Prospective fecal microbiomic biomarkers for chronic wasting disease.

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the United States. Here, we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the USA compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer. Through machine-learning, these taxa and taxa pairs at each rank level could facilitate identification of around 70% of both the CWD-negative and the CWD-positive samples. Our results provide a potential tool for diagnostics and surveillance of CWD in the wild, as well as conceptual advances in our understanding of the disease.IMPORTANCEThis is a comprehensive study that tests the connection between the composition of the gut microbiome in deer in response to chronic wasting disease (CWD). We analyzed 50 fecal samples obtained from CWD-positive animals compared to 50 CWD-negative controls to identify CWD-dependent changes in the gut microbiome, matched with the analysis of fecal metabolites. Our results show promising trends suggesting that fecal microbial composition can directly correspond to CWD disease status. These results point to the microbial composition of the feces as a potential tool for diagnostics and surveillance of CWD in the wild, including non-invasive CWD detection in asymptomatic deer and deer habitats, and enable conceptual advances in our understanding of the disease.

Measurement of 8-hydroxy-2'-deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia.

BACKGROUND: Eight-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative damage evaluated in human neurodegenerative disease, has potential to correlate with postmortem diagnosis of neuroaxonal dystrophy/degenerative myeloencephalopathy (NAD/DM) in horses. HYPOTHESIS: We hypothesized that 8-OHdG will be higher in CSF and serum from NAD/DM horses compared with horses with other neurologic diseases (CVSM, EPM) and a control group of neurologically normal horses. We also hypothesized that 8-OHdG will be higher in CSF compared with serum from NAD/DM horses. ANIMALS: Fifty client-owned horses with postmortem diagnoses: 20 NAD/DM, 10 CVSM, 10 EPM, and 10 control horses. Serum and CSF samples were obtained between November 2010 and March 2022. METHODS: Case-control study using biobanked samples was performed and commercial competitive ELISA kit (Highly Sensitive 8-OHdG Check ELISA) utilized. Concentration of 8-OHdG was quantitated in both CSF and serum and compared between groups. RESULTS: No correlation was established between the measures of 8-OHdG in serum and CSF and group. CSF median [8-OHdG] for NAD/DM was 169.9 pg/mL (IQR25-75 : 67.18-210.6), CVSM 157.1 pg/mL (IQR25-75 : 132.1-229.1), EPM 131.4 pg/mL (IQR25-75 : 102.1-193.2), and control 149.8 pg/mL (IQR25-75 : 113.3-196.4). Serum median [8-OHdG] for NAD/DM was 130 pg/mL (IQR25-75 : 51.73-157.2), CVSM 125.8 pg/mL (IQR25-75 : 62.8-170.8), EPM 120.6 pg/mL (IQR25-75 : 87.23-229.7), and control 157.6 pg/mL (IQR25-75 : 97.15-245.6). Poisson regression analysis showed no difference established once confounding variables were considered. CONCLUSIONS: Eight-OHdG did not aid in antemortem diagnosis of NAD/DM in this cohort of horses. At the time of diagnosis horses with NAD/DM do not have ongoing oxidative stress.

Clinical and histopathological features in horses with neuroaxonal degeneration: 100 cases (2017-2021).

BACKGROUND: Adult horses with proprioceptive ataxia and behavior changes that have histologic lesions consistent with neurodegenerative disease have been increasingly recognized. HYPOTHESIS/OBJECTIVES: Describe the history, clinical findings and histopathologic features of horses presented to a referral institution with neuroaxonal degeneration. ANIMALS: One hundred horses with a necropsy diagnosis of neuroaxonal degeneration compatible with neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM). METHODS: Retrospective study of horses presented to the University of Pennsylvania, New Bolton Center, between 2017 and 2021 with a necropsy diagnosis of eNAD/EDM. RESULTS: Affected horses had a median age of 8 years (range, 1-22), and the majority were Warmbloods (72). Sixty-eight horses had behavioral changes, and all 100 had proprioceptive ataxia (median grade, 2/5). Fifty-seven horses had abnormal findings on cervical vertebral radiographs, and 14 had myelographic findings consistent with compressive myelopathy. No antemortem diagnostic test results were consistently associated with necropsy diagnosis of neurodegenerative disease. All 100 horses had degenerative lesions characteristic of eNAD in the brainstem gray matter, and 24 had concurrent degenerative features of EDM in the spinal cord white matter. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histopathologic findings in this large group of horses with neurodegenerative disease were most consistent with eNAD/EDM, but with a different signalment and clinical presentation from earlier descriptions. The increasing occurrence of neurodegenerative disease in horses and the safety risk posed emphasize the importance of focused research in affected horses.

Subarachnoid hemorrhage and axonal degeneration after C1-C2 cervical centesis in 2 horses.

Ultrasound-guided cervical centesis has gained popularity as a method for collecting cerebrospinal fluid (CSF) from standing horses. There are anecdotal reports of neck stiffness, regional swelling, sensitivity to palpation, and fever after the procedure. We report 2 horses with complications that occurred within days of C1-C2 centesis and ultimately resulted in euthanasia. Both C1-C2 centesis were performed routinely, with CSF cytologic analysis providing no evidence of blood contamination. Post-mortem examination revealed equine degenerative myeloencephalopathy as the primary disorder causing Horse 1's initial neurologic deficits, whereas Horse 2 did not have a distinct lesion explaining the horse's deficits. Both horses had evidence of subarachnoid hemorrhage at or near the centesis site with Wallerian axonal degeneration in the cranial cervical spinal cord. Although hemorrhage with associated axonal degeneration at the cervical centesis site appears to be rare, this complication of C1-C2 centesis should be considered as this technique gains popularity.

Multiple Sex Cord-stromal Tumors in a Standardbred Stallion Testis.

A 12-year-old Standardbred stallion presented with a 5-month history of a growing mass in the left testis as well as an overall decrease in left testicular size. Palpation and ultrasonography of the left testis revealed a firm, hypoechoic, clearly delineated soft tissue mass in the craniolateral portion of the testis that measured 2.5 × 2.3 × 1.9 cm. Two smaller, hypoechoic regions also were visible ultrasonographically in the left testis, suggesting the presence of multifocal/multicentric neoplasia. The affected testis was very small (testicular volume of 40.3 cm3). The right testis was significantly larger (144.3 cm3), and the parenchyma was ultrasonographically normal. Due to the concern that these findings could indicate the presence of a more aggressive tumor type, unilateral orchiectomy was performed. Multiple soft tissue masses were identified grossly, and histopathologic evaluation identified the larger mass as a Sertoli cell tumor and the two smaller masses as mixed sex cord-stromal tumors with Sertoli cell and Leydig cell differentiation. To our knowledge, this the first report of concurrent Sertoli cell and mixed sex cord-stromal tumors in a single descended equine testis.

Onchocerca sp. in an imported Zangersheide gelding causing suspensory ligament desmitis.

A 5-year-old imported Zangersheide gelding was evaluated for SC swellings over both forelimbs and lameness localized to the distal metacarpus. Ultrasound examination of the SC masses was compatible with verminous granulomas. Linear hyperechoic foci were present within the suspensory ligament branches of both forelimbs, suggestive of ligamentous parasitic infiltrates. A diagnosis of onchocerciasis was confirmed on biopsy of a SC mass. The gelding was treated with ivermectin and a tapering course of PO dexamethasone but was eventually euthanized. Necropsy confirmed the presence of SC eosinophilic granulomas and degenerative suspensory ligament desmitis, both with intralesional nematodes. Given the location and appearance of the nematode, a diagnosis of Onchocerca sp., most likely O. reticulata, was made. Onchocerciasis should be included as a differential diagnosis for multifocal suspensory ligament desmitis with these sonographic characteristics when paired with SC masses in imported European Warmbloods.

Intracranial medulloblastoma as the cause of progressive ataxia in a 6-month-old draft horse cross gelding.

We describe the unique clinical presentation of a central nervous system neoplasm in a 6-month-old draft horse cross gelding. Based on the neurologic examination at admission, neurolocalization was most consistent with a mildly asymmetric cervical, multifocal, or diffuse myelopathy. Mild vestibular involvement also was considered, but no cranial nerve deficits were observed. The gelding was negative for Sarcocystis neurona or Neospora hughesi based on paired serum and cerebrospinal fluid (CSF) samples analyzed, with no evidence of cervical compression based on contrast myelography. The horse was euthanized because of progression of clinical signs. At necropsy, a mass was identified associated with the cerebellum, and histopathology was consistent with medulloblastoma, which has not been reported previously in the horse.

Percutaneous doxycycline sclerotherapy in a horse with a mandibular aneurysmal bone cyst.

OBJECTIVE: To describe the use and outcome of sclerotherapy with intralesional doxycycline foam in a horse with a mandibular aneurysmal bone cyst. STUDY DESIGN: Case report. ANIMALS: Client-owned 1 year old Standardbred filly. METHODS: The horse presented for progressive mandibular swelling. A 10 mg/mL doxycycline foam was prepared for intralesional injection. Three doses were injected into the lesion under computed tomographic guidance at 6 and 15 weeks after initial treatment. Volume reduction was monitored after each treatment with 3D volumetric rendering and region of interest segmentation using commercially available software. RESULTS: The volume of the lesion decreased from 458.7455 cm3 before treatment, to 363.3101 cm3 at 6 weeks, 273.5855 cm3 at 15 weeks, and 247.2316 cm3 6 months later, resulting in a total reduction of 54% of the initial volume. Bone formation was noted in the lesion. No adverse effects related to doxycycline foam injections were noted. The mandibular swelling was resolved after treatment. CONCLUSION: Intralesional doxycycline sclerotherapy was shown to be efficacious in reducing the volume of the aneurysmal bone cyst in the horse presented in this report. There was complete resolution of mandibular swelling with no side effects related to the intralesional injections.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Phlegmonous gastritis in 2 yearling horses.

Phlegmonous gastritis was diagnosed in 2 yearling fillies that were presented with a 1-wk history of fever, lethargy, and hypoproteinemia, associated with a previous diagnosis of equine proliferative enteropathy based on clinical signs and PCR assay detection of Lawsonia intracellularis in fecal samples. Abdominal ultrasound revealed enlargement of the stomach and expansion of its submucosal layer with hypoechoic fluid, as well as thickened hypomotile small intestinal segments. Given the poor prognosis and poor response to treatment, both horses were euthanized, one on the day of presentation and the other after 3 wk of intensive medical management including a combination of antimicrobials, analgesics, and intravenous colloids. At autopsy, acute mural gastritis characterized by severe submucosal edema with suppurative inflammation (i.e., phlegmonous gastritis) and necroulcerative enteritis compatible with the necrotizing form of equine proliferative enteropathy were identified in both horses. The gastric inflammation was associated with thrombosis and mixed bacterial populations, including Clostridium perfringens, that were confined to the submucosa without evidence of mucosal involvement; toxin genes compatible with C. perfringens type C were identified in one case. Human phlegmonous gastritis is an uncommon, often-fatal pyogenic infection that is often associated with mucosal injury, bacteremia, or immunocompromise. Our finding of this unusual gastric lesion in 2 horses with similar signalment, clinical disease, and spectrum of postmortem lesions suggests a similar etiopathogenesis that possibly involves local, regional, or distant hematogenous origin, and should be considered a potential complication of gastrointestinal mucosal compromise in horses.

A Subset of Equine Gastric Squamous Cell Carcinomas Is Associated With Equus Caballus Papillomavirus-2 Infection.

Squamous cell carcinoma (SCC) is the most common neoplasm of the equine stomach. However, the mechanisms underlying malignant transformation are unknown. As Equus caballus papillomavirus-2 (EcPV-2) is a likely cause of some genital SCCs, we hypothesized that EcPV-2 is associated with a subset of equine gastric SCCs. To this aim, we performed polymerase chain reaction (PCR) and in situ hybridization (ISH) for EcPV-2 E6/ E7 oncogenes on 11 gastric SCCs and on gastric samples from 15 control horses with no SCC. PCR for EcPV-2 was positive in 7/11 (64%) gastric SCCs; non-SCC gastric samples were all negative. Intense hybridization signals for EcPV-2 E6/E7 nucleic acid were detected by ISH within tumor cells in 5/11 (45%) gastric SCCs, including distant metastases. No hybridization signals were detected within any of the non-SCC gastric cases. This study provides support for a potential association between EcPV-2 infection and a subset of equine gastric SCC.

Neoplasia of the tubular genital tract in 42 goats.

OBJECTIVE: To assess the prevalence of tubular genital tract neoplasia in does evaluated at 2 veterinary teaching hospitals; describe the main clinical, surgical, and histopathologic or necropsy findings in affected does; and assess factors potentially associated with short-term prognosis in these animals. ANIMALS: 42 does. PROCEDURES: Medical records of 2 veterinary teaching hospitals were searched to identify does with neoplasia of the tubular genital tract. Signalment; history; physical and diagnostic imaging results; biopsy, surgery, and necropsy findings; and short-term outcome were recorded. Age and breed frequencies for the sample were compared with those of the overall hospital population, and variables of interest were tested for associations with a diagnosis of adenocarcinoma and with short-term outcome by statistical methods. RESULTS: Median age at hospital admission (10 years) was greater for the study sample than for the general hospital population (2 years). Pygmy goats were overrepresented (22/42 [52%]). Common reasons for evaluation were bloody vaginal discharge or hematuria and abdominal straining. Adenocarcinoma (13/42 [31%]), leiomyoma (13 [31%]), and leiomyosarcoma (11 [26%]) were the most common tumors. Does with distant metastasis had greater odds of a diagnosis of adenocarcinoma (OR, 40.5) than does without distant metastasis. In the analysis adjusted for hemorrhagic discharge, odds of euthanasia for does with straining were 13 times those for does without straining. In the analysis adjusted for straining status, does with hemorrhagic discharge had almost 7 times the odds of euthanasia for does without this finding. The survival-to-discharge rate was low (13/42 [31%]). CONCLUSIONS AND CLINICAL RELEVANCE: The frequency of adenocarcinomas in the study sample was unexpectedly high. Further research is needed to confirm the study findings.

Pyrethroid toxicity in a Vietnamese pot-bellied pig.

A 7-month-old spayed female Vietnamese pot-bellied pig (VPBP) was presented for diffuse muscle fasciculations and seizure-like activity that had started 4 hours before presentation. The pig was stuporous and displayed diffuse involuntary gross motor movement and muscle fasciculations, as well as hypertonicity of all 4 limbs. Hematologic analysis revealed hemoconcentration, severe hyperlactatemia, moderate metabolic acidosis, increased creatine kinase and gamma-glutamyltransferase. The pig failed to respond to diazepam, propofol, methocarbamol, and supportive care, followed by general anesthesia, and was euthanized. Bifenthrin, a pyrethroid insecticide, was identified by gas chromatography-mass spectrometry of stomach contents. Pyrethroid insecticide toxicity in VPBP may result in clinical signs similar to those seen in companion animals.

Toxicité pyréthroïde chez un Cochon vietnamien. Une truie stérilisée Cochon vietnamien (CV) âgée de 7 mois a été présentée pour des fasciculations des muscles diffus et une activité s'apparentant à des crises d'épilepsie qui avaient commencé 4 heures avant la présentation. Le cochon était stuporeux et manifestait des mouvements moteurs diffus et des fasciculations musculaires involontaires ainsi que de l'hypertonicité des quatre membres. Une analyse hématologique a révélé l'hémoconcentration, une hyperlactatémie grave, une acidose métabolique modérée, une kinase de créatine et une gamma-glutamyltransférase élevées. Le cochon n'a pas répondu au diazépam, au propofol, au méthocarbamol et à des soins de soutien suivis de l'anesthésie générale et a été euthanasié. Le bifenthrine, un insecticide de pyréthroide, a été identifié par chromatographie en phase gazeuse et spectométrie de masse du contenu de l'estomac. La toxicité de l'insecticide de pyréthroïde chez le Cochon vietnamien peut donner des signes cliniques semblables à ceux observés chez les animaux de compagnie.(Traduit par Isabelle Vallières).

Myocardial infarct associated with a partial thickness left atrial tear in a dog with mitral insufficiency.

A 10-year-old male neutered cavalier King Charles Spaniel with a 1-year history of degenerative mitral valve disease presented for dyspnea and severe weakness. He was diagnosed with congestive heart failure, systolic dysfunction, presumptive myocardial infarction and a left atrial thrombus based on thoracic radiographs, electrocardiogram and echocardiographic findings. Clinical signs also suggested right foreleg embolism. The dog was euthanized due to the grave prognosis and a postmortem evaluation was performed. The postmortem examination confirmed myocardial infarction and was thought to be due to embolic showering from the thrombus attached to a partial thickness left atrial endocardial tear.

Murine coronavirus receptors are differentially expressed in the central nervous system and play virus strain-dependent roles in neuronal spread.

Coronavirus infection of the murine central nervous system (CNS) provides a model for studies of viral encephalitis and demyelinating disease. Mouse hepatitis virus (MHV) neurotropism varies by strain: MHV-A59 causes mild encephalomyelitis and demyelination, while the highly neurovirulent strain JHM.SD (MHV-4) causes fatal encephalitis with extensive neuronal spread of virus. In addition, while neurons are the predominant CNS cell type infected in vivo, the canonical receptor for MHV, the carcinoembryonic antigen family member CEACAM1a, has been demonstrated only on endothelial cells and microglia. In order to investigate whether CEACAM1a is also expressed in other cell types, ceacam1a mRNA expression was quantified in murine tissues and primary cells. As expected, among CNS cell types, microglia expressed the highest levels of ceacam1a, but lower levels were also detected in oligodendrocytes, astrocytes, and neurons. Given the low levels of neuronal expression of ceacam1a, primary neurons from wild-type and ceacam1a knockout mice were inoculated with MHV to determine the extent to which CEACAM1a-independent infection might contribute to CNS infection. While both A59 and JHM.SD infected small numbers of ceacam1a knockout neurons, only JHM.SD spread efficiently to adjacent cells in the absence of CEACAM1a. Quantification of mRNA for the ceacam1a-related genes ceacam2 and psg16 (bCEA), which encode proposed alternative MHV receptors, revealed low ceacam2 expression in microglia and oligodendrocytes and psg16 expression exclusively in neurons; however, only CEACAM2 mediated infection in human 293T cells. Therefore, neither CEACAM2 nor PSG16 is likely to be an MHV receptor on neurons, and the mechanism for CEACAM1a-independent neuronal spread of JHM.SD remains unknown.

Pathogenesis of murine coronavirus in the central nervous system.

Murine coronavirus (mouse hepatitis virus, MHV) is a collection of strains that induce disease in several organ systems of mice. Infection with neurotropic strains JHM and A59 causes acute encephalitis, and in survivors, chronic demyelination, the latter of which serves as an animal model for multiple sclerosis. The MHV receptor is a carcinoembryonic antigen-related cell adhesion molecule, CEACAM1a; paradoxically, CEACAM1a is poorly expressed in the central nervous system (CNS), leading to speculation of an additional receptor. Comparison of highly neurovirulent JHM isolates with less virulent variants and the weakly neurovirulent A59 strain, combined with the use of reverse genetics, has allowed mapping of pathogenic properties to individual viral genes. The spike protein, responsible for viral entry, is a major determinant of tropism and virulence. Other viral proteins, both structural and nonstructural, also contribute to pathogenesis in the CNS. Studies of host responses to MHV indicate that both innate and adaptive responses are crucial to antiviral defense. Type I interferon is essential to prevent very early mortality after infection. CD8 T cells, with the help of CD4 T cells, are crucial for viral clearance during acute disease and persist in the CNS during chronic disease. B cells are necessary to prevent reactivation of virus in the CNS following clearance of acute infection. Despite advances in understanding of coronavirus pathogenesis, questions remain regarding the mechanisms of viral entry and spread in cell types expressing low levels of receptor, as well as the unique interplay between virus and the host immune system during acute and chronic disease.

CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors.

Immunotherapy of established solid tumors is rarely achieved, and the mechanisms leading to success remain to be elucidated. We previously showed that extended control of advanced-stage autochthonous brain tumors is achieved following adoptive transfer of naive C57BL/6 splenocytes into sublethally irradiated line SV11 mice expressing the SV40 T Ag (T Ag) oncoprotein, and was associated with in vivo priming of CD8(+) T cells (T(CD8)) specific for the dominant epitope IV (T Ag residues 404-411). Using donor lymphocytes derived from mice that are tolerant to epitope IV or a newly characterized transgenic mouse line expressing an epitope IV-specific TCR, we show that epitope IV-specific T(CD8) are a necessary component of the donor pool and that purified naive epitope IV-specific T(CD8) are sufficient to promote complete and rapid regression of established tumors. While transfer of naive TCR-IV cells alone induced some initial tumor regression, increased survival of tumor-bearing mice required prior conditioning of the host with a sublethal dose of gamma irradiation and was associated with complete tumor eradication. Regression of established tumors was associated with rapid accumulation of TCR-IV T cells within the brain following initial priming against the endogenous T Ag in the peripheral lymphoid organs. Additionally, persistence of functional TCR-IV cells in both the brain and peripheral lymphoid organs was associated with long-term tumor-free survival. Finally, we show that production of IFN-gamma, but not perforin or TNF-alpha, by the donor lymphocytes is critical for control of autochthonous brain tumors.

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia.

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR(-/-)) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-beta in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-beta production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-beta production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-beta mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-beta by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.

Priming of CD8+ T cells during central nervous system infection with a murine coronavirus is strain dependent.

Virus-specific CD8(+) T cells are critical for protection against neurotropic coronaviruses; however, central nervous system (CNS) infection with the recombinant JHM (RJHM) strain of mouse hepatitis virus (MHV) elicits a weak CD8(+) T-cell response in the brain and causes lethal encephalomyelitis. An adoptive transfer model was used to elucidate the kinetics of CD8(+) T-cell priming during CNS infection with RJHM as well as with two MHV strains that induce a robust CD8(+) T-cell response (RA59 and SJHM/RA59, a recombinant A59 virus expressing the JHM spike). While RA59 and SJHM/RA59 infections resulted in CD8(+) T-cell priming within the first 2 days postinfection, RJHM infection did not lead to proliferation of naïve CD8(+) T cells. While all three viruses replicated efficiently in the brain, only RA59 and SJHM/RA59 replicated to appreciable levels in the cervical lymph nodes (CLN), the site of T-cell priming during acute CNS infection. RJHM was unable to suppress the CD8(+) T-cell response elicited by RA59 in mice simultaneously infected with both strains, suggesting that RJHM does not cause generalized immunosuppression. RJHM was also unable to elicit a secondary CD8(+) T-cell response in the brain following peripheral immunization against a viral epitope. Notably, the weak CD8(+) T-cell response elicited by RJHM was unique to CNS infection, since peripheral inoculation induced a robust CD8(+) T-cell response in the spleen. These findings suggest that the failure of RJHM to prime a robust CD8(+) T-cell response during CNS infection is likely due to its failure to replicate in the CLN.