Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.

Immunocytochemical analysis of equine synovial membranes revealed presence of several neuropeptides, including substance P (SP), neurokinin A, and neuropeptide Y, in nerves of the radiocarpal, middle carpal, and metacarpophalangeal (fetlock) joints. Within the subsynovium, these neuropeptides were located perivascularly, whereas in the fronds, only neuropeptide Y was restricted to the vessels of the synovial membrane. Only SP and neurokinin A were found in the intimal layer. The intimal layer of the metacarpophalangeal joint contained more SP-immunoreactive fibers than were observed in the intimal layer of the radiocarpal joint. Substance P also was detected in the synovial fluid from all 3 joints, but mean +/- SD concentrations were significantly different only between the middle carpal joint (37.56 +/- 5.48 fmol/ml; n = 6) and the metacarpophalangeal joint (55.80 +/- 8.33 fmol/ml; n = 5) and between the middle carpal joint and the radiocarpal joint (52.43 +/- 14.60 fmol/ml; n = 7).

Kidney tissues were removed from euthanatized mature White Leghorn chickens 4 days after iv inoculation with type A influenza virus. The kidney tissues were then fixed at -70 C, using a freeze substitution technique. Type A influenza virus nucleoprotein was readily detected in the nuclei and cytoplasm of the proximal and distal tubular epithelial cells by immunocytochemistry, and the sharpness of the immunomarker in the cells indicated minimal antigen migration during fixation and tissue section preparation. This tissue fixation technique also resulted in good preservation of cellular morphology. The freeze substitution technique of tissue fixation is an excellent alternative to cryostat-cut acetone-fixed tissue sections or conventional chemical fixation of paraffin-embedded tissues for in situ immunocytochemical localization of type A influenza virus nucleoprotein antigen.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr. These proviruspositive, antigen-negative cases may represent infection with latent or replication-defective FeLV.

Two types of sensory receptors were located in the equine foot, using anatomic techniques. Histologic examination of stained hoof sections revealed lamellated corpuscles in the hoof dermis, which had many of the morphologic characteristics of Pacinian corpuscles. These sensory receptors were restricted to the palmar (caudal) aspects of the solar dermis of the heel. A second type of receptor was detected by use of immunocytochemistry, indicating apparently naked nerve endings containing the neuropeptide calcitonin gene-related peptide-like immunoreactivity in skin, solar dermal tubules, and the digital cushion. This peptide is an example of a sensory neurotransmitter contained in dorsal root ganglion cells and is believed to exist only in unmyelinated sensory nerve fibers. These 2 morphologic structures may be used for detection of sensory stimuli, such as pressure (or vibratory senses) and pain, respectively, in horses during various locomotory gaits.

Immunoreactivity for 28 kd vitamin D-dependent calcium-binding protein (calbindin-D28k) has been localized in the germinal epithelium and cells surrounding oogonia and oocytes (future granulosa cells) of developing and growing ovaries of chicken embryos. The protein first appeared prominently in the germinal epithelium of the developing left ovary in 8-day embryos. At the twelfth day of incubation, cells surrounding oogonia and oocytes reacted intensely for calbindin-D28k. The number and intensity of calbindin-D28k-containing cells increased in both types of cells as the embryos further developed. Calbindin-D28k remained in the germinal epithelium throughout the study period observed (up to 10 weeks). However, the protein was present transiently in the future granulosa cells. It gradually decreased after hatching, and was virtually absent from granulosa cells in a 10-week old chicken. Compared with the known process of onset of sexual development, these results indicated possible involvement of calbindin-D28k in the early phases of oogenesis in chicken ovaries.