Clinical, histopathological, and molecular characterization of canine pigmented viral plaques.

Canine pigmented viral plaques (PVPs) are proliferative epidermal lesions caused by canine papillomaviruses (CPVs). Although the lesions are benign, neoplastic transformation has been reported. Cases reported in the literature are few and mainly focused on genome sequencing. The aim of this study was to collect data on the epidemiology, clinicopathological features, and genotyping of PVPs. Fifty-five canine PVPs were retrospectively retrieved and histologically evaluated. Follow-up was available for 33 cases. The median age was 6.5 years and pugs were the most represented breed (25%). There were 4 clinical presentations: a single lesion (24%), multiple lesions (75%) in one (41%) or different sites (34%), and generalized lesions all over the body (24%). The abdomen and axillae were the most common sites. In single lesions, no recurrence was observed after conventional surgery, whereas different medical treatments reported for multiple lesions were not successful. Spontaneous regression was reported in 3 cases. Neoplasia in contiguity with PVPs was seen in 5 of 55 lesions (9%), and 1 dog was euthanized due to invasive squamous cell carcinoma (SCC). The most useful histopathological features for diagnosis were scalloped profile, epidermal spikes, hypergranulosis, and hyperpigmentation. L1 immunolabeling was present in 14 of 16 cases (87%). Sequencing revealed that 10 of 16 cases were associated with CPV-9 (71%), 2 cases were associated with CPV-4 (14%), and 2 cases were associated with CPV-8 (14%). In conclusion, this represents a large cohort study on canine PVPs reporting data on clinicopathological features, therapy, outcome, and the type of CPV involved for the first time in Italy.

Feline Parvovirus Lethal Outbreak in a Group of Adult Cohabiting Domestic Cats.

Feline panleukopenia is a highly contagious and often fatal disease in cats. The virus, known as feline panleukopenia virus (FPV), primarily affects kittens and unvaccinated cats. It is transmitted through contact with infected cats or their bodily fluids, as well as contaminated objects and environments. The diagnosis of FPV infection can be confirmed through a combination of clinical signs, blood tests, and fecal testing. Prevention through vaccination is recommended for all cats. This case report describes an outbreak of feline panleukopenia in a group of unvaccinated domestic cats that resulted in acute mortality. The lesions were evaluated using histopathology, and the specific viral strain was characterized using molecular techniques. The clinical course of the outbreak was peracute, with a hemorrhagic pattern and 100% of lethality. The observed clinical-pathological pattern was unusual; nevertheless, molecular studies did not highlight peculiar genomic features of the parvovirus isolate. The outbreak affected 3 out of 12 cats in a very short time. However, the prompt application of biosecurity measures and vaccination resulted in an effective interruption of virus spread. In conclusion, we could assume that the virus found the ideal conditions to infect and replicate at high titers, resulting in a particularly aggressive outbreak.

Contamination of Zophobas morio Larvae Rearing Substrate with Listeria monocytogenes: A Preliminary Study.

The interest in edible insects is continuously increasing due to their environmental, nutritional, and productive features. The aim of this study was to evaluate the effects and survival of Listeria monocytogenes in Zophobas morio rearing, using two different bacterial loads (2 and 6 log CFU/g). We also considered the effect of washing, fasting, and cooking treatments on the larvae. During the experimental trial, no mortality was observed among the larvae. The L. monocytogenes loads decreased over time, and it was below the detection limit for crates inoculated with 2 log CFU/g, while the substrate inoculated with 6 log CFU/g reached loads of 4.26 (without larvae) and 2.83 log CFU/g (with larvae). Due to the absence of L. monocytogenes in the larvae on day seven, it was not possible to assess the fasting treatment or the washing and the cooking. However, when looking at the total microbial count, significant effects were revealed for all treatments. The unfasted larvae showed no effect of washing, while the total microbial counts decreased after washing in the fasted larvae. This proves that fasting is a good treatment in terms of hygiene assurance for the consumer.

Serological, Virological Investigation and Hepatic Injury Evaluation for Hepatitis E Virus in Hunting Dogs.

Hepatitis E virus (HEV) is a quasi-enveloped single-stranded positive-sense RNA virus belonging to the Orthohepevirus A genus within the Hepeviridae family. The most common transmission route of this virus is fecal-oral, although zoonotic transmission by contact with infected animals has also been described. In this study, 80 sera and rectal swabs were collected from dogs during the 2019/2020 and 2020/2021 wild boar hunting season in Tuscany. All dogs were submitted for serological screening to detect the presence of anti-HEV antibodies. To evaluate the circulation of HEV, rectal swabs from both seropositive dogs and dogs living in the same kennels were examined by One-Step RT-qPCR. In addition, the presence of markers of hepatic damage in dogs' sera was investigated. Results indicated the presence of anti-HEV antibodies in 4/80 subjects (5%). However, neither HEV RNA nor signs of hepatic damage were found. In conclusion, although HEV can stimulate a specific immuno-response in dogs, this species does not seem to play an important role in HEV epidemiology.

Molecular and Pathological Detection of Hepatitis E Virus in Roe Deer (Capreolus capreolus) and Fallow Deer (Dama dama) in Central Italy.

Hepatitis E virus (HEV) is a common causative agent of acute hepatitis in the world, with a serious public health burden in both developing and industrialized countries. Cervids, along with wild boars and lagomorphs, are the main wild hosts of HEV in Europe and constitute a documented source of infection for humans. The aim of this study was to evaluate the presence of HEV in roe deer (Capreolus capreolus) and fallow deer (Dama dama) living in Tuscany, Central Italy. Liver samples from 48 roe deer and 60 fallow deer were collected from carcasses during the hunting seasons. Following the results obtained from molecular and histopathologic studies, 5/48 (10.4%) roe deer and 1/60 (1.7%) fallow deer liver samples were positive for the presence of HEV RNA. All PCR-positive livers were also IHC-positive for viral antigen presence, associated with degenerative and inflammatory lesions with predominantly CD3+ cellular infiltrates. This study represents the first identification in Italy of HEV RNA in roe and fallow deer and the first study in literature describing liver alterations associated with HEV infection in cervids. These results demonstrate that HEV is present in wild cervid populations in Italy and confirm the potential zoonotic role of these species.

Antiviral effect of a commercially phytotherapeutic compound against feline immunodeficiency virus.

The feline immunodeficiency virus (FIV) is a widespread lentivirus of felids. Due to its worldwide diffusion and the lack of an effective preventive and therapeutic protocol, it has a high impact on the cats' health. Several therapeutical protocols have been proposed, among those, phytotherapeutic compounds have been tested with the purpose to find a possible natural treatment. The most studied active compounds are derived from Ganoderma lucidum, Cordyceps sinensis, and Trametes versicolor. The present study aims to investigate in vitro antiviral effects of a commercially available compound HELP-TH1 (Camon, S.p.A., Italy) against FIV. The antiviral effect of HELP-TH1 was evaluated by quantifying and comparing the viral load of control groups, infected and not-treated cells, vs both experimental groups, infected and treated cells. Our data indicate that HELP-TH1 reduce the viral load in the experimental conditions demonstrating its antiviral effect.

In vitro anthelmintic activity of an aqueous extract of Glycyrrhiza glabra and of glycyrrhetinic acid against gastrointestinal nematodes of small ruminants.

This study evaluated the in vitro anthelmintic activity of a liquorice (Glycyrrhiza glabra) root aqueous extract and of glycyrrhetinic acid at 30, 10, 5, 1, and 0.5 mg/mL against sheep gastrointestinal nematodes (GINs), using the egg hatch test (EHT), the larval development test (LDT), and the larval migration inhibition test (LMIT). The compounds were applied on a mixture of GIN eggs and larvae, mainly Trichostrongylus spp. and Teladorsagia/Ostertagia spp. Cytotoxicity assays were also performed. In the EHT, both candidates showed significant concentration-dependent efficacy and were significantly more effective (p < 0.001) at the highest concentrations (30 and 10 mg/mL) than the lowest ones. In the LDT, only G. glabra showed a concentration-dependent effect (R2 = 0.924), but glycyrrhetinic acid (R2 = 0.910) had significantly higher efficacy than G. glabra root extract. Moreover, the efficacy of glycyrrhetinic acid at 30, 10, and 5 mg/mL was significantly higher (p < 0.001) than at lower concentrations. In the LMIT, G. glabra showed concentration-dependent efficacy (R2 = 0.971), while considerably reduced efficacy was observed for glycyrrhetinic acid (R2 = 0.855) at the lowest concentrations. These data suggest that the two compounds may have different mechanisms of action. In the LMIT, the 50% lethal concentration (LC50) of glycyrrhetinic acid (~5.12 mg/mL) was > 2.0-fold lower when compared to G. glabra (12.25 mg/mL). Analysis and previous findings indicated low toxicity for both compounds. The results obtained encourage in vivo studies aimed at evaluating the potential use of the tested compounds as natural de-wormers in ruminants.

TITLE: Activité anthelminthique in vitro d'un extrait aqueux de Glycyrrhiza glabra et de l'acide glycyrrhétinique contre les nématodes gastro-intestinaux des petits ruminants. ABSTRACT: Cette étude a évalué l'activité anthelminthique in vitro d'un extrait aqueux de racine de réglisse (Glycyrrhiza glabra) et de l'acide glycyrrhétinique à 30, 10, 5, 1 et 0,5 mg/mL contre les nématodes gastro-intestinaux (NGI) du mouton, en utilisant le test d'éclosion des œufs (TEO), le test de développement larvaire (TDL) et le test d'inhibition de la migration larvaire (TIML). Les composés ont été appliqués sur un mélange d'œufs et de larves de NGI, principalement Trichostrongylus spp. et Teladorsagia/Ostertagia spp. Des tests de cytotoxicité ont également été effectués. Dans le TEO, les deux candidats ont montré une efficacité concentration-dépendante significative et ont été significativement plus efficaces (p < 0,001) aux concentrations les plus élevées (30 et 10 mg/mL) qu'aux plus faibles. Dans le TDL, seul G. glabra a montré un effet concentration-dépendant (R2 = 0,924), mais l'acide glycyrrhétinique (R2 = 0,910) avait une efficacité significativement plus élevée que l'extrait de racine de G. glabra. De plus, l'efficacité de l'acide glycyrrhétinique à 30, 10 et 5 mg/mL était significativement plus élevée (P < 0,001) qu'à des concentrations plus faibles. Dans le TIML, G. glabra a montré une efficacité concentration-dépendante (R2 = 0,971), tandis qu'une forte réduction d'efficacité a été observée pour l'acide glycyrrhétinique (R2 = 0,855) aux concentrations les plus faibles. Ces données peuvent suggérer que les deux composés peuvent avoir des mécanismes d'action différents. Dans le TIML, la concentration létale à 50% (CL50) de l'acide glycyrrhétinique (~ 5,12 mg/mL) était > 2,0 fois inférieure à celle de G. glabra (12,25 mg/mL). L'analyse et les résultats précédents ont indiqué une faible toxicité pour les deux composés. Les résultats obtenus encouragent les études in vivo visant à évaluer l'utilisation potentielle des composés testés ici comme anthelminthique naturels chez les ruminants.

First seroprevalence investigation of epizootic haemorrhagic disease virus in Libya.

Background: Epizootic haemorrhagic disease (EHD) is a vector-borne viral disease of domestic and wild ruminants. Epizootic haemorrhagic disease virus (EHDV) is transmitted by Culicoides spp. EHDV is a member of the Orbivirus genus within the Reoviridae family. It shares many morphological and structural characteristics with other members of the genus, such as the bluetongue virus, African horse sickness virus, and equine encephalosis virus. Aims: The purpose of our study was to investigate the epidemiological situation of EHDV in Libya in order to gain some knowledge about the presence of this virus in the country. Methods: In this study, we investigated the seroprevalence of EHDV in Libya, testing 855 blood samples collected during 2015. The samples were collected from domestic ruminants (cattle, sheep, and goats) originating from 11 provinces of Libya. Sera were tested by competitive enzyme-linked immunosorbent assays and positive samples confirmed by serum neutralization test. Results: The overall seroprevalence of EHDV was estimated to be 4% (95% confidence intervals = 2.8%-5.4%). Small ruminant seroprevalence was significantly (p = 0.016) higher than that found in cattle. Neutralizing antibodies against EHDV-6 were detected in a sheep from the western region of Libya. Conclusion: This study suggests that EHDV has circulated or is circulating in Libya, and sheep could play an important role in the epidemiology of EHDV, and the virus may still be circulating in North Africa.

Hepatitis E Virus RNA Presence in Wild Boar Carcasses at Slaughterhouses in Italy.

Hepatitis E virus (HEV) is a waterborne and foodborne pathogen largely spread around the world. HEV is responsible for acute hepatitis in humans and it is also diffused in domestic and wild animals. In particular, domestic pigs represent the main reservoir of the infection and particular attention should be paid to the consumption of raw and undercooked meat as a possible zoonotic vehicle of the pathogen. Several studies have reported the presence of HEV in wild boar circulating in European countries with similar prevalence rates. In this study, we evaluated the occurrence of HEV in wild boar hunted in specific areas of Tuscany. Sampling was performed by collecting liver samples and also by swabbing the carcasses at the slaughterhouses following hunting activities. Our data indicated that 8/67 (12%) of liver samples and 4/67 (6%) of swabs were positive for HEV RNA. The presence of HEV genome on swabs indicates the possible cross-contamination of carcass surfaces during slaughtering procedures. Altogether, our data indicated that it is essential to promote health education programmes for hunters and consumers to limit the diffusion of the pathogen to humans.

Detection and Characterization of Viral Pathogens Associated with Reproductive Failure in Wild Boars in Central Italy.

Wild boar and domestic swine share several pathogens, including viruses responsible for reproductive failures, representing an important sanitary and economic risk for the swine industry. Among them, suid herpesvirus 1 (SuHV-1), porcine circovirus 2 (PCV2) and porcine parvovirus 1 (PPV1) are widely diffused in the wild boar population. Unfortunately, little is known about their pathogenetic mechanisms and impact on the reproductive parameters of wild animals. This study aims to investigate the presence of viruses responsible for reproductive failure in pregnant wild boar sows and their foetuses. The investigation was conducted on 46 pregnant wild boar and their foetuses by molecular analysis; a phylogenetic study was performed on the positive samples. All of the investigated pathogens were identified in sows, while only herpesvirus and circovirus were detected in the tissues of their foetuses. Phylogenetic analysis revealed that the viral sequences obtained from the positive wild boars were closely related to those previously identified in domestic swine belonging to the same study areas. The results suggest that SuHV-1 and PCV2 can infect wild boar foetuses, with a possible impact on wild boar reproductive performance. Moreover, our data highlight the importance of continuous monitoring of swine pathogens circulating in wild environments, so as to carry out adequate sanitary actions.

Next generation sequencing study on RNA viruses of Vespa velutina and Apis mellifera sharing the same foraging area.

The predator Asian hornet (Vespa velutina) represents one of the major threats to honeybee survival. Viral spillover from bee to wasp has been supposed in several studies, and this work aims to identify and study the virome of both insect species living simultaneously in the same foraging area. Transcriptomic analysis was performed on V. velutina and Apis mellifera samples, and replicative form of detected viruses was carried out by strand-specific RT-PCR. Overall, 6 and 9 different viral types were reported in V. velutina and A. mellifera, respectively, and five of these viruses were recorded in both hosts. Varroa destructor virus-1 and Cripavirus NB-1/2011/HUN (now classified as Triato-like virus) were the most represented viruses detected in both hosts, also in replicative form. In this investigation, Triato-like virus, as well as Aphis gossypii virus and Nora virus, was detected for the first time in honeybees. Concerning V. velutina, we report for the first time the recently detected honeybee La Jolla virus. A general high homology rate between genomes of shared viruses between V. velutina and A. mellifera suggests the efficient transmission of the virus from bee to wasp. In conclusion, our findings highlight the presence of several known and newly reported RNA viruses infecting A. mellifera and V. velutina. This confirms the environment role as an important source of infection and indicates the possibility of spillover from prey to predator.

In Vitro Antibacterial Activity of Manuka (Leptospermum scoparium J.R. et G. Forst) and winter Savory (Satureja montana L.) Essential Oils and Their Blends against Pathogenic E. coli Isolates from Pigs.

Neonatal diarrhoea (ND), post-weaning diarrhoea (PWD) and oedema disease (OD) are among the most important diseases affecting pig farming due to economic losses. Among the main aetiological agents, strains of Escherichia coli are identified as the major responsible pathogens involved. Several strategies have been put in place to prevent these infections and, today, research is increasingly studying alternative methods to antibiotics to reduce the antibiotic resistance phenomenon. Essential oils (EOs) are among the alternative tools that are being investigated. In this study, the in vitro effectiveness of winter savory and manuka essential oils and their mixtures in different proportions against strains of E. coli isolated from episodes of disease in pigs was evaluated. The EOs alone demonstrated slight antibacterial effectiveness, whereas the blends, by virtue of their synergistic action, showed remarkable activity, especially the 70%-30% winter savory-manuka blend, showing itself as a potential tool for prevention and therapy.

Identification of two divergent swine Noroviruses detected at the slaughterhouse in North East Italy.

Norovirus (NoV) has emerged as one of the major causative agents of non-bacterial, food- and water-borne gastroenteritis in humans, with the main genogroup involved in human outbreaks (GII), which has been detected worldwide in different animal species including swine. A four-month investigation at the slaughterhouse aiming to examine the presence of NoV in the swine in North-Eastern Italy, enabled the detection of two divergent Noroviruses (NoVs) (GII.P11) in two swine farms. This represents the first study in the swine population of North-Eastern Italy, which has paved the way for future integrated virological and epidemiological investigations on swine NoVs.

Effect of Oral Administration of 1,3-1,6 ß-Glucans in DWV Naturally Infected Newly Emerged Bees (Apis mellifera L.).

Honeybee pathogens have an important role in honeybee colony mortality and colony losses; most of them are widely spread and necessitate worldwide solutions to contrast honeybee's decline. Possible accepted solutions to cope with the spread of honeybee's pathogens are focused on the study of experimental protocols to enhance the insect's immune defenses. Honeybee's artificial diet capable to stimulate the immune system is a promising field of investigation as ascertained by the introduction of 1,3-1,6 ß-glucans as a dietary supplement. In this work, by collecting faecal samples of honeybees exposed to different dietary conditions of 1,3-1,6 ß-glucans (0.5% and 2% w/w), it has been possible to investigate the Deformed wing virus (DWV) viral load kinetic without harming the insects. Virological data obtained by a one-step TaqMan RT-PCR highlighted the ability of 1,3-1,6 ß-glucans to reduce the viral load at the 24th day of rearing. The results indicated that the diet supplemented with 1,3-1,6 ß-glucans was associated with a dose-dependent activation of phenoloxidase. The control group showed a higher survival rate than the experimental groups. This research confirmed 1,3-1,6 ß-glucans as molecules able to modulate honeybees' defense pathways, and this is the first report in which the kinetic of DWV infection in honeybee faeces has been monitored by a RT-qPCR.

Detection of Pseudorabies Virus in Wild Boar Foetus.

Pseudorabies, or Aujeszky's disease, is a notifiable worldwide infection of domestic and feral swine that causes economic losses for the swine industry. In domestic pigs, the virus is responsible for nervous and/or respiratory symptoms; in pregnant sows, it is one of the major causes of stillbirth, mummification, embryonic death, and infertility (SMEDI). It is known that PRV infection in wild boar is associated with low pathogenicity and attenuated or absent symptomatology, but limited information is available about the ability of the virus to infect the foetuses of infected wild boar pregnant sows. Due to scarce information about the reproductive consequences, we investigate the possible intrauterine vertical transmission of the virus in wild boar pregnant sow living in a highly infected area. A number of 54 hunted wild boar were sampled during 2018-2019, and blood, genital and nasal swabs, placenta, and fetuses were collected for serological and molecular investigations. A seroprevalence of 74% (40/54) was detected, while 1/24 pregnant sow and 1/24 pooled foetuses tested positive by PCR (gene gB). This is the first evidence of viral detection in foetuses from seropositive pregnant wild boar. This finding suggests the possible pathogenetic role of PRV on pregnancy in wild boar and the existence of an additional transmission route.

Serological Survey on Bacterial and Viral Pathogens in Wild Boars Hunted in Tuscany.

Wild boar (Sus scrofa) is one of the large mammals most spread worldwide, including Italy. This animal is highly adaptable, and its population has rapidly increased in many areas in Europe. Central Italy, as well as Tuscany region, is an area particularly suitable for wild boar. In order to verify the role of this animal species in the epidemiology of some important infectious diseases for livestock and humans, a seroepidemiological survey on Brucella spp., Leptospira spp., Mycoplasma hyopneumoniae, Pseudorabies virus (PrV), and Hepatitis E virus (HEV) has been performed on 374 sera collected from wild boar during 2015/2016 and 2016/2017 hunting seasons. Overall, 2 out of 374 sera (0.53%) tested positive to Brucella spp., 33 out of 374 sera (8.82%) tested positive for Leptospira spp., while 79 out of 374 (21.12%) were positive for M. hyopneumoniae. Considering viral pathogens, serology indicated that 107 out of 374 (28.60%) samples scored positive for PrV, while 186 out of 374 (49.73%) for HEV. This investigation indicated that wild boar free ranging in the study area are potential hosts for different etiological agents. This animal could contribute to maintaining and/or disseminating some bacterial or viral pathogens to humans (especially hunters) and domestic animals, especially in free range farms.

Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis.

BACKGROUND: Felid herpesvirus type 1 (FHV-1)-associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. OBJECTIVE: To evaluate the realibility of qRT-PCR-2- ΔΔC q and RNAscope in situ hybridization (RNA-ISH) methods to diagnose FHV-1-associated dermatitis, in formalin-fixed paraffin-embedded (FFPE) tissues. ANIMALS: Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. METHODS AND MATERIALS: Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2- ΔΔC q method was used to estimate the "upregulation" of each FHV-1 target viral gene copies (glycoprotein-B and thymidine-kinase) relative to reference gene. Detection of FHV-1 mRNA was performed using the RNAscope 2.5 detection kit. RESULTS: By 2- ΔΔC q analysis, upregulation of both FHV-1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. CONCLUSIONS AND CLINICAL IMPORTANCE: QRT-PCR 2-ΔΔCq analysis and RNA-ISH can identify the FHV-1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR, and may be proposed for research and diagnostic purposes.

Detection of replicative Kashmir Bee Virus and Black Queen Cell Virus in Asian hornet Vespa velutina (Lepelieter 1836) in Italy.

Information concerning the pathogenic role of honey bee viruses in invasive species are still scarce. The aim of this investigation was to assess the presence of several honey bee viruses, such as Black Queen Cell Virus (BQCV), Kashmir Bee Virus (KBV), Slow Paralysis Virus (SPV), Sac Brood Virus (SBV), Israeli Acute Paralysis Virus (IAPV), Acute Bee Paralysis Virus (ABPV), Chronic Bee Paralysis Virus (CBPV), in Vespa velutina specimens collected in Italy during 2017. Results of this investigation indicate that among pathogens, replicative form of KBV and BQCV were detected, assessing the spillover effect of both these viruses from managed honey bees to hornets.

Felis catus Papillomavirus Types 1, 2, 3, 4, and 5 in Feline Bowenoid in Situ Carcinoma: An In Situ Hybridization Study.

Several studies based on histopathology or molecular investigations suggest a causal relation between Felis catus papillomavirus (FcaPV-2) infection and bowenoid in situ carcinoma (BISC) in cats. Nevertheless, data on distribution of viral DNA for different F. catus papillomavirus types (FcaPV-1, 2, 3, 4, 5) in precancerous skin lesions are lacking. In this study, incisional and excisional skin biopsies from 18 cats with BISC were investigated for the presence of FcaPV DNA by quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH) using specific probes to detect each of the FcaPVs that have been identified so far. By qPCR analysis, 15 of 18 samples were positive for FcaPV-2, 2 were positive for FcaPV-4, and 1 sample was negative for all FcaPVs studied. Two cases were positive for FcaPV-5 by qPCR only. FcaPV-1 and FcaPV-3 were not detected by either method. CISH positivity for FcaPV-2 and FcaPV-4 was 100% concordant with qPCR. FcaPV-2 CISH signal was observed as nuclear dots within grouped neoplastic keratinocytes in 12 BISCs and in the perilesional skin of 9 biopsies. In 3 of these 9 cases, the signal was not observed within the BISC. FcaPV-4 CISH positivity was detected only within BISCs in 2 cases. The overall rate of concordance for FcaPV detection between PCR and CISH was 97.8%. This study suggests that CISH is a reliable method to detect FcaPV-2 and FcaPV-4 infection in cats and provides useful information on the type, rate, and localization of infected cells.