P‐53 Histopathologic evaluation of feline plasmacytic pododermatitis

The purpose of this study was to evaluate the histopathologic changes seen in feline plasmacytic pododermatitis in an attempt to correlate clinical response with any specific histopathologic feature, and to evaluate these specimens for possible infectious organisms. Fourteen skin biopsies from cats with plasmacytic pododermatitis and a clinical follow‐up of 12–36 months were included in the study. Sections were stained with H&E as well as with a polyclonal anti‐Mycobacterium bovis [Bacille Calmette–Guerin (BCG)] antibody cross‐reactive to a broad spectrum of fungi and bacteria. Sections stained with the anti‐BCG antibody were graded as positive or negative. Inflammatory cell types and density, overall pattern, and the presence or involvement of adnexal structures were assessed in sections stained with H&E. All sections were negative for organisms within the actual footpad tissue with the anti‐BCG antibody stains. Two samples revealed a few foci of cocci on the outer surface of the ulcers. Funding: Self‐funded.     

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.     

Characterisation of lymphosarcomas in Australian cats using polymerase chain reaction and immunohistochemical examination

Objective To examine tumour tissue of cats with lymphosarcoma for the presence of feline leukaemia virus and feline immunodeficiency virus and analyse the immunophenotype of the tumours.
Design A retrospective study of feline lymphosarcoma cases.
Methods Formalin-fixed, paraffin-embedded tumour tissue of 14 feline lymphosarcomas was examined for the presence of feline leukaemia virus and feline immunodeficiency virus by polymerase chain reaction and immunohistochemistry. Using polyclonal and monoclonal antibodies against T and B lymphocytes, the phenotypic expression of the tumours was characterised.
Results No feline leukaemia virus antigen or proviral sequences were detected. Feline immunodeficiency virus proviral sequences were detected in two cases by polymerase chain reaction. Immunophenotyping of all 14 cases resulted in seven cases being classified as B-cell phenotype, four as T-cell phenotype, and the remaining three undetermined.
Conclusions In contrast to previous reports overseas, our results suggest that feline leukaemia virus infection appears to be an infrequent cause of lymphosarcoma in the cats that were necropsied. Feline immunodeficiency virus may have a role in lymphomagenesis. The potential role of feline immunodeficiency virus needs to be explored in more depth. Compared with most previous reports, B-cell tumours were more common than T-cell tumours in this series of cats.     

Identification of Pentatrichomonas hominis in feline fecal samples by polymerase chain reaction assay

Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some investigators presumptively identified P. hominis as a causative agent of feline diarrhea. However, molecular identification of P. hominis infection in the cat has not been reported. Another trichomonad, Tritrichomonas foetus, is recognized as an intestinal pathogen in cats and often presumptively diagnosed on the basis of the presence of trichomonads in diarrheic feces. It is of importance to determine if cats are natural hosts for P. hominis, as the presence of this organism could result in inaccurate assumption of T. foetus infection. In this study, we used a species-specific PCR assay to identify P. hominis 18S rRNA genes in fecal samples collected from a convenience population of cats in which a high prevalence of T. foetus infection had been previously identified (cat show) or suspected (submitted for T. foetus diagnostic testing). The prevalence of T. foetus infection in these samples was 31% and 28.6%, respectively. P. hominis infection was identified by PCR of DNA extracted from feces of five cats (1.9% and 2.1% of fecal samples, respectively). All cats in which P. hominis was identified were also infected with T. foetus. PCR identification of P. hominis infection in the cat should facilitate future studies to determine the pathogenicity of this species and enable differentiation of P. hominis from other known or as-yet unidentified species of trichomonads that may infect cats.     

Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.     

Accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats

OBJECTIVE: To determine the sensitivity, specificity, and overall diagnostic accuracy of polymerase chain reaction (PCR) assays offered by commercial diagnostic laboratories for diagnosis of FIV infection in cats. DESIGN: Prospective clinical trial. ANIMALS: 124 cats. PROCEDURE: Blood was collected from cats that were neither infected with nor vaccinated against FIV, uninfected cats that were vaccinated with a licensed FIV vaccine, and cats experimentally and naturally infected with FIV representing subtypes A, B, and C. Coded blood samples were submitted to 3 laboratories in the United States and Canada offering PCR assays for diagnosis of FIV infection to veterinary practitioners. All laboratories tested fresh blood samples, and 1 laboratory also tested samples submitted as dried blood smears. The FIV infection status in all cats was confirmed by virus isolation. Sensitivity, specificity, and correct results were calculated for each PCR assay. RESULTS: Sensitivity ranged from 41% to 93%. Specificity ranged from 81% to 100% in unvaccinated cats and 44% to 95% in cats vaccinated against FIV. Correct results were obtained in 58% to 90% of 124 cats tested. All tests misidentified both uninfected and infected cats. False-positive results by all laboratories were higher in cats vaccinated against FIV than in unvaccinated cats, suggesting that vaccination interferes with the performance or interpretation of PCR assays used for diagnosis of FIV infection. CONCLUSIONS AND CLINICAL RELEVANCE: PCR assays used for diagnosis of FIV infection presently marketed to veterinary practitioners in North America vary significantly in diagnostic accuracy and did not resolve the diagnostic dilemma resulting from vaccination of cats against FIV.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.     

Effects of temperature and time in transit on polymerase chain reaction detection of feline herpesvirus DNA.

The purpose of this study was to report methods currently recommended by commercial laboratories for collecting, shipping, and processing of samples for feline herpesvirus type 1 (FHV-1) testing using polymerase chain reaction (PCR) and to determine the effect of temperature and time on the ability of 1 PCR method to detect FHV-1 DNA in experimental and clinical samples. Eleven laboratories offering FHV-1 PCR were surveyed. There was notable variation in sample types and shipping conditions recommended and PCR protocols used by these laboratories. Subsequently, using a single PCR method, FHV-1 DNA was detected in samples exposed to various temperatures within the laboratory. Finally, FHV-1 PCR was performed on paired clinical samples collected from 25 cats and shipped at ambient temperatures via US Postal Service (USPS) or with an ice pack via a courier. Samples sent by USPS were exposed to significantly longer transit time and arrived at significantly higher temperature than did samples sent by courier. Despite this, all sample pairs yielded concordant results when tested for FHV-1 DNA using this PCR method. Although it may not be necessary for samples collected for detection of FHV-1 DNA using this PCR method to be shipped under the most expedient or temperature-controlled conditions, this should be verified for a variety of PCR assays and sample types.     

Lack of detection of feline leukemia and feline sarcoma viruses in diffuse iris melanomas of cats by immunohistochemistry and polymerase chain reaction.

Diffuse iris melanoma was confirmed by light-microscopic examination in 10 formalin-fixed, paraffin-embedded globes from 10 cats. To determine if feline leukemia virus or a replication defective feline leukemia virus, feline sarcoma virus, was present in these anterior uveal melanomas, immunohistochemistry and polymerase chain reaction for feline leukemia virus were utilized. Immunohistochemical staining for feline leukemia virus glycoprotein 70 was performed on all 10 tumors using an avidin-biotin complex technique. The DNA was extracted from each specimen and a 166-base pair region of the feline leukemia virus long terminal repeat was targeted by polymerase chain reaction. Immunohistochemical staining for feline leukemia virus glycoprotein 70 and polymerase chain reaction amplification of a feline leukemia virus long terminal repeat region were negative in all cases. Feline leukemia virus/feline sarcoma virus was not detected in any neoplasms and therefore was unlikely to play a role in the tumorigenesis of these feline diffuse iris melanomas.     

Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization.

An adult domestic shorthair cat had severe chemosis due to purulent and necrotizing blepharitis and conjunctivitis. Purulent rhinitis, necrotizing glossitis, and dermatitis were also diagnosed. The cat was positive for feline immunodeficiency virus and feline leukemia virus. Histologically, intranuclear Cowdry type A inclusions were found within numerous epithelial cells adjacent to the lesions in skin, conjunctiva, and tongue. Electron microscopic examination revealed herpesviral particles within the lesions. Paraffin-embedded skin and tongue tissues were processed in a polymerase chain reaction, using primers to amplify a 306-bp region of the thymidine kinase gene of feline herpesvirus type 1, resulting in a distinct amplification product of the predicted size. The distribution of feline herpesvirus was demonstrated by immunohistochemistry and nonradioactive in situ hybridization. Positive immunostaining was found in nuclei and cytoplasm of numerous epithelial cells within and next to the lesions, whereas in situ hybridization, performed with a digoxigenin-labeled double-stranded DNA probe, revealed hybridization signal only in nuclei of intact epithelial cells. Neither immunohistochemistry nor in situ hybridization showed feline herpesvirus type 1 in tissues of lungs, liver, spleen, intestine, or brain.     

Detection of feline immunodeficiency proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction.

Feline immunodeficiency virus (FIV) proviral DNA was detected by the polymerase chain reaction method (PCR). PCR products were detected by gel electrophoresis and ethidium bromide staining. The P-10, P-15 and P-24 regions of the gag gene of FIV were chosen as the target sequences for amplification, and three primer pairs were prepared. The PCR products subjected to amplification with each primer pair were found to possess sites of digestion by a restriction enzyme, as hypothesized. They did not react with feline leukemia virus (FeLV)-infected or feline syncytium-forming virus (FeSFV)-infected cell-derived DNA, and specifically amplified FIV-infected cell-derived DNA. FIV proviral DNA was detected by the PCR method with either primer pair (one-step amplification: single PCR) in DNA derived from peripheral blood lymphocytes (PBL) from 7 of 12 FIV antibody-positive cats. When PCR products in each of the 12 cats were subjected to a second amplification using the same primer pair (two-step amplification: double PCR), FIV proviral DNA was detected in all of the cats. When PBL samples collected from three cats that were negative and three that were positive in the single PCR were cultured for a few weeks in the presence of interleukin 2, FIV proviral DNA was detected in all six cats by the single PCR method. The results suggest that either the use of cultured PBL as the sample or the performance of the double PCR method enables simple and specific detection of FIV proviral DNA in PBL.     

Detection of feline coronavirus infection in captive cheetahs (Acinonyx jubatus) by polymerase chain reaction.

Feline coronavirus genetic elements were detected by polymerase chain reaction from blood, fecal samples, and effusive fluid collected from 33 cheetahs in the U.S.A. Feline coronavirus-specific serum antibodies were also measured by indirect immunofluorescence. Ten cheetahs were positive for viral shedding by polymerase chain reaction, whereas 13 were seropositive by immunofluorescence. Results of serology did not consistently correlate with shedding of virus, and the capture antigen used for detection of feline coronavirus-specific antibodies had a significant impact on results. Testing of samples from one population over a 1-yr period indicated chronic infection in some animals. These relatively healthy carrier animals were a source of virus for contact animals. Screening programs in cheetah populations for feline coronavirus infection may be most reliable if a combination of serologic analysis and viral detection by polymerase chain reaction is used.     

Detection of pigeon circovirus by polymerase chain reaction

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.