Impact of different methodologies on the detection of point mutations in routine air-dried fine needle aspiration (FNA) smears.

Currently the best method to select suspicious thyroid nodules for surgery is fine needle aspiration (FNA) cytology. However, FNA cytology has some inherent limitations, which can partly be overcome by molecular analysis. Therefore, molecular testing for somatic mutations has emerged as the most promising approach for molecular FNA diagnostics. The objective of this methodological study was to evaluate the feasibility of detecting BRAF, NRAS, HRAS, and KRAS mutations from routine air-dried thyroid FNA smears, and to find an optimal method for detecting these mutations in FNA samples. DNA was extracted from 110 routine air-dried FNA smears and the corresponding surgically obtained formalin-fixed paraffin-embedded tissues. The presence of BRAF, NRAS, HRAS, and KRAS mutations was assessed by real-time PCRs and high resolution melting analysis, and/or pyrosequencing in comparison to real-time PCRs using hybridization probes and fluorescence melting curve analysis. The high-resolution melting-PCRs revealed a significantly lower number of PCR failures and questionable results, and detected more mutations than the PCRs using hybridization probes. The number of PCR failures ranging from 14-16% by high-resolution melting-PCRs could be further reduced to 5-14% by adding pyrosequencing assays. Moreover, pyrosequencing increased the specificity of the assays, up to 98-100%, while the sensitivity ranged between 32-63%. In summary, the mutation detection, especially in air-dried FNA samples, improves when using PCR assays in combination with high resolution melting analysis. Additional improvement can be obtained by subsequent pyrosequencing in comparison to previously described real-time PCRs using hybridization probes and fluorescence melting curve analysis.

Osteochondrosis in feedlot cattle.

Beginning in summer 1977 and continuing through four consecutive seasons, we examined lame limb joints from 106 partially fattened cattle and 28,235 pairs of occipital condyles from fully fattened cattle for osteochondrosis. Of the 106 lame cattle, nine (8.5%) had characteristic lesions, usually in their stifle joints, and of the 28,235 atlanto-occipital joints, 1063 (3.8%) had lesions on their condyles. Slices of affected articular cartilage and subjacent bone were studied radiographically and histologically. Radiographs showed subchondral defects and epiphyseal "cyst-like" lesions, and histologic sections showed disarrayed and degenerated chondrocytes. Splits occurred along osteochondral junctions or in adjacent cartilage or bone. Some bone trabeculae had been replaced by fibrous tissue that, in some areas, extended deep into epiphyses.

Laryngeal diphtheria and papillomatosis in feedlot cattle.

In a random sample of 30,444 larynges from slaughtered beef cattle, 441 (1.4%) had chronic diphtheria and 1,345 (4.4%) had papillomatosis. Both types of lesions were centered in mucous membranes over the vocal processes and medial angles of the arytenoid cartilages. Since they are found in the same anatomical locations as laryngeal contact ulcers, we hypothesize that ulcers are primary and diphtheria and papillomatosis are secondary.

Laryngeal contact ulcers in feedlot cattle.

Of 30,444 larynges from fattened cattle at three companies, 3,985 (13.1%) had contact ulcers or ulcer scars in the mucous membranes over the vocal processes and medial angles of the arytenoid cartilages. The incidence was higher in cattle fed during fall than in cattle fed during other seasons. We hypothesize: 1. that some feedlot cattle develop acute mucositis from mixed infections with species of Pasteurella, Haemophilus, Mycoplasma, and viruses in the nose, pharynx, and larynx; 2. that reflex coughing and swallowing accelerate the rate of larynx closure; and 3. that the closures erode the swollen membrane over the vocal processes and medial angles of the arytenoid cartilages and thus produce primary contact ulcers.

Limb arthropathies and periarticular injuries in feedlot cattle.

Beginning summer 1977 and continuing through 4 consecutive seasons, we examined samples of at least 25, and a total of 106, lame limbs or joints from slaughtered beef cattle. Of this number about 64 (60%) were articular and 42 (40%) periarticular lesions. Fifty-three were caused by trauma and 9 by osteochondrosis. Stifle joints (40) and digital joints (19) were the most frequently affected articulations, and the humerus and femur were the most frequently fractured bones. Twenty joints, mostly stifles, had fibrinous arthritis, and Mycoplasma spp. were isolated from 11.