Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples.

The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.

Antitumor effect of the vascular-disrupting agent ZD6126 in a murine renal cell carcinoma model.

ZD6126 is a vascular-disrupting agent that affects the endothelial tubulin cytoskeleton causing selective occlusion of tumor vasculature and extensive tumor cell necrosis. The present study evaluated the antitumor and antivascular activities of ZD6126 in the clinically relevant murine renal cell carcinoma (RENCA) model and also evaluated biological response to therapy using color Doppler imaging as biomarker. Mice were implanted with RENCA tumor cells (day 0) and established tumors were treated with ZD6126 (100 mg/kg i.p.) or vehicle with repeated intermittent doses on day 10, 14 and 18. ZD6126 treatment led to a significant reduction in tumor size and was associated with extensive tumor necrosis and a reduction in tumor blood flow versus controls. MVD increased with intermittent treatment (day 10, 14 and 18). In an additional study, animals were treated at day 19 and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Corrosion casting showed that tumor vessel architecture is affected by treatment, whereas pre-existing vessels in control tissues are practically not affected. Inter-vessel and inter-branch distances as well as vessel diameters are influenced by treatment. In conclusion, ZD6126 showed potent antitumor efficacy in the RENCA model and our data suggest that decrease in tumor blood flow may be a useful surrogate marker of treatment effect.

Angiogenesis and the ET-1/ETA receptor system: immunohistochemical expression analysis in bone metastases from patients with different primary tumors.

Angiogenesis is necessary for the growth of primary tumors and the formation of metastases. It is well known that vascular endothelial growth factor (VEGF) and its receptors play a major role in this process. To date, the formation of bone metastases has been poorly understood. Tumor cells must interact with the microenvironment of the bone and new blood vessels must spread. The ET/ET(A) (endothelin) receptor system seems to play an important role in this process. Specimens from metastatic bone lesions and non-malignant bone tissue were analyzed by histological and immunohistochemical staining. Sections were stained with antibodies against CD31, Flt-1, KDR, endothelin-1 (ET-1) and endothelin receptor A (ET(A)). Our studies show that there is an increased microvessel density (MVD) in metastatic bone lesions from different primary tumors in contrast to normal bone tissue. In nearly all tumor formations of the bone, ET-1 and its receptor ET(A) was found by immunohistochemistry. We also performed immunohistochemical staining for the VEGF-receptors Flt-1 and KDR. In conclusion, there is an increased vessel density in metastatic bone lesions in contrast to normal bone tissue. The ET/ET(A) system can be detected in nearly all bone specimens and is upregulated in metastatic bone lesions in contrast to healthy bone tissue.

Risk factors for glaucoma filtering bleb infections.

OBJECTIVE: To determine risk factors for bleb-related ocular infection after glaucoma filtering surgery. METHODS: A case-control study comparing all consecutive cases of glaucoma filtering bleb-related infections (55 eyes of 55 patients) with matched control eyes between January 1, 1990, and June 30, 1998, was performed. Bleb-related infection was classified as blebitis when a mucopurulent infiltrate was identified within the bleb and associated with mild to moderate anterior segment inflammation. Eyes with endophthalmitis had hypopyon, cells in the anterior vitreous cavity, or a positive vitreous biopsy sampling result. A control was selected for each case based on matching of the surgeon, date and type of glaucoma surgery, and type of antifibrotic agent used. Multivariate, matched, case-control logistic regression analysis was performed using age, race, sex, diagnosis, number of previous incisional operations, filtering bleb location, and presence of bleb leak to determine which variables were associated with bleb-related infection. RESULTS: The odds of an eye with a bleb-related infection being seen with a concomitant late-onset bleb leak are 25.8 times the odds of a noninfected eye having a late-onset bleb leak at any time in the postoperative period (P<.001; 95% confidence interval, 2.3-294.1). Other risk factors for bleb-related infection included younger age (P = .05), black race (P = .03), diagnosis of primary open-angle glaucoma (P = .03), and inferior location of the filtering bleb (P = .04). CONCLUSIONS: Late-onset bleb leakage is a significant risk factor for bleb-related infection. The risk of infection may warrant closure of late-onset bleb leaks in selected eyes.

Vertical strabismus resulting from an anomalous extraocular muscle.

Unusual ocular motility abnormalities have been rarely reported to result from anomalous extraocular structures. These structures, which may be either muscular or fibrotic, attach to the globe and produce a mechanical restriction resulting in incomitant strabismus. To our knowledge, we report the first patient with an anomalous extraocular muscle in whom the clinical, radiologic, surgical, and histopathologic findings are described.

Bilateral optic neuritis originating in a single chiasmal lesion. A case report.

A case of bilateral retrobulbar optic neuritis with profound visual loss is described. Magnetic resonance imaging (MRI) with fat-saturation suppression and gadolinium enhancement done 15 days after the onset of symptoms demonstrated bilateral posterior optic nerve disease in a pattern similar to that seen in previously reported cases. However, subsequent retrospective review of an MRI study done at a different hospital during the first week of disease demonstrated a single focus of gadolinium enhancement within the body of the chiasm. This observation suggests that bilateral demyelination of the posterior optic nerves may begin as a single focus of chiasmal disease that then spreads to involve the proximal portions of the optic nerves.

Merkel cell carcinoma of the eyelid.

PURPOSE: To demonstrate the clinical variability and histologic characteristics of Merkel cell carcinoma of the eyelid. METHODS: We investigated two cases of Merkel cell carcinoma of the eyelid in a 67-year-old man and an 83-year-old woman, respectively. Both lesions were initially misdiagnosed as benign tumors on clinical examination. RESULTS: After the correct diagnosis was made, both lesions were treated with wide resection aided by frozen sections, and reconstructive surgery. CONCLUSIONS: Merkel cell carcinoma is an aggressive tumor with variable clinical manifestation. Radical surgical therapy and close follow-up are indicated.

Bilateral macular lesions in incontinentia pigmenti. Bloch-Sulzberger syndrome.

BACKGROUND: Incontinentia pigmenti (IP) is an inherited disorder with many systemic and ocular manifestations. Abnormalities of the retinal pigment epithelium, including peripheral hypopigmented areas, have been reported occasionally in patients with IP. These have not been symmetric, and they have not involved the fovea. The visual function in affected areas has not been described. METHODS: The ophthalmic examination and results of fluorescein angiography are described in a patient with IP and bilateral discrete hypopigmented macular lesions. RESULTS: The patient had normal vision. Fluorescein angiography of the macular lesions revealed transmission of fluorescence in the early phase and staining in the late phase. CONCLUSION: The authors believe that this is the first report of bilateral macular hypopigmented lesions in a patient with IP, and it is the first to document normal vision in an affected area.

Transplantation of corneal endothelial cells using a cell carrier device.

Penetrating keratoplasty is currently the only treatment for corneal endothelial dysfunction. Although corneal transplantation has a high success rate, a few problems still remain, such as the limited availability of donor grafts, the change in refraction after penetrating keratoplasty, and the higher chance of immune rejection. In this study, a coated hydrogel lens (Chiron Ophthalmics Inc., Irvine CA, U.S.A.) has been used as a carrier to transplant cultured homologous kitten and rabbit corneal endothelial cells into adult cats and rabbits. The transplantation procedure was the same in both species. Corneal endothelial cells from homologous rabbits or cats were seeded on coated hydrogel lenses and cultured until they reached a complete monolayer with an average cell density of 2,500 cells/mm2. Five weeks before transplantation surgery, corneal endothelial cells were scraped to induce corneal edema. The cell carrier device was then transplanted as follows: a trephine cut (7.7 mm) was made into the stroma, producing an outer corneal plug. The inner cornea was then cut by using a 5.5-mm trephine, and this inner plug was discarded. The implant was inserted and the outer corneal plug was sutured back into place. Corneas cleared completely within 3 days in both rabbits and cats, and stayed clear for an average of 40 days in rabbits and 50 days in cats. The histopathological evaluation of the rejected grafts showed vascularized retrocorneal membrane formation in cats, whereas in rabbits severe cellular infiltration of the stroma with neovascularization occurred without retrocorneal membrane formation.

Effects of growth factors on wound healing in serum-deprived kitten corneal endothelial cell cultures.

The influence of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF) I and II on wound healing was investigated in a corneal endothelial system with minimal mitotic activity, using serum-deprived kitten corneal endothelial-cell cultures. After wounding, growth factors were added and wound diameter was evaluated. The DNA synthesis was determined by 3H-thymidine labeling. Wounds did not close in the control cultures grown in serum-free medium without growth factors. The IGF I or II, alone (10 and 100 ng/ml) or added (10 ng/ml) to EGF or bFGF, had no significant effect on wound closure or thymidine uptake. With EGF or bFGF (10 ng/ml), wounds closed after 15 days. Wounds closed after 10 days with EGF or bFGF (100 ng/ml) alone or with the combination of EGF and bFGF (each at 10 ng/ml). Combined EGF and bFGF (each at 100 ng/ml) did not enhance wound closure further. Thymidine uptake was significantly higher in cultures treated with EGF or bFGF (10 ng/ml) than in controls. The uptake could be increased, if both growth factors were combined, but only to the same level achieved with a single factor at 100 ng/ml. This study showed that EGF and bFGF, but not IGF I or II, enhanced wound closure and DNA synthesis in a corneal endothelial cell system that had minimal mitotic activity.

Isolation of plasma membrane domains from bovine corneal endothelial cells.

Corneal endothelial cells are polarized cells responsible for pump, barrier and transport functions, which are carried out by proteins located on the apical or basolateral plasma membrane domain. Apical, basolateral, and total plasma membranes of cultured bovine corneal endothelial cells were isolated to characterize their protein constituents. Apical membranes were isolated by applying charged beads onto the cell monolayer, and harvesting the membranes by washing off the beads. Total cell membranes were isolated in cell/bead suspensions followed by sonication and vortexing. Basolateral plasma membranes were scraped off culture dishes after disrupting cells with distilled water. A variety of marker enzyme assays demonstrated, that the plasma membrane domains could be isolated and separated with relative purity, and with only little contamination by intracellular membrane proteins. SDS-polyacrylamide gel electrophoresis showed approximately 50 protein bands for total plasma membranes, 35 protein bands for apical plasma membranes and approximately 45 bands for basolateral plasma membranes.

[Amyloidosis of the vitreous body. Possibilities of diagnosis]. / Amyloidose des Glaskörpers. Möglichkeiten der Diagnostik.

Vitreous amyloidosis is often the presenting clinical manifestation of type I, type II or Jewish-type familial amyloid polyneuropathy (FAP). FAP is an autosomal dominant inherited disorder. It is caused by systemic deposition of variants of transthyretin (TTR), formerly called prealbumin. TTR is a tetrameric protein with beta pleated sheets (mol wt = 56,000 dalton). In two cases we were able to confirm the clinical diagnosis of vitreous amyloidosis. Immunohistochemistry revealed TTR in vitreous samples after therapeutic pp vitrectomy for vitreous opacity. The same result was found in samples of rectal mucosa. Amyloid was not found in skin. Isoelectrical focusing disclosed that TTR in the serum was the Portuguese (TTR-Met 30) variant. Together with polyneuropathy of the lower limbs, a diagnosis of FAB type I was made. In the second generation of the first patient's family the normal variant was found (the pathologic gene was not inherited). In the second case the pathologic variant was detected in the second generation, but without any pathologic clinical features. The third generation showed the normal variant. The disorder was detectable before any clinical signs were present. These findings are also important for genetic counseling.

Incidence of posterior capsule opacification after extracapsular cataract extraction in diabetic patients.

Posterior capsule opacification (PCO) is the most frequent complication following extracapsular cataract extraction (ECCE). To evaluate the rate of PCO we examined 939 eyes (786 patients) following ECCE after a period of 26.3 months on the average. In 202 eyes (21.5%) cataract was associated with a clinically manifest type I or type II diabetes mellitus. The overall incidence of PCO was 28.1% (264 eyes). In patients with no other diseases (371 eyes, 100%) it was 33.4% (124 eyes). Out of 202 eyes (100%) associated with diabetes PCO was seen in 21.8% (44 eyes). This difference was statistically significant (p = 0.05). Average age and sex did not differ significantly in both groups. In diabetic patients the lens epithelial cells show accumulation of sorbitol and fructose which is proposed to contribute to cataract formation by disturbing cellular metabolism. This mechanism may explain our findings of a lower incidence of PCO possibly caused by reduced proliferation of lens epithelial cells.

Molecular cloning and physical mapping of the genome of insect iridescent virus type 6: further evidence for circular permutation of the viral genome.

A defined and complete gene library of the Chilo iridescent virus (CIV) genome was established. The CIV DNA was cleaved with restriction endonucleases EcoRI, NcoI, SphI, and BamHI or double digested with BamHI/SalI and the resulting DNA fragments were inserted into the corresponding sites of the bacterial vectors pACYC184, pKm2, pL-ES-C3, and pAT153 using T4 DNA ligase. All cloned fragments were identified by digestion of the recombinant plasmids with different restriction enzymes and checked by hybridization of recombinant plasmid to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned into the EcoRI site of pACYC184. Although the CIV genome is linear, all 32 EcoRI fragments have been cloned directly. This suggests that the CIV genome is circularly permuted. In addition, NcoI(72%), SphI(40.7%), BamHI (11.6%), and BamHI/SalI(39.7%) DNA fragments of the viral genome were inserted into the corresponding sites of pKm2, pL-ES-C3, and pAT153, respectively. The physical map of the viral genome was constructed using the established gene library for restriction enzymes ApaI, BamHI, EcoRI, NcoI, SalI, and SmaI. Although the CIV genome is linear, this analysis revealed that the restriction maps of the viral genome are circular. This finding supports the hypothesis that the CIV genome is circularly permuted.