Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis.

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1). Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

A disparate trace element metabolism in zebu (Bos indicus) and crossbred (Bos indicus × Bos taurus) cattle in response to a copper-deficient diet.

Copper deficiency is a commonly diagnosed problem in cattle around the globe. In Jimma, Ethiopia, 8 zebu (Bos indicus) and 8 zebu ×: Holstein Friesian cross (Bos taurus ×: Bos indicus) heifers were used in an 11-wk study to investigate breed type differences and effects of Cu deficiency on concentrations of trace elements in plasma and edible tissues as well as mRNA expression of Cu-related genes. Heifers were fed a grass diet (6.4 ± 0.2 [SEM] mg Cu/kg DM) supplemented with 1 mg Mo/kg DM in wk 1 to 4 and 2 mg Mo/kg DM in wk 5 to 11, with blood samples collected every 2 wk and tissue collection postmortem. Plasma, liver, kidney, and semitendinosus and cardiac muscle were analyzed for Zn, Cu, Fe, Se, Mo, Co, and Mn. Expression of mRNA Cu-related genes was measured in aorta (lysyl oxidase [LOX]), liver (Cu transporting ß-polypeptide [Atp7b], Cu chaperone for superoxide dismutase [CCS], cytochrome c oxidase assembly homolog 17 [Cox17], Cu transporter 1 homolog [Ctr1], and superoxide dismutase 1 [Sod1]), and duodenum (diamine oxidase [DAO] and metallo-thionein-1A [Mt1a]) as well as the Se-related glutathione peroxidase 1 (Gpx1). Zebu cattle maintained initial plasma Cu concentrations just below the threshold value for deficiency, whereas crossbred cattle gradually became severely Cu deficient over time (P < 0.001). In contrast, plasma Zn and Co were greater in zebu cattle at the onset of the trial but became similar to crossbred cattle towards the end of the trial (P < 0.001). Liver Cu (P = 0.002) and Fe (P ≤ 0.001), kidney Se (P < 0.001), and kidney and cardiac muscle Co (P ≤ 0.001) concentrations were greater in zebu than in crossbred cattle. Increased hepatic mRNA expression of the Cu regulatory genes Atp7b, Ctr1 (P = 0.02), CCS (P = 0.03), and Cox17 (P = 0.009) and Cu-related Sod1 (P = 0.001) as well as the Se-related Gpx1 (P ≤ 0.001) were greater in zebu than in crossbred cattle. However, duodenal mRNA expression of DAO (P = 0.8) and Mt1a (P = 0.2) and aortic expression of LOX (P = 0.8) were not different. Both the differences in Cu status indices (plasma and liver concentrations) and hepatic mRNA expression of Cu regulatory genes point to the possibility of a more efficient use of dietary Cu in B. indicus as compared to B. taurus ×: B. indicus cattle resulting in greater sensitivity to Cu deficiency in B. taurus crossbred cattle.

Measurement of IL-12 (p40, p35), IL-23p19, and IFN-γ mRNA in duodenal biopsies of cats with inflammatory enteropathy.

BACKGROUND: Dietary hypersensitivity and inflammatory bowel disease (IBD) are important causes of chronic vomiting and diarrhea in cats. IL-23 has been recently found to be a key factor in the immunopathogenesis of IBD in humans but the involvement in IBD has not been investigated in cats. HYPOTHESIS/OBJECTIVES: Expression of genes encoding Il-12p35 and p40, IL-23p19, and IFN-γ may be up-regulated in duodenal biopsy specimens taken from cats with histologic evidence of inflammation. ANIMALS AND METHODS: Duodenal biopsy specimens were collected from control cats (n = 21) and cats with inflammatory enteropathy (n = 13). Routine histopathology, immunohistochemistry (IHC), and qRT-PCR were used to assess expression of MHC class II and to measure gene transcripts encoding the p35, p40, and p19 subunits of the IL-12 family of cytokines and IFN-γ. RESULTS: There were significant differences in expression of mRNA encoding IL-12p35 and IL-23p19 between healthy cats and cats with inflammatory enteropathy. IL-12p35 mRNA was lower in the duodenal mucosa of cats with inflammatory enteropathy compared with the mucosa of healthy cats (P = .001). In contrast, IL-23p19 mRNA expression was higher in duodenal biopsy specimens from cats with inflammatory enteropathy than in those from healthy controls (P = .001). There was no difference in expression of IL-12p40 and IFN-γ mRNA (P > .05). The majority of cats with inflammatory enteropathy had histologic evidence of moderate to severe colitis (score 2). CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this preliminary study suggest that IL-23 plays a role in the pathogenesis of feline inflammatory enteropathy.

Analysis of gene expression in canine idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) in dogs is a rare disease of unknown aetiology, seen in terrier breeds, particularly the West Highland white terrier (WHWT). The aim of this study was to determine pulmonary gene expression in canine IPF in order to gain insights into the pathogenesis of the disease and to identify possible biomarkers. Microarray analyses were conducted to determine gene expression profiles in the lungs of dogs with IPF and control dogs of various breeds. More than 700 genes were identified as having greater than two-fold difference in expression between the two groups. The significant biological functions associated with these genes were related to cellular growth and proliferation, developmental processes, cellular movement, cell to cell signalling and interaction, and antigen presentation. Altered levels of expression were confirmed by quantitative reverse transcriptase PCR for genes encoding chemokine (C-C) ligand (CCL) 2 (+4.9 times), CCL7 (+6.8 times), interleukin 8 (+4.32 times), chemokine (C-X-C) ligand 14 (+3.4 times), fibroblast activation protein (+4.7 times) and the palate, lung and nasal associated protein (PLUNC, -25 times). Serum CCL2 concentrations were significantly higher in WHWTs with IPF (mean 628.1 pg/mL, interquartile range 460.3-652.7 pg/mL) than unaffected WHWTs (mean 344.0 pg/mL, interquartile range 254.5-415.5 pg/mL; P=0.001). The results support CCL2 as a candidate biomarker for IPF in dogs.

Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood mononuclear cells in dogs with sino-nasal aspergillosis.

The causal agent of sino-nasal aspergillosis is usually Aspergillus fumigatus, which is a saprophytic and ubiquitous fungus that causes a severe rhinosinusitis in apparent healthy dogs. Affected dogs do not have systemic immuno-suppression. It has been shown previously that dogs affected by this disease have local over-expression of interleukin (IL)-10 and Th1 cytokines in nasal mucosal tissue. The aim of the present study was to assess the response of peripheral blood mononuclear cells (PBMC) from affected and unaffected dogs to antigen-specific stimulation with heat-inactivated Aspergillus spp. conidia, by quantifying gene expression for specific Th1, Th2, Th17 and Treg cytokines and their related transcription factors. Quantification of IL-4 and IFN-γ protein in culture supernatant was performed by enzyme-linked immunosorbent assay (ELISA). PBMC from dogs with SNA produced adequate mRNA encoding IFN-γ and IFN-γ protein. The expression of IL-17A mRNA was significantly greater in PBMC of affected compared with unaffected dogs. The amount of IL-10 mRNA in PBMC from affected dogs decreased after antigen-specific challenge. These results suggest that the incapacity of affected dogs to clear these fungal infections is not related to a defect in Th1 immunity or to an overwhelming regulatory reaction, but rather to an uncontrolled pro-inflammatory reaction driven by Th17 cells.

Potential role of Alternaria and Cladosporium species in canine lymphoplasmacytic rhinitis.

OBJECTIVES: To evaluate the possible role of Alternaria and Cladosporium species in the pathogenesis of canine lymphoplasmacytic rhinitis by comparing the amount of specific fungal DNA in nasal mucosal biopsies between dogs without nasal neoplasia and those with lymphoplasmacytic rhinitis or nasal neoplasia. METHODS: Quantitative real-time polymerase chain reaction (qPCR) assays detecting DNA from Alternaria and Cladosporium fungi were applied to nasal mucosal biopsies collected from dogs with lymphoplasmacytic rhinitis (n = 8), dogs with nasal neoplasia (n = 10) and control animals (n = 10). A copy number for each sample was calculated using a standard curve of known copy number and differences amongst groups were assessed using Kruskal-Wallis tests. RESULTS: No significant difference was found between the groups. Low levels of Alternaria DNA (10-100 copies/PCR) were detected in one sample; very low levels of DNA (<10 copies/qPCR) were detected in 6 samples, and 21 samples were negative. Low levels of Cladosporium DNA were detected in 2 samples; very low levels of DNA in 18; and 8 were negative. CLINICAL SIGNIFICANCE: Results of this study reveal that Alternaria and Cladosporium species are part of the canine nasal flora, and that these fungi are probably not involved in the pathogenesis of lymphoplasmacytic rhinitis.

Toll- and NOD-like receptor mRNA expression in canine sino-nasal aspergillosis and idiopathic lymphoplasmacytic rhinitis.

The pathogenesis of canine sino-nasal aspergillosis (SNA) and lymphoplasmacytic rhinitis (LPR) remains poorly understood. The innate immune system is implicated in the etiology of human chronic rhinosinusitis. Therefore, we hypothesized that dysfunction in innate immunity could be implicated in the pathogenesis of SNA and LPR. Expression of messenger RNA (mRNA) encoding Toll-like receptors (TLRs) 1-10 and NOD-like receptors (NODs) 1 and 2 in nasal mucosal biopsies from SNA or LPR dogs was compared with mucosa from healthy controls. Gene expression was quantified using quantitative real-time reverse transcriptase polymerase chain reaction normalized against multiple housekeeper genes. All TLR and NOD genes were quantified in all samples. SNA was associated with significantly increased expression of TLRs 1-4, 6-10; and NOD2, relative to controls. LPR was associated with significantly increased expression of TLRs 1, 2, 6-8, relative to controls. There was significantly more expression of TLRs 1, 4, 6-10 and NOD2 in SNA dogs than in LPR dogs. The significance of these differences in the pathogenesis of these diseases is yet to be determined.

Comparison of the value of measurement of serum galactomannan and Aspergillus-specific antibodies in the diagnosis of canine sino-nasal aspergillosis.

Serology is currently used for the diagnosis of canine sino-nasal aspergillosis (SNA). However, the accuracy of serological testing using commercially available, standardized purified antigen preparations of Aspergillus (CAPurAspAg) has only been poorly documented. The aim of the present study was to assess the diagnostic value of an agar-gel double immunodiffusion (AGDD) test and an anti-Aspergillus IgG ELISA, using CAPurAspAg and the commercially available Platelia test for the detection of serum galactomannan. Sera from 17 dogs with SNA, 18 dogs with a nasal tumour (NT), 11 dogs with lymphoplasmacytic rhinitis (LPR) and 33 control dogs were tested with the 3 methods. AGDD result was positive in 76.5% of dogs with SNA, whereas all sera from dogs with non-fungal nasal disease and control dogs were negative. A positive IgG ELISA result was obtained in 88% of dogs with SNA and in 18% of dogs with LPR. All patients with NT and control dogs had a negative IgG ELISA result. The Platelia test was positive in 24% of dogs with SNA, 11% of dogs with NT, 9% of dogs with LPR and 24% of control dogs. The results of this study suggest that (1) the detection of serum Aspergillus-specific antibodies with AGDD or ELISA, using CAPurAspAg, provides excellent specificity and good sensitivity, (2) the specificity is higher for AGDD (100%) than for ELISA (96.8%) while sensitivity is higher for ELISA (88.2%) than for AGDD (76.5%) and (3) serum galactomannan quantification with the Plateliat test is unreliable for the diagnosis of canine SNA.

Early increase in pulmonary vascular reactivity with overexpression of endothelin-1 and vascular endothelial growth factor in canine experimental heart failure.

Heart failure is a cause of pulmonary vasoconstriction and remodelling, leading to pulmonary hypertension (PH) and decreased survival. The pathobiology of PH in heart failure remains incompletely understood. We investigated pulmonary vascular function and signalling molecules in early stage PH secondary to experimental heart failure. Eight beagle dogs with overpacing-induced heart failure underwent haemodynamic assessment and postmortem pulmonary arterial reactivity, morphometry and quantification of genes encoding for factors involved in vascular reactivity and remodelling: endothelin-1 (ET-1), ETA and ETB receptors, vascular endothelial growth factor (VEGF), VEGF receptors 1 and 2 (VEGFR1 and VEGFR2), endothelial nitric oxide synthase, angiopoietin-1, bone morphogenetic protein receptors (BMPR1A and BMPR2), serotonin transporter (5-HTT) and the 5-HT(2B) receptor. Overpacing was associated with a decrease in cardiac output and an increase in pulmonary vascular pressures. However, there were no changes in pulmonary vascular resistance or in arteriolar medial thickness. There were increased expressions of genes encoding for ET-1, ETB, VEGF and VEGFR2, while expression of the other genes analysed remained unchanged. In vitro, pulmonary arteries showed decreased relaxation and increased reactivity, while systemic mammary arteries were unaffected. Early PH in heart failure is characterized by altered vasoreactivity and increased ET-1/ETB and VEGF/VEGFR2 signalling.

Distinct tissue cytokine and chemokine mRNA expression in canine sino-nasal aspergillosis and idiopathic lymphoplasmacytic rhinitis.

Idiopathic lymphoplasmacytic rhinitis (LPR) and sino-nasal aspergillosis (SNA) are among the most common causes of nasal discharge in dogs. The pathogenesis of both diseases is poorly understood. Some have proposed that LPR is a chronic inflammatory response to an inhaled irritant, pollutant or allergen, but others suggest that most cases of LPR constitute undiagnosed cases of SNA. Local immune dysfunction is thought to permit opportunist infection in canine SNA. This study investigates the nature of the local tissue immune response mounted in canine LPR and SNA in order to determine whether these diseases have similar or distinct pathogenesis. Quantitative reverse transcriptase polymerase chain reaction was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify messenger RNA (mRNA), encoding a panel of cytokines and chemokines. SNA was associated with significantly increased expression of mRNA encoding interleukin (IL)-6, IL-8, IL-10, IL-12p19, IL-12p35, IL-12p40, IL-18, IFN-gamma, TNF-alpha, TGF-beta, eotaxin-2 and all four monocyte chemoattractant proteins (MCPs) relative to controls. LPR was associated with significantly increased expression of mRNA encoding IL-5, IL-8, IL-10, IL-12p19, IL-12p40, IL-18, TNF-alpha, TGF-beta, MCP-2 and MCP-3 relative to controls. There was significantly more expression of mRNA encoding IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-18, IFN-gamma, TNF-alpha, TGF-beta and all MCPs, and significantly less expression of IL-5 in dogs with SNA than in dogs with LPR. Thus, the profile of cytokine and chemokine gene expression in the nasal mucosa is different in dogs with LPR when compared to dogs with SNA. A partial Th2 immune response appears to be mounted in the nasal mucosa of dogs with LPR, whereas the mucosal immune response in canine SNA is of the Th1 type. Increase in IL-10 and TGF-beta transcripts in dogs with SNA is thought to be implicated in the failure to clear the Aspergillus infection. These results constitute the first evidence that the pathogenesis of canine LPR and SNA is distinct.

Real-time RT-PCR quantification of mRNA encoding cytokines, CC chemokines and CCR3 in bronchial biopsies from dogs with eosinophilic bronchopneumopathy.

Idiopathic canine eosinophilic bronchopneumopathy (EBP) is a disease characterized by eosinophilic infiltration of the pulmonary interstitium and bronchial mucosa, a cause for which has not yet been discovered. A recent study, examining the relative proportion of various lymphocyte cell subsets within bronchoalveolar lavage fluid from dogs with EBP, has shown a selective increase in CD4(+) T-cells and a selective decrease in CD8(+) T-cells, suggesting that a similar Th2 immune response might occur in EBP. The aim of the present study was to determine the profile of cytokine, chemokine and CC chemokine receptor 3 (CCR3) messenger RNA (mRNA) expression in bronchial tissue from dogs with EBP. Real-time RT-PCR assays were used for the quantification of mRNA encoding for a panel of cytokines, CC chemokines and CCR3 in perendoscopic bronchial biopsies from eight dogs with EBP and seven age-matched control dogs. Messenger RNA transcribed from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used for normalisation of the threshold cycle in order to determine the relative copy numbers of the transcripts. No significant difference in the expression of any cytokine, MCP-1, -2, -4 and CCR3 was found between control and EBP dogs. The expression of transcript for MCP-3, eotaxin-2 and -3 was significantly greater in bronchial biopsies from dogs with EBP than in samples from control dogs while there was significantly less mRNA encoding RANTES in the mucosa of dogs with EBP. In conclusion, the cytokine mRNA expression profile in perendoscopic bronchial biopsies is similar in dogs with EBP and dogs without respiratory disease. Further studies on the quantification of mRNA encoding cytokines in isolated T lymphocytes from bronchoalveolar lavage fluid or bronchial biopsies are needed before any conclusion on the cytokine profile in canine EBP can be drawn. Eotaxin-2, -3 and MCP-3 appear to be implicated in the pathogenesis of the disease.

Detection of allelic variants of the canine IGHA gene by fluorescence resonance energy transfer melting temperature examination.

The fluorescence resonance energy transfer (FRET) dual hybridisation probe system has been used for the detection of the accumulation of target DNA during real-time PCR and for the identification of nucleotide polymorphisms through examination of melt curves. This system involves the use of two oligonucleotide probes which are located close to each other and are complementary to an internal segment of a target DNA of interest. Four allelic variants of the gene encoding the hinge region of the immunoglobulin A (IgA) heavy chain (IGHA) have been so far identified in the dog and this variability is due to a combination of single nucleotide polymorphisms and insertion/deletion of nucleic acid motifs. An individual dog may be homozygous or heterozygous for these allelic variants. The purpose of this study was to develop a FRET-based dual probe melting temperature assay to identify the alleles present within an individual dog and to use this assay to determine the frequency of the four allelic variants in different breeds within the canine population. A single pair of oligonucleotide probes were designed that were able to discriminate between the four allelic variants in both homozygous and heterozygous individuals. The genotype of 96 DNA samples obtained from various purebreeds of dogs was determined using this FRET assay. The frequency of each allele differed between the breed groups. The results of this study indicate that it is possible to distinguish relatively complex gene polymorphisms using a single set of oligonucleotide probes. Furthermore, any future comparison of IGHA genotypes between normal and diseased dog populations must take into account the breed variation in allelic frequency.

Real-time RT-PCR quantification of mRNA encoding cytokines and chemokines in histologically normal canine nasal, bronchial and pulmonary tissue.

Cytokines and chemokines are likely to be involved in the pathogenesis of inflammatory diseases of the canine respiratory tract. The roles and relative amounts of these molecules have not yet been defined in the respiratory mucosa of normal dogs or dogs with naturally acquired respiratory inflammation. In the present study, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays were employed to quantify messenger RNA (mRNA) encoding the chemokines monocyte chemotactic protein (MCP)-2, eotaxin-2 and eotaxin-3, and the cytokines interleukin-4 (IL-4), IL-5, IL-6, IL-10, IL-12p40, IL-18, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in normal nasal, bronchial and pulmonary tissues from puppies (n = 4) and from adult dogs (n = 7). There was no significant difference in the expression of any transcript between puppies and adult dogs at any of the anatomical sites examined. The expression of mRNA encoding eotaxin-2 and eotaxin-3 increased significantly with progression from the nasal mucosa to pulmonary parenchyma but expression of MCP-2 mRNA did not show this trend. At all levels of the respiratory mucosa, the most abundant transcripts were those encoding IL-18 and TGF-beta. Transcripts encoding IL-6, IL-10, IL-12 and TNF-alpha were approximately ten-fold less abundant, and IL-4, IL-5 and IFN-gamma were the least abundant templates. There was significantly different amount of mRNA encoding IL-5, IL-18 and TNF-alpha between particular anatomical levels of the respiratory mucosa while the mRNA expression of the other cytokines was similar at all anatomical sites. The results of the present study will enable comparisons to be made with results obtained from similar samples obtained from dogs with nasal, bronchial or pulmonary diseases.

Effect of intestinal inflammation on fecal elastase concentration in dogs.

BACKGROUND: A commercially available ELISA kit for fecal elastase measurement can be used in the diagnosis of exocrine pancreatic insufficiency (EPI) in dogs. However, other causes of diarrhea also may affect fecal elastase concentration. OBJECTIVE: This study was undertaken to determine whether intestinal inflammation alters fecal elastase concentration in dogs. METHODS: Fecal elastase concentration was measured with an ELISA kit in the following groups of dogs: group 1 (n=16), control dogs, without gastrointestinal disease; group 2 (n=14), dogs with diarrhea and no histopathologic evidence of intestinal inflammation; and group 3 (n=12), dogs with diarrhea and histopathologic evidence of intestinal inflammation. Serum trypsin-like immunoreactivity (TLI) was determined in dogs with diarrhea to rule out EPI. RESULTS: All dogs in groups 2 and 3 had serum TLI concentrations >5 microg/L, ruling out EPI. No statistically significant difference was found in fecal elastase concentration among the 3 groups of dogs (P=.969). CONCLUSIONS: The results indicate that intestinal inflammation does not affect fecal elastase concentration, such that test results may be used to exclude a diagnosis of EPI even in animals with inflammatory bowel disease.

Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR.

CD4(+) T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. The role that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-gamma, TNF-alpha and TGF-beta in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-beta and TNF-alpha were found to be the most abundant transcripts, with IL-10 and IFN-gamma present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. The methods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.

Measurement of immunoglobulin concentrations in the feces of healthy dogs.

Selective immunoglobulin A (IgA) deficiency is the most common primary immunodeficiency in humans and may be associated with chronic gastrointestinal disease. This observation has led to the suggestion that the high susceptibility of German shepherd dogs (GSD) to chronic enteropathies is related to a deficiency in mucosal IgA production. Relative deficiencies of IgA has been reported in the serum, saliva, tears, and feces of GSD both with and without alimentary disease; however, the findings of different studies are not consistent. The aim of this study was to confirm whether a relative deficiency of IgA exists in the feces of GSD. Feces were collected from healthy GSD (n = 209), Labrador retrievers (n = 96), beagles (n = 19), and miniature schnauzers (n = 32). Fecal IgA, IgM, and IgG were measured by capture enzyme-linked immunosorbent assays. Fecal IgG concentrations in the four breed groups were not significantly different. IgA concentrations were significantly greater in miniature schnauzers than in GSD (P = 0.0003) and Labradors (P = 0.0004) but not significantly different from those in beagles. IgM concentrations were significantly greater in miniature schnauzers than in GSD (P < 0.0001), Labradors (P < 0.0001), and beagles (P = 0.0098). These findings do not support the hypothesis that GSD have a relative deficiency in fecal IgA. The differences in immunoglobulin concentrations measured from a single defecation, between individuals of the same breed and between breeds, as well as the lack of an internal control molecule, make the determination of a normal reference range for all dogs impossible. Therefore, the usefulness of fecal immunoglobulin quantification for the assessment of intestinal immunoglobulin secretion in dogs is limited.

Real-time RT-PCR: considerations for efficient and sensitive assay design.

Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taq-man probes with a deoxyguanosine base at the 5' end fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer-dimer formation associated with RT enzymes is described.