Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species.

Over the past decade, several Helicobacter species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents. In this report, we describe a novel urease-negative helicobacter, Helicobacter typhlonius sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections. H. typhlonius is genetically most closely related to H. hepaticus. Like H. hepaticus, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H. hepaticus. Notably, H. typhlonius does not produce urease or H(2)S nor does it hydrolize indoxyl-acetate. Compared to other Helicobacter species that commonly colonize rodents, H. typhlonius was found to be less prevalent than H. hepaticus and H. rodentium but as prevalent as H. bilis. H. typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species.

BACKGROUND AND PURPOSE: Several rodent helicobacters have been associated with chronic active hepatitis or inflammatory bowel disease. Severe combined immunodeficient (SCID) mice appear to be inherently susceptible to disease attributable to these emerging pathogens. With the advent of polymerase chain reaction (PCR) analysis, it has become clear that several as yet unidentified Helicobacter species may also colonize rodents, but their capacity to cause disease is unknown. METHODS: A Helicobacter species isolated from feces of a BALB/c mouse and provisionally named "H. typhlonicus" was used to inoculate helicobacter-free 4-week-old SCID mice (n = 11 males and 11 females). At various weeks after inoculation, mice were sacrificed and liver and intestinal specimens were collected for histologic examination and PCR analyses. RESULTS: The C.B-17 scid/scid mice inoculated with "H. typhlonicus" developed moderate to severe proliferative typhlocolitis, similar to that seen in SCID mice infected with H. hepaticus or H. bilis. However, in contrast to mice infected with H. hepaticus or H. bilis, lesions of chronic active hepatitis were not detected in mice inoculated with "H. typhlonicus." A similar disease syndrome developed in SCID mice cohabitated with B6D2F1 mice naturally infected with a novel Helicobacter species that was genetically identical to "H. typhlonicus." CONCLUSION: "Helicobacter typhlonicus" joins a growing list of helicobacters that are capable of inducing enteric disease in immunodeficient mice.

Cloning and expression of an immunogenic membrane-associated protein of Helicobacter hepaticus for use in an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera from H. hepaticus-infected mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacter infection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum and sera from mice free of Helicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.

Enterohepatic lesions in SCID mice infected with Helicobacter bilis.

Helicobacter bilis is a recently identified species that colonizes the intestine and liver of mice. In immunocompetent mice, infections have been associated with mild hepatitis, and in immunocompromised mice, inflammatory bowel disease has been induced by intraperitoneal inoculation of the organism. We report inoculation of 6-week-old C.B-17 scid/scid mice by gastric gavage with approximately 10(7) H. bilis colony-forming units. Groups of mice were euthanized and necropsied 12, 24, and 36 weeks after inoculation. Mild to moderate proliferative typhlitis was evident in all mice at 12 and 36 weeks after inoculation and in most mice 24 weeks after inoculation. Mild to severe chronic active hepatitis was detected in 10 of 10 male mice and 3 of 10 female mice. These results indicate that H. bilis can cause moderate to severe enterohepatic disease in immunocompromised mice.

Evaluation of hyperplastic goiter in a colony of Syrian hamsters (Mesocricetus auratus).

Hyperplastic goiter was diagnosed during routine health monitoring of a closed Syrian hamster colony (SG). Adult and juvenile hamsters were affected at a prevalence of 45%. Histologic examination of the enlarged thyroid gland revealed marked follicular cell hyperplasia. Because prevalence of thyroid hyperplasia in this colony exceeded the 6 to 7% prevalence expected in aged hamsters, additional studies were performed to investigate the pathogenesis of this condition. Juvenile male SG hamsters and age- and sex-matched Syrian hamsters that did not have increased prevalence of goiter were obtained from an unrelated source (Fredrick Cancer Research and Development Center [FCRDC]). The thyroid glands of hamsters were evaluated by 123I radionuclide imaging. Eight of 18 SG hamsters and none of the FCRDC hamsters had a diagnosis of enlarged thyroid gland. Serum baseline and post-thyrotropin thyroxine concentrations in SG hamsters were not statistically different from those in FCRDC hamsters. To investigate whether diet played a role in development of hyperplastic goiter, for 6 months 15 FCRDC hamsters were fed the diet that had been fed to SG hamsters (mouse breeder diet), and five were fed a control diet. To determine whether dietary change would result in resolution of goiter, affected SG hamsters were fed a control diet for 3 months. At the end of each feeding trial, thyroid gland uptake of 123I was reevaluated. The amount of 123I taken up by the thyroid glands of FCRDC hamsters fed the mouse breeder diet was not significantly different from that of controls. In contrast, thyroid gland uptake of 123I remained high for all affected SG hamsters fed the control diet. On the basis of results of these investigations, diet was ruled out as the cause of goiter. Also, a diagnosis of euthyroid hyperplastic goiter was made for the SG hamsters. A genetic cause is suspected to play a role in the increased prevalence of goiter in SG hamsters.

Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a newly recognized bacterium associated with chronic active hepatitis, hepatic carcinoma, and inflammatory bowel disease in mice. Currently, fecal or tissue PCR, fecal culture, or histologic examination of silver-stained liver sections is used to diagnose H. hepaticus infection. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of H. hepaticus infection in mice with a membrane digest preparation of H. hepaticus as the antigen. Sera from mice positive for H. hepaticus by PCR or histologic examination (n = 88), positive for Helicobacter bilis by PCR (n = 13), positive for other helicobacters (not identifiable to species level) by PCR (n = 25), or negative for all Helicobacter species by PCR (n = 162) were used to evaluate the ELISA. Results indicated that ELISA provided 93.2% sensitivity, 94% specificity, 87.2% positive predictive value, and 96.9% negative predictive value. Cross-reactive antibodies were detected in some mice infected with helicobacters not identifiable to species level. To further define ELISA sensitivity and specificity, groups of 10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter muridarum, or H. bilis. Sera were collected and examined by the ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and maintained ELISA reactivity throughout the 18-week study, while mice infected with H. muridarum and H. bilis were negative by ELISA. These results indicate that this reported ELISA is highly sensitive and specific for the serodiagnosis of H. hepaticus infection in mice.

Isolation of a novel Helicobacter species, Helicobacter cholecystus sp. nov., from the gallbladders of Syrian hamsters with cholangiofibrosis and centrilobular pancreatitis.

A filamentous, gram-negative, motile bacterium with a single polar sheathed flagellum was isolated from gallbladders of hamsters with cholangiofibrosis and centrilobular pancreatitis. Bacteria grew under microaerophilic conditions at 37 and 42 degrees C, were oxidase, catalase, arginine aminopeptidase, and L-arginine arylamidase positive, reduced nitrate to nitrite, were resistant to cephalothin, and exhibited intermediate susceptibility to nalidixic acid. Sequence analysis of the 16S rRNA gene indicated that the bacterium was a novel member of the Helicobacter genus, most closely related to Helicobacter pametensis. We propose to name this bacterium Helicobacter cholecystus. In epidemiologic studies, isolation of H. cholecystus correlated strongly with the presence of cholangiofibrosis and centrilobular pancreatitis; however, further studies are needed to define the role of this bacterium in pathogenesis.

A novel presentation of Clostridium piliforme infection (Tyzzer's disease) in nude mice.

Clostridium piliforme infection (Tyzzer's disease) was diagnosed in a colony of nude mice. Because spontaneous Tyzzer's disease had not been reported in nude mice, a study was undertaken to better define the clinicopathologic features of this disease outbreak. Sixty homozygous nude (nu/nu) females, 10 nu/nu males, and 10 heterozygous nude (nu/+) females were observed for signs of disease. Over a 3-month period, 43% of the nu/nu mice died or manifested clinical signs of disease and were euthanized, but nu/+ mice remained healthy. Clinical signs of disease were infrequently observed in nu/nu mice and, when evident, were followed by rapid deterioration and death. Gross and histologic lesions, including severe hepatic and intestinal necrosis associated with C. piliforme, were observed only in clinically affected animals. Clostridium piliforme isolated from diseased livers had marked cytotoxicity in in vitro assays. This outbreak is unique in that, contrary to a previous experimental report, nu/nu mice had increased susceptibility to Tyzzer's disease, suggesting that T cells may play an important role in host defenses against C. piliforme infection. In addition, this is the first report of a toxigenic isolate of C. piliforme recovered from mice. The cytotoxin produced by the isolate may have contributed to the severity of clinical disease and lesions.

Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity.

Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a beta-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A(1,2)) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C(2,1) was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3-galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues.

Host-selective toxins and their role in plant diseases.

Toxins with unusual characteristics are involved in some destructive diseases of plants. Certain parasitic fungi produce toxins of low molecular weight that selectively affect the host plant; nonhosts are tolerant. These toxins have diverse structures, including cyclic peptides and linear polyketols. Genetic and other data show that resistance to each fungus is based on tolerance to its toxin. The same fungal genes control toxin production and ability to cause disease. Little is known about toxic action, although one toxin selectively affects mitochondria. Plant cell membranes are affected; this may allow the fungus to colonize tissues. Resistant cells may lack toxin receptor sites.

Conversion of Helminthosporium sacchari Toxin to Toxoids by beta-Galactofuranosidase from Helminthosporium.

Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as ;toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with beta-galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. beta-Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. beta-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No beta-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum.

Isolation and characterization of host-selective toxin from Helminthosporium sacchari.

Helminthosporium sacchari infects certain clones of sugar cane and produces a toxin with the same plant selectivity as the fungus itself. The toxin was purified by use of activated charcoal plus thin layer, gel, and ion exchange chromatography. Gas chromatography (GC) of a trimethylsilyl derivative of toxin gave a single peak. Toxin was characterized by GC, mass spectroscopy (MS), and NMR spectroscopy. The spectra of hydrolytic products showed that toxin contains galactose plus a C15H21 moiety which appears to be a sesquiterpene. Spectral data and methylation procedures showed that toxin contains an oligosaccharide composed of beta, 1 leads to 5 galactofuranose units (probably 5 units). Several interconvertible forms of the C15H21 moiety were evident after acid hydrolysis. Toxin was separated from 3 closely related, nontoxic compounds ("noxins"), which contained galactose plus the C15H21 moiety. Comparative data show that the toxin examined in this study is the same as the toxin described by Steiner and Strobel (Steiner, G. W., and Strobel, G. A. (1971) J. Biol. Chem. 246, 4350-4357). The data also show that the previously proposed structure is incorrect.