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.

Analysis of national serological surveys for the documentation of freedom from porcine reproductive and respiratory syndrome in Switzerland.

Results of national serological surveys for porcine reproductive and respiratory syndrome (PRRS) conducted in Switzerland in 2001 and 2004 were analyzed. In 2001, 41,124 breeding sows from 2,540 herds out of 6,406 were sampled, and in 2004, 7,498 animals were sampled from 1,074 herds out of 5,320. All serum samples were tested for PRRS using an ELISA developed at the Institute of Virology and Immunoprophylaxis (IVI), Switzerland with a sensitivity (Se) and specificity (Sp) of 94 and 97%, respectively. Positive samples were re-tested with a commercial ELISA (IDEXX) with Se of 100% and Sp of 99%. Samples positive in the second test were confirmed with the fluorescent antibody test (FAT). A stochastic model using data from the main survey conducted in 2001 was done to verify whether the sampling scheme used could detect at least one infected herd with 99% confidence level if the herd designed prevalence was at 0.1 or 0.2%. Additionally, a Bayesian approach was conducted to calculate the post-survey probability of freedom from PRRS using data from the 2001 and 2004 surveys. A Monte Carlo simulation with 5000 iteration was run for each model. Eleven samples in 2001 and six in 2004, all from different farms, could not be conclusively confirmed as negative by the FAT. All other samples were negative. Truly infected animals and herds were not predicted by a stochastic model at the 99% confidence level and 0.1% herd prevalence using data from the 2001 survey. However, it was demonstrated that the prior probability of freedom from PRRS increased from 89.3 to 99.2% after the 2001 survey. Upon completion of the 2004 survey, the probability of freedom from PRRS reached a value of 99.7%. Based on our results, we could conclude that the pig industry in Switzerland is free of PRRS virus with this level of confidence. Restricted import activities over the last decades are a possible explanation for the continuing absence of PRRS-infection in the Swiss swine population. Import requirements defined by the pig industry minimize the risk of introduction of PRRS-infected animals in the future.

Assessment of classical swine fever diagnostics and vaccine performance.

Rapid and accurate diagnosis is of the utmost importance in the control of epizootic diseases such as classical swine fever (CSF), and efficacious vaccination can be used as a supporting tool. While most of the recently developed CSF vaccines and diagnostic kits are mostly validated according to World Organisation for Animal Health (OIE) standards, not all of the well-established traditional vaccines and diagnostic tests were subject to these validation procedures and requirements. In this report, data were compiled on performance and validation of CSF diagnostic tests and vaccines. In addition, current strategies for differentiating infected from vaccinated animals are reviewed, as is information on the control of CSF in wildlife. Evaluation data on diagnostic tests were kindly provided by National Reference Laboratories for CSF in various European countries.

Assessment of the use of gross lesions at post-mortem to detect outbreaks of classical swine fever.

The performance of pathological findings as a diagnostic tool for the detection of classical swine fever (CSF) outbreaks during the 1997/1998 CSF-epidemic in The Netherlands was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. This was done at the individual pig level and at the submission level (a group of pigs from the same herd submitted together for post-mortem investigation). At post-mortem examination, the tonsils, spleen, ileo-caecal valve and renal pelvis were sampled, sent to the reference laboratory, and tested by means of a CSF-specific fluorescent antibody test in combination with a confirmatory test. This resulted in an infection status at the individual pig level. The infection status and pathological findings of 1072 individual pigs from a total of 230 infected herds were included in this analysis. We also included submissions of pigs from herds that were sent to post-mortem examination because of a clinically CSF-suspect situation but afterwards were concluded to be from non-infected herds. Infection status and pathological findings of 1224 individual pigs from a total of 241 non-infected herds were included in the analysis. Pneumonia, pleuritis, chronic bronchitis, pulmonary oedema, chronic gastric ulceration, dry faecal contents in the colon, conjunctivitis, haemorrhages in the renal pelvis, renal haemorrhages, splenic enlargement, haemorrhages in the urinary bladder, haemorrhagic and enlarged lymph nodes were the most frequently recorded pathological findings during a post-mortem examination of pigs submitted in a CSF-suspect clinical situation. However, some of these pathological findings (e.g. pneumonia, pleuritis) were almost evenly distributed in infected and in non-infected pigs, resulting in a high sensitivity combined with a low specificity. The area under the ROC curve of pathological findings at the individual pig level and at the submission level was 0.720 and 0.782, respectively, which was significantly (P<0.0001) larger than the area under the random ROC curve. It was concluded that, although gross pathology is a legitimate test, its quantitative contribution to the detection of CSF is limited.

Prevalence of Cryptosporidium and Giardia infections on two Ohio pig farms with different management systems.

The prevalence of Cryptosporidium and Giardia infections in pigs was investigated by the use of a direct immunofluorescence assay on two Ohio farms with different management systems. Cryptosporidium and Giardia infections were detected only in weanlings on the farm with slotted and wire floors, but in both weanlings and nursing piglets on the farm with porous concrete floors. Giardia infection was also detected in sows on the latter farm. The farm with porous concrete floors had a significantly higher Cryptosporidium infection rate in nursing piglets and Giardia infection rates in weanlings than the farm with slotted and wire floors. Sows were implicated as the source of both Cryptosporidium and Giardia infections for nursing piglets.