Canine Haematopoietic Chimerism Analyses by Semiquantitative Fluorescence Detection of Variable Number of Tandem Repeat Polymorphism

Canine models are successfully applied to the study of haematopoietic stem cell transplantation (HSCT). Monitoring of haematopoietic donor/recipient chimerism is of major significance in detecting and quantifying engraftment or graft rejection of the donor-derived haematopoietic cells after transplantation. Radioactive analyses of polymorphic microsatellite markers are commonly used for chimerism analyses. We describe an improved, non-isotopic method that is based on the analysis of microsatellite markers in donor and recipient cells using capillary electrophoresis and fluorescence detection. Artificial mixtures of donor and recipient DNA that were generated from peripheral blood mononuclear cells from dog leukocyte antigen-identical siblings were used to analyse the sensitivity of the assay. DNA from dogs that had received HSCT were also analysed in order to demonstrate the feasibility of the method in vivo. For chimerism analyses, six different microsatellite loci were systematically amplified using fluorescent PCR primer. The fluorescent polymerase chain reaction products were separated by capillary electrophoresis using POP4 on a 310 ABI Prism Genetic Analyzer. After electrophoresis, fluorescence signals were automatically sized and quantified using GeneScan software. The method described provides an accurate assessment of haematopoietic chimerism in the canine model with significantly reduced hands-on time compared to conventional gel electrophoresis.     

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).     

Biological characteristics of fish germ cells and their application to developmental biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.     

同源异体骨髓间充质干细胞移植治疗犬急性肝功能损伤

探讨同源异体骨髓间充质干细胞(BMMSCs)移植对犬急性肝功能损伤的治疗作用,为犬干细胞治疗相关疾病提供理论基础和技术支持。对患急性肝损伤的10只病犬经超声引导腹腔内肝门附近移植BMMSCs,移植后检测血常规、肝功能和肝脏B超,观察同期的症状体征和不良反应情况。结果显示,干细胞移植7天后可使患犬各项肝功能指标明显改善,10d后B超结果显示肝组织回声均匀,无明显异常,14d后患犬的精神、体力、食欲明显好转,干细胞移植21d后血常规各项指标恢复正常,患犬基本康复,移植干细胞的患犬均未发现有不良反应。说明相对于常规治疗,犬BMMSCs同源异体移植治疗急性肝损伤可明显缩短治疗时间,显著提高患犬生活质量。     

Bone marrow transplantation for canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain which is a subunit of the receptors of IL-2, IL-4, IL-7, IL-9 and IL-15. Bone marrow transplantation (BMT) of human XSCID patients without pretransplant conditioning (cytoablation) results in engraftment of donor T-cells and reconstitution of T-cell function but engraftment of few, if any, donor B cells with resultant poor reconstitution of humoral immune function. In this study, we show that XSCID dogs can be transplanted with allogeneic bone marrow cells resulting in engraftment of both donor B and T cells and reconstitution of full systemic immune function including normal humoral immune function without the need for cytoablation.     

Allogeneic tooth transplantation in the dog

Allogeneic tooth transplantation was evaluated as a functional and aesthetic treatment for dental fracture in the dog. Of 7 dogs that received tooth transplants, 5 were research animals and 2 were clinical patients. Canine teeth were transplanted immediately after extraction in the research dogs. Endodontic therapy had been performed on the affected canine tooth of one clinical patient. The other clinical patient had bilateral maxillary canine fractures 2 months earlier. One of the research dog transplants failed at 3 weeks as a result of improper surgical technique. Four of the research dogs had a solid implant for 18 months, after which time the dogs were euthanatized serially. All transplanted teeth were anchored firmly into the alveoli, but were nonviable. Root resorption, with bone replacement, was first noticed at 24 months. The transplanted tooth in the first clinical patient remained functional for 3 months, after which time the tooth was fractured. The right canine transplant in the second clinical patient failed by 3 months, probably because of preexisting periapical inflammation. The left transplanted tooth remains stable at 38 months. It was concluded that allogeneic tooth transplantation may have merit as a rapid and inexpensive method for replacement of fractured teeth in the dog. Function is compromised gradually as a result of root resorption and ankylosis, with tooth fracture likely to occur after 2 years.     

Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.     

犬细小病毒环介导等温扩增快速检测方法的初步建立

为建立犬细小病毒(CPV)环介导等温扩增(LAMP)检测方法,实现CPV的早期快速诊断,本研究根据GenBank登录的CPV VP2基因序列,在其序列保守区域设计LAMP引物,利用CPV基因组DNA为模板进行扩增。结果表明:LAMP方法检测灵敏度达到10-1TCID50/mL;并且与其它细小病毒等无特异性扩增,表现出良好的特异性。与PCR技术相比,LAMP法操作更加简单方便,更适合基层和实验室的快速检测。     

Detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive, and rapid technique for detection of genomic DNA. The end-product of the technique is a white precipitate of magnesium pyrophosphate that is visible without the use of gel electrophoresis. The LAMP method was applied to the detection of canine parvovirus (CPV) genomic DNA. A set of 4 primers, 2 outer and 2 inner, were designed from CPV genomic DNA targeting the VP2 gene. The optimal reaction time and temperature for LAMP were determined to be 60 minutes and 63 degrees C. On the basis of results for 50 canine fecal samples using polymerase chain reaction (PCR) analysis as the gold standard, the relative sensitivity of LAMP was 100% and the relative specificity was 76.9%. The detection limit of the LAMP method was 10(-1) median tissue culture infective doses (TCID50)/ml, compared with 10 TCID50/ml for PCR analysis. In addition to the advantage resulting from visual detection of the end product, the LAMP method is very rapid, requiring only 1 hour to complete. This assay would be a viable alterative to PCR analysis for diagnosis of CPV infection in dogs. The LAMP method holds promise for use as a diagnostic assay for CPV detection in a clinical setting.     

GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas

The diagnosis of canine lymphoma is achieved using morphological and immunological methods. In a certain percentage of cases, difficulties in making a definitive diagnosis of lymphoproliferative disorders may occur despite extensive immunophenotyping. Therefore, additional diagnostics, such as molecular assessment of Ig/TCR gene rearrangements clonality, may confirm the final diagnosis. Polyacrylamide gel electrophoresis and heteroduplex analysis have already been proven to be suitable for detecting clonality but are cumbersome and labor-intensive. In the present study, GeneScanning analysis of PCR products originating from different primer sets targeting different regions of Ig and TCR was validated in improving sensitivity as well as in reducing the turnaround time of gene rearrangement assays. GeneScanning exploits 5' fluorescently labelled primers for the automated and fast analysis of PCR products either as singleplex or multiplex runs. Initially, the assay was set up using DNA purified from normal tissues (n=6), hyperplastic/reactive tissues (n=10) and a small set of immunophenotyped lymphoma samples (n=12). The optimized methods were then used in a large set of 96 canine lymphoma samples. Normal and hyperplastic/reactive lymphoid tissues showed typically polyclonal or, occasionally, oligoclonal PCR products. Lymphoma samples showed monoclonal peaks arranged as a single or, occasionally, a double narrow base peak sometimes embedded in a polyclonal background. In all immunophenotyped cases, an Ig or TCR clonal finding corresponded to B- and T-cell lymphomas, respectively. Overall, 94/96 (97.9%) samples showed clonal Ig/TCR clonal rearrangements among which clonal Ig was found in 61/96 (63.5%) of samples and clonal TCR in 33/35 Ig negative samples (34.4% of all cases). In one out of ten randomly chosen cases, both Ig and TCR clonal gene rearrangements were found. Among the factors affecting assay accuracy, DNA quality has been shown to be critical and the amplification of DNA controls of different size are recommended to evaluate DNA integrity. Frozen material such as that which remained inside the hub of the needle used for diagnostic procedures is optimal for the analysis herein described. In conclusion, GeneScanning represents a versatile tool for routinely assessing Ig/TCR clonal rearrangements and supporting the diagnostic protocol of canine lymphomas.     

Upfront denileukin diftitox as in vivo regulatory T-cell depletion in order to enhance vaccination effects in a canine allogeneic hematopoietic stem cell transplantation model

Denileukin Diftitox (ONTAK^®, DAB₃₈₉ IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_reg). Elimination of immunosuppressive T_reg by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T_reg depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18 μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide^® ISA 51 were locally applied. In vitro studies demonstrated that canine T_reg are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T_reg was abolished by addition of Denileukin Diftitox (10 nM). An increase of proliferation of median 300% (range: 200%–425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T_reg followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.     

Analysis of genetic variations in the exon 27 region of the canine BRCA2 locus

Mammary tumors are the most common tumor type in women as well as in female dogs. The BRCA2 gene encodes a large nuclear protein that is involved in DNA repair, and mutations in the human BRCA2 confer an increased risk of female mammary tumors. The BRCA2 protein acts as a tumor suppressor, and inactivation of BRCA2 by loss of heterozygosity is implicated in mammary carcinogenesis. In this study, to establish an appropriate polymorphic marker for loss of heterozygosity analysis of the canine BRCA2, we analyzed the genomic sequences of the exon 27 regions of 30 mammary-tumor-bearing and 21 tumor-free dogs. In addition to 10204ins/delAAA, which is the only polymorphism previously identified for the canine BRCA2 locus, we discovered four novel single nucleotide polymorphisms. The analysis of these five polymorphisms revealed the presence of four allele types. Since 10204ins/delAAA was the most common of the five polymorphisms identified, we developed a PCR-based assay method to assay for this polymorphism. We believe that this method is valuable for loss of heterozygosity analysis of the canine BRCA2 gene in tumor pathogenesis.     

A Case of Leucocyte Chimerism (78,XX/78,XY) in a Dog with a Disorder of Sexual Development

A 1‐year‐old Shih Tzu dog was presented for examination because of abnormal external genitalia. A residual penis with a prepuce was located in a position typical of a male. The dog had no palpable testicles or scrotum. The ultrasound examination revealed the presence of the prostate, but the gonads remained undetectable. Cytogenetic analysis performed on chromosome preparations obtained from lymphocyte culture showed two cell lines – 78,XX and 78,XY. Molecular analysis of 14 polymorphic microsatellite markers allowed us to distinguish leucocyte chimerism from whole body chimerism. The presence of 3 or 4 alleles was confirmed in DNA isolated from blood, while in DNA isolated from hair follicles only 1 or 2 alleles were detected. The case was classified as leucocyte 78,XX/78,XY chimerism. Our study showed that XX/XY leucocyte chimerism might be associated with disorder of sexual development in dogs. Furthermore, it is emphasized that the use of cytogenetic study, in combination with analysis of polymorphic markers in DNA isolated from different somatic cells, facilitates distinguishing between leucocyte and whole body chimerism.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.     

人Bcl-2乳腺定位表达转基因小鼠的建立

以 p CAT、p WB和 p WC为原始质粒 ,构建 WAP启动子调控的 Bcl- 2乳腺组织特异性表达载体 p WBS。载体用Bgl +Sal +Pvu 酶切 ,回收 3.0 kb的基因片段 WAP- Bcl- 2 - SV40 Poly A,通过显微注射方法导入 C5 7BL / 6 J×DBA/ 2 JF1代小鼠受精卵的雄原核内。共注射 10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 5 5只。PCR检测 ,阳性鼠 12只。Southern blot检测 ,阳性鼠 4只 ,其中公、母鼠各 2只 ,在饲养过程中 ,2只公鼠均意外死亡。Western blot证实 ,1只母鼠 Bcl- 2表达阳性 ,其 F1代的 40只小鼠中 ,有 18只呈 PCR阳性 ,其中母鼠8只 ,在 5个月的反复怀孕过程中 ,未观察到乳房有任何肿瘤或肿块的出现 ,证实 Bcl- 2单独在转基因鼠乳腺中表达不会引起转基因鼠乳腺癌的发生。     

人c-myc乳腺定位表达转基因小鼠的建立

以含有 1.6 kb的小鼠乳清酸蛋白 (WAP)上游调序列的 p CAT- WAP和含有人 c- myc c DNA的 p WM为原始质粒 ,构建了 WAP启动子调控下的 c- myc乳腺定位表达载体 p WCS。载体用 Bgl Bam H 酶切 ,回收 3.5 kb的基因片段 WAP- c- myc- SV40 Poly A,通过显微注射方法导入 C5 7BL/ 6 J× DBA/ 2 JF1 代小鼠受精卵的雄原核内。共注射10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 45只。PCR检测 ,阳性鼠 9只 ,South-ern blot检测 ,阳性鼠 3只 ,其中 1只母鼠 ,2只公鼠 ,在饲养过程中 ,1只母鼠意外死亡。对 2只转基因公鼠的 F1代PCR检测表明 ,仅 1只公鼠具有遗传性 ,在所生的 2 7只 F1代小鼠中有 13只为 PCR阳性。     

Multiorgan engraftment of human somatic cells in swine foetuses after intra-blastocyst transplantation

Adult human stem cells, mainly from hematopoietic lineage, have been injected into developing pre-immune animal foetuses, and xenogenic engraftment of liver and other organs has been reported. We isolated a rare cell population from adult human liver, fat and skin. Colonies with few cells became visible as early as 2-3 days, and a fully formed colony took 10-14 days to form. These colonies were named as liver-derived cell lines (LDCs), fat-derived cell lines (FDCs) and skin-derived cell lines (SDCs). All these cells express few pluripotency markers like Klf4, c-myc and Sox2. Pig blastocysts were injected with LDCs, FDCs and SDCs and transferred to recipient pigs. We achieved an overall pregnancy rate of 71.4% at day 35. The foetuses were analysed for human cell chimerism in liver, kidney and heart both by RT-PCR and real-time PCR using primers specific to human and pig mitochondrial DNA. The percentage of foetuses showing chimerism was 17.4% (4/23), 12.5% (2/16) and 11.1% (1/9) for LDCs, FDCs and SDCs, respectively. Of these, 42.9% (three out of seven) showed chimerism in liver and 71.4% (five out of seven) showed kidney chimerism. However, we did not detect any chimerism in the heart. The level of chimerism varied and was in the range of one human cell per one hundred thousand to one million pig cells.     

Optimization of a species-specific polymerase chain reaction assay for identification of Pentatrichomonas hominis in canine fecal specimens

OBJECTIVE: To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces. SAMPLE POPULATION: DNA extracted from feces of 4 dogs with diarrhea from which trichomonads were observed, 81 dogs that had feces submitted to a diagnostic laboratory, and 19 dogs residing in a laboratory animal facility. PROCEDURES: Optimum reaction conditions and absolute and practical detection limits of 2 P hominis 18S species-specific primer pairs were determined by use of an in vitro cultivated canine isolate of P hominis in the presence and absence of canine feces. The optimized PCR assay was applied to amplification of P hominis 18S rRNA genes from DNA extracted from the feces of dogs. RESULTS: Under optimized conditions, a primer pair was identified as able to detect as few as 1 P hominis organism/180-mg fecal sample. The PCR assay identified P hominis in diarrheic feces of 4 dogs in which trichomonads were seen by light microscopy. The P hominis genes were not amplified from other fecal samples examined. CONCLUSIONS AND CLINICAL RELEVANCE: Molecular identification of P hominis in feces of 4 dogs with trichomonosis and diarrhea reported here validates the identity of this species in such infections. Sensitive and specific PCR amplification of P hominis 18S rRNA genes from DNA extracted from feces will directly facilitate studies examining pathogenicity of this trichomonad and enable differentiation of P hominis from other known or novel species of trichomonads that may infect the gastrointestinal tract of dogs.     

Establishment and Application of A Double TaqMan MGB Real-time PCR Assay for Detection of Canine Distemper Virus and Canine Parvovirus

This study was aimed to establish a double TaqMan MGB Real-time PCR assay to simultaneously and specifically detect canine distemper virus (CDV) and canine parvovirus (CPV) in one reaction.Two pairs of specific primers for CDV and CPV,along with two TaqMan MGB probes for each virus were designed in the assay basing on CDV H gene and CPV VP2 gene sequences.The specificity,sensitivity and repetition of the double TaqMan MGB Real-time PCR assay were tested,and 48 samples taken from clinic suspicious CDV and CPV infected canines had been testified by the established double TaqMan MGB Real-time PCR.The results indicated that the doulde TaqMan MGB Real-time PCR assay was successfully established,and the number of standard curve correlation (R²) of CDV and CPV were 0.997 and 0.993,respectively.The specificity of the double TaqMan MGB Real-time PCR assay revealed that amplifications were showed on CDV and CPV samples,but other pathogens and negative controls had no amplifications;The sensitivity of CDV and CPV were both 10 copies/μL.Meanwhile,14 CDV positive samples,19 CPV positive samples and 4 CDV/CPV double positive samples were detected,which were consistent with the results of the sequencing.Therefore,the established double TaqMan MGB Real-time PCR assay had high sensitivity,specificity and flux accurate quantitative,which could be applied to clinical CDV/CPV infection each periods.     

Detection of novel papillomaviruslike sequences in paraffin-embedded specimens of invasive and in situ squamous cell carcinomas from cats

OBJECTIVE: To detect and partially characterize papillomavirus (PV) DNA in squamous cell carcinoma (SCC) tumor specimens from cats. SAMPLE POPULATION: 54 formalin-fixed paraffinembedded skin biopsy specimens were examined. Specimens originated from Bowenoid in situ SCC (BISC; n = 21), invasive SCC (22), and skin affected by miscellaneous nonneoplastic conditions (11). PROCEDURES: Samples from each tissue block underwent DNA extraction after deparaffinization, and PCR assays were performed. Two sets of primers derived from PV E1 were used. The first set of primers was designed for the narrow-range PCR assay and was able to generate amplification products of feline PV (FePV), canine oral PV, or closely related PVs. The second set of primers was selected for the broad-range PCR assay because of its ability to amplify DNA from 64 human PVs. Sequence analysis of each amplified DNA was performed. RESULTS: 1 of the 21 specimens of BISC was positive for PV DNA on the basis of narrow-range PCR assay results, whereas all the other specimens (BISC, invasive SCC, and controls) had negative results for PV DNA. In contrast, 5 of 21 BISC specimens and 4 of 22 invasive SCC specimens were positive for PV DNA on the basis of broad-range PCR assay results. Sequence analysis revealed that only 1 specimen was infected by a virus closely related to classic FePV. In the 8 other specimens positive for PV DNA, DNA of unknown PVs was uncovered. CONCLUSIONS AND CLINICAL RELEVANCE: Bowenoid in situ SCC and invasive SCC of cats may be associated with PVs of genetic diversity.