicidin are the most frequently studied human AMPs. An increase in AMPs in atopic humans has been reported recently. Our goals were to determine the distribution of AMPs and evaluate their mRNA and protein expression in non-lesional (Day 0), acute lesional skin (Day 3) and post-challenged skin after resolution of skin lesions (Day 10) using a canine model of atopic dermatitis (AD). All dogs were environmentally challenged for three consecutive days with house dust mite. Clinical evaluation of atopic beagles was performed using a CADESI score at each time point before and after environmental challenge. Skin biopsies were taken from six healthy and seven atopic beagles before and after allergen challenge (Day 0, Day 3 and Day 10). The transcription of canine cathelicidin (cCath) and beta-defensins (cBD)-1, -2 and -3 mRNA was quantified using quantitative-RT-PCR while the protein distribution of cBD2, cBD3 and cCath was detected by indirect immunofluorescence. A significant effect, over-time, was seen in CADESI score in AD beagles with an increase score after challenge (Day 3). Quantitative analysis showed a significant difference in mRNA transcript levels between groups (with atopic dogs having more than controls) for all AMPs but cBD2. No effect over time was evident for either group. No significant differences were seen for the AMP protein patterns of distribution (homogenous distribution). Although, these results showed no differences in AMP's localization after allergen exposure in each group; atopic dogs had a higher mRNA expression of AMPs when compared with healthy dogs, a similar finding to humans.

Estradiol deficiency during development modulates the expression of circadian and daily rhythms in male and female aromatase knockout mice.

Gonadal steroids modify the phase, amplitude and period of circadian rhythms. To further resolve the role of estradiol, we examined daily patterns of activity, circadian free running period and behavioral responses to light pulses using aromatase deficient (ArKO) mice. These animals lack the enzyme necessary to produce estradiol. We hypothesized that circulating estrogens during development and adulthood modulate the amount of activity, the temporal relationship of activity patterns relative to a light:dark cycle, and the free running period. Intact and gonadectomized male and female ArKO and wildtype (WT) littermates were used. WT males, but not ArKO males, retained the ability to respond to steroid hormones; the time of activity onset, free running period in constant darkness, and total daily activity were significantly different in gonadectomized compared to intact males. In contrast, gonadectomy did not alter the expression of these variables in ArKO males. ArKO females had a longer free running period in constant darkness compared to WT females regardless of gonadal state. Ovariectomized ArKO females had a significantly delayed activity onset when compared to intact ArKO females and ovariectomized WT females, despite all 3 groups being estrogen deficient. Phase shifts in response to light pulses given at different times of the day revealed an interaction between genotype, sex, and circulating steroids. These results from ArKO animals strongly suggest an organizational effect of estradiol during a critical period of development on the expression of biological rhythms.

Expression and distribution of antimicrobial peptides in the skin of healthy beagles.

Antimicrobial peptides (AMPs) are small proteins involved in defense against pathogenic organisms. Defensins and cathelicidin are the most frequently studied human AMPs. Our goals were to determine the distribution of AMPs and evaluate their mRNA expression in normal canine skin. Skin biopsies were taken from six healthy beagles. The relative transcript level of canine cathelicidin (cCath) and ß-defensin (cBD)-1, cBD2 and cBD3 mRNA was quantified using quantitative real-time polymerase chain reaction. Indirect immunofluorescence (IIF), using polyclonal antibodies against cBD2, cBD3 and cCath, was used to evaluate protein localization in the skin of healthy dogs. The Pfaffl method, using experimentally determined primer efficiencies of amplification, was used to determine the expression level of cCath, cBD1 and cDB3 relative to cDB2. The levels of cCath, cBD3 and cBD1 mRNA were 358, 296 and 177 times higher than those of cBD2, respectively. Using IIF, cBD2 and cBD3 protein fluorescence was detected in all layers of the epidermis, whereas cCath was detected predominantly in the stratum granulosum and corneum. In addition, antimicrobial peptide detection was limited to the infundibular portion of the pilosebaceous units. We have validated useful methods to evaluate AMPs in canine skin. Further studies are needed to compare AMP expression in healthy dogs with that of dogs with inflammatory skin conditions.

Expression of basigin in reproductive tissues of estrogen receptor-{alpha} or -{beta} null mice.

Basigin plays important roles in both male and female reproduction because basigin (Bsg) null male and female mice are infertile. The aim of the present study was to determine whether basigin expression in reproductive organs requires estrogen receptor-alpha (ESR1, ERalpha) or -beta (ESR2, ERbeta). Expression of basigin protein in the testis, ovary, and male and female reproductive tracts was studied in adult wild-type (WT), Esr1-null (alphaERKO), and Esr2-null (betaERKO) mice by immunohistochemistry and immunoblotting. Basigin mRNA levels in ovary and uterus were examined by quantitative RT-PCR. In females, basigin protein expression was observed mainly in granulosa and interstitial cells of the ovary and epithelial cells of the proximal oviduct in all genotypes. Basigin protein was also expressed in the uterine epithelium at proestrus and estrus in WT and betaERKO mice but not in alphaERKO mice. However, a higher level of basigin mRNA was observed in uteri of alphaERKO mice compared with WT and betaERKO mice. In males, basigin was expressed in Leydig cells and all germ cells except spermatogonia in all genotypes. Basigin was present in epithelial cells lining the efferent ductules in WT and betaERKO mice, but expression was greatly reduced in alphaERKO mice. In epididymal ducts, basigin expression was observed in epithelial cells in the caput and cauda in all genotypes. These data suggest that expression of basigin protein requires ESR1, but not ESR2, in the uterus and efferent ductules, but is independent of estrogen receptor in the ovary, oviduct, testis, and epididymis.

Morphological comparison of the testis and efferent ductules between wild-type and estrogen receptor alpha knockout mice during postnatal development.

Estrogen and the estrogen receptor (ER)alpha play an important role in the male reproductive tract and in fertility. Previous studies demonstrated that disruption of ERalpha function resulted in abnormal morphology of the testis and efferent ductules (EDs) of adult mice. However, the effect of a lack of a functional ERalpha during early postnatal development has not been determined. The present study is an evaluation of morphological changes effected by a lack of ERalpha in the male reproductive tract during the postnatal period. Age-equivalent wild-type and ERalpha knockout (alphaERKO) mice at 10, 18, 35 and 60 days of age after birth were used for morphological comparison of the testes and ED. Light microscopic examination of the testes of the alphaERKO mouse revealed a dramatic dilation of the rete testis as early as 10 days of age, premature lumen formation, reduced epithelial height and greatly dilated lumen of seminiferous tubules as early as 18 days of age. The proximal ED of the alphaERKO mouse showed lumen dilation, reduction of epithelial height and a decrease of nuclear height as early as 10 days of age. Similar, but somewhat less severe, morphological abnormalities were observed in the distal ED of the alphaERKO mouse. These results indicate that a lack of functional ERalpha leads to morphological changes of the testis and ED of the early postnatal developing mouse. Based on these observations, we conclude that ERalpha plays an important role in normal development of the testis and ED, not only during adulthood but also during the entire postnatal period and presumably during fetal development.

Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus.

Our objective was to determine if vaccination with killed avian infectious bronchitis virus (AIBV) causes epididymal calcium stones in the rooster as is seen following vaccination with live attenuated AIBV. Specific-pathogen-free roosters were divided into three groups: nonvaccinated (NONVAC), live attenuated AIBV-vaccinated (LVAC), and killed AIBV-vaccinated (KVAC) groups. Roosters were vaccinated at 2, 6, 10, and 14 wk of age and the epididymal region was observed at 27 wk of age. Epididymal stones were present in 13% of NONVAC, 50% of KVAC, and 64% of LVAC roosters. Histologically, immune cells were seen in the interstitium of efferent ductules containing stones. We conclude that use of a killed vaccine does not reduce the incidence of epididymal stones.

Gene expression profiling of 17beta-estradiol and genistein effects on mouse thymus.

Estrogen regulates thymic development and involution and modulates immune function. Despite its critical role in thymus, as well as in autoimmune disorders, the mechanism by which estrogen affects the thymus is not well understood. We previously reported that the estrogenic soy isoflavone genistein, as well as 17beta-estradiol (E2), could induce thymic involution, but genistein effects were only partially mediated through estrogen receptors. To provide insights into mechanisms of estrogenic effects in the thymus, we investigated thymic gene expression changes induced by E2 (125 ng/day) and genistein (1500 ppm in feed) in weanling mice using high-density DNA arrays. We identified several E2-responsive genes involved in thymic development and thymocyte signaling during selection and maturation. Functional characterization indicated effects on genes involved in transcription, apoptosis, and the cell cycle. This study also identified changes in several E2-regulated transcripts essential to maintain immune self-tolerance. E2 upregulated more genes than genistein, while genistein downregulated more genes than E2. Though each treatment regulated several genes not altered by the other, there was considerable overlap in the genes regulated by E2 and genistein. Changes in transcription factors and cell cycle factors were consistent with decreases in cell proliferation induced by both genistein and E2. As indicated by the regulation of non-E2-responsive genes, genistein also induced unique effects through non-estrogenic mechanisms. The specific downregulation of the CD4 coreceptor transcript by genistein was consistent with the decline of CD4+ thymocytes in genistein-treated mice in our previous study. This is the first study identifying E2 and genistein target genes in the thymus. These findings provide new mechanistic insights toward explaining estrogen action on thymocyte development, selection, and maturation, as well as the effects of genistein on prenatal and neonatal thymic development and function.

The soy isoflavone genistein decreases adipose deposition in mice.

Adipose tissue is responsive to estrogen and expresses both estrogen receptor alpha and beta. To test the hypothesis that the estrogenic soy isoflavone genistein can have effects on adipose tissue, juvenile or adult C57/BL6 mice were ovariectomized and given daily injections of vehicle, 17beta-estradiol (5 microg/kg.d) or genistein (8-200 mg/kg.d) sc for 21-28 d. To test effects of dietary genistein, 25- to 27-d-old mice were fed diets containing 0-1500 parts per million (ppm) genistein for 12 d. Mice were killed and fat pads weighed. Parametrial fat pads were used for morphometric and Northern analysis. Genistein injections decreased adipose weight and adipocyte circumference at higher doses; effects in adult and juvenile mice were similar. Genistein decreased lipoprotein lipase mRNA, which may be a critical aspect of its adipose effects. Juveniles fed 500-1500 ppm dietary genistein had dose-responsive decreases in fat pad weights of 37-57%, compared with controls; 300 ppm genistein did not cause decreases. Genistein doses of 300, 500, 1000, and 1500 ppm produced serum genistein concentrations of 1.02 +/- 0.14 microM, 1.79 +/- 0.32 microM, 2.55 +/- 0.18 microM, and 3.81 +/- 0.39 microM, respectively. These results indicate dietary genistein at 500-1500 ppm produces antilipogenic effects in mice at serum levels that humans are realistically exposed to.