Household Transmission of SARS-CoV-2 from Humans to Pets, Washington and Idaho, USA.

SARS-CoV-2 likely emerged from an animal reservoir. However, the frequency of and risk factors for interspecies transmission remain unclear. We conducted a community-based study in Idaho, USA, of pets in households that had >1 confirmed SARS-CoV-2 infections in humans. Among 119 dogs and 57 cats, clinical signs consistent with SARS-CoV-2 were reported for 20 dogs (21%) and 19 cats (39%). Of 81 dogs and 32 cats sampled, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive. This discordance might be caused by delays in sampling. Respondents commonly reported close human‒animal contact and willingness to take measures to prevent transmission to their pets. Reported preventive measures showed a slightly protective but nonsignificant trend for both illness and seropositivity in pets. Sharing of beds and bowls had slight harmful effects, reaching statistical significance for sharing bowls and seropositivity.

Household transmission of SARS-CoV-2 from humans to pets in Washington and Idaho: burden and risk factors.

SARS-CoV-2 is believed to have emerged from an animal reservoir; however, the frequency of and risk factors for inter-species transmission remain unclear. We carried out a community-based study of pets in households with one or more confirmed SARS-CoV-2 infection in humans. Among 119 dogs and 57 cats with completed surveys, clinical signs consistent with SARS-CoV-2 were reported in 20 dogs (21%) and 19 cats (39%). Out of 81 dogs and 32 cats sampled for testing, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive; this discordance may be due to delays in sampling. Respondents commonly reported close human-animal contact and willingness to take measures to prevent transmission to their pets. Reported preventative measures showed a slightly protective trend for both illness and seropositivity in pets, while sharing of beds and bowls had slight harmful effects.

Rhodococcus equi-specific hyperimmune plasma administration decreases faecal shedding of pathogenic R. equi in foals.

Rhodococcus equi is the most common cause of pneumonia in young foals. Pneumonic foals are an important source of environmental contamination as they shed higher amounts of R. equi in their faeces than unaffected foals. As R. equi-specific hyperimmune plasma (HIP) lessens clinical pneumonia, we hypothesise that its use would result in decreased faecal shedding of R. equi by foals. Neonatal foals were either given HIP (n=12) or nothing (n=9, control) shortly after birth and were then experimentally infected with R. equi Faeces were collected before and on weeks 2, 3, 5 and 7 after infection. Presence of virulent R. equi was tested using qPCR. There was strong evidence of an association between HIP administration and a decrease in faecal shedding of virulent R. equi (P=0.031 by Pearson chi-squared test). Foals in the control shed significantly more R. equi (colony-forming units/ml) than foals that received HIP (P=0.008 by Mann-Whitney rank-sum test). While our study is the first to report this additional benefit of HIP administration, future studies are needed to evaluate the implications of its use under field conditions.

Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia.

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor ß-induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

hTERT-immortalized ovine microglia propagate natural scrapie isolates.

Ex vivo propagation of natural prion isolates (i.e., propagated solely in the natural host) is crucial for the characterization and study of transmissible spongiform encephalopathies (TSEs). Several well-established, prion-permissive cell culture systems are available; however, only a few cell lines are permissive to natural prion isolates and these cells are not pathophysiologically relevant (e.g., renal epithelium and fibroblast-like cells). Therefore, a pathophysiologically relevant cell line derived from a natural TSE host could be used for propagation of natural prion isolates. In this study, ovine brain macrophages (microglia) were immortalized by transfection with the human telomerase reverse transcriptase (hTERT) gene to identify cell lines (hTERT-microglia) permissive to natural scrapie prion isolates. Following transfection, hTERT-microglia were passaged up to 100 times and their lifespan was significantly longer compared to parental cells (Fisher's exact test, P<0.001). Multiple sublines were permissive to cell culture-adapted prions; two sublines were also permissive to natural scrapie isolates (i.e., derived from brain homogenates of sheep infected with scrapie). Prion infectivity and partial protease resistance of the prion protein were maintained in hTERT-microglia. Comparisons between scrapie-permissive and non-permissive hTERT-microglia sublines revealed that overall quantity of the normal cellular prion protein was not associated with prion permissiveness. The use of hTERT-microglia in future TSE studies may be more germane to the characterization of the cellular and subcellular pathophysiology of natural scrapie prion isolates and to investigate host-specific factors involved in prion replication.

Francisella tularensis infection without lesions in gray tree squirrels (Sciurus griseus): a diagnostic challenge.

Fifteen cases of Francisella tularensis infection (tularemia) were identified in western gray (Sciurus griseus) and eastern gray (Sciurus carolinensis) squirrels submitted to the Washington Animal Disease Diagnostic Laboratory between 2008 and 2011. All of the squirrels originated in Washington State, a geographical area with endemic tularemia in wildlife. Nine of the 15 squirrels with F. tularensis infection had gross (2/15) or microscopic (9/15) multifocal necrotizing lesions in the spleen, liver, or lymph nodes, typical of tularemia. Special stains did not reliably identify intralesional bacteria microscopically. Six of the 15 squirrels infected with F. tularensis lacked gross and microscopic lesions typical of tularemia. All 15 squirrels with F. tularensis infection were identified by polymerase chain reaction tests on the spleen, liver, or lymph node (including all 6 squirrels without typical tularemia lesions); 8 out of 9 squirrels were positive by direct fluorescent antibody test of tissues, and 5 out of 15 squirrels were positive by culture of tissues. The findings underscore the importance of considering tularemia as a possible cause of death when no lesions of tularemia can be identified at necropsy. Furthermore, the findings suggest the possibility of subclinical infections in gray squirrels, and the importance of molecular diagnostics for definitive diagnosis of F. tularensis infection in wild squirrels.

Discovery of a novel, monocationic, small-molecule inhibitor of scrapie prion accumulation in cultured sheep microglia and Rov cells.

Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C)) to disease-associated prion protein (PrP(Sc)). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrP(Sc)-specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrP(Sc) accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrP(C) expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrP(Sc) accumulation in the two cell systems and confirmed that the inhibition of PrP(Sc) accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrP(C) expression is not the mechanism of PrP(Sc) inhibition. PrP(Sc) accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrP(Sc) activity was independent of anti-pestivirus activity. The anti-PrP(Sc) selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrP(Sc) accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.

A novel papillomavirus isolated from proliferative skin lesions of a wild American beaver (Castor canadensis).

Cutaneous papillomatosis was diagnosed in an adult American beaver (Castor canadensis). Gross lesions included numerous exophytic, roughly circular, lightly pigmented lesions on hairless areas of fore and hind feet and the nose. The most significant histopathologic findings were multifocal papilliform hyperplasia of the superficial stratified squamous epithelium, with multifocal koilocytes, and multiple cells with large, darkly basophilic intranuclear inclusion bodies. A virus with properties consistent with papillomavirus (PV) was recovered by virus isolation of skin lesions, utilizing rabbit and feline kidney cell lines. The presence of the virus was confirmed by PV-specific polymerase chain reaction. The partial sequences of E1 and L1 genes did not closely match those of any PVs in GenBank, suggesting that this might be a new type of PV. Partial E1 and L1 nucleotide sequences of the beaver papillomavirus (hereafter, ARbeaver-PV1) were used to create a phylogenetic tree employing the complete E1 and L1 open reading frame nucleotide sequences of 68 PVs. The phylogenetic tree placed the ARbeaver-PV1 in a clade that included the Mupapillomavirus (HPV1 and HPV63) and Kappapapillomavirus (OcPV1 and SfPV1) genera. The present article confirms the papillomaviral etiology of cutaneous exophytic lesions in the beaver.

Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale.

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale.

An outbreak of Chlamydophila psittaci in an outdoor colony of Magellanic penguins (Spheniscus magellanicus).

An outbreak of Chlamydophila psittaci occurred in an outdoor colony of 63 Magellanic penguins (Spheniscus magellanicus) at the San Francisco Zoo. Affected penguins presented with inappetence, lethargy, and light green urates. Hematologic and serum biochemical findings were consistent with chronic inflammation. Penguins did not respond to initial supportive and antimicrobial therapy, and 3 died. Necropsy results of the 3 birds revealed hepatomegaly and splenomegaly, and histologic lesions included necrotizing hepatitis, splenitis, and vasculitis. Chlamydophila psittaci infection was confirmed by results of Gimenez staining, immunohistochemistry, and tissue polymerase chain reaction assay. As additional birds continued to present with similar clinical signs, the entire colony of penguins was prophylactically treated with a 30-day minimum course of doxycycline, administered orally or intramuscularly or as a combination of both. Despite treatment, 9 additional penguins died during a 3-month period. Pathologic results from these birds revealed renal and visceral gout (n = 4), cardiac insufficiency (n = 2), sepsis from a suspected esophageal perforation (n = 2), and no gross lesions (n = 1). During the outbreak, 4 birds presented with seizures, 5 developed dermatitis, and nearly 90% of birds in the colony showed severe keratoconjunctivitis, believed to be related to drug therapy with doxycycline. We report the clinical and pathologic features of Chlamydophila psittaci infection in an outdoor colony of penguins and the associated challenges of treatment.

Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

Evaluation of an arthroscopic approach for transection of the equine collateral sesamoidean ligament.

OBJECTIVE: To evaluate: (1) an arthroscopic technique for transection of the collateral sesamoidean ligament (CSL); and (2) the healing response using magnetic resonance (MR) and microscopic examination. STUDY DESIGN: Experimental study. ANIMALS: Adult horses (n=6). METHODS: Six sound horses with normal front foot radiographic and MR examinations were used. Lameness examination was performed before surgery and monthly for 12 months. Front foot radiography was performed at 180 and 360 days after surgery. Front foot MR was performed before, and at 7, 90, 180, and 360 days after surgery. Arthroscopic CSL desmotomy was performed on 1 forelimb. Gross and microscopic examination was performed on the CSL from both forelimbs at 360 days after surgery. Lameness scores were compared over time using the nonparametric Friedman's test for paired groups. CSL measurements were compared using paired t-tests with a 2-tailed significance level of P<.05. RESULTS: Radiographs remained normal throughout study period. Surgery resulted in lameness on the operated limb for up to 2 months, after which all horses returned to soundness. CSL transection was confirmed during arthroscopy and with MR examination 7 days after surgery. Gross and microscopic evaluation confirmed ligament healing. CONCLUSIONS: CSL desmotomy resulted in short-term lameness after surgery followed by healing of the CSL confirmed by gross and microscopic analysis.

A Babesia bovis gene syntenic to Theileria parva p67 is expressed in blood and tick stage parasites.

Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species' unique host and life-cycle.

Coxiella burnetii infection of a Steller sea lion (Eumetopias jubatus) found in Washington State.

A pregnant sea lion stranded in the State of Washington was found to have placentitis caused by a unique strain of Coxiella burnetii. This is the first description of coxiellosis in a sea lion and suggests that exposure to sea lions may be a risk factor for contracting Q fever.

Identification of Anaplasma marginale proteins specifically upregulated during colonization of the tick vector.

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.

Limited transcriptional response of ovine microglia to prion accumulation.

The conversion of normal cellular prion protein to disease-associated prion protein (PrP(Sc)) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion conversion and the impact of PrP(Sc) accumulation on cellular biology are not fully understood. To further define the molecular changes associated with PrP(Sc) accumulation in cultured cells, the transcriptional profile of PrP(Sc)-accumulating primary ovine microglia was compared to the profile of PrP(Sc)-lacking microglia using the Affymetrix Bovine Genome Array. The experimental design included three biological replicates, each with three technical replicates, and samples that were collected at the point of near maximal PrP(Sc) accumulation levels as measured by ELISA. The array analysis revealed only 19 upregulated genes and 30 downregulated genes in PrP(Sc)-accumulating microglia. The results support the hypothesis that chronic PrP(Sc) accumulation in cultured microglia results in a limited transcriptional response.

Quantitative differences in salivary pathogen load during tick transmission underlie strain-specific variation in transmission efficiency of Anaplasma marginale.

The relative fitness of arthropod-borne pathogens within the vector can be a major determinant of pathogen prevalence within the mammalian host population. Strains of the tick-borne rickettsia Anaplasma marginale differ markedly in transmission efficiency, with a consequent impact on pathogen strain structure. We have identified two A. marginale strains with significant differences in the transmission phenotype that is effected following infection of the salivary gland. We have proposed competing hypotheses to explain the phenotypes: (i) both strains are secreted equally, but there is an intrinsic difference in infectivity for the mammalian host, or (ii) one strain is secreted at a significantly higher level and thus represents delivery of a greater pathogen dose. Quantitative analysis of pathogen replication and secretion revealed that the high-efficiency St. Maries strain replicated to a 10-fold-higher titer and that a significantly greater percentage of infected ticks secreted A. marginale into the saliva and did so at a significantly higher level than for the low-efficiency Israel vaccine strain. Furthermore, the transmission phenotype of the vaccine strain could be restored to that of the St. Maries strain simply by increasing the delivered pathogen dose, either by direct inoculation of salivary gland organisms or by increasing the number of ticks during transmission feeding. We identified morphological differences in the colonization of each strain within the salivary glands and propose that these reflect strain-specific differences in replication and secretion pathways linked to the vector-pathogen interaction.

Small-ruminant lentivirus enhances PrPSc accumulation in cultured sheep microglial cells.

Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C) [C superscript stands for cellular]) to disease-associated prion protein (PrP(Sc) [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrP(Sc), sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrP(Sc) accumulation over time. The accumulated PrP(Sc) demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrP(Sc) demonstrated an approximately twofold increase in PrP(Sc) accumulation compared to that of primary microglia infected with PrP(Sc) alone. The results demonstrate the in vitro utility of PrP(Sc)-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

A species barrier limits transmission of chronic wasting disease to mink (Mustela vison).

Transmissible mink encephalopathy (TME) occurs as sporadic outbreaks associated with ingestion of feed presumably contaminated with some type of prion disease. Mink lack a species barrier to primary oral challenge with bovine spongiform encephalopathy, whereas they have a barrier to such challenge with scrapie. We investigated whether mink have a species barrier to chronic wasting disease (CWD) by performing primary intracerebral (IC) and primary oral challenge with CWD-positive elk brain. Primary IC challenge resulted in clinical disease in two of eight mink at 31-33 months incubation. Affected mink had spongiform vacuolation and astrocytosis within the central nervous system and immunoreactivity to disease-associated prion protein (PrP(d)) in brain, retina and lymph node. CWD IC recipients had significantly lower brain vacuolation and PrP(d) deposition scores, significantly lower cerebrocortical astrocyte counts and significantly higher hippocampal astrocyte counts than TME IC recipients. Primary oral challenge with CWD-positive elk brain (n=22) or with CWD-negative elk brain given IC (n=7) or orally (n=23) did not result in clinical or microscopic abnormalities during 42 months observation. Novel prion gene polymorphisms were identified at codon 27 (arginine/tryptophan) and codon 232 (arginine/lysine). This study shows that, whilst CWD can cause disease when given IC to mink, the lesions are not characteristic of TME, transmission is inefficient compared with TME and oral challenge does not result in disease. The demonstration of a species barrier in cervid-to-mustelid prion transmission indicates that mink are unlikely to be involved in natural CWD transmission.

Bovine immune response to inoculation with Neospora caninum surface antigen SRS2 lipopeptides mimics immune response to infection with live parasites.

Infection of cattle with Neospora caninum protozoa, the causative agent of bovine protozoal abortion, results in robust cellular and humoral immune responses, particularly CD4(+) T-lymphocyte activation and gamma interferon (IFN-gamma) secretion. In the present study, N. caninum SRS2 (NcSRS2) T-lymphocyte-epitope-bearing subunits were incorporated into DNA and peptide preparations to assess CD4(+) cell proliferation and IFN-gamma T-lymphocyte-secretion immune responses in cattle with predetermined major histocompatibility complex (MHC) genotypes. In order to optimize dendritic-cell processing, NcSRS2 DNA vaccine was delivered with granulocyte macrophage-colony-stimulating factor and Flt3 ligand adjuvant. The synthesized NcSRS2 peptides were coupled with a palmitic acid molecule (lipopeptide) and delivered with Freund's adjuvant. Cattle vaccinated with NcSRS2 DNA vaccine alone did not induce T-lymphocyte activation or IFN-gamma secretion, whereas subsequent booster inoculation with NcSRS2-lipopeptides induced robust NcSRS2-specific immune responses. Compared to the response in control animals, NcSRS2-lipopeptide-immunized cattle had significantly increased NcSRS2-specific T-lymphocyte proliferation, numbers of IFN-gamma-secreting peripheral blood mononuclear cells, and immunoglobulin G1 (IgG1) and IgG2a antibody levels. The findings show that N. caninum NcSRS2 subunits bearing T-lymphocyte epitopes induced cell-mediated immune responses similar to the protective immune responses previously described against live parasite infection, namely T-lymphocyte activation and IFN-gamma secretion. The findings support the investigation of NcSRS2 immunogens for protection against N. caninum-induced fetal infection and abortion in cattle.