Detection of human papillomavirus DNA in feline premalignant and invasive squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most common malignant cutaneous and oral neoplasm of cats. Papillomavirus (PV) DNA has been identified in a proportion of feline Bowenoid in situ carcinomas (BISCs), cutaneous SCCs and a single oral SCC, but its exact role in the pathogenesis remains unknown. In humans, it has been suggested that ultraviolet (UV) light and human PV (HPV) may act as cofactors in cutaneous SCC carcinogenesis. Little is known about the influence of UV light on PV prevalence in feline cutaneous lesions, including actinic keratosis (AK). Additionally, PV prevalence in noncutaneous feline lesions, including oral SCC, is largely not known. This study aimed to determine the presence of PV in 84 cats with premalignant and invasive SCC from cutaneous and noncutaneous sites using polymerase chain reaction and to investigate an association with UV light. Papillomaviral DNA was amplified from two of 12 cases of AK, seven of 22 BISCs, nine of 39 cutaneous SCCs and two of 35 non‐cutaneous SCCs. Of the PV DNA sequenced, 50% was most similar to HPV of the genus Betapapillomavirus, while the other 50% was most similar to Felis domesticus PV type 2. Exposure to UV was not associated with an increase in PV for cutaneous SCC. The results of this study suggest that in the cat, HPV DNA may be detectible within a higher percentage of squamous lesions than previously demonstrated, UV exposure may not be a confounder for PV presence, and noncutaneous lesions may have a low prevalence of PV.     

Amplification of papillomaviral DNA sequences from a high proportion of feline cutaneous in situ and invasive squamous cell carcinomas using a nested polymerase chain reaction

Squamous cell carcinomas (SCCs) are common skin tumours of cats. Previous studies have suggested that papillomaviral (PV) DNA is detectible within some feline SCCs. A PV DNA sequence has been previously amplified from five feline bowenoid in situ carcinomas (BISCs). Primers specific for this sequence were used in a nested polymerase chain reaction to compare PV detection rates in SCCs to rates within non-SCC skin lesions. Papillomaviral DNA was amplified from 20 of 20 BISC, 17 of 20 invasive SCC and 3 of 17 non-SCC controls. The rate of PV amplification from feline cutaneous SCCs was significantly higher than from non-SCC lesions. These results confirm that feline cutaneous SCCs are associated with PV infection. In humans, there is evidence that PVs promote SCC development within sun-exposed skin. The demonstrated association between PVs and feline cutaneous SCCs suggests, but does not prove, that PVs may also promote feline SCC development. If PVs are oncogenic in cats, prevention of PV infection may reduce feline cutaneous SCC development. To the authors' knowledge, this is the first time that PV DNA has been amplified from a non-SCC sample of feline skin.     

Papillomaviral DNA and increased p16CDKN2A protein are frequently present within feline cutaneous squamous cell carcinomas in ultraviolet-protected skin

Squamous cell carcinomas (SCCs) are common feline skin tumours. While exposure to ultraviolet (UV) light causes some SCCs, a subset develop in UV-protected skin. In cats, papillomaviruses (PVs) cause viral plaques and Bowenoid in situ carcinomas (BISCs). As both may progress to SCC, it was hypothesized that SCCs in UV-protected skin may represent neoplastic transformation of a PV-induced lesion. To investigate this hypothesis, PCR was used to amplify PV DNA from 25 UV-protected and 45 UV-exposed SCCs. Oncogenic human PVs cause neoplasia by mechanisms that also increase p16(CDKN2A) protein (p16). As increased p16 is present in feline viral plaques and BISCs, immunohistochemistry was used to detect p16 within the SCCs. Papillomaviral DNA was amplified from 76% of UV-protected SCCs, but only 42% of UV-exposed SCCs. Increased p16 was present in 84% of UV-protected SCCs, but only 40% of UV-exposed SCCs. The more frequent detection of PV DNA and increased p16 within UV-protected SCCs supports the hypothesis that some develop from a PV-induced plaque or BISC. Felis domesticus PV-2 is thought to cause viral plaques and BISCs. This PV was detected most frequently within the UV-protected SCCs, supporting development from a PV-induced lesion. Increased p16 and PV DNA were less frequent within UV-exposed SCCs, presumably because these developed from actinic keratosis rather than a PV-induced lesion. The results support the hypothesis that some feline cutaneous SCCs are caused by PV infection and suggest that PVs may cause neoplasia by mechanisms that also increase p16.     

Frequent detection of papillomavirus DNA in clinically normal skin of cats infected and noninfected with feline immunodeficiency virus

Feline cutaneous squamous cell carcinomas (SCCs) often contain felis domesticus papillomavirus type 2 (FdPV‐2) DNA. While this may suggest FdPV‐2 causes feline SCC development, the proportion of cats that are asymptomatically infected by this PV is unknown. Infection by feline immunodeficiency virus (FIV) is associated with high rates of cutaneous SCC development, possibly due to increased PV infection. This study examines the frequency of cutaneous asymptomatic FdPV‐2 infections in cats and compares the rate of FdPV‐2 infection in 22 FIV‐positive cats with that in 22 FIV‐negative cats. FdPV‐2 sequences were detected in 39% of skin swabs. One or both swabs contained FdPV‐2 DNA from 52% of the cats. FIV status, age or sex of the cat did not significantly influence FdPV‐2 infection. Cats that shared a household with a PV‐infected cat could remain uninfected suggesting infection depends more on host factors than exposure to the PV. These results indicate that asymptomatic FdPV‐2 infections are common in cats, but do not provide evidence that FdPV‐2 causes feline SCC development.     

Detection of novel papillomaviruses in canine mucosal, cutaneous and in situ squamous cell carcinomas

Papillomavirus (PV) DNA is frequently uncovered in samples of human skin squamous cell carcinomas (SCC). However, the role of these viruses in the development of such cancers in canine species remains controversial. While approximately 100 human PVs are known, only one single canine oral PV (COPV) has been identified and studied extensively. Therefore, we applied a narrow-range polymerase chain reaction (PCR) suitable for the detection of classical canine and feline PVs, as well as a broad-range PCR, which has been used for the detection of various novel PVs in humans, in order to analyse 42 paraffin-embedded samples, representing three different forms of canine SCCs. Ten samples of skin tissues with various non-neoplastic conditions served as controls. While none of the negative controls reacted positively, PV DNA was discovered in 21% of the tested SCC samples. Interestingly, the classical COPV was amplified from only one sample, while the other positive cases were associated with a variety of thus far unknown PVs. This study suggests that a fraction of canine SCC is infected with PVs and that a genetic variety of canine PVs exists. Therefore, these results will facilitate the future study of the role of PVs in the development of canine skin cancers.     

Cutaneous squamous cell carcinomas in domestic rabbits (Oryctolagus cuniculus): 39 cases (1998-2019)

BackgroundDomestic rabbits (Oryctolagus cuniculi) can develop a variety of cutaneous neoplasms, including squamous cell carcinoma (SCC). A detailed review of gross and microscopic pathology and response to treatment of spontaneously arising SCCs in domestic rabbits has not been published.MethodsA retrospective survey study of the clinical characteristics and response to treatment in 39 cases of spontaneous SCC in pet rabbits was performed in an attempt to better characterize the typical presentation, prognosis, and therapeutic response of SCCs in domestic rabbits. Sixteen of these cases were also selected for papillomavirus testing using a generic polymerase chain reaction.ResultsSCC was identified in rabbits between 2 and 10 years of age, with a median age of 7 years. The neoplasm has a predilection for ears and feet and the conventional subtype is most commonly diagnosed microscopically. Lighter colored rabbits may be predisposed to developing SCC. The majority of cases examined were found in rabbits housed primarily indoors. Only one SCC tested positive for papillomavirus and was located in the oral cavity. Sequencing of the detected PCR product detected 98.75% similarity to human papillomavirus type 120. The significance of this virus for tumorigenesis is unknown.Conclusions and clinical relevanceAggressive surgical resection provided the most successful therapeutic option and proved curative in 12 of 23 rabbits. Papillomavirus likely does not play a major role in the development of spontaneous SCCs in pet rabbits. More research is needed to investigate the use of adjunctive therapies in treatment of this neoplasm in pet rabbits.     

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.     

A feline hemoplasma, 'Candidatus Mycoplasma haemominutum', detected in dog in Japan

We examined for 'Candidatus Mycoplasma haemominutum' infection in 167 blood samples collected from domestic dogs between 2008 and 2009 in the Tohoku area, Japan, and found 5 (3.0%) were positive by PCR assay. This is the first demonstration of 'Candidatus Mycoplasma haemominutum', a feline haemotropic mycoplasma, in the dogs raised in Japan.     

Identification of bap and icaA genes involved in biofilm formation in coagulase negative staphylococci isolated from feline conjunctiva

Bap and icaA genes are commonly known to be involved in the biofilm formation. The prevalence of bap and icaA genes and biofilm formation was determined in conjunctival isolates of coagulase negative staphylococci (CNS) collected from cats. The study was conducted on 90 archival CNS isolates collected from feline conjunctiva obtained from clinically healthy cats and cats with ocular problems. Biofilm formation was examined using the microtiter plate (MTP) method. The prevalence of icaA and bap genes was determined using polymerase chain reaction (PCR). Genetic profiles of the bap-positive isolates were examined using the modified random amplified polymorphic DNA (RAPD) method. Of the 90 CNS isolates investigated, 58.9 % (53/90) were confirmed to form biofilms on a polystyrene plate after 24 h, and the intensity of the biofilm production varied strongly between positive strains. Among the biofilm-producing isolates, 24.5 % (13/53) carried the icaA gene and 3.8 % (2/53) carried the bap gene. Among the isolates that did not produce biofilms, the icaA gene and bap gene were detected in 8.1 % (3/37) and 2.7 % (1/37) of isolates, respectively. This is the first report demonstrating that CNS isolated from feline conjunctiva can potentially be a bap gene reservoir. Preliminary comparison of the genetic profiles of three bap-positive isolates collected from cats showed that each of the isolates has a different genetic background with a high similarity with the human strain of S. epidermidis.     

Prevalence and identity of cdt-related sequences in necrotoxigenic Escherichia coli

The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in necrotoxigenic E. coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. coli.     

EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa

Squamous cell carcinoma (SCC) represents the most common genital malignant tumor in horses. Similar to humans, papillomaviruses (PVs) have been proposed as etiological agents and recently Equine papillomavirus type 2 (EcPV2) has been identified in a subset of genital SCCs. The goals of this study were (1) to determine the prevalence of EcPV2 DNA in tissue samples from equine genital SCCs, penile intraepithelial neoplasia (PIN) and penile papillomas, using EcPV2-specific PCR, (2) to examine the prevalence of latent EcPV2 infection in healthy genital mucosa and (3) to determine genetic variability within EcPV2 and to disentangle phylogenetic relationships of EcPV2 among PVs. EcPV2 DNA was detected in all but one penile SCC (15/16), in all PIN lesions (8/8) and penile papillomas (4/4). Additionally, EcPV2 DNA was demonstrated in one of two metastasized lymph nodes, one contact metastasis in the mouth, two vaginal and one anal lesion. In healthy horses, EcPV2 DNA was detected in 10% (4/39) of penile swabs but in none of vulvovaginal swabs (0/20). This study confirms the presence of EcPV2 DNA in equine genital SCCs and shows its involvement in anal lesions, a lymph node and contact metastases. Latent EcPV2 presence was also shown in normal male genital mucosa. We found that different EcPV2 variants cocirculate among horses and that EcPV2 is related to the Delta+Zeta PVs and is only a very distant relative of high-risk human PVs causing genital cancer. Thus, similar viral tropism and similar malignant outcome of the infection do not imply close evolutionary relationship.     

Normal somatic cell count and subclinical mastitis in Murrah buffaloes

This study was conducted to investigate the normal somatic cell count (SCC) and to define subclinical mastitis in Murrah buffaloes. Data were collected from 60 clinically normal buffaloes stationed at five farms of Chitwan Nepal and Buffalo Research Center, Hissar, India. Somatic cell count was measured using the Newman-Lampert staining technique. The upper limit of SCC was determined >or=200 000/ml of milk based on the mean +/- 2SD of a total SCC. Abnormal data of the SCC was repeatedly removed, which lie beyond the values of more than mean + 2SD until all the data come to lie within (mean + 2SD). Averages of SCC of right front and right hind quarters were significantly higher than left front and left hind quarters. Nearly 94% of California mastitis test (CMT) negative quarters were having somatic cells >or=200 000/ml. The mean SCC of CMT positive quarter was significantly higher (P < 0.01) than CMT negative quarters. Subclinical mastitis was diagnosed on the basis of samples with SCCs >or=200 000/ml with positive bacterial cultures. Subclinical mastitis was found in 21.7% buffaloes and 8% of the quarter foremilk samples. Neutrophil counts were significantly higher in subclinical mastitis milk.     

Prevalence of selected rickettsial infections in cats in Southern Germany

Prevalence of Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia DNA in blood of 479 cats collected in different veterinary clinics in Southern Germany was determined using a previously published conventional PCR using 16S-23S intergenic spacer primers (5′ CTG GGG ACT ACG GTC GCA AGA C 3′ – forward; 5′ CTC CAG TTT ATC ACT GGA AGT T 3′ – reverse). Purified amplicons were sequenced to confirm genus and species. Associations between rickettsial infections, and feline immunodeficiency virus (FIV), as well as feline leukemia virus (FeLV) status were evaluated. Rickettsial prevalence was 0.4% (2/479; CI: 0.01–1.62%). In the two infected cats, Anaplasma phagocytophilum DNA was amplified. These cats came from different environment and had outdoor access. Both were ill with many of their problems likely related to other diseases. However, one cat had neutrophilia with left shift and the other thrombocytopenia potentially caused by their A. phagocytophilum infection. There was no significant difference in the FIV and FeLV status between A. phagocytophilum-negative and -positive cats. A. phagocytophilum can cause infection in cats in Southern Germany, and appropriate tick control is recommended.     

Canine parvovirus in asymptomatic feline carriers

Canine parvovirus (CPV) and feline panleukopaenia virus (FPLV) are two closely related viruses, which are known to cause severe disease in younger unvaccinated animals. As well as causing disease in their respective hosts, CPV has recently acquired the feline host range, allowing it to infect both cats and dogs. As well as causing disease in dogs, there is evidence that under some circumstances CPV may also cause disease in cats. This study has investigated the prevalence of parvoviruses in the faeces of clinically healthy cats and dogs in two rescue shelters. Canine parvovirus was demonstrated in 32.5% (13/50) of faecal samples in a cross sectional study of 50 cats from a feline only shelter, and 33.9% (61/180) of faecal samples in a longitudinal study of 74 cats at a mixed canine and feline shelter. Virus was isolated in cell cultures of both canine and feline origin from all PCR-positive samples suggesting they contained viable, infectious virus. In contrast to the high CPV prevalence in cats, no FPLV was found, and none of 122 faecal samples from dogs, or 160 samples collected from the kennel environment, tested positive for parvovirus by PCR. Sequence analysis of major capsid VP2 gene from all positive samples, as well as the non-structural gene from 18 randomly selected positive samples, showed that all positive cats were shedding CPV2a or 2b, rather than FPLV. Longitudinally sampling in one shelter showed that all cats appeared to shed the same virus sequence type at each date they were positive (up to six weeks), despite a lack of clinical signs. Fifty percent of the sequences obtained here were shown to be similar to those recently obtained in a study of sick dogs in the UK (Clegg et al., 2011). These results suggest that in some circumstances, clinically normal cats may be able to shed CPV for prolonged periods of time, and raises the possibility that such cats may be important reservoirs for the maintenance of infection in both the cat and the dog population.     

Relationships between the phagocytic ability of milk macrophages and polymorphonuclear cells and somatic cell counts in uninfected cows

Phagocyte numbers and activities were compared in milk from 2 groups of uninfected mammary-gland quarters from 3 cows each: 6 quarters with a high (> or = 200 000/mL) somatic cell concentration (SCC), analyzed as 4 individual quarters and 1 pooled sample; and 12 quarters with a low SCC (< 200 000/mL), analyzed as 6 paired samples. The concentrations and ability of macrophages and polymorphonuclear (PMN) cells to phagocytize fluorescent microspheres were determined by flow cytometry after exposure of the cells to the microspheres. The macrophages and PMNs contained 2 major subpopulations, characterized by low phagocytic (LP) or high phagocytic (HP) ability. The quarters with high SCCs had significantly lower percentages of HP cells than did the quarters with low SCCs (P < 0.01). Whether mammary-gland quarters or cows were the unit of analysis, the HP/LP ratio was negatively related to the SCC (P < 0.04), which explained more than 50% of the SCC variability. Thus, poor bovine mammary-gland phagocytic ability may be associated with high SCC. Longitudinal studies are suggested to further explore and characterize these relationships.     

Prevalence and anatomopathological characterization of cutaneous squamous cell carcinomas with regional and distant metastases in dogs and cats: 20 cases (1985–2020)

The aim of this study was to assess the prevalence of regional and distant metastases from cutaneous squamous cell carcinomas (SCCs) in dogs (n = 11) and cats (n = 9) in a retrospective case series over 36 years (1985–2020), as well as to characterize its macroscopic aspects (location and size), degree of differentiation (well, moderately and poorly differentiated [WD, MD and PD, respectively]) and the rate of cell proliferation, by counting the AgNORs. Immunohistochemistry (IHC) was used to identify patterns of tumour migration and invasion (islands, ribbons, cords, small aggregates, individual cells [fusiform and amoeboid]) and to evaluate the intensity of desmoplasia and the amount of myofibroblasts. The prevalence of metastatic SCCs was 4.39% (21/478), being 3.8% in dog (12/309) and 5.3% in cat (9/169). Metastases affected lymph nodes in all dogs and 66% (6/9) of cats, and less frequently distant organs. Primary tumours predominantly affected the abdominal skin in dogs and the nasal planum in cats. Among the 20 cases, 52% were MDs, 34% were WDs, and 14% were PDs. Histological lesions suggestive of exposure to chronic solar radiation were present in 57% (8/14). The main patterns of tumour migration and invasion were islands for WD SCCs and individual cells for PD SCCs. MD SCCs had a mix of patterns. In cats, individual spindle cells were restricted to PDs. A marked desmoplastic reaction was more associated with PD SCCs and often with MDs. This study highlights that the prevalence of SCC metastases in dogs and cats is predominantly regional. The IHC was essential in the identification of individual fusiform keratinocytes, whose presence in surgical margins may represent a greater risk of recurrence. Although the presence of myofibroblasts was observed in all infiltrative and metastatic tumours, further studies evaluating these cells may be important to better understand their role in the tumour microenvironment of cutaneous SCCs with metastasis in dogs and cats.     

Molecular detection and genotype identification of E. cuniculi from pet rabbits

Encephalitozoon cuniculi is a microsporidian which is frequently reported from rabbits. This microorganism can either ravage rabbit farms or transmit to humans from pet rabbits. This study aimed to investigate the prevalence and the genotype distribution of E. cuniculi among pet rabbits. In this study urine samples were collected from 50 pet rabbits, aged 2 months to 3 years, admitted to teaching veterinary hospital. Four races Lop, Dutch, Mix, and Angora were screened for E. cuniculi. The clinical symptoms were recorded and total DNA was extracted from urine samples. E. cuniculi was identified using amplification of the small subunit ribosomal RNA (ssu rRNA) gene and its genotypes were characterized using PCR/sequencing of the polar tube protein (PTP) gene. Phylogenetic tree was drawn to confirm the characterized genotypes. Out of 50 samples, 41 (82 %) of rabbits were asymptomatic, while nine (18 %) had at least one of symptoms including head-tilt, circling, and ataxia. A statistical correlation was seen between mean age + SD and symptoms (P-value = 0.039). The presence of E. cuniculi was confirmed in 16/50 (32 %) rabbits and all of them were identified as the genotype I. Our findings represented no consistency between E. cuniculi PCR – positive and the presence of symptoms (P-value = 0.318). Our results showed positive correlation between symptoms and age; however, the lack of correlation between PCR results with age may signify the latent infection in younger rabbits. All identified E. cuniculi were the genotype I, which is reported from rabbits and humans, highlighting the zoonotic concern for this genotype, particularly among subjects who keep pet rabbits.