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.

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.

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.

Culture and comparison of feline myeloid dendritic cells vs macrophages.

To gain insight into the role of dendritic cells (DCs) in feline immunodeficiency virus (FIV) infection and immunity, methods were developed to culture feline myeloid DCs from CD14(+) monocytes with a combination of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and interleukin-4 (hrIL-4). These cells were compared with feline macrophages cultured in the presence of hrGM-CSF. As with DCs in other species, feline DCs showed uniformly high MHC class II expression, moderate B7.1 expression, potent induction of the allogeneic mixed leucocyte reaction (MLR), and moderate uptake of fluorescein isothiocyanate-dextran (FITC-DX) in the endocytic assay. In comparison with feline macrophages, DCs showed higher expression of MHC class II, similar expression of B7.1, CD14, CXCR4 and CD1a, and lower expression of CD11b. When placed on alcian blue-coated glass slides, DCs differed from macrophages in showing a greater tendency to spread out; they also had characteristic fine cytoplasmic processes instead of the broader pseudopodia of macrophages. Basal IL-12 mRNA expression and FITC-DX uptake were greater in DCs than in macrophages. Unlike feline DCs, feline macrophages exhibited a dose-dependent suppressive effect in the MLR. Feline DCs propagated in vitro should prove useful in the development of DC-mediated vaccination and therapy for infectious and neoplastic feline diseases. Additionally, macrophages cultured with GM-CSF provide a potential means of studying the mechanism of immunosuppression in cats.

Feline immunodeficiency virus cell entry.

The process of feline immunodeficiency virus (FIV) cell entry was examined using assays for virus replication intermediates. FIV subtype B was found to utilize the chemokine receptor CXCR4, but not CCR5, as a cellular receptor. Zidovudine blocked formation of late viral replication products most effectively, including circular DNA genome intermediates. Our findings extend the role of CXCR4 as a primary receptor for CD4-independent cell entry by FIV.

Feline immunodeficiency virus Gag- and Env-specific immune responses after vaginal versus intravenous infection.

To better understand the correlation of mucosal and systemic immune responses with lentiviral containment, we contrasted the early mucosal and systemic immune responses induced by vaginal versus intravenous exposure of cats to feline immunodeficiency virus (FIV) isolates of differing pathogenicity and clade (i.e., FIV-B-2542 and FIV-A-PPR). We found that despite divergence in viral genotype, the mucosal and systemic immune responses induced differed more with route of exposure than virus isolate. In intravenously exposed cats, Gag-specific antibody (both IgG and IgA isotype) predominated in the serum, saliva, and vaginal wash fluid irrespective of infecting virus isolate. While Env-specific responses were more variable, they were more often detected in vaginally infected cats. Both IgG and IgA directed against Gag and Env were consistently present in vaginal wash fluids independent of route of infection or virus isolate. FIV Gag- and Env-specific cytotoxic lymphocytes (CTLs) were detected in blood and tissue lymphocytes of cats infected with either virus strain but were greatest in intravenously infected animals. Likewise, FIV-specific CTLs were detected in CD8(+) vaginal lymphocytes of animals infected by either route but were also more frequent in intravenously inoculated animals. In summary, we found qualitative differences in the immune responses following vaginal infection but no evidence (1) that mucosal immune responses were enhanced in vaginally exposed cats, (2) that local mucosal infection led to measurably greater immune responses in either compartment; or (3) that more prominent immune responses correlated with lower viral burden.

Relationship of lymphoid lesions to disease course in mucosal feline immunodeficiency virus type C infection.

Feline immunodeficiency virus (FIV) infection typically has a prolonged and variable disease course in cats, which can limit its usefulness as a model for human immunodeficiency virus infection. A clade C FIV isolate (FIV-C) has been associated with high viral burdens and rapidly progressive disease in cats. FIV-C was transmissible via oral-nasal, vaginal, or rectal mucosal exposure, and infection resulted in one of three disease courses: rapid, conventional/slow, or regressive. The severity of the pathologic changes paralleled the disease course. Thymic depletion was an early lesion and was correlated with detection of FIV RNA in thymocytes by in situ hybridization. The major changes in thymic cell populations were depletion of p55+/S100+ dendritic cells, CD3- cells, CD4+/CD8- cells, and CD4+/CD8+ cells and increases in apoptosis, CD45R+ B cells, and lymphoid follicles. In contrast to thymic depletion, peripheral lymphoid tissues often were hyperplastic. Mucosally transmitted FIV-C is thymotropic and induces a spectrum of lymphoid lesions and disease mirroring that seen with the human and simian immunodeficiency virus infections.

Feline immunodeficiency virus clade C mucosal transmission and disease courses.

The transmissibility and pathogenicity of a clade C feline immunodeficiency virus (FIV-C) was examined via the oral-nasal, vaginal, or rectal mucosa. FIV-C was transmissible by all three mucosal routes. Vaginal transmission was most efficient (100%), oral exposure resulted in a 80% infection rate, and rectal transmission was least effective (44%). In contrast to previous intravenous passage studies, a broader range of host-virus relationships was observed after mucosal exposure. Three categories of FIV-C infection were defined: (1) rapidly progressive infection marked by high virus burdens and rapid CD4+ cell depletion (43% of vaginally exposed animals); (2) conventional (typical) infection featuring slowly progressive CD4+ cell decline (61% of all exposed animals); and (3) regressive (transient) infection marked by low and then barely detectable virus burdens and no CD4+ cell alterations (22% of rectally inoculated cats). These disease courses appear to have parallels in mucosal HIV and SIV infections, emphasizing the importance of the virus-mucosa interface in lentiviral pathogenesis.

Neuron loss and axon reorganization in the dentate gyrus of cats infected with the feline immunodeficiency virus.

The pathophysiological bases of cognitive, motor, and behavioral abnormalities in patients infected with the human immunodeficiency virus (HIV-1) remain largely unknown. To test the possibility that changes in hippocampal neuronal structure may contribute to these neurologic abnormalities, we examined the brains of cats infected with the feline immunodeficiency virus (FIV), an animal model of HIV-1 infection. We evaluated the dentate gyrus by using Timm's staining to estimate the extent of granule cell axon reorganization and by using Nissl staining, immunocytochemistry, and the optical fractionator method to estimate changes in the number of different neuronal subtypes. FIV-infected cats had abnormally high amounts of Timm's staining in the inner molecular layer and granule cell layer and loss of Nissl-stained, somatostatin-immunoreactive, and parvalbumin-immunoreactive neurons in the hilus. An inverse correlation existed between hilar neuron numbers and extent of aberrant Timm's staining. Increased Timm's staining and hilar neuron loss occurred throughout the septotemporal axis of the hippocampus. This type of neuronal loss and synaptic reorganization may provide an anatomic basis for some of the neurologic symptoms found in FIV-infected cats and HIV-infected humans.

In vivo monocyte tropism of pathogenic feline immunodeficiency viruses.

Virus-infected monocytes rarely are detected in the bloodstreams of animals or people infected with immunodeficiency-inducing lentiviruses, yet tissue macrophages are thought to be a major reservoir of virus-infected cells in vivo. We have identified feline immunodeficiency virus (FIV) clinical isolates that are pathogenic in cats and readily transmitted vertically. We report here that five of these FIV isolates are highly monocytotropic in vivo. However, while FIV-infected monocytes were numerous in the blood of experimentally infected cats, viral antigen was not detectable in freshly isolated cells. Only after a short-term (at least 12-h) in vitro monocyte culture were FIV antigens detectable (by immunocytochemical analysis or enzyme-linked immunosorbent assay). In vitro experiments suggested that monocyte adherence provided an important trigger for virus antigen expression. In the blood of cats infected with a prototype monocytotropic isolate (FIV subtype B strain 2542), infected monocytes appeared within 2 weeks, correlating with high blood mononuclear-cell-associated viral titers and CD4 cell depletion. By contrast, infected monocytes could not be detected in the blood of cats infected with a less pathogenic FIV strain (FIV subtype A strain Petaluma). We concluded that some strains of FIV are monocytotropic in vivo. Moreover, this property may relate to virus virulence, vertical transmission, and infection of tissue macrophages.

Virus threshold determines disease in SIVsmmPBj14-infected macaques.

Simian immunodeficiency virus (SIV) variant SIVsmmPBj14 is unique in producing an acutely lethal enteropathic syndrome in pigtail macaques. To determine whether the nature of the PBj14 disease would be attenuated by decreasing virus input and to relate tissue virus burden to the severity of disease, we infected pigtail macaques with serial 10-fold doses of SIVsmmPBj14 clone bcl.3 spanning 10(-2) through 10(4)TCID50. The results revealed a strikingly narrow difference between minimum infectious and fatal disease-inducing doses and a close association between enteric lymphoid tissue virus burden and disease. All animals infected with as much as 10(4) TCID50 through as little as 100 TCID50 of virus died of the lethal PBj14 syndrome between 7 and 13 days postinfection. Animals receiving 10(-1) TCID50 became infected (PCR+) but did not develop clinical disease. Animals receiving 10(-2) TCID50 did not become infected. The clinical syndrome was surprisingly similar in all affected macaques, although the time to disease onset and total survival time increased slightly as virus input decreased from 10(4) to 10 degrees TCID50. Highest terminal virus loads in plasma, gut-associated lymphoid tissue (GALT), and lymph nodes and greatest lesion severity were attained at intermediate levels of virus input (10(1) to 10(2) TCID50), probably owing to optimal time for virus amplification in target tissues. The present study reinforces others on the PBj14 system, suggesting that once a threshold level of virus replication is attained in intestinal lymphoid tissues, the cascade of events precipitating the lethal PBj14 syndrome is triggered irreversibly.

In vivo cell and tissue tropism of SIVsmmPBj14-bcl.3.

To gain insight into the unique pathogenicity of simian immunodeficiency virus (SIV) variant PBj14, which produces an acutely lethal enteropathic syndrome in infected pigtail macaques, we investigated the cell and tissue tropisms of a highly pathogenic biologic clone (bcl.3) of SIVsmmPBj14. To compare the relative amount of viral antigen in lymphoid organs of infected macaques we used an objective semiquantitative immunohistochemistry (sQIHC) assay. We found that in all animals viral antigen load was greater in alimentary-associated lymphoid tissues (gut-associated lymphoid tissue [GALT], tonsil, mesenteric and retropharyngeal lymph nodes) than in non-alimentary-associated lymphoid tissues (spleen, thymus, inguinal and axillary lymph nodes). Moreover, in six of nine animals examined, virus load in GALT was greater than that in any other lymphoid tissue. To determine whether the acute pathogenicity and prolific replication of SIVsmmPBj14 might be explained by a broader in vivo cell tropism than is typical of SIVs, we used cell subset separation and nested PCR. We found that the primary target cells in mesenteric lymph node for SIVsmmPBj14 were CD4+ T lymphocytes. However, the virus also infected macrophages, as well as CD8+ T cells and B cells, albeit at low frequencies. These results suggest that alimentary lymphoid tissue localization rather than unusual cell phenotype tropism distinguishes the singular pathogenesis of SIVsmmPBj14.

Maternal-fetal feline immunodeficiency virus transmission: timing and tissue tropisms.

The feline immunodeficiency virus (FIV) model of vertical human immunodeficiency virus type 1 transmission was used to explore the timing and tissue tropisms associated with intrauterine lentivirus infection. Cats chronically infected with FIV-B-2542 and their cesarean-derived fetuses and placentas were assayed by polymerase chain reaction and coculture at defined gestational intervals. Prevalence of fetal FIV infection was 0 at 3 weeks, 5% at 5 weeks, 38% at 7 weeks, and 60% at 9 weeks (term). Fetal tissues exhibiting the highest viral tropism were blood mononuclear cells and brain (each containing virus in 60% of FIV-positive fetuses) and thymus (47%). Maternal hematologic and virus load markers did not vary substantially with gestational stage. Therefore, fetal and/or placental maturation may determine the timing of lentivirus transmission. FIV infection prevalence in term fetuses was equivalent to that seen previously in vaginally delivered offspring, suggesting that most vertical FIV transmission occurs late in utero rather than intrapartum.

Cross virus neutralizing antibodies against feline immunodeficiency virus genotypes A, B, C, D and E.

Feline immunodeficiency virus (FIV) is classified into five genotypes, A, B, C, D and E, based on the phylogenetic analysis of the env V3-V5 region. However, whether there is correlation between phylogenetic and antigenic diversities remains unknown. In this study, we examined the cross virus neutralization of FIV genotypes A through E by sera from cats infected with a single genotype. The results indicated some relationships between phylogenetic genotype and neutralization serotype, and that cross-clade virus neutralization is possible. For example, serum from a cat infected with genotype E virus neutralized all five FIV genotypes. Our results suggest that the FIV subtyping according to the sequence diversity is partially reflected by antigenic diversity and serum neutralization.

Axonal sprouting in hippocampus of cats infected with feline immunodeficiency virus (FIV).

Neurologic dysfunction and neuropathology are common findings in patients infected with HIV and in cats infected with feline immunodeficiency virus (FIV). The pathogenesis of lentivirus-associated alterations in the central nervous system (CNS) is multifactorial. Because seizures, alterations in memory, and behavioral changes are clinical manifestations in adults and children infected with HIV, we explored the possibility that changes in neuronal structure may occur in the hippocampus. To do this, we examined the dentate gyrus of FIV-infected cats, an animal model of HIV infection. Neuropathologic findings included gliosis within the hilus of the dentate gyrus and granule cell axonal sprouting. Using the Timm's method, which labels axons of dentate gyrus granule cells, abnormally high amounts of staining were observed in the inner one third of the molecular layer in 45% of FIV-infected cats (n = 11) and in none of the controls (n = 19). Prominent axonal sprouting was seen in three FIV-infected cats that were infected as kittens, suggesting that younger cats may be more susceptible. Axon reorganization of the dentate granule cells has been hypothesized to underlie complex partial seizure activity in human temporal lobe epilepsy. These results suggest that FIV infection causes granule cell axon reorganization in the hippocampus of cats. A similar neuropathogenetic mechanism may contribute to neurologic dysfunction in HIV-infected patients.

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades.

For the rapid genetic analysis of feline immunodeficiency virus (FIV), we developed a heteroduplex mobility assay (HMA) that utilizes a PCR-amplified fragment of the FIV envelope gene spanning the third and fourth variable regions of the envelope surface protein coding sequence. Viral sequences were successfully amplified from blood specimens from 98 naturally infected cats from Australia, Canada, Germany, Italy, South Africa, and the United States. Eighty were clearly assignable to the A or B envelope sequence subtypes. Three belonged to subtype C, one was dually infected with viruses harboring the A and B env subtypes, and several were categorized as outliers to any of the established subtypes or as probable intersubtype recombinants. Some geographic clustering was evident, with subtypes A and B found in greater frequency in the western and eastern regions of the United States, respectively. Subtypes A, B, and C were found on more than one continent, and countries with more than two samples analyzed contained at least two subtypes. The broadest representation of subtypes was found in Munich, Germany, where three subtypes and one virus that was not classifiable by HMA were found. Thirteen samples were selected for DNA sequence determination over the same region of env used for HMA. Analysis of all available FIV env sequences from this and previous studies revealed the existence of recombinant viruses generated from subtype A/B, B/D, and A/C envelope gene sequences. Subtype A env sequences were less diverse than subtype B sequences, although both groups had well-supported clusters. Furthermore, the mutational pattern giving rise to diversification in the two subtypes differed, with the subtype A viruses showing half as many synonymous site mutations compared to subtype B yet showing similar levels of nonsynonymous site changes. These results are consistent with the hypothesis that FIV-B is an older virus group and is possibly more host adapted than FIV-A.

Regression of feline immunodeficiency virus infection.

Regression of feline immunodeficiency virus (FIV) infection was observed in seven of nine vertically infected kittens born to two chronically infected mother cats. Both provirus and nonmaternal FIV antibody were detected in all kittens by 4 weeks of age but only three of the seven kittens were positive by blood mononuclear cell coculture. Between 10 and 14 months of age blood mononuclear cells from each of the seven cats were negative at least once by polymerase chain reaction (PCR), but evidence of virus infection was detected by coculture and/or PCR in biopsied lymph node or bone marrow from five of the seven cats. Despite this evidence of persistent tissue provirus, antibody production did not persist in any of the cats beyond 1 year of age. All seven cats remained asymptomatic although CD4 and CD8 T cell counts were in the low normal range throughout the study. By contrast, two additional perinatally infected littermates that were persistently virus isolation positive developed rapid CD4 depletion and progressed to terminal immunodeficiency by 9 weeks of age. Thus FIV infection can be downregulated and/or sequestered to extremely low levels barely detectable with the assays available, although absolute clearance of virus may not occur. These observations are relevant to human immunodeficiency virus (HIV) infection in paralleling both the apparent "regression" of HIV infection reported in some perinatally infected infants and the low-level, apparently stable, infection established by attenuated simian immunodeficiency viruses.