Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.     

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells.The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.     

Puma lentivirus is controlled in domestic cats after mucosal exposure in the absence of conventional indicators of immunity

A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.     

Oral transmission of chronic wasting disease in captive Shira's moose

Three captive Shira's moose (Alces alces shirasi) were orally inoculated with a single dose (5 g) of whole-brain homogenate prepared from chronic wasting disease (CWD)-affected mule deer (Odocoileus hemionus). All moose died of causes thought to be other than CWD. Histologic examination of one female moose dying 465 days postinoculation revealed spongiform change in the neuropil, typical of transmissible spongiform encephalopathy. Immunohistochemistry staining for the proteinase-resistant isoform of the prion protein was observed in multiple lymphoid and nervous tissues. Western blot and enzyme-linked immunosorbent assays provided additional confirmation of CWD. These results represent the first report of experimental CWD in moose.     

Exposure to FIV and FIPV in wild and captive cheetahs

Two RNA-containing viruses, feline infectious peritonitis virus (FIPV) and feline immunodeficiency virus (FIV), have been observed to infect cheetahs. Although both viruses cause lethal immunogenetic pathology in domestic cats, only FIPV has documented pathogenesis in cheetahs. We summarize and update here a worldwide survey of serum and plasma from cheetah and other nondomestic felids for antibodies to FIV and FIPV, based on Western blot and immunofluorescence assays. FIPV exposure shows an acute pattern with recognizable outbreaks in several zoological facilities, but is virtually nonexistent in sampled free-ranging populations of cheetahs. FIV is more endemic in certain natural cheetah populations, but infrequent in zoological collections. FIV exposure was also seen in lions, bobcats, leopards, snow leopards, and jaguars. FIV causes T-cell lymphocyte depletion and associated diseases in domestic cats, but there is little direct evidence for FIV pathology in exotic cats to date. Because of the parallels with a high incidence of simian immunodeficiency virus in free-ranging African primates without disease, the cat model may also reflect historic infections that have approached an evolutionary balance between the pathogen and immune defenses of their feline host species. Published 1993 Wiley-Liss, Inc.     

Interspecies Transmission of Feline Immunodeficiency Virus from the Domestic Cat to the Tsushima Cat (Felis bengalensis euptilura) in the Wild

Feline immunodeficiency virus (FIV) was isolated from a wild-caught Tsushima cat (Felis bengalensis euptilura), an endangered Japanese nondomestic subspecies of leopard cat (F. bengalensis). Phylogenetic analysis of the env gene sequences indicated that the FIV from the Tsushima cat belonged to a cluster of subtype D FIVs from domestic cats. FIVs from both the Tsushima cat and the domestic cat showed similar levels of replication and cytopathicity in lymphoid cell lines derived from these two species. The results indicated the occurrence of interspecies transmission of FIV from the domestic cat to the Tsushima cat in the wild.     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects.

The natural occurrence of lentiviruses closely related to feline immunodeficiency virus (FIV) in nondomestic felid species is shown here to be worldwide. Cross-reactive antibodies to FIV were common in several free-ranging populations of large cats, including East African lions and cheetahs of the Serengeti ecosystem and in puma (also called cougar or mountain lion) populations throughout North America. Infectious puma lentivirus (PLV) was isolated from several Florida panthers, a severely endangered relict puma subspecies inhabiting the Big Cypress Swamp and Everglades ecosystems in southern Florida. Phylogenetic analysis of PLV genomic sequences from disparate geographic isolates revealed appreciable divergence from domestic cat FIV sequences as well as between PLV sequences found in different North American locales. The level of sequence divergence between PLV and FIV was greater than the level of divergence between human and certain simian immunodeficiency viruses, suggesting that the transmission of FIV between feline species is infrequent and parallels in time the emergence of HIV from simian ancestors.     

Applicability of current bovine spongiform encephalopathy (BSE) diagnostic procedures for chronic wasting disease (CWD)

Chronic wasting disease (CWD) in cervids is one of the transmissible spongiform encephalopathies ; however, its risk to humans is still obscure. An increase in number of diseased deer in North America has raised concerns regarding the CWD risk to humans. We demonstrated that the con-firmatory procedures and the commercial diagnostic kits for bovine spongiform encephalopathy (BSE) can be adopted for the diagnosis of CWD. No CWD case was confirmed in the surveillance of 558 cervids that were examined between 2003 and 2006 in Japan.     

Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer,elk, sheep,and cattle

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.     

Alteration of the chronic wasting disease species barrier by in vitro prion amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrP(RES)) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrP(RES) deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.     

Molecularly cloned feline immunodeficiency virus NCSU1 JSY3 induces immunodeficiency in specific-pathogen-free cats.

A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low-virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1.     

Kinetics of Replication of a Partially Attenuated Virus and of the Challenge Virus during a Three-Year Intersubtype Feline Immunodeficiency Virus Superinfection Experiment in Cats

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4⁺ T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.     

Feline leukemia virus in a captive bobcat

An 11-mo-old captive-bred male neutered bobcat (Felis rufus) presented with lethargy, anorexia, leukopenia, neutropenia, lymphopenia, and nonregenerative anemia. The animal was diagnosed as feline leukemia virus (FeLV) positive by immunofluorescent antibody and enzyme-linked immunosorbant assay (ELISA) testing. It died despite supportive care. Pathologic examination revealed multifocal non-suppurative encephalitis, diffuse interstitial pneumonia, multifocal hepatocellular necrosis, non-suppurative peritonitis, and lymphoid depletion. FeLV was isolated from peripheral blood mononuclear cells, bone marrow, spleen, and lymph node. FeLV-specific gag sequences were amplified by DNA polymerase chain reaction (PCR) and aligned with known domestic cat FeLV's. The source of the virus was speculated to be a domestic cat that was a surrogate nurse. Case reports of FeLV in nondomestic felids are few, and FeLV does not appear to be enzootic in wild felids, except European wildcats (Felis silvestris) in France and Scotland. Introduction of FeLV into free-living and captive nondomestic felid populations could have serious consequences for their health and survival. Measures to prevent the introduction of this virus to nondomestic felids are warranted.     

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

Background

Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.

Results

Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.

Conclusion

The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.     

A serologic survey of wild felids from central west Saudi Arabia

Forty-five wildcats (Felis silvestris), 17 sand cats (Felis margarita), and 17 feral domestic cats were captured in central west Saudi Arabia, between May 1998 and April 2000, with the aim to assess their exposure to feline immunodeficiency virus/puma lentivirus (FIV/PLV), feline leukaemia virus (FeLV), feline herpesvirus (FHV-1), feline calicivirus (FCV), feline coronavirus (FCoV), and feline panleukopenia virus (FPLV). Serologic prevalence in wildcats, sand cats, and feral domestic cats were respectively: 6%, 0%, 8% for FIV/PLV; 3%, 8%, 0% for FeLV; 5%, 0%, 15% for FHV-1; 25%, 0%, 39% for FCV; 10%, 0%, 0% for FCoV; and 5%, 0%, 8% for FPLV. We recorded the first case of FeLV antigenemia in a wild sand cat. Positive results to FIV/PLV in wildcats and feral cats confirmed the occurrence of a feline lentivirus in the sampled population.     

Frequent Cross-Species Transmission of Parvoviruses among Diverse Carnivore Hosts

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus (“FPV-like”) or canine parvovirus (“CPV-like”). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.     

Retroviruses and sexual size dimorphism in domestic cats (Felis catus L.).

Hochberg and co-workers have predicted that an increase in host adult mortality due to parasites is balanced by an earlier age at first reproduction. In polygynous species we hypothesize that such a pattern would lead to diverging selection pressure on body size between sexes and increased sexual size dimorphism. In polygynous mammals, male body size is considered to be an important factor for reproductive success. Thus, under the pressure of a virulent infection, males should be selected for rapid growth and/or higher body size to be able to compete successfully as soon as possible with opponents. In contrast, under the same selection pressure, females should be selected for lighter adult body size or rapid growth to reach sexual maturity earlier. We investigated this hypothesis in the domestic cat Felis catus. Orange cats have greater body size dimorphism than non-orange cats. Orange females are lighter than non-orange females, and orange males are heavier than non-orange males. Here, we report the extent to which orange and non-orange individuals differ in infection prevelance for two retroviruses, feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). FIV is thought to be transmitted almost exclusively through aggressive contacts between individuals, whereas FeLV transmission occurs mainly through social contacts. The pattern of infection of both diseases is consistent with the higher aggressiveness of orange cats. In both sexes, orange cats are significantly more infected by FIV, and tend to be less infected by FeLV than other cats. The pattern of infection is also consistent with an earlier age at first reproduction in orange than in non-orange cats, at least for females. These results suggest that microparasitism may have played an important role in the evolution of sexual size dimorphism of domestic cats.     

Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.