Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450?nm (OD₄₅₀) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.

RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD₄₅₀≥0.41 for samples positive, and OD₄₅₀<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).

CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.

CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.     

Identification of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula)

A novel, fatal neurological disease of the Australian brushtail possum (Trichosurus vulpecula) was first identified in 1995 in a research facility and subsequently in free-living possums in New Zealand and termed wobbly possum disease (WPD). The results of previous transmission studies suggested that the aetiological agent of WPD is most likely a virus. However, the identity of the presumed viral agent had not been elucidated. In the current report, we describe identification of a novel virus from tissues of WPD-affected possums using a combination of next generation sequencing and traditional molecular methods. The proportion of possums positive for the novel virus by PCR was significantly higher (p<0.0001) among animals with WPD than clinically healthy possums, strongly suggesting an aetiological involvement of the virus in WPD. Analysis of the partial genomic sequence of the putative WPD virus indicated that it is a novel nidovirus, most closely related to the current members of the family Arteriviridae.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

Routes of transmission of wobbly possum disease

AIMS: To determine the routes of transmission of wobbly possum disease (WPD) virus and whether or not these would favour further examination of its potential for biological control of possums. METHODS: A standard inoculum, prepared as a tissue suspension from possums which had been infected with WPD, was titrated in vivo. Possums were challenged with this inoculum by the intra-gastric, intra-tracheal and intra-dermal routes. Further possums were challenged with blood by the intra-dermal and intra-peritoneal routes, with urine by the intraperitoneal route and with homogenised mites (Trichosurolaelaps crassipes) by the intra-dermal route. Transmission was investigated between possums in closely-adjacent, individual cages and between possums in a group enclosure. RESULTS: Possums developed WPD following administration of the standard inoculum by all of the above routes, following administration of blood by the intra-peritoneal and intra-dermal routes, following administration of urine by the intraperitoneal route and following administration of homogenised mites by the intra-dermal route. Individually caged control possums did not contract WPD. All non-inoculated adult possums in the group enclosure and many joeys in direct contact with infected possums contracted WPD. CONCLUSION: WPD was efficiently transmitted by close contact. Without such contact transmission did not occur. Infectivity was demonstrated in tissue suspensions, blood, urine and mites. Given the routes by which possums are susceptible to these substances and the need for direct contact, infection may be spread in the wild by several mechanisms, including aggressive encounters in which blood is exchanged, contamination of wounds with urine, ingestion of contaminated food, transfer of mites during den-sharing, and other social encounters. WPD has potential as a biological control agent for possums on the basis that it is readily transmitted between individuals in close contact.     

Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses,and contact rates between possums and carcasses

Abstract

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses.

METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5–1 g with 0.2 mL liquid culture containing approximately 5 x 10⁷ cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present.

The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A ‘contact’ was recorded if these possums came within 40–50 cm of proximity loggers fitted to possum carcasses.

RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter.

In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4–6.7) days after placement.

CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.     

Samples sizes required to accurately quantify viral load and histologic lesion severity at the maternal–fetal interface of PRRSV-inoculated pregnant gilts

Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal–fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2–infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

An in situ single-pass perfusion model for assessing absorption across the intestinal mucosa of the brushtail possum

AIM: To develop an in situ animal model for assessing absorption of molecules across the intestinal mucosa of possums.

METHODS: A surgical preparation was used to perfuse known concentrations of reference compounds (fluorescein and luteinising hormone-releasing hormone; LHRH) through measured sections of selected regions (jejunum, caecum, proximal colon) of the intestinal tract of 19 possums, over a 2-h period. Plasma concentrations of the compounds, which were perfused either with or without co-administration of a permeation enhancer (sodium deoxycholic acid; SDA), were determined in the perfusion effluent, peripheral and in some instances in the pre-hepatic circulation by spectrofluorometry (fluorescein) or radio-immunoassay (LHRH). Pharmacokinetic parameters of both compounds in the possum were determined over a period of up to 4 h in a further 30 animals (fluorescein, n=6; LHRH n=24), from their plasma profiles following intravenous (I/V) administration of a bolus dose.

RESULTS: In animals perfused with 25 mg/ml fluorescein (Perfusion Experiment (PE) 1), the mean plasma concentration was 2.8 (SE 0.12) µg/ml in post-hepatic blood samples. When possums were perfused with 2.5 mg/ml fluorescein and 7 µg/ml LHRH (PE 2), mean plasma concentrations were 0.3 (SE 0.01) and 7.8 (SE 1.64) µg/ml fluorescein and 0.1 (SE 0.02) and 6.3 (SE 0.45) ng/ml LHRH, in the absence and presence of permeation enhancer, respectively. There was a poor correlation between pre-hepatic and post-hepatic concentrations.

CONCLUSIONS: The single-pass in situ perfusion technique provided a useful model for investigating basic information on the absorption of biocontrol agents across the intestinal tract of possums, but had limitations that must be recognised.     

Mycobacterium avium subsp. paratuberculosis infection in wildlife on three deer farms with a history of Johne's disease

Abstract

AIM: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock.

METHODS: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map.

RESULTS: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples.

CONCLUSIONS: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits.

CLINICAL RELEVANCE: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in NewZealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.     

Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula).

METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA.

RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins.

CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.     

Aleutian Disease (Plasmacytosis) of Mink: II. Responses of Mink to Formalin-Treated Diseased Tissues and to Subsequent Challenge With Virulent Inoculum

An experiment was carried out to examine the responses of Aleutian and standard dark types of mink to inoculations of formalintreated suspensions of tissues of mink with experimentally transmitted plasmacytosis. Control groups of mink received similar injections of normal Aleutian mink tissues or diseased tissues without formalin treatment. A second experiment was conducted to test the formalinized diseased tissue suspension for immunogenic value. Groups of mink which received one, two, or three doses of “vaccine” were later challenged with virulent inocula. Additional groups of mink served as unvaccinated and environmental controls.

Treatment with 0.3% formalin with fine trituration and incubation at 37°C was effective in preventing the development of plasmacytosis in inoculated mink. These mink remained susceptible to subsequent challenge with untreated diseased tissue suspensions. No immunity was demonstrated in the vaccinated mink. Mink inoculated with normal mink tissues did not develop plasmacytosis, nor did uninoculated environmental controls.

     

Shedding of feline leukemia virus RNA in saliva is a consistent feature in viremic cats

The purpose of this investigation was to characterize the shedding pattern of feline leukemia virus (FeLV) RNA in saliva, and to correlate it with the proviral load in whole blood, viral load in plasma, levels of p27 in saliva and plasma, the isolation of infectious FeLV from saliva, and the titers of FeLV-specific antibodies of the IgG and IgA isotypes. We evaluated 24 experimentally FeLV-infected cats for these parameters using real-time RT-PCR and PCR, cell culture assay and sandwich ELISA. We observed that shedding of viral RNA in saliva was a consistent feature in viremic cats. Latently FeLV-infected cats, displaying a very low proviral load, did not shed infectious virus in saliva, but occasionally shed viral RNA. Consequently, salivary shedding of FeLV RNA may not necessarily indicate a transmission potential for susceptible cats. This study also confirmed previous results from our laboratory, showing that a negative result for p27 in plasma, or for viral RNA in plasma or saliva does not exclude FeLV infection, considering that blood cells from those cats contained provirus. We also showed that FeLV RNA and DNA were stable for more than 64 days in saliva samples stored at room temperature. We conclude that the detection of FeLV RNA in saliva may be a useful indicator of viremia, and that the detection of salivary viral RNA by RT-PCR could become a reliable tool for the diagnosis of FeLV infection, which is facilitated by the low invasive method of collection of the samples.     

A behaviour study on the potential for direct transmission of tuberculosis from possums (Trichosura vulpecula) to alpacas (Lama pacos), and the converse from alpacas to possums

Aims. To evaluate the potential for the direct transmission of tuberculosis from possums to alpacas, and vice versa.

Methods. A field study was conducted on an alpaca farm in Northland, New Zealand on 7 January 1999. Observations were recorded on the interaction of one group of male alpacas with a simulated dead possum, one male and one female alpaca group with a simulated terminally tuberculous possum, and one group of male alpacas with a normal possum in an enclosure from which the animals could not escape. The possum was sedated with ketamine as hydrochloride to simulate death (inactive; no movement), and terminal illness (active, into-ordinated movement around the paddock). The observations were based on the focal animal sampling technique, they were un-replicated, and recorded visually and manually with intermittent still photography.

Results. Both male and female alpacas showed strongly inquisitive interaction with the possum. They clustered around the possum (focal animal) very soon after it was observed by the first member of the group. The interest of the majority of both sex groups remained high for the observation periods of approximately 30 minutes, and most individuals remained within 5 metres of the possum for that time. Approximately 50% of the alpacas were within possible aerosol transmission distance of 2 metres from the sedated, erratically mobile possum with their heads towards it for approximately 50% of the two observation periods. Aggressive behaviour was recorded for a young male with stamping on the moving possum. Similar, but more vigorous and prolonged stamping behaviour was recorded for a female with a young (<1 week) offspring (cria).The stamping behaviour was accompanied by very close nose to nose contact of the alpaca and possum. At one point the female threw the possum approximately 1.5 metres in the air with her teeth. The group of male alpacas placed in an enclosure with an unsedated normal possum generally moved away from the possum during its rapid active attempts to escape. When it became inactive their approaches were cautious and only once elicited a defence reaction from the possum, from which they recoiled. One male made one attempt to stamp on the active possum. Soon after the possum became inactive in a small loose hay pile, the alpacas lost interest in it.

Conclusions. The alpaca / possum behavioural interactions show there is potential for direct aerosol transmission of tuberculosis from possums to alpacas, but probably not from alpacas to possums.     

Lung worm (Marsupostrongylus spp.) infection in common brushtail possums (Trichosurus vulpecula)

Marsupostrongylus spp. are the metastrongyloid nematodes most commonly associated with verminous pneumonia in Australian marsupials. Currently, there is a scarcity of information regarding this parasite in the common brushtail possum (Trichosurus vulpecula). Thirty-four free-living possums submitted to two wildlife hospitals in Sydney, Australia, between 2008 and 2015 were diagnosed with verminous pneumonia on postmortem examination. The majority of possums presented ill with multiple comorbidities. However, only five cases had clinical signs of respiratory disease. Necropsy and histopathology revealed extensive lung lesions characterised by diffuse, mixed interstitial infiltrates of macrophages, lymphocytes and plasma cells with mild to marked concentrations of eosinophils. Bronchopneumonia, pulmonary oedema, interstitial fibrosis, atelectasis and type II pneumocyte hyperplasia were also present in most cases. Adult nematodes, first-stage larvae and embryonating eggs were present in the large airways and alveolar spaces. The parasites were definitively identified as Marsupostrongylus spp. in eight cases with presumptive diagnoses based on histopathological characteristics reached in a further 26 cases. Twenty-nine of the 34 affected possums were adults with no sex predisposition. A review of the brushtail possum records at Taronga Wildlife Hospital from 1999 to 2015 revealed no lungworm infections were reported in the 45 possums examined before 2008. However, between 2008 and 2015, 30 of 47 possums (63.8%) examined were diagnosed with metastrongyloid lungworms. This case series is the first detailed report of Marsupostrongylus nematodes in common brushtail possums and highlights the clinical and pathological features, along with epidemiological findings.     

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.     

Comparison of Selected Nonlethal Samples from Adult Steelhead for Detection of Infectious Hematopoietic Necrosis Virus Using Cell Culture

Abstract

Nonlethal sampling techniques have previously been evaluated for detection of a variety of viral salmonid pathogens. However, many of these studies have used molecular assays in lieu of widely accepted cell culture techniques to evaluate the sampled tissues. Samples were collected from female steelhead Oncorhynchus mykiss broodstock using three potential nonlethal sampling methods (mucus/skin scrape, pectoral fin clip, and gill tissue biopsy) and evaluated for the presence of infectious hematopoietic necrosis virus (IHNV) via cell culture techniques. The results were compared with those from samples collected using a standard lethal sampling method (pooled anterior kidney and spleen tissues) applied to the same fish. Of the three nonlethal sampling techniques that were evaluated, fin clipping was the easiest and least invasive method. Furthermore, fin tissue was as sensitive as or more sensitive than kidney/spleen tissue for detecting IHNV in this population of fish. However, with the exception of gill tissue, the nonlethal samples did not appear to be appropriate surrogates for lethally collected tissues with regard to identifying an active infection in a particular fish. Nevertheless, nonlethal sampling coupled with cell culture appears to be suitable for helping to define the IHNV status of a steelhead population.

Received July 27, 2016; accepted December 11, 2016 Published online March 21, 2017     

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease

Reasons for performing study: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV‐1, BPV‐2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. Hypothesis: Given the pathogenic role of BPV‐1 and BPV‐2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. Methods: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid‐bearing horses and one donkey. Viral load was estimated via quantitative real‐time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV‐1/‐2 genome for one randomly selected lesion per horse and correlated with disease severity. Results: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0–556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild‐type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. Conclusions: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. Potential relevance: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.