Comparative assessment of a canine-specific medium to support colony formation from canine hair follicular keratinocytes.

BACKGROUND: Follicular stem cells and their progeny are responsible for the cyclical renewal of hair follicles and maintenance of the hair coat. The understanding of pathways involved in this process is essential to elucidate the pathogenetic mechanisms of primary alopecia. Stem cells and their direct descendants are located in the bulge region of the isthmus of hair follicles. Although these cells have been studied extensively in mice and humans, data for canine isthmic keratinocyte activation and proliferation are not available. HYPOTHESIS/OBJECTIVES: The aim was to establish an accurate and reliable in vitro system to study the growth potential of canine isthmic keratinocytes. We assessed the colony-promoting capability of a commercially available canine-specific medium, CELLnTEC (CnT-09), compared with a well-established home-made medium, complete FAD (cFAD). The CnT-09 medium is specific for the growth of canine keratinocytes, while the cFAD medium can support growth and colony formation of keratinocytes from several species. ANIMALS: Skin biopsies were obtained from 15 recently euthanized dogs of various breeds with no skin abnormalities. METHODS: The isthmic region of compound hair follicles was isolated by microdissection and cell growth monitored using several parameters with colony-forming assays. RESULTS: The CnT-09 and cFAD media provided similar growth as measured by the total number and size of colonies, as well as rate of cell differentiation. CONCLUSIONS: The commercial canine-specific CnT-09 medium was comparable to the home-made cFAD medium in supporting the growth and proliferation of canine follicular keratinocytes in vitro. The CnT-09 medium should be a viable alternative growth medium for molecular studies of alopecic disorders in dogs.

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

Quantitative assessment of physiological and behavioural parameters in healthy dairy cows evoked by transcutaneous electrical nerve stimulation of the udder.

The pain and distress associated with transcutaneous electrical nerve stimulation (TENS) of the udder was evaluated by treating 20 healthy dairy cows with an electrical udder stimulator. This generated a sequence of pulses (frequency: 160±10% impulses per second, duration 250 µs) and provided voltage ranges from 0 to 10 volts (±10%). Trials took place on three consecutive days, twice daily after morning and evening milking. Daily sessions were divided into two periods: (1) control (sham treatment) and (2) treatment (real treatment). Physiological (heart rate, respiratory rate, and plasma cortisol concentration) as well as ethological parameters (kicking, weight shifting, and looking backwards to udder) were defined as pain-indicating parameters and observed. Evaluation of data showed that only one parameter (kicking) was significantly increased during real treatment compared to sham treatment. It is concluded that the TENS therapy tested in this study can evoke changes in behaviour (increased kicking) consistent with an experience of pain in some cows.

Using scenario tree modelling for targeted herd sampling to substantiate freedom from disease.

BACKGROUND: In order to optimise the cost-effectiveness of active surveillance to substantiate freedom from disease, a new approach using targeted sampling of farms was developed and applied on the example of infectious bovine rhinotracheitis (IBR) and enzootic bovine leucosis (EBL) in Switzerland. Relevant risk factors (RF) for the introduction of IBR and EBL into Swiss cattle farms were identified and their relative risks defined based on literature review and expert opinions. A quantitative model based on the scenario tree method was subsequently used to calculate the required sample size of a targeted sampling approach (TS) for a given sensitivity. We compared the sample size with that of a stratified random sample (sRS) with regard to efficiency. RESULTS: The required sample sizes to substantiate disease freedom were 1,241 farms for IBR and 1,750 farms for EBL to detect 0.2% herd prevalence with 99% sensitivity. Using conventional sRS, the required sample sizes were 2,259 farms for IBR and 2,243 for EBL. Considering the additional administrative expenses required for the planning of TS, the risk-based approach was still more cost-effective than a sRS (40% reduction on the full survey costs for IBR and 8% for EBL) due to the considerable reduction in sample size. CONCLUSIONS: As the model depends on RF selected through literature review and was parameterised with values estimated by experts, it is subject to some degree of uncertainty. Nevertheless, this approach provides the veterinary authorities with a promising tool for future cost-effective sampling designs.

The canine hair cycle - a guide for the assessment of morphological and immunohistochemical criteria.

The hair follicle has a lifelong capacity to cycle through recurrent phases of controlled growth (anagen), regression (catagen) and quiescence (telogen), each associated with specific morphological changes. A comprehensive classification scheme is available for mice to distinguish the cycle stages anagen I-VI, catagen I-VIII and telogen. For dogs, such a classification system does not exist, although alopecia associated with hair cycle arrest is common. We applied analogous morphological criteria and various staining techniques to subdivide the canine hair cycle stages to the same extent as has been done in mice. Of all the staining techniques applied, haematoxylin and eosin stain, Sacpic, Masson Fontana and immunohistochemistry for vimentin and laminin proved to be most useful. To evaluate the applicability of our criteria, we investigated skin biopsies from healthy beagle dogs (n=20; biopsies from shoulder and thigh) kept in controlled conditions. From each biopsy, at least 50 hair follicles were assessed. Statistical analysis revealed that 30% of the follicles were in anagen (12% early and 18% late), 8% in catagen (2% early, 5% late and 1% not determinable) and 27% in telogen. Thirty-five per cent of hair follicles could not be assigned to a specific cycle stage because not all follicles within one biopsy were oriented perfectly. In conclusion, this guide will not only be helpful for the investigation of alopecic disorders and possibly their pathogenesis, but may also serve as a basis for research projects in which the comparison of hair cycle stages is essential, e.g. comparative analysis of gene expression patterns.