An evaluation of the Irish Single Reactor Breakdown Protocol for 2005-2008 inclusive and its potential application as a monitor of tuberculin test performance

The number of recognized papillomavirus (PV) species and potential PV genera has dramatically been increasing throughout the past decade. It seems that every host species might potentially harbour a large set of PVs, while the PVs of each species appear to belong to only a few genera. In horses at least three conditions beside the equine sarcoid have been described, being supposedly PV induced namely classical equine papillomas, genital papillomas and aural plaques. We were able to identify the DNA of novel equine PVs (EcPVs) in the two latter disorders where PV involvement had been predicted. Both PV genomes were entirely cloned and sequenced. Both EcPV genomes, one derived from a penile papilloma, the other derived from an ear papilloma contain the characteristic open reading frames (ORFs) E6, E7, E1, E2, L2 and L1, a large non-coding region between the late and early region as well as a small non-coding region between the early and the late region. The viruses were consequently designated as EcPV2 and EcPV3. The genomes of the three equine PVs were analysed and compared with each other and further PVs. Upon phylogenetic analyses the equine PVs group well together. Pairwise alignment of the L1 nucleotide sequences reveals that EcPV1 shares 54.9% identities with EcPV2 and 53.2% with EcPV3. EcPV2 and EcPV3 share 51.3% identities. As the three EcPVs share less than 60% of nucleotide identities in L1, they may be regarded as belonging to different genera.     

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.     

Evaluation of equine papillomas, aural plaques, and sarcoids for the presence of Equine papillomavirus DNA and Papillomavirus antigen

Immunohistochemical (IHC) testing and electron microscopy have implicated Papillomavirus (PV) as the etiologic agent for equine papillomas and aural plaques, but Equine papillomavirus (EPV) DNA has yet to be demonstrated in these lesions by polymerase chain reaction (PCR). The purpose of this study was to evaluate formalin-fixed, paraffin-embedded tissues from naturally occurring cases of equine papillomas, aural plaques, and sarcoids for the presence of EPV DNA by means of PCR and for the presence of PV antigen by means of IHC testing. We used EPV-specific primers that amplified a region of 384 base pairs (bp) spanning the E4 and L2 genes of the EPV genome and consensus PV primers that amplified a 102-bp region of the L1 gene. Group-specific PV structural antigens were detected with the use of a streptavidin-biotin-alkaline phosphatase IHC stain. With IHC testing, 23 of 38 papillomas, 4 of 9 aural plaques, and 0 of 10 sarcoids were positive for PV antigen; EPV DNA was found in 20 of the 38 papillomas and 1 of the 10 sarcoids but 0 of the 9 aural plaques. The consensus primers did not amplify novel PV DNA in any of the tissues. Nucleotide sequencing of viral DNA from 7 papillomas amplified with EPV-specific primers revealed DNA fragments that were 96% to 99% identical to known EPV sequences. Some samples had nucleotide substitutions in common, which suggests infection with related strains. Together, EPV DNA or PV antigen (or both) was demonstrated in 26 (68%) of the 38 equine papillomas. Although aural plaques contained PV antigen, they were negative for EPV DNA; therefore, we hypothesize that aural plaques contain a PV distinct from EPV.     

Equus caballus papillomavirus‐2 (EcPV‐2): An infectious cause for equine genital cancer?

Reasons for performing study: The aetiology of genital squamous cell carcinoma (SCC) in horses remains unknown, but the similarity to the disease in man, for which papillomavirus infection has been shown to be a causal factor, requires to be investigated in horses. Hypothesis: One or more novel papillomaviruses cause equine genital SCC and its associated premalignant lesions. Methods: DNA was extracted from samples of equine genital SCC and performed rolling circle amplification, in order to identify closed circular DNA viral genomes within the samples. The amplified DNA was subcloned and sequenced and the DNA sequence compared to that of other papillomavirus genomes. Using PCR primers developed from these genomic DNA sequences, studies were then carried out in order to identify the frequency at which the viral DNA could be identified in equine genital cancer samples from horses in both the UK, Australia and Austria. Finally, in situ hybridisation using specific probes developed from this DNA sequence were used to confirm the presence of the viral RNA sequences in the neoplastic cells in these lesions. Results: The full length genome of a novel papillomavirus species was characterised from the equine genital SCC tissue and termed Equus caballus papillomavirus‐2 (EcPV‐2). Viral DNA and RNA was identified in the genital tumour samples, but not in the adjacent histologically normal tissue. EcPV‐2 DNA could not be identified in equine ocular or nasal carcinomas or within the scrotal skin or in most smegma samples obtained from tumour‐free horses. Sequencing of amplicons, generated from the archived equine genital tumours, identified variations within E1 and E6 on DNA and predicted protein level. Conclusions: A novel papillomavirus, EcPV‐2, is likely to play a causal role in the pathogenesis of equine genital epithelial tumours. Potential relevance: Identification of a papillomavirus causal for genital carcinomas in horses may lead to development of a vaccine that could be used to prevent this serious disease in horses. This would be analogous to man, where vaccination against oncogenic papillomavirus species is currently being used to help prevent cervical cancer.     

Novel papillomavirus isolated from the oral mucosa of a polar bear does not cluster with other papillomaviruses of carnivores

Papillomatosis has been documented in several carnivores, and papillomavirus (PV) types have been characterized from lesions in a number of carnivore species: the canine oral PV (COPV), the Felis domesticus PV type 1 (FdPV-1) isolated from a Persian cat, the Procyon lotor PV type 1 (PlPV-1) isolated from a raccoon, the canine PV type 2 (CPV-2) from a dog's foot pad lesion and the canine PV type 3 (CPV-3) associated with a canine epidermodysplasia verruciformis - like disease. A tissue sample was taken from a papillomatous lesion on the oral mucosa of a polar bear (Ursus maritimus). Extracted DNA was used as a template for multiply primed rolling-circle amplification (RCA), and restriction enzyme analysis of the RCA product indicated the presence of papillomaviral DNA. The genome of this PV was cloned and the complete genomic sequence was determined. The Ursus maritimus PV type 1 (UmPV-1) genome counts 7582 basepairs and is smaller than that of other papillomaviruses from carnivore species. UmPV-1 contains the typical noncoding region NCR1, but unlike the carnivore PVs of the Lambda genus, UmPV-1 does not possess a second noncoding region NCR2. Phylogenetic analysis based on a nucleotide sequence alignment of the L1 ORF of UmPV-1 and 51 other PV types indicates that UmPV-1 does not cluster with any of the other carnivore PVs, but branches off near the root of the common branch of the genus Alphapapillomavirus.     

Infrequent detection of papillomaviral DNA within canine cutaneous squamous cell carcinomas, haemangiosarcomas and healthy skin on the ventrum of dogs

Background – Canine squamous cell carcinomas (SCCs) most frequently develop on the ventral abdomen and are thought to be caused by ultraviolet (UV) light. Papillomaviruses (PVs) have been associated with cutaneous SCCs in multiple species, including dogs. Hypothesis – That PVs act as cofactors in canine UV‐induced SCCs. Animals – The study was performed on skin from the ventrum of 60 dogs. These samples included 20 SCCs, 20 haemangiosarcomas and 20 samples of clinically normal skin. Two canine viral plaques were included as positive controls for PV. Methods – PCR was used to amplify PV DNA from all samples. Primers used included two sets of consensus primers and two sets of primers that were designed specifically to amplify PV DNA sequences detected in the viral plaques. Results – The MY09/11 consensus primers amplified PV DNA from both viral plaques. One plaque contained a DNA sequence (CfPV‐JM) that had been previously reported from a dog with multiple cutaneous SCCs. The other plaque contained a previously unreported PV DNA sequence. No PV DNA was amplified by either consensus primer from any of the ventrum skin samples. Primers designed specifically to amplify the CfPV‐JM sequence amplified DNA from one SCC, but no other sample. No PV DNA was amplified using the other specific PCR primer set. Conclusions and clinical importance – These results do not support a significant role for PVs in SCC development from the ventrum of dogs. However, they contribute another PV sequence to the list of PVs that have been associated with viral plaque development in dogs.     

The efficacy of imiquimod 5% cream (Aldara®) in the treatment of aural plaque in horses: a pilot open‐label clinical trial

Aural plaques affect at least 22% of horses and can be asymptomatic or cause ear sensitivity. Immunohistochemical and electron microscopy studies have shown a strong association between aural plaques and papilloma virus. The purpose of this study was to investigate the efficacy of imiquimod 5% cream, an immune response modifier with potent antiviral activity, in the treatment of equine aural plaques. Twenty‐one horses were enrolled and 16 completed the study. Imiquimod 5% cream was applied three times a week, every other week. When both ears were affected only the worst affected ear was treated. Adverse effects in all horses included marked local inflammation, exudation and thick crust formation at the site of treatment and the adjacent skin. Removal of the crust before treatment was painful and required sedation in most horses. Complete resolution of lesions was noted in all horses immediately post‐treatment and the long‐term resolution rate was 87.5%. Duration of therapy ranged from 1.5 to 8 months (median: 2.9 mean: 3.5). All horses were followed‐up for 12–22 months after treatment was discontinued and only two horses had a recurrence of lesions. Clinical signs related to the aural plaques prior to treatment were reported in 11 of 16 (68.8%) horses and included resistance to touching the ears and bridling. Complete resolution of these signs was reported by the owners in all of the horses followed‐up for at least 12 months. In conclusion, the topical application of imiquimod 5% cream is an efficacious treatment for aural plaques in horses.     

Co-occurrence of papillomas related to Equus caballus papillomavirus type 2 and cutaneous habronemiasis

The present report describes a case of cutaneous papillomatosis related to equine papillomavirus type 2 (EcPV2) with concomitant cutaneous habronemiasis. A 2-year-old female Pura Raza Española horse was referred for a dermatological evaluation because of the onset of cutaneous multifocal, nodular lesions on the face characterised by a warty and partially ulcerated overlying epidermis. Multiple biopsies were taken from these lesions, and cutaneous viral papillomas with a concomitant severe focal eosinophilic dermatitis with intralesional Habronematidae nematode larvae were diagnosed. Immunohistochemistry revealed papillomaviral capsid protein L1 in the epidermal proliferative lesion, confirming the presence of a productive infection. Rt-qPCR confirmed the presence of EcPV type 2. To the authors’ knowledge, this is the first report of EcPV2-related papillomas with concurrent habronemiasis.     

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.     

Novel betapapillomavirus associated with hand and foot papillomas in a cynomolgus macaque

Betapapillomavirus is a genus of papillomaviruses (PVs) commonly found in human skin and associated with both benign and malignant skin lesions. Only 2 previous beta-PVs have been fully characterized in nonhuman species. This report describes a novel beta-PV, named Macaca fascicularis PV type 2 (MfPV2), isolated from exophytic skin papillomas on the hands and feet of a 2-year-old male cynomolgus monkey (M. fascicularis). On histology the papillomas were composed of diffusely thickened epidermis with superficial foci of cytomegaly, cytoplasmic pallor, marginalized chromatin, and rare eosinophilic intranuclear inclusion bodies. Positive immunostaining for p16 and the proliferation marker Ki67 was present multifocally within affected epidermis, most prominently within basal-type cells. Complete sequence identity (100%) was noted between PV genomes fully sequenced from hand and foot lesions. The MfPV2 genome was 7632 base pairs in length and included putative open reading frames (ORFs) for E1, E2, E4, E6, E7, L1, and L2 genes, similar to other PVs. The closest relatives to MfPV2 based on the L1 ORF sequence were all beta-PVs. These included human PV (HPV) 9, HPV115, HPV76, HPV75, and MfPV1 (60-70% pairwise identity for all), the latter of which was also isolated from hand and foot papillomas in a cynomolgus macaque. Phylogenetic analysis placed MfPV2 in a new species group (beta-6), distinct from HPVs (beta-1 to beta-5) and MfPV1 (beta-1). These findings characterize a new nonhuman beta-PV and provide additional support for the idea that tissue tropism among ancestral primate PVs developed prior to divergence of certain Old World primate lineages.     

Detection of a novel papillomavirus in pigmented plaques of four pugs

Pugs are predisposed to the development of deeply pigmented, slightly elevated hyperkeratotic noncancerous plaques. Polymerase chain reaction amplification of a papillomavirus (PV)-like DNA fragment from such lesions suggested that PV may be responsible for them, although the predicted virus has not yet been identified. The goal of the present study was to make use of pigmented plaques from four pugs to identify and sequence the predicted virus. Taking advantage of the circular nature of PV DNA, the entire viral genome was amplified by rolling circle amplification and restriction enzyme analysis disclosed the same pattern in all four cases. Sequencing of one of the amplificates revealed a novel canine PV, termed CPV4, related to the recently described CPV3 but clearly distinct from canine oral PV and CPV2. Thus, a novel canine PV and a method for its future diagnosis are described.     

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.     

Evidence of host adaptation in Lawsonia intracellularis infections

Background

Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.

Materials and methods

Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10⁹L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.

Results

Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.

Conclusions

Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.     

Feline papillomas and papillomaviruses

Papillomaviruses (PVs) are highly species- and site-specific pathogens of stratified squamous epithelium. Although PV infections in the various Felidae are rarely reported, we identified productive infections in six cat species. PV-induced proliferative skin or mucous membrane lesions were confirmed by immunohistochemical screening for papillomavirus-specific capsid antigens. Seven monoclonal antibodies, each of which reacts with an immunodominant antigenic determinant of the bovine papillomavirus L1 gene product, revealed that feline PV capsid epitopes were conserved to various degrees. This battery of monoclonal antibodies established differential expression patterns among cutaneous and oral PVs of snow leopards and domestic cats, suggesting that they represent distinct viruses. Clinically, the lesions in all species and anatomic sites were locally extensive and frequently multiple. Histologically, the areas of epidermal hyperplasia were flat with a similarity to benign tumors induced by cutaneotropic, carcinogenic PVs in immunosuppressed human patients. Limited restriction endonuclease analyses of viral genomic DNA confirmed the variability among three viral genomes recovered from available frozen tissue. Because most previous PV isolates have been species specific, these studies suggest that at least eight different cat papillomaviruses infect the oral cavity (tentative designations: Asian lion, Panthera leo, P1PV; snow leopard, Panthera uncia, PuPV-1; bobcat, Felis rufus, FrPV; Florida panther, Felis concolor, FcPV; clouded leopard, Neofelis nebulosa, NnPV; and domestic cat, Felis domesticus, FdPV-2) or skin (domestic cat, F. domesticus, FdPV-1; and snow leopard, P. uncia, PuPV-2).     

Clinical, immunophenotypic and functional characterisation of T-cell leukaemia in six horses

REASON FOR PERFORMING STUDY: Lymphoid leukaemia (LL) is rare in equids. In man, immunophenotypic classification identifies distinct leukaemic types with different treatment strategies. Improved understanding and classification of equine LL may allow similar advances. OBJECTIVES: To document the clinical, immunophenotypic and functional characteristics in 6 cases of equine LL of T-cell origin. METHODS: The clinical records and pathological findings from 6 cases of equine LL were analysed. Immunohistochemistry to identify T or B lymphocytes was performed on paraffin embedded tissues in 4 cases. Peripheral blood mononuclear cells (PBMC) were phenotyped for expression of CD4, CD8, MHC class I and II and B-cell antigens in 4 cases using monoclonal antibodies (mAbs) and flow cytometry. Neoplastic lymphocytes from 4 horses were stimulated with mitogens. RESULTS AND CONCLUSIONS: Six horses of various breeds were identified with LL of T-cell origin. The clinical course and presenting signs varied. Neoplastic lymphocytes were identified in peripheral blood samples from all horses and tissue invasion was confirmed at examination post mortem in 4 horses. Immunophenotyping identified a predominance of CD3+ T-cells in lymphoid tissues and CD4+ T-cells in circulating peripheral blood mononuclear cells (PBMC) in the affected horses. Neoplastic lymphocytes from the 4 cases that were tested failed to proliferate in response to mitogens. POTENTIAL RELEVANCE: Characterisation of the clinical, pathological and immunological findings in 6 horses with LL has added to reports of this rare condition, characterised it in greater detail and therefore provides a starting point for further investigations.     

Enthesophytosis and Impingement of the Dorsal Spinous Processes in the Equine Thoracolumbar Spine

Impingement of the dorsal spinous processes (DSPs) is a common cause of pain and poor performance in sport horses, but there is limited information regarding regional differences in the prevalence and severity of DSP osseous lesions in the equine thoracolumbar spine. It was hypothesized that lesion severity would increase with horse age and height, and that severe lesions would be more prevalent in the mid-caudal thoracic region. The thoracolumbar spines of 33 horses were removed postmortem, disarticulated, and boiled out. The thoracic and lumbar DSPs were examined for the presence of proliferative or lytic osseous lesions of the DSPs. Age and height of the horses were recorded, and severity of pathologic changes at each vertebral level was scored using an ordinal grading system (grades 0–3) and a continuous visual analog scale (VAS). Osseous lesions of the DSPs were present at every vertebral level from C7–T1 to L6–S1, and 70% of horses had at least one lesion of severity grade 2 or higher. Grade 3 lesions were found in the cranial thoracic (T2–T4), mid-thoracic to cranial lumbar (T11–L1) and mid-lumbar (L4–L5) segments. Analysis of VAS data using analysis of variance indicated that increasing age and height were associated with more severe osseous lesions (P < .001). DSP osseous lesions occur frequently in horses with more severe lesions in the cranial thoracic, mid to caudal thoracic, and mid-lumbar regions. Lesions in the cranial thoracic and lumbar regions present a challenge for diagnostic imaging and may be underdiagnosed clinically.     

Characterization of a novel papillomavirus species (ZcPV1) from two California sea lions (Zalophus californianus)

A seven-year old California sea lion (Zalophus californianus) presented with focally extensive, bilaterally symmetric, proliferative axillary skin lesions and preputial lesions. A second California sea lion in the same population presented with similar proliferative lesions on the underside of the tail. Histopathology revealed epidermal hyperplasia with severe hyperkeratosis, with proliferating keratinocytes forming broad, branching pegs that extended into the dermis. Pan-papillomaviral consensus PCR was used to obtain initial E1 sequence template and the complete genome was determined using a combination of rolling circle amplification and specific-primer PCR. Analysis revealed a novel papillomavirus, Zalophus californianus papillomavirus 1 (ZcPV1), with seven open reading frames encoding five early proteins (E6, E7, E1, E2 and E4) and two late proteins (L1 and L2). Phylogenetic analysis revealed that (ZcPV1) is most closely related to Equine papillomavirus 1 (EcPV1) in the genus Zetapapillomavirus, and Canine papillomaviruses 3 and 4 (CPV3, CPV4) in the genus Chipapillomavirus. The lesions regressed without intervention over a period of several months.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

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.