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Abstract

Objective

To use a nested polymerase chain reaction (PCR) to detect feline herpesvirus (FHV-1) DNA in conjunctiva or cornea from clinically normal cats and cats with conjunctivitis or corneal sequestra.

Samples

Conjunctival snip biopsy specimens from 50 cats with conjunctivitis and 50 clinically normal cats; 28 keratectomy specimens from 26 cats with sequestra, and 13 specimens from clinically normal cats.

Procedure

Tissue specimens were digested, and FHV-1 DNA was amplified, using a double round of PCR. Products were visualized by use of agarose gel electrophoresis.

Results

Polymerase chain reaction was positive in 27 of 50 (54%) conjunctival specimens from cats with conjunctivitis and 6 of 50 (12%) specimens from clinically normal cats. Difference in the results between cats with conjunctivitis and clinically normal cats was statistically significant. Polymerase chain reaction was positive in 5 of 28 (18%) corneal specimens from cats with sequestra and 6 of 13 (46%) clinically normal cats. Distribution of positive results between clinically normal cats and those with sequestra was not significant.

Conclusion

Cats with conjunctivitis were more likely to have a positive PCR result than were clinically normal cats, making it likely that FHV-1 was associated with the disease state. Herpesvirus DNA could not be detected in most corneas from cats with sequestra.

Clinical Relevance

Polymerase chain reaction is a useful clinical test for identifying FHV-1 DNA in cats with conjunctivitis, yielding greater sensitivity over that of currently available tests. Herpesvirus may be less of a cause of corneal sequestration in the commonly affected breeds, Himalayan and Persian, than other factors, such as lagophthalmos or corneal metabolic defects. (Am J Vet Res 1997;58:338–342)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To use transient and stable transfection of Chinese hamster ovary cells to clone the gene encoding feline erythropoietin (feEPO) protein, characterize the expressed protein, and assess its biological activity.

Sample Population—Cultures of Chinese hamster ovary or TF-1 cells.

Procedure—The gene encoding feEPO was cloned into a eukaryotic expression plasmid. Chinese hamster ovary cells were transiently or stably transfected with the plasmid. Expressed recombinant feEPO (rfeEPO) protein was purified from transiently transfected cells. The protein was characterized by use of SDS gel electrophoresis and western blot analysis. Biological activity was assessed by measuring thymidine incorporation by TF-1 erythroleukemic cells.

Results—Purified rfeEPO from supernatants of transiently transfected cells was determined to be 34 to 40 kilodaltons (kd) by use of SDS gel electrophoresis, whereas the molecular weight predicted from the amino acid sequence was 21.5 kd. The banding pattern and high molecular weight suggested the protein was glycosylated. The rfeEPO proteins derived from transient or stable transfections subsequently were determined to be biologically active in vitro.

Conclusions and Clinical Relevance—The gene encoding feEPO can be transfected into eukaryotic cells, and the expressed rfeEPO protein is biologically active in vitro. Cats with chronic renal failure often are anemic as a result of reduced expression of erythropoietin (EPO). Treatment with human-derived EPO stimulates RBCs in anemic cats; however, treatment is often limited by the development of antibodies directed against the recombinant human protein, which can then cross-react with endogenous feEPO. Recombinant feEPO may prove beneficial for use in cats with chronic renal failure. (Am J Vet Res 2003; 64:1465–1471)

Full access
in American Journal of Veterinary Research