CD56+ B-cell Neurolymphomatosis in a Cat.

A 16-year-old male Russian blue cat was presented with acute onset of paraparesis of the forelimbs that progressed to tetraparesis. Neurological examination revealed non-ambulatory tetraparesis with decreased postural reactions in all four limbs. Magnetic resonance imaging revealed multifocal nerve root swelling on the right at C6/C7 and C7/T1, while ultrasonography demonstrated swelling of the right brachial plexus. To understand the cause of the nerve swelling, the right musculocutaneous nerve arising from the brachial plexus and the pectoralis muscle were biopsied. Histologically, there was evidence of neurolymphomatosis (neurotropic lymphoma) with Wallerian degeneration and denervation atrophy of myofibres. The neoplastic lymphoid cells expressed CD79a, CD20 and CD56. Based on these findings, a diagnosis of B-cell neurolymphomatosis was made. Expression of CD56, synonymous with neural cell adhesion molecule, is rare in B-cell lymphomas and has not been reported in feline B-cell lymphomas or feline neurolymphomatosis. CD56 expression was suspected to have played an important role in neurotropism of the neoplastic cells in this case.

Emergence of a sylvatic enzootic formosan ferret badger-associated rabies in Taiwan and the geographical separation of two phylogenetic groups of rabies viruses.

Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation.

Areca nut extract suppresses T-cell activation and interferon-gamma production via the induction of oxidative stress.

Areca quid chewing is a major risk factor associated with oral submucous fibrosis (OSF) and oral cancer. Experimental evidence indicates that immune deterioration is associated with the pathophysiology of OSF and oral cancer. In addition, reactive oxygen species (ROS) is shown to play a role in the cytotoxic and genotoxic effect induced by areca nut extracts (ANE) in oral cells. The present studies investigated the effects of ANE on T-cell reactivity and the role of ROS in ANE effects. Treatment of splenocytes with ANE induced a marked cytotoxic effect, and suppressed the production of IL-2 and IFN-gamma, whereas the production of IL-4 was unaffected. The ANE-mediated cytotoxicity, and suppression of IFN-gamma and IL-2 production were attenuated by the presence of antioxidant N-acetyl-l-cysteine (NAC). Moreover, flow cytometric analysis demonstrated an increase in cellular ROS levels in splenic T-cells treated with ANE, which was also attenuated by the presence of NAC. Concordantly, the cellular level of glutathione was diminished by ANE in splenic T-cells pretreated with NAC. Collectively, these results demonstrated that ANE markedly suppressed T-cell activation and Th1 cytokine production, which was mediated, at least in part, by the induction of oxidative stress.

Vaccine-associated rhabdomyosarcoma with spinal epidural invasion and pulmonary metastasis in a cat.

A 7-year-old, female, domestic medium-haired cat had a recurrent deep dermal mass in the interscapular region after initial surgical removal 3 months earlier. The cat had received a killed rabies vaccine and a five-in-one vaccine in the same area about 2 months prior to the first surgery. The relapsed mass was diagnosed as vaccine-associated sarcoma. The cat was euthanized 2 months later because of hind limb paralysis. At necropsy, multiple, poorly demarcated, nodular masses were seen in the muscles around the shoulders, neck, and thoracic vertebrae. Pulmonary metastasis and spinal epidural invasion at T1-T3 with regional cord compression and malacia were observed. Microscopically, the masses consisted of interwoven bundles of spindle cells with prominent multinucleated giant cell formation. The neoplastic cells stained strongly positive for myoglobin, and moderately but variably positive for vimentin, desmin, and alpha- smooth muscle actin. Phosphotungstic acid-hematoxylin staining revealed cytoplasmic striations in scattered tumor cells. The tumor was considered a vaccine-associated rhabdomyosarcoma.

Bovine ephemeral fever in Taiwan.

Bovine ephemeral fever (BEF) is a vector-borne disease of cattle, spanning tropical and subtropical zones of Asia, Australia, and Africa, caused by Ephemerovirus of the Rhabdoviridae. Taiwan has had 3 BEF epizootics, occurring in 1989, 1996, and 1999, since the vaccination regimen was initiated in 1984, given once a year in the spring with a single-dose formaldehyde-inactivated vaccine using the 1983 isolate as the seed virus. This study evaluated the 1999 population immunity against BEF virus in Taiwanese dairy cows with a neutralization test and whether the recent BEF virus isolates have mutated significantly from the vaccine virus. In March 1999, before vaccination, 94% of the animals studied were already seropositive, suggestive of an endemic or persistent infection from the previous year. By June 1999, when 51% of herds had been vaccinated, the antibody level rose, and by September 1999, the serum-neutralizing antibody (SNA) level fell to a minimum, preceding the outbreak of BEF in October 1999, during which the antibody levels of vaccinated cows continued to decline while those of unvaccinated cows rose sharply. The results suggest that, in 1999, vaccine-induced immunity was partially protective against BEE Because the current single-dose vaccination regimen resulted in minimal population immunity by September, a booster vaccination given in late summer may be advisable for future disease control. Analysis of the glycoprotein gene of Taiwanese isolates between 1983 and 1999 showed a 97.4-99.6% homology, with an alteration of 4 amino acids in antigenic sites G1, G3b, and G3c. Phylogenetic analysis of Taiwanese isolates revealed at least 2 distinct clusters: the 1983-1989 isolates and the 1996-1999 isolates. Both were distinct from 2 Japanese strains and the Australian BB7721 strain. Thus, at least 2 distinct BEF viruses, which had diverged before 1983, existed in Taiwanese dairy cows.

Hydatidosis in a Chapman's zebra (Equus burchelli antiquorum).

A 14-year-old male Chapman's zebra had been treated for a cervical granulomatous cellulitis for 3 months prior to sudden death associated with myocardial fibrosis. Incidental findings at necropsy included multiple 3-8-cm unilocular cysts in the liver and lungs. Cysts contained either a clear fluid with or without hydatid sand or a gelatinous substance with foci of mineralization. Light and scanning electron microscopic examination of the hydatid sand revealed free protoscolices as well as intact and ruptured brood capsules with protoscolices attached to the germinal membrane. The protoscolices had 2 rows of 36-38 rostellar hooks with a length of 25-30 microm. The cyst wall consisted of inner germinal, intermediate laminated, and outer adventitial layers. Hydatidosis caused by Echinococcus granulosus was diagnosed based on the unilocular cysts, multiple protoscolices formed in a brood capsule, typical trilayered cyst wall, and herbivorous intermediate host. This is the first reported case of animal hydatidosis in Taiwan. The infection is thought to have been established 12 years ago in South Africa prior to importation.

A primary retroperitoneal seminoma invading the kidneys of a cryptorchid dog.

The relationship between cryptorchidism and testicular tumors has been well established in canines, and the tumor has been proposed as a model for studying its human counterparts. Herein we report canine malignant retroperitoneal seminoma in a 4-year-old castrated Basset hound, most likely without testicular involvement, similar to that of the classic seminoma of humans.

A renal hemangiosarcoma causing hematuria in a dog.

A 14.5-year-old male dog was presented with stranguria and hematuria of 1-month duration. Hematology and blood chemistry revealed a neutrophilia, mild azotemia and a mild decrease in the packed cell volume. Urinalysis showed high specific gravity (> 1.040 g/mL), hematuria, proteinuria and mild bilirubinuria. On physical examination, a firm oval mass located caudal to the distended urinary bladder, was palpated. Differential diagnoses included prostatitis, prostatic neoplasm, prostatic hyperplasia, and abscess. The enlarged prostate was suspected to be the cause of hematuria, and a total prostatectomy was performed. Histologically, the prostate was affected by a prostatitis with cystic papillary hyperplasia of the epithelium. The dog's condition continued to deteriorate, and death occurred 1 week later. Necropsy showed a tumor mass, approximately 5 x 4 x 3 cm in size, between the abdominal aorta and the left kidney, where the adrenal glands were embedded. Lesions were found in the kidneys, adrenal gland, lungs, heart, liver, intestine, and serosa of viscera, while the spleen was spared. This hemangiosarcoma most likely arose from the renal arteries, resulting in diffuse lesions in the kidneys thought to be the cause of hematuria.

A peritoneal mesothelioma in a captive aardwolf (Proteles cristatus).

A 10-year-old male aardwolf (Proteles cristatus) was presented abdominal distention and emaciation for 3 months. Physical examination revealed firm abdominal masses with effusions. Cytologic assessment of the effusion showed uniform round tumor cells with a surface brush border. Necropsy showed white velvety masses covering the peritoneal surface of the liver, gall bladder, stomach, omentum, mesentery, spleen, intestine, abdominal wall and diaphragm. Histologic examination demonstrated papillary projections, lined with cuboidal tumor cells supported by fibrous connective tissue cores, arising from the serosa of visceral organs. Cytoplasmic vacuolation and a surface brush border were evident on some cells under light microscopy. Tumor cells stained positive for both cytokeratin (AE1/AE3) and vimentin. Electron microscopy showed prominent surface microvilli, rough endoplasmic reticulum, mitochondria and desmosomes in tumor cells. This may be the first reported case of peritoneal mesothelioma in a captive wild aardwolf.

In vitro migratory responses of swine neutrophils to actinobacillus pleuropneumoniae.

Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed. About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control. The migration of APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration. When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively. Heat-killed APP or E. coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC). Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP. The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC.

Orbital adenocarcinoma of lacrimal gland origin in a dog.

A 13-year-old intact female mixed-breed dog was presented for a progressive enlargement of the right eye, which had been treated previously for conjunctivitis. A round, firm mass, approximately 4 cm in diameter, was protruding from the superotemporal aspect of the right orbit, displacing the eyeball anteriorly and ventromedially. The mass was encapsulated, distinct from the eyeball, and not associated with the eyelids. On cut surface, there was a pale multilobulated periphery, with a dark red, soft, and depressed core. Histologically, tumor cells formed cords and tubules, which were stained with mouse anti-human cytokeratin antibody AE1/AE3. Residual glands were serous, and the majority of tumor cells were negative for mucin. The supraorbital location, encapsulation, and residual serous glands suggest that this mass was a low-grade adenocarcinoma of the lacrimal gland.

Unilateral concurrence of pyelocaliceal diverticula and intracapsular angiomyolipoma in the kidney of a cat.

A 7-month-old cat was examined for progressive abdominal distension. Radiography showed a fluid-containing cyst, which had subsequently ruptured as the result of a fall. Nephrectomy was performed, and examination revealed the coexistence of pyelocaliceal diverticula with a cystic intracapsular angiomyolipoma (mesenchymal hamartoma) in the left kidney. The diverticula were present on both cranial and caudal poles of the kidney and were lined by transitional epithelium. The hamartoma was characterized by the presence of multiple mesenchymal tissues, including thick-walled blood vessels, smooth muscle, and adipose tissue.

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.

Minimal residues of porcine reproductive and respiratory syndrome virus in pig carcases and boar semen.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease that affects pigs of all ages worldwide. One of the key features of PRRS pathogenesis is the prolonged viremia in which the virus coexists with antibody. Prolonged viremia raises the concern that porcine products and boar semen may be contaminated by residues of the PRRS virus serving as vehicles for spreading of the virus. To answer this question, we sampled blood, muscles and viscera organs from market pigs slaughtered using an on-the-rail system and utilized a direct reverse transcription-polymerase chain reaction (RT-PCR) to detect residual viral RNA. We found that the overall seropositive rate of the market pigs was 85.4% (205/240), yet only 7.9% (11/140) of the blood samples contained detectable amounts of virus residues, and all tested carcase specimens (n = 472) were negative. The clinical sensitivity of this PCR varied with the tissues tested, with 5 TCID50 per 50 microliters of serum, as determined by means of a spiking experiment. Semen samples (n = 38) of clinically healthy seropositive and seronegative boars were collected from six herds which were routinely subjected to artificial insemination for production purposes. None of the seminal plasma or sperm-rich fractions contained PCR detectable virus residues. However, the possibility of PRRS virus present in semen cannot be totally excluded. We conclude that in naturally infected albeit clinically healthy pigs, the amounts of PRRS virus residues in carcases or semen are minimal. Thus, the risk of these products causing animal health hazards is low.

Sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus Taiwan MD-001 strain.

The 3'-portion of the genome from a Taiwan isolate of porcine reproductive and respiratory syndrome (PRRS) virus, strain MD-001, was cloned and sequenced. The resultant 549 nucleotides contained an open reading frame with a coding capacity of 123 amino acids (predicted Mr 13,600). The predicted protein corresponds to the nucleocapsid protein, the gene product of ORF7. Comparative sequence analysis of several known PRRSV strains indicated that this protein showed the highest degree of amino acid similarity to the US VR2332 and the Canadian IAF-Exp91 strains (92.7%) and the least to the Dutch Lelystad strain (56.5%). The phylogenic trees constructed on the basis of the known PRRSV nucleotide sequences indicated that MD-001 strain belongs to the North American strain cluster and that it is distinct from the European virus.

Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression.

The hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) is an optional envelope protein present in only some MHV isolates. Its expression is regulated by the copy number of a UCUAA pentanucleotide sequence present in the leader sequence of the viral genomic RNA. The functional significance of this viral protein so far is not clear. In this report, we compared the neuropathogenicity of two MHV isolates, JHM(2) and JHM(3), which express different amounts of HE protein. Intracerebral inoculation of these two viruses into C57BL/6 mice showed that JHM(2), which expresses an abundant amount of HE protein, was more neurovirulent than JHM(3), which expresses very little HE. Histopathology showed that early in infection, JHM(2) infected primarily neurons, while JHM(3) infected mainly glial cells. JHM(3) eventually infected neurons and caused a delayed death relative to JHM(2)-infected mice, suggesting that the progression of JHM(3) infection in the central nervous system was slower than JHM(2). In vitro infection of JHM(3) in primary mixed glial cell cultures of astrocyte-enriched cultures yielded higher virus titers than JHM(2), mimicking the preferential growth of JHM(3) in glial cells in vivo. These findings suggest that the reduced neuropathogenicity of JHM(3) may correlate with its preferential growth in glial cells. Sequence analysis showed that the S genes of these two viruses are identical, thus ruling out the S gene as the cause of the difference in neuropathogenicity between these two viruses. We conclude that the HE protein contributes to viral neuropathogenicity by influencing either the rate of virus spread, viral cell tropism or both.

The role of lymphotoxin in the IL-2-driven differentiation of human lymphokine-activated T-killer (T-LAK) cells in vitro.

Brief stimulation of human peripheral blood mononuclear cells with PHA and subsequent coculture with IL-2 results by 5 days in cultures of human lymphokine-activated killer (T-LAK) cells. While IL-2 drives the proliferation of these cells in vitro, their maturation into functional effector cells capable of cytokine secretion and cell cytokines depends on the presence of other cytokines. The role of LT in the differentiation and proliferation of human T-LAK cells in vitro was investigated. Higher levels of LT than TNF were secreted by T-LAK cells during the first 5 days of the primary culture, then secretion levels dropped sharply. Human T-LAK cells cultivated with anti-LT rabbit antisera showed a slight reduction in growth compared to normal rabbit serum controls. In contrast, phenotypic analysis by FACS showed a decrease in CD4+ and an increase in CD8+ populations of T-LAK cells in the treated cultures. Addition of LT from the beginning of the T-LAK cell culture resulted in an increase in CD4+ and a decrease in CD8+ cell populations at day 7. In addition, the cytolytic activity of non-MHC-restricted cytotoxicity and NK-like activity of anti-LT cultured T-LAK cells was also effected. These data indicated that LT may have a role in differentiation of IL-2 stimulated human T-LAK cells in vitro.

Interaction of immune and central nervous systems: contribution of anti-viral Thy-1+ cells to demyelination induced by coronavirus JHM.

The murine coronavirus JHM (JHMV or MHV-4) has been intensively studied as an experimental model of viral-induced demyelination; nonetheless, the degree to which demyelination results from direct viral cytolysis of oligodendroglia or immunological mechanisms remains controversial. To examine the contribution of immunity to the pathogenesis of JHMV in the central nervous system (CNS), mice were exposed to immunosuppressive doses of x-irradiation 3 days post infection and observed for clinical and pathological evidence of acute and subacute demyelination. Irradiated mice were found to have a nearly thousand-fold increase in central nervous system virus titer, as well as the presence of both abundant virus and viral antigen in white matter cells with the morphological characteristics of oligodendrocytes. Nonetheless, infected, irradiated mice had little or no evidence of demyelination or destruction of CNS cells. Adoptive transfers of spleen cells from syngeneic JHMV-immunized donors into irradiated JHMV-infected mice were carried out in order to determine the effect of immune reconstitution on pathogenesis. Splenocytes from JHMV-immune donors, but not naive donors or donors immunized with irrelevant antigen, completely restored demyelination in irradiated, JHMV-infected recipients. Depletion of Thy-1+ cells by treatment with monoclonal antibody and complement abolished the ability to transfer demyelination. We conclude that: 1) JHMV infection of the CNS does not result in acute or subacute demyelination in the absence of an intact immune response, and 2) viral-specific Thy-1+ cells are an essential element in the induction of demyelinating CNS lesions that result from JHMV infection.

Interaction of pseudorabies virus with porcine peripheral blood lymphocytes.

To examine effects of pseudorabies virus (PrV) on immune cells, we investigated the ability of PrV to infect and replicate in porcine peripheral blood leukocytes (PBLs). Flow cytometric analysis revealed a leukopenia after challenge, with loss of 40% of circulating monocytes and 50% of circulating lymphocytes. Virus was isolated from PBLs of challenged pigs by cocultivation with porcine kidney cells, indicating that PBLs were infected in vivo. Presence of virus in PBLs coincided with the appearance of neurological signs 1 to 2 days prior to death. Lymphocytes stimulated with mitogens and infected in vitro sustained a low-level infection (10(5) median tissue culture infective dose per 2 x 10(6) cells). In vivo challenge perturbed the CD4/CD8 ratio of circulating lymphocytes. Survival was associated with low CD4/CD8 ratios and high levels of CD8+ cells. Mortality was associated with low levels of CD8+ cells and CD4/CD8 ratios greater than one. A maturational deficiency of CD8+ cells was found in young pigs. Our results support a mechanism of PrV immunosuppression through direct infection of circulating lymphocytes, with CD8+ T lymphocytes being important for survival.

Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.

An antigenic variant of the neurotropic murine coronavirus JHMV, designated 2.2-V-1, causes marked demyelination in the relative absence of encephalitis. It is thus useful for the study of the pathogenesis of demyelinating lesions. To better understand the sequential events leading to demyelination, we have examined murine brain and spinal cord tissue at daily intervals after intracerebral inoculation, evaluating them for the distribution of viral antigen, leukocyte infiltration, and demyelination. Immunohistochemical staining indicated that virus established primary infection in the ependymal cells in both brain and spinal cord before spreading into nearby structures and along white matter tracts by cell-to-cell contact. Spread from brain to spinal cord appeared to occur via cerebrospinal fluid. Viral replication was focally cytocidal for ependymal cells, and essentially noncytocidal for other neural cells including glia. In brain, viral antigen and inflammation reached a peak at day 5 postinfection, and rapidly subsided by day 10 postinfection. In spinal cord, viral antigen was less abundant than in brain and was maximal between days 7 and 9 postinfection. The inflammatory response and demyelination, however, were more severe persisting from day 7 through day 19. In the spinal cord, demyelinating lesions developed initially in areas closer to the central canal and were detected most prominently in the anterior funiculi. This finding suggests that the permissiveness of the ependymal cell is crucial to viral entry and that sequential infection of glial cells leads to the characteristic distribution of demyelination.