Lesion profiling and subcellular prion localization of cervid chronic wasting disease in domestic cats.

Chronic wasting disease (CWD) is an efficiently transmitted, fatal, and progressive prion disease of cervids with an as yet to be fully clarified host range. While outbred domestic cats (Felis catus) have recently been shown to be susceptible to experimental CWD infection, the neuropathologic features of the infection are lacking. Such information is vital to provide diagnostic power in the event of natural interspecies transmission and insights into host and strain interactions in interspecies prion infection. Using light microscopy and immunohistochemistry, we detail the topographic pattern of neural spongiosis (the "lesion profile") and the distribution of misfolded prion protein in the primary and secondary passage of feline CWD (Fel(CWD)). We also evaluated cellular and subcellular associations between misfolded prion protein (PrP(D)) and central nervous system neurons and glial cell populations. From these studies, we (1) describe the novel neuropathologic profile of Fel(CWD), which is distinct from either cervid CWD or feline spongiform encephalopathy (FSE), and (2) provide evidence of serial passage-associated interspecies prion adaptation. In addition, we demonstrate through confocal analysis the successful co-localization of PrP(D) with neurons, astrocytes, microglia, lysosomes, and synaptophysin, which, in part, implicates each of these in the neuropathology of Fel(CWD). In conclusion, this work illustrates the simultaneous role of both host and strain in the development of a unique Fel(CWD) neuropathologic profile and that such a profile can be used to discriminate between Fel(CWD) and FSE.

Management of chronic wasting disease in ranched elk: conclusions from a longitudinal three-year study.

Chronic wasting disease is a fatal, horizontally transmissible prion disease of cervid species that has been reported in free-ranging and farmed animals in North America, Scandinavia, and Korea. Like other prion diseases, CWD susceptibility is partly dependent on the sequence of the prion protein encoded by the host's PRNP gene; it is unknown if variations in PRNP have any meaningful effects on other aspects of health. Conventional diagnosis of CWD relies on ELISA or IHC testing of samples collected post-mortem, with recent efforts focused on antemortem testing approaches. We report on the conclusions of a study evaluating the role of antemortem testing of rectal biopsies collected from over 570 elk in a privately managed herd, and the results of both an amplification assay (RT-QuIC) and conventional IHC among animals with a several PRNP genotypes. Links between PRNP genotype and potential markers of evolutionary fitness, including pregnancy rates, body condition, and annual return rates were also examined. We found that the RT-QuIC assay identified significantly more CWD positive animals than conventional IHC across the course of the study, and was less affected by factors known to influence IHC sensitivity - including follicle count and PRNP genotype. We also found that several evolutionary markers of fitness were not adversely correlated with specific PRNP genotypes. While the financial burden of the disease in this herd was ultimately unsustainable for the herd owners, our scientific findings and the hurdles encountered will assist future CWD management strategies in both wild and farmed elk and deer.

Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk.

Chronic wasting disease is a progressively fatal, horizontally transmissible prion disease affecting several members of the cervid species. Conventional diagnosis relies on ELISA or IHC evaluation using tissues collected post-mortem; however, recent research has focused on newly developed amplification techniques using samples collected antemortem. The present study sought to cross-validate the real-time quaking-induced conversion assay (RT-QuIC) evaluation of rectal biopsies collected from an elk herd with endemic CWD, assessing both binary positive/negative test results as well as relative rates of amplification between laboratories. We found that results were correlative in both categories across all laboratories performing RT-QuIC, as well as to conventional IHC performed at a national reference laboratory. A significantly higher number of positive samples were identified using RT-QuIC, with results seemingly unhindered by low follicle counts. These findings support the continued development and implementation of amplification assays in the diagnosis of prion diseases of veterinary importance, targeting not just antemortem sampling strategies, but post-mortem testing approaches as well.

Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats.

A replication-defective variant of feline leukemia virus was molecularly cloned directly from infected tissue and found to induce a rapid and fatal immunodeficiency syndrome in cats. Studies with cloned viruses also showed that subtle mutational changes would convert a minimally pathogenic virus into one that would induce an acute form of immunodeficiency. The data suggest that acutely pathogenic viruses may be selected against by current methods for isolation of the human and simian immunodeficiency viruses.

Experimental chronic wasting disease (CWD) in the ferret.

Chronic wasting disease (CWD), a prion disease of North American deer, elk and moose, affects both free-ranging and captive cervids. The potential host range for CWD remains uncertain. The susceptibility of the ferret to CWD was examined experimentally by administering infectious brain material by the intracerebral (IC) or oral (PO) route. Between 15 and 20 months after IC inoculation, ferrets developed neurological signs consistent with prion disease, including polyphagia, somnolence, piloerection, lordosis and ataxia. Upon first sub-passage of ferret-adapted CWD, the incubation period decreased to 5 months. Spongiform change in the neuropil was most marked in the basal ganglia, thalamus, midbrain and pons. The deposition of PrP(CWD) was granular and was occasionally closely associated with, or localized within, neurons. There were no plaque-like or perivascular PrP aggregates as seen in CWD-infected cervids. In western blots, the PrP(CWD) glycoform profile resembled that of CWD in deer, typified by a dominant diglycosylated glycoform. CWD disease in ferrets followed IC but not PO inoculation, even after 31 months of observation. These findings indicate that CWD-infected ferrets share microscopical and biochemical features of CWD in cervids, but appear to be relatively resistant to oral infection by primary CWD inoculum of deer origin.

Determinants of susceptibility and resistance to feline leukemia virus infection. I. Role of macrophages.

The role of autochthonous peritoneal feline macrophages (M theta) in the age-related resistance of cats to feline leukemia virus (FeLV) was investigated by a study of the functional properties and FeLV susceptibility of M theta from kittens and adult cats and the effect of hydrocortisone (HC) and silica on M theta-FeLV interactions. Although the phagocytic functions of isolated M theta from kittens and adults were equivalent, the mean FeLV susceptibility of M theta from kittens was five times that of M theta from adult cats, thus establishing a direct correlation between the age-related susceptibility of cats and M theta from cats to FeLV. M theta of viremic cats were found to be infected with FeLV in vivo; virus titers were slightly higher than those obtained after in vitro infection of M theta, M theta from cats that had experienced regressive FeLV infection were not significantly more resistant to FeLV infection in vitro than were M theta from naive adult specific-pathogen-free cats. HC, which has been shown to enhance the in vivo FeLV susceptibility of cats, also enhanced the permissiveness of M theta from cats to FeLV in vitro (600-fold for M theta from adult cats and 200-fold for M theta) from kittens. M theta permissiveness to FeLV was highly sensitive to HC and occurred in M theta infected in vivo or in vitro. In parallel with the effect of HC on the natural resistance of cats to FeLV, administration of silica before virus inoculation also markedly enhanced the FeLV susceptibility of adult cats. Silica was toxic for isolated M theta but not for lymphocytes in vitro, and silica produced monocytopenia and neutrophilia, delayed skin allograft rejection, and augmented feline oncovirus-associated cell membrane antigen antibody responses in vivo. These experiments indicate that M theta were linked to the natural resistance of cats to FeLV and that the temporary elimination of M theta functions (e.g., by silica) and/or the conversion of the M theta-FeLV relationship from a nonpermissive to a permissive state (e.g., by corticosteroids) resulted in failure of early virus containment, in persistent virus amplification in hemolymphatic tissues, and in subsequent FeLV-related proliferative or antiproliferative disease.

Determinants of susceptibility and resistance to feline leukemia virus infection. II. Susceptibility of feline lymphocytes to productive feline leukemia virus infection.

The interplay between feline leukemia virus (FeLV) and feline lymphocytes (lc) infected in vitro or in vivo was investigated. Surface marker analysis and viral infectivity (VI) assays of lc populations were used to determine susceptibility of lc subsets to FeLV. The principal FeLV-replicating cell in the mesenteric lymph node of persistently infected, preleukemic cats was a nonadherent, complement receptor (CR)-bearing lc (B-cell). The lymph nodes of preleukemic cats also had increased numbers of uninfected T-cells [cells forming rosettes with guinea pig erythrocytes (GPE)] and cells with receptors for the Fc portion of 7S IgG (Fc gamma R cells) as compared with lymph nodes of age-matched specific-pathogen-free (SPF) cats. The induction of productive infection of feline peripheral blood mononuclear leukocytes (PBL) in vitro depended on a 48-hour in vitro preincubation period before virus exposure. The equivalent susceptibility of whole and adherent cell-depleted PBL to productive infection and the failure of hydrocortisone to enhance viral infection were compatible with identification of the FeLV-replicating cell as an lc. Furthermore, lc from susceptible SPF kittens replicated 50 times as much FeLV as did lc from resistant adult SPF cats. The Ic productively infected with FeLV after in vitro exposure were more precisely identified with the use of Ficoll-Isopaque density gradient separations of rosetted and nonrosetted lc. Whole PBL, GPE rosette-positive PBL (T-cells), and CR-positive PBL (B-cells) were permissive to FeLV infection, and maximal VI was evident at 14 days after exposure. The substantial (1,325-fold) increment in VI found in the Fc gamma R-depleted PBL suggested a role for Fc gamma R cells in the containment of FeLV infection. Unstimulated mononuclear leukocytes from blood, spleen, lymph node, thymus, and marrow were susceptible to productive FeLV infection after in vitro exposure. The degree of spontaneous DNA synthesis in marrow, thymus, and spleen but not lymph node or PBL was inversely related to permissiveness to viral replication. Mitogen activation of lc was associated with decreased viral replication when either T-cell mitogens (concanavalin A, phytohemagglutinin, or pokeweed mitogen) or a B-cell mitogen (dextran sulfate) was used. Virus production by spleen cells and PBL was enhanced twofold to tenfold by prior lc stimulation by the B-cell mitogen, lipopolysaccharide, or protein A-bearing Staphylococcus aureus, a mitogen for feline T-cells with Fc gamma R. Both productively infected (preincubated) and nonproductively infected (freshly isolated) PBL transferred infectious FeLV to autochthonous peritoneal macrophages (M theta); most of the virus in PBL-peritoneal M theta cocultures was produced by adherent cells, irrespective of whether the adherent or nonadherent cell population was inoculated originally.

Horizontal transmission of feline leukemia virus under experimental conditions.

Thirty-seven specific-pathogen-free (SPF) cats ranging from newborn to 1 year were inoculated with the Rickard strain of feline leukemia virus (FeLV). Each inoculated cat shared a cage with a control SPF cat for 40 weeks post inoculation. After 4-5 weeks, 20 of the inoculated cats became group-specific antigen (gsa)-positive; the other 17 remained gsa-negative but developed virus neutralizing and feline oncornavirus cell membrane-associated antigen antibody titers. Seventeen of the control cats in contact with the gsa-positive cats developed evidence of FeLV infection 4-18 weeks after virema was detected in their inoculated cage mates. Of the control cats in contact with inoculated cats that remained gsa-negative, none developed evidence of FeLV infection. Data indicated that the gsa-positive state in cats inoculated with FeLV correlated with the capacity for horizontal transmission of the virus.

Feline leukemia virus infection: age-related variation in response of cats to experimental infection.

Sixty-seven specific-pathogen-free cats of various ages (newborn, 2 wk, 1 mo, 2 mo, 4 mo, and 1 yr) were inoculated ip with either the Rickard (R) or the Kawakami-Theilen (KT) strain of feline leukemia virus (FeLV). Susceptibility to FeLV was judged by induction of a) FeLV group-specific antigens (gsa) in leukocytes, b) FeLV-related disease, c) antibody to feline oncornavirus-associated cell membrane antigen (FOCMA), and d) virus-neutralizing (VN) antibody. Susceptibility to FeLV-decreased with age. Persistent viremia and FeLV-related disease developed in 100% of cats inoculated as newborns, in 85% of cats inoculated at 2 weeks to 2 months of age, and in 15% of cats inoculated at 4 months or 1 year of age. Cats susceptible to FeLV leukemogenesis became persistently FeLV gsa-positive (viremic) at 4 weeks post inoculation and thereafter and produced little or no FOCMA or VN antibody. Cats that resisted leukemogenesis by FeLV all developed persistent FOCMA and VN titers and never became FeLV gsa-positive. The disease in inoculated cats was influenced by virus strain; FeLV-R induced predominantly thymic lymphosarcoma, whereas FeLV-KT caused fatal nonregenerative anemia without concurrent neoplasia.

Characterization of feline T-and B-lymphocytes and identification of an experimentally induced T-cell neoplasm in the cat.

Membrane markers of feline T- and B-lymphocytes were identified for further investigation of leukemogenesis in the cat. Feline T-cells formed spontaneous erythrocyte rosettes with guinea pig (GPE) and rat erythrocytes (RE). The receptors for GPE and RE were separate entities and expressed independently on lymphoid cell membranes. The RE receptor appeared to be present only on more mature or differentiated T-cells, whereas the GPE receptor reacted with a broader population that included less differentiated T-cells. Feline B-cells bore a complement receptor that was detected by adherence of SE coated with antibody and complement. Malignant lymphoblasts obtained from thoracic fluid of cats with feline leukemia virus (FeLV)-induced thymic lymphosarcomas, as well as FL-74 cells (a FeLV-transformed feline lymphoblastoid cell line) expressed T-cell markers. These results provided definitive evidence for markers of feline T- and B-cells and identified an experimentally induced T-cell lymphosarcoma.

Lymphocyte mitogen reactivity and enumeration of circulating B- and T-cells during feline leukemia virus infection in the cat.

Mitogen-induced blast transformation of peripheral blood lymphocytes and quantitative changes in circulating T- and B-cells were studied serially in cats inoculated with feline leukemia virus (FeLV). Concanavalin A-induced blast transformation sharply declined beginning at 5 weeks post inoculation (Pl) in FeLV-infected cats when compared to age-matched uninfected control cats. Similar but less consistent changes were seen in responses to pokeweed mitogen-induced stimulation. In most infected kittens this defect persisted until they died from thymic lymphosarcoma, 15-24 weeks Pl. An early lymphopenia, due primarily to a decrease in circulating B-cells, occurred in infected cats 5-8 weeks Pl. Following a return of total and B-lymphocytes to control values, infected cats developed increased numbers of T-cells at 16 or more weeks Pl, which correlated with circulating lymphoblastic lymphocytes bearing T-cell markers. These results correlated neoplasia arising in a thymus-derived lymphocyte population with mitogenic hyporeactivity in the preneoplastic period and suggested that FeLV-induced immune alterations may be a necessary antecedent of leukemogenesis in the cat.

Feline oncornavirus-associated cell membrane antigen. VI. Cytotoxic antibody in cats exposed to feline leukemia virus.

Feline leukemia virus (FeLV)-infected specific pathogen-free (SPF) cats, normal uninfected SPF cats, and healthy cats from leukemic households were tested for antibody reactive to the feline oncornavirus-associated cell membrane antigen (FOCMA)-containing target cell line FL-74 by microcytotoxicity and indirect membrane immunofluorescence. Of the infected SPF animals, 81% showed concordant reactivity for the two tests. In contrast, only 55% of the healthy cats known to be naturally exposed to FeLV for long periods showed such concordance. FOCMA antibody could not be detected in normal SPF cats by either indirect membrane immunofluorescence or microcytotoxicity. Most cats in the FeLV-infected SPF group developed antibody detectable by both procedures by the fifth week post inoculation. Antibody detectable by membrane immunofluorescence persisted in a high percentage (75-90%) of the animals throughout the observation period of 19 weeks; after 9 weeks, fewer cats had antibody that was also detectable by microcytotoxicity.

Influence of antibody infusion on pathogenesis of experimental feline leukemia virus infection.

For examination of the influence of antibody on the pathogenesis of feline leukemia virus (FeLV) infection, 12 weanling specific-pathogen-free cats were inoculated with isolates of FeLV and were treated beginning at 7, 19, 21, 24, 34, or 49 days post inoculation (DPI) with feline anti-FeLV hyperimmune serum (10 infusions, 37 mg globulin/kg each at 48-hr intervals). Anti-FeLV serum infusion initiated at 7 DPI prevented the onset of hematopoietic cell infection and viremia. Antibody treatment initiated at 19 or 24 DPI abrogated recently established FeLV viremia and extinguished p27 expression in bone marrow and blood cells. Viremia established for longer periods was refractory to antibody infusion despite establishment of enzyme-linked immunosorbent assay antibody titers of 1:80 to 1:320 in the treated cats. Latent FeLV infection was a sequel to antibody-induced curtailment of viral replication in bone marrow cells and was able to reactivate spontaneously in vivo as well as in vitro.

Preparation of cell-free feline oncornavirus-associated cell membrane antigen.

Cell-free feline oncornavirus-associated cell membrane antigen (FOCMA) was prepared from a feline lymphoblastoid cell line of tumor origin (FL-74). Membrane fractions, separated on sucrose density gradients, and papain-solubilized products were found to contain FOCMA as determined by their capacity to inhibit reference cytotoxic cat antisera.

Infection of feline embryo adherent cells with feline leukemia virus: feline oncornavirus-associated cell membrane antigen expression and morphologic transformation.

Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

Inhibition of normal lymphocyte mitogenic reactivity by serum from feline leukemia virus-infected cats.

The effect of serum from 12 cats with lymphosarcoma induced by feline leukemia virus (FeLV) on the response of normal cat peripheral blood lymphocytes to phytomitogen-induced blastogenesis was studied. The majority of FeLV sera, when tested at a concentration of 20% of the incubation medium, caused a 40 to 70% reduction in the mean blastogenic response to concanavalin A compared to the response obtained with a similar concentration of normal feline serum. Results with pokeweed mitogen were similar, but the depression in blastogenesis was less than with concanavalin A. Further studies showed that the blastogenic inhibitory activity of FeLV serum (a) was heat labile at 56 degrees for 30 min, (b) could not be overcome by greater concentrations of mitogens, (c) was proportional to the concentration of FeLV serum in the incubation medium, and (d) could not be demonstrated when lymphocytes were preincubated in FeLV serum followed by washing and reincubating in normal feline serum. The results suggested that a substance present in the serum of FeLV-infected cats contributes to altered immunological reactivity during leukemogenesis in the cat.

Relationship between feline leukemia virus antigen expression and viral infectivity in blood, bone marrow, and saliva of cats.

Correlation was greater than 90% between feline leukemia virus (FeLV), group-specific antigen (GSA) in leukocytes, and viral infectivity (VI) in serum or plasma from 132 cats infected with either the Rickard strain of FeLV, the Snyder-Theilen strain of feline sarcoma virus, or field strains of FeLV. Detection of GSA in blood cells was at least as sensitive as detection of VI in serum. In 45% of FeLV GSA-positive cats inoculated with FeLV-Rickard strain, VI was detected in saliva. No saliva samples from GSA-negative cats had VI. Sequential bone marrow biopsies from 34 cats inoculated with Snyder-Theilen feline sarcoma virus indicated that the correlation between FeLV GSA in bone marrow cells and blood cells was virtually 100%. FeLV GSA appeared in bone marrow leukocyte precursors 1 week before its appearance in peripheral blood leukocytes in 50% of the cats. The FeLV GSA-positive state was transient (3 to 6 weeks) in 34% of the Snyder-Theilen feline sarcoma virus-inoculated cats.

Influence of adrenal corticosteroids on the susceptibility of cats to feline leukemia virus infection.

The natural resistance of adult specific-pathogen-free cats to feline leukemia virus (FeLV) was abrogated by treatment with various doses of a synthetic corticosteroid, methylprednisolone acetate (MPA), prior to either oral-nasal or i.p. inoculation of FeLV. Persistent viremia was induced in 82% (18 of 22) of MPA-treated cats versus 11% (1 of 9) of age-matched control cats. MPA-treated FeLV-inoculated cats developed prolonged lymphopenia (2 to 8 weeks postinoculation) and a delayed antibody response to the feline oncornavirus-associated cell membrane antigen. The distribution of FeLV group specific antigen in tissues of MPA-treated, FeLV-inoculated cats suggested that corticosteroids enhanced susceptibility to FeLV by impairing early viral containment in the reticuloendothelial and lymphoid tissues.

Increased susceptibility to feline leukemia virus infection in cats exposed to methylnitrosourea.

Exposure of adult specific-pathogen-free cats to methylnitrosourea resulted in increased susceptibility to infection by feline leukemia virus. A greater proportion of cats exposed to methylnitrosourea and feline leukemia virus (69%) became persistently viremic than those exposed to feline leukemia virus alone (17%). Segmented neutrophils were reduced by 90 to 99% within 3 days following exposure to methylnitrosourea, (15 to 20 mg/kg) whereas the effects on lymphocytes and erythrocytes, although less obvious, were also detected.

Immunization against feline oncarnavirus disease using a killed tumor cell vaccine.

Specific pathogen-free cats were immunized with an inactivated feline oncornavirus tumor cell vaccine. Immunized cats produced high antibody titers to the feline oncornavirus-associated cell membrane antigen and were protected from oncogenic feline sarcoma virus challenge. However, immunization did not produce virus-neutralizing antibody nor did it prevent viremia.