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Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro., (Copyright 2003 Elsevier Science (USA))

Cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, extracellular bacterium that causes chronic respiratory tract disease in rodents. Infected mice develop microscopic lesions characterized by a primary lymphocytic response followed by macrophage and neutrophilic infiltration. To characterize the lymphocytic subsets that respond to CAR bacillus infection, BALB/c mice were inoculated with 10(5) CAR bacillus bacteria. At seven weeks after inoculation, mice were euthanized and the tracheobronchiolar and hilar lymph nodes were collected and stained for cell surface markers to T cells (CD3, CD4, and CD8), B cells (B220, CD5), natural killer (NK) cells (pan-NK) and intracellular interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). Flow cytometric analysis of lymph nodes from CAR bacillus-infected mice revealed 11% increase in frequency of B cells (R220+), 12% increase in the frequency of double-negative (CD4-CD8-CD3+) T cells, and slight increase in the B-1 subset of B cells (B220+CD5+). There was no change in the frequency of NK cells. The CAR bacillus-infected mice had an overall decrease in the frequency of T cells. Intracellular cytokine staining revealed distinct populations of T cells producing IL-10 and IFN-gamma, and IL-10 production from B cells; NK cells were not a substantial source of IFN-gamma. To our knowledge, this is the first characterization of lymphocytic responses and suggestion that B cells and double-negative T cells may be principally responsible for the lesions associated with CAR bacillus infection.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, extracellular bacterium that causes persistent respiratory tract infections in rodents. We have previously demonstrated that BALB/c mice are more susceptible to CAR bacillus-induced disease than resistant C57BL/6 mice, with elevations in pulmonary gamma interferon (IFN-gamma) and interleukin (IL)-4. IL-10 is a type 2 cytokine that can increase host susceptibility to bacterial diseases through its anti-inflammatory effects, including suppression of macrophage function. The purpose of this study was to further describe the cytokine profiles associated with histologic lesions in CAR bacillus-infected mice and to assess the effects of cytokine depletion on the pathogenesis of disease. Six-week-old female BALB/c and C57BL/6 mice and mice with targeted mutations in IFN-gamma and IL-4 were inoculated intratracheally with 10(5) CAR bacillus organisms, and samples were collected at 6 to 7 weeks postinoculation. Lung samples were collected for histopathologic examination and analysis of cytokine mRNA. IFN-gamma, IL-10, and IL-4 mRNA levels in the lungs of infected mice were semiquantitatively measured using a reverse transcriptase-mediated PCR assay and compared to those in uninfected control animals of each strain. BALB/c mice infected with CAR bacillus had a median lung lesion score of 6 and IL-10 and IL-4 mRNA levels were significantly elevated. The majority of C57BL/6 mice were resistant to disease characterized by lung lesions scores of 2 or less and a dominant IFN-gamma mRNA cytokine profile. A few C57BL/6 mice with lesions scores of 5 or greater had elevations in all three cytokines and were susceptible to disease. C57BL/6 IFN-gamma knockout mice had increased disease with elevations in IL-10 and IL-4 mRNA, while BALB/c IL-4 knockout mice infected with CAR bacillus had a mild decrease in lesion severity and an attenuated IL-10 mRNA expression compared to wild-type BALB/c mice. These data indicate that IL-10 and IL-4 predominate in CAR bacillus-induced histologic lesions in mice, while IFN-gamma may play a role in resistance to disease.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.

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.

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.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein., (Copyright 1999 Academic Press.)

Clostridium piliforme induces enterohepatic disease in many domestic and laboratory animal species. Susceptibility to infection is known to vary with the immune status and strain of the host, but little is known about specific immune mechanisms that regulate this disease. To evaluate host control of C. piliforme infection, we examined the role of interleukin-12 (IL-12) both in the control of and in the response to murine C. piliforme infection. For this study, 3-week-old C. piliforme-resistant C57BL/6 or -susceptible DBA/2 mice were infected intravenously with either the toxic H1 or the nontoxic M1 C. piliforme isolate. Serum and liver samples were collected prior to C. piliforme inoculation (day 0) and at days 1, 3, 7, 14, and 28 postinoculation. Evaluation of hepatic IL-12 p40 mRNA expression by reverse transcription-PCR and of total-IL-12 protein levels in serum by enzyme-linked immunosorbent assay revealed that C. piliforme induced elevations in both hepatic p40 mRNA and serum total-IL-12 levels at all times postinoculation. Elevations were similar with both toxic and nontoxic C. piliforme isolates. Levels of total IL-12 in serum were significantly (P < 0.05) higher in C57BL/6 mice than in DBA/2 mice. Additional experiments were performed in which polyclonal antibody treatment was used to neutralize IL-12 in mice of both strains prior to intravenous inoculation with toxic C. piliforme H1. IL-12 neutralization increased the severity of Tyzzer's disease at day 3 postinoculation in both mouse strains, but the degree of increase was greater in C57BL/6 mice than in DBA/2 mice.

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.

Mouse monoclonal antibodies (MAbs) developed to a rat isolate (R-3) of cilia-associated respiratory (CAR) bacillus were used to assess antigenic relationships among three rat and five rabbit CAR bacillus isolates. Evaluation of MAbs by enzyme-linked immunosorbent assays (ELISAs) indicated that 87 of 241 hybridomas secreted CAR bacillus-reactive antibodies that could be grouped into four major groups. Group-I MAbs reacted with epitopes expressed by all CAR bacillus isolates and at least two or more nonrelated species of bacteria. Group-II, -III, and -IV MAbs reacted with only one or more of the rat CAR bacillus isolates; no MAbs reacted only with rat and rabbit CAR bacillus isolates. Western blot analyses indicated that 41-, 50-, and 105-kDa peptides of rat CAR bacillus isolates expressed rat CAR bacillus group- and isolate-specific epitopes. Hyperimmune anti-CAR bacillus antiserum and serum specimens from a CAR bacillus histologically positive mouse and rat also reacted with the 41-, 50-, and 105-kDa peptides. Sera from CAR bacillus histologically negative rats did not react with these peptides. These results suggest that the 41-, 50-, and 105-kDa peptides may represent suitable antigens for development of a specific ELISA for detection of rodent CAR bacillus infections. Furthermore, these data indicate that use of crude CAR bacillus preparations for either rat or rabbit CAR bacillus ELISAs is inappropriate.

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.

Clostridium piliforme infection (Tyzzer's disease) induces enterohepatic disease in many domestic and laboratory animals. Murine susceptibility to Tyzzer's disease varies with host strain, age, and immune status However, little is known about the role of the immune system in control of this disease. To investigate the role of host immunity in Tyzzer's disease, mice were depleted of either neutrophils, natural killer cells, or macrophages by antibody administration or chemotherapy. After depletion, DBA/2 mice, which are naturally susceptible to C. piliforme, or naturally resistant C57BL/6 mice were inoculated intravenously with C. piliforme. Animals were euthanized 3 days postinoculation and evaluated for gross and histologic lesions and hepatic bacterial load. In juvenile DBA/2 or C57BL/6 mice, depletion of either neutrophils or natural killer cells increased severity of disease. In adult mice, depletion of natural killer cells significantly increased severity of Tyzzer's disease in the resistant (C57BL/6) but not in the susceptible (DBA/2) strain. Macrophage depletion did not alter the course of infection in either mouse strain. These studies indicate an important role for neutrophils and natural killer cells in the pathogenesis of murine Tyzzer's disease. The role of macrophages in murine C. piliforme infection will require further evaluation.

A fecal PCR assay for detection of Helicobacter infections in laboratory rodents was developed. DNA was isolated from murine fecal pellets, and a region of the 16S rRNA gene conserved among murine Helicobacter species was amplified. The fecal PCR was sensitive and specific. This assay does not require euthanasia of rodents, which is especially important for valuable rodents, such as transgenic mice.

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.

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.

Ninety-seven BALB/c mice were inoculated intravaginally with 8.0 x 10(5) Tritrichomonas foetus organisms, using either isolate ATCC 30003 or field isolate MU Y22 2 days after estrogenization with 15 micrograms 17 beta-estradiol. Reproductive tracts were examined at several time points post-inoculation to determine gross and histologic responses to trichomonad infection as compared to estrogenized, uninfected control animals. The two isolates varied greatly in ability to maintain chronic infection; no ATCC 30003-inoculated animals remained culture-positive beyond 7 weeks post-inoculation, whereas MU Y22-inoculated animals were infected for greater than 26 weeks. Lesions were seen in 40-60% of animals prior to 10 weeks post-inoculation and included moderate uterine dilation and glandular atrophy, uterine gland abscesses, pyometra, intramural perivascular lymphoid infiltrates, and ovarian bursitis. The severity of lesions was independent of the T. foetus isolate. Lesions became more severe at 10 weeks post-inoculation, and at 10 and 26 weeks post-inoculation, lesions were seen in 60% and 75% of animals, respectively. In addition to lesions described above, epithelial changes were marked at these late necropsies, including ulceration, flattening, hypertrophy, and squamous metaplasia. The lesions seen in these mice closely resemble those described in natural bovine infection, suggesting that the estrogenized BALB/c mouse is an excellent model for study of bovine trichomoniasis.

Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).

Three murine helicobacter species have recently been identified: Helicobacter hepaticus, Helicobacter muridarum, and Helicobacter bilis. Infections with H. hepaticus and H. bilis have been associated with hepatitis and hepatic neoplasia. In this study, oligonucleotide primers were designed from regions of the 16S rRNA gene that are conserved among members of the Helicobacter genus. The assay amplified the expected 374-bp product from all three rodent Helicobacter species and was able to detect as little as 5 pg of H. hepaticus, H. bilis, or H. muridarum DNA. The specificity of the reaction was determined by testing cecal DNA from uninfected mice and mice with documented Helicobacter infections and by testing DNA from other bacterial genera. A product of the expected size was generated with cecal DNA from Helicobacter-infected mice but not with DNA from uninfected mice. With the exception of that of "Flexispira rappini, " which is closely related to the Helicobacter genus, DNA from other bacterial genera was not amplified with the Helicobacter genus-specific primers. MboI, MaeI, and HhaI restriction enzyme analyses of the amplified product were able to differentiate among the murine Helicobacter species but could not differentiate H. bilis from "F. rappini." To distinguish H. bilis, a reverse primer based on H. bilis 16S rRNA sequence was designed. PCR with the H. bilis-specific reverse primer (Hbr) and the Helicobacter genus-specific forward primer (H276f) amplified H. bilis DNA but not DNA from "F. rappini" or other rodent helicobacters. Examination of a large number of murine cecal tissues with this combination of PCR assays and restriction enzyme analyses indicated that H. hepaticus and H. bilis infections are widespread in laboratory mouse and rat colonies.

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.

A recombinant baculovirus containing the NS1 gene of minute virus of mice was constructed. Optimal expression of the recombinant NS1 protein (rNS1) was achieved by infecting Trichoplusa ni High Five cells at a multiplicity of 10 and incubating them for 72 h postinfection. An enzyme-linked immunosorbent assay (ELISA) with rNS1 as the antigen was evaluated for serologic testing of laboratory rodents. The rNS1 ELISA proved to be a more sensitive method for the detection of antibodies to recently recognized rodent parvovirus species (mouse orphan parvovirus and rat orphan parvovirus) and prototypic parvovirus species (minute virus of mice, Kilham's rat virus, and H-1) than were conventional parvovirus ELISAs that use whole parvovirus virions.

Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.

Previous studies indicated that Tritrichomonas foetus isolate ATCC 30003 was capable of maintaining only short-term genital infection in estrogenized BALB/c mice. In the present study, the ability of eight T. foetus isolates to establish and maintain infections in intravaginally inoculated estrogenized BALB/c mice was examined. All isolates were found to be equally capable of establishing genital infections but varied greatly in ability to maintain infections. One isolate maintained infection for less than 7 weeks, four isolates maintained intermediate infections lasting less than 13 weeks, and three isolates maintained chronic infections of greater than 26 weeks. Varying the number of trophozoites inoculated intravaginally decreased the ability of isolates to establish infection but did not affect maintenance of infection. Prolonged passage of T. foetus isolates either in vivo in an estrogenized nu/nu BALB/c mouse or by in vitro culture failed to affect their ability to maintain infection, suggesting that virulence was parasite-dependent and not related to environment-induced changes. Co-infection of estrogenized mice with isolates ATCC 30003 and MU Y32 failed to increase the length of ATCC 30003 infections or decrease the length of isolate MU Y32 infections. Taken together these results indicate that T. foetus isolates vary greatly in virulence in estrogenized BALB/c mice and provide evidence suggesting that maintenance of infection is a parasite-controlled factor.

The most common diagnostic technique for the detection of Sendai virus infection in rodents is serological evaluation by enzyme-linked immunosorbent assay (ELISA) with semipurified preparations of whole virions as antigens. This assay often suffers from a lack of specificity. The goal of the present project was to develop more specific antigens for use in diagnostic testing by producing recombinant antigens in insect cells. To identify viral proteins immunoreactive in multiple laboratory rodent species, Western blots (immunoblots) of viral polypeptides were probed with immune sera from mice, rats, and hamsters. The nucleocapsid protein (NP) reacted with immune sera from all species tested. Therefore, the NP gene was selected for cloning and expression in a baculovirus. To construct the recombinant, complementary DNA was synthesized by reverse transcription PCR from Sendai virus RNA with primers from the 5' and 3' termini of the NP-coding region. Amplified DNA was cloned into a baculovirus transfer vector (pBlueBacHis A) and was cotransfected with wild-type baculovirus into insect cells. Baculovirus recombinants containing the NP gene were identified by PCR. Evaluation of the recombinant proteins expressed in insect cells by Western blot analysis revealed specific reactivity with immune sera. In comparison with conventional ELISAs that use whole virions as the antigen, ELISAs that use recombinant NP were more specific.

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.

Four guinea pigs from a colony of approximately 50 animals were examined for progressive neurologic disease of 5 days' duration. Signs of neurologic dysfunction included cachexia, stupor, hyperexcitability, lateral recumbency, and opisthotonos. Results of gross pathologic, microbiologic, and serologic examinations were unremarkable. Histologic examination of cerebral and cerebellar sections revealed multifocal malacia and regions of eosinophilic granulomatous inflammation. Cross-sections of nematode larvae, identified as Baylisascaris sp., most likely B. procyonis, the raccoon ascarid, were seen in the brain of some affected animals. An intact Baylisascaris larva was recovered from a symptomatic animal when cerebral tissue was processed by the Baermann extraction technique. Results of further investigation indicated that wood shavings used for the guinea pigs had been contaminated by raccoon feces, some of which contained numerous B. procyonis eggs. The bedding source for this colony was changed and, to date, no new cases of neurologic disease have been seen. This report emphasizes the potential insidious entrance of B. procyonis into well-managed laboratory animal facilities.

The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative bacterium that has been implicated as an etiologic agent of respiratory tract disease in laboratory rodents. A morphologically and antigenically similar organism has been identified in rabbits and is thought to be a related bacterium, although clinical signs of disease and histologic lesions are absent in infected rabbits. To compare the pathogenicity of rat- and rabbit-origin CAR bacillus isolates in rabbits, neonatal rabbits were experimentally infected with CAR bacillus isolates obtained from an infected rat and rabbit. Rabbits experimentally inoculated with rabbit-origin CAR bacillus had a nasal discharge, seroconverted and developed histologic lesions, whereas rabbits inoculated with rat-origin CAR bacillus seroconverted but did not have evidence of colonization of the respiratory tract. The CAR bacillus isolates were further examined at the genetic level by sequencing 1,261 base pairs of the 16S rRNA gene from six CAR bacillus isolates obtained from infected rabbits. A consensus sequence was obtained and compared with the analogous gene sequence data from rat-origin CAR bacillus isolates. Results indicated that these two organisms are distinctly different, with only 48.8% sequence homology. Comparison of the rabbit-origin 16S rRNA gene sequence with the database Genbank indicated that the organism is most closely related to members of the genus Helicobacter. Bacteria with the highest percentage of similarity with the rabbit-origin CAR bacillus were Helicobacter sp. strain Seymour and H. felis, with 91.1 and 90.8%, respectively. Findings of this study indicate that CAR bacillus isolates from rats and rabbits are host-specific and are different bacteria that belong to distinct genera.

Tyzzer's disease, a well-recognized syndrome in numerous laboratory animal species, is caused by the obligate intracellular bacterium, Clostridium piliforme. Distinct isolates of C. piliforme from various laboratory animal species have been identified based on protein and antigenic heterogeneity. The goal of this study was to examine the host specificity of three well-characterized isolates of C. piliforme. Groups of mice, rats, and hamsters were experimentally infected with isolates obtained from a naturally infected mouse (M1), a naturally infected rat (R1), and a naturally infected hamster (H2). To assess infection status, animals were monitored serologically for antibody to C. piliforme over a 12-week period. Evaluation of results indicated that the M1 isolate infected rats and mice but not hamsters, whereas the R1 and H2 isolates infected only the host species from which the isolates were originally obtained. These findings suggest that C. piliforme isolates can be categorized into two types: 1) cross-infective isolates, such as M1, which can infect more than one laboratory animal species, and 2) isolates, such as R1 and H2, which have a more limited host range within laboratory animal species. These results emphasize the need to consider the host specificity of C. piliforme isolates when investigating outbreaks of Tyzzer's disease.

Studies were initiated to establish and maintain intravaginal Tritrichomonas foetus infections in female BALB/c mice as a model for elucidation of parasite and host factors that affect the course of vaginal protozoan infections. Results of these studies indicated that T. foetus infections could only be established in mice in which estrus was induced and maintained. Over a period of several weeks, mice induced to estrus by weekly administration of estradiol cypionate exhibited purulent vaginal discharge and perivulvar abscesses. Implantation of silastic tubing containing 15 micrograms of estradiol-17 beta proved effective in induction and maintenance of estrus and avoided the animal health problems associated with estradiol cypionate treatment. Results of quantitative experiments indicated that the duration of trichomonad infection was influenced by initial colonization of the vagina, i.e., mice with high numbers of vaginal trichomonads at 7 days after infection maintained infections longer than did mice with lower numbers of vaginal parasites. Weekly administration of either 2 or 4 mg of methylprednisolone acetate to estrogenized mice did not extend the duration of T. foetus infections, thereby suggesting that the immune response did not limit the establishment and maintenance of primary vaginal trichomonad infections. Study of estrogenized BALB/c nu/nu mice supported these observations in that establishment of T. foetus infections was difficult in nu/nu mice and that, in most nu/nu mice (76%), the course of infection was not lengthened (mean, 1.9 weeks). When examined by electron microscopy, the earliest lesions were characterized by degeneration and necrosis of chondrocytes, along with degradation of cartilage matrix. These findings confirm that quinolone arthropathy develops in juvenile rabbits and is similar to quinolone arthropathy in other laboratory animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, motile bacterium that has been implicated as an etiologic agent of respiratory disease in laboratory rodents. In the present study, approximately 1,200 bases of the 16S rRNA gene from three CAR bacillus isolates were sequenced. CAR bacillus-specific primers were designed on the basis of the 16S rRNA gene sequence and used in a PCR assay. The PCR assay detected as little as 500 fg of purified CAR bacillus DNA. The expected 267-bp DNA fragment was amplified from respiratory tissue of frozen, formalin-fixed, and paraffin-embedded samples from experimentally and naturally infected rats and mice. In contrast, no product was amplified from respiratory tissues of sham-infected experimental animals or animals that were serologically or histopathologically negative for the CAR bacillus. Our findings indicate that this PCR assay is a rapid, specific, and sensitive detection method for the diagnosis of CAR bacillus infection in rats and mice.

Isolates of the cilia-associated respiratory (CAR) bacillus were harvested from the trachea of three naturally infected rats and five naturally infected rabbits and were grown on 3T3 mouse fibroblast cells. Isolates were compared by growth characteristics in mammalian cell culture, by use of protein and DNA analyses, and by experimental infections of BALB/c mice. Examination of CAR bacillus isolates by transmission electron microscopy indicated that organisms from rats and rabbits were similar in appearance and had an acidic mucopolysaccharide layer. In culture, the isolates from rats appeared larger than the rabbit isolates and formed large multiorganism aggregates, whereas isolates obtained from rabbits did not. Protein and antigenic analyses and DNA ribotyping revealed minor differences between isolates but could not be used to distinguish the rat from the rabbit isolates. Mice experimentally inoculated with CAR bacillus of rat origin developed interciliary colonization, seroconverted, and developed microscopic pulmonary lesions. Mice inoculated with isolates of rabbit origin did not display intraciliary colonization, seroconvert, or develop pulmonary disease. The findings of this study indicate that CAR bacillus isolates of rat and rabbit origins may be distinct strains and suggest that, in mice, isolates of rat origin may be more virulent than those of rabbit origin.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect Bacillus piliformis isolate-specific antibodies in serum specimens from rats and gerbils experimentally infected with B. piliformis isolates R1, R2, or M. Detection was based on the ability of serum antibodies to block binding of B. piliformis isolate-specific monoclonal antibodies to purified B. piliformis flagella. Application of this assay to serum specimens collected from sham-infected or experimentally infected rats and gerbils demonstrated that the serum specimens were capable of specifically inhibiting the binding of B. piliformis isolate-specific monoclonal antibodies to homologous flagella preparations (> 70% inhibition) only when the serum specimens were from animals infected with the homologous B. piliformis isolate. Only one false-negative and false-positive result were obtained when 80 serum specimens were tested by this competitive inhibition ELISA. In addition, we demonstrated that little nonspecific inhibition of monoclonal antibody binding occurred (< 30% inhibition) in this immunoassay specific inhibition of monoclonal antibody binding by serum was due to serum antibody and a serum's ability to inhibit binding of monoclonal antibodies to purified B. piliformis flagella was correlated with antibody reactivity with B. piliformis flagella but not with serum antibody reactivity to whole B. piliformis organisms. These results suggest that this monoclonal antibody-based competitive inhibition assay could be successfully applied to the serologic identification of isolates involved in naturally occurring B. piliformis infections in laboratory animals.

We compared antibody levels among serum, egg yolk extract, and granuloma fluid in chickens immunized with bovine serum albumin (BSA). One group of hens was immunized by intramuscular and subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, followed by two subsequent booster injections in incomplete Freund's adjuvant. Two other groups were surgically implanted with plastic, perforated wiffle balls (subcutaneous chambers). After a 30-day recovery period, one of the groups with subcutaneous chambers was immunized with BSA in sterile water with two subsequent boosts. The other group was injected with only sterile water. Serum samples, eggs, and granuloma fluid were collected biweekly and analyzed to determine specific IgG, total IgG, and total protein. The subcutaneous chambers were well tolerated. Quantitative ELISAs of serum, egg yolk extract, and granuloma fluid specimens disclosed that specific antibody levels were present in all specimens by 2 weeks after primary immunization. During the course of the experiment, specific antibody levels of serum and egg yolk specimens were significantly higher than those of granuloma fluid (P less than 0.05). However, an additional injection of antigen into the subcutaneous chambers resulted in specific antibody levels in granuloma fluid specimens that were comparable to those of serum and egg yolk extract. Use of subcutaneous chambers in chickens may be a viable alternative to routine antibody production methods.

Previous studies have demonstrated pathologic similarities between human melanoma and the spontaneous melanoma in the Sinclair miniature swine (SMS) with respect to cutaneous histologic features and patterns of metastasis. The current biopsy series, correlating growth curves and histopathologic features of cutaneous melanomas, was undertaken for documentation of the histologic events associated with the successful regression of melanoma in the SMS. Cutaneous growth and regression was characterized by a series of cellular events that eventually led to depigmentation and scar formation. Mononuclear inflammatory infiltrates, seen in over half of the 104 biopsies, showed several temporal and topographic distribution patterns, similar to that described in human melanoma. Histopathologic observations in the SMS confirm clinical observations that the host can, with consistent effectiveness, react with the tumors to modify their biologic aggressiveness. Although regression is associated with lymphocytic and macrophage infiltration, the exact role of the immune response in the regression of the cutaneous melanoma remains to be elucidated.

Specimens from seven Sinclair swine melanomas were transplanted to the cheek pouches of Syrian Golden hamsters. The specimens were taken from young swine and were derived from raised tumors that either were present at birth or developed after birth from flat melanocytic lesions as well as from apparently normal skin. All seven specimens grew in the hamster cheek pouch. One lesion, derived from a 3-day-old piglet, exhibited the most aggressive growth in the hamster and was successfully transferred to other hamster cheek pouches. These results confirm the malignancy of Sinclair swine melanoma and indicate that tumors of neonatal swine contain more malignant cells than those of older animals.

In a substrain of Sinclair miniature black swine, bred for increasing incidence of cutaneous malignant melanomas, tumor regression occurs spontaneously and is accompanied by depigmentation of the skin, hair, and eyes. We conducted a 12-month longitudinal study of the ocular phenomena in 30 swine beginning at 3 weeks of age. The clinically observed sequence of depigmentation of the fundus and iris was correlated with histopathologic changes in selected enucleated eyes. Normal melanocytes of the uveal tract are destroyed between the 4th and 16th week of life. Melanocyte destruction is preceded by an invasion of the uveal tract by mononuclear cells having the ultrastructural features of lymphocytes and monocytes. Melanin and other cellular debris of ruptured melanocytes are ingested by macrophages which then migrate to the walls of blood vessels. Cataracts and band keratopathy develop secondary to the uveitis in some animals. Pilot electroretinograms show diminished electrical activity in photoreceptors of totally depigmented eyes possibly indicating ischemic or toxic damage to the retina. The retinal pigment epithelium remains essentially normal during the acute stages of uveal inflammation; later some damage and reparative hyperplasia may occur. The death of normal uveal melanocytes that occurs during the systemic attack on the cutaneous malignant melanomas appears to be an "innocent bystander" error in the immune recognition mechanism. The antigenic basis of this immunologic cross reaction is under investigation.

Sinclair swine melanoma usually regresses in vivo. In the present study, swine melanoma cells were adapted to long-term growth in culture. The morphology of cultured melanoma cells ranged from dendritic to cuboidal, similar to that described for human melanoma cells. Doubling times of the swine melanoma cells were also similar to those of human melanoma cells in vitro. 3,4-Dihydroxy-L-phenylalanine oxidase-positive cells were detected by light microscopy, and melanin and premelanosomes were detected by electron microscopy. Cell cultures could be propagated from progressing, partially regressed, and primary cutaneous lesions, as well as from visceral metastases. Thus, it appears that, under these cell culture conditions, Sinclair swine melanoma cells can be adapted to prolonged growth in vitro.

A Campylobacter-like organism was isolated from the ilea of normal hamsters. The organism was isolated from an ileal homogenate which was passed through a filter (0.65-micron pore size) and cultured on blood-agar plates in a microaerophilic atmosphere at 37 degrees C. Pinpoint translucent colonies were first observed after 120 h of incubation. The isolated organism measured 2.0 to 3.5 microns in length (excluding flagella) by 0.17 to 0.25 micron in width and typically had a single terminal sheathed flagellum. The organism was oxidase, catalase, and urease positive, reduced nitrates, and was susceptible to nalidixic acid (30-micrograms disk) and resistant to cephalothin (30-micrograms disk). Unlike Campylobacter pylori subsp. mustelae, this organism did not hydrolyze indoxylacetate. Immunofluorescence studies with a Campylobacter species-specific monoclonal antibody (8322-2E6) revealed the presence of numerous positively stained organisms within the crypt epithelial cells of the hamsters from which this organism was isolated. The role of this organism in the pathogenesis of proliferative ileitis in hamsters is uncertain, as is the taxonomic relationship of this organism to other members of the genus Campylobacter.

The role of Campylobacter jejuni in the pathogenesis of proliferative ileitis of Syrian hamsters (Mesocricetus auratus) has been uncertain. C. jejuni has been implicated as the etiologic agent on the basis of the campylobacter-type morphology of the intracellular organism and the repeated microbiologic isolation of C. jejuni from hamsters with proliferative ileitis. The inability to reproduce the disease with pure culture inocula, coupled with immunohistochemical studies, however, has suggested that although C. jejuni may be present in the ilea of infected hamsters, its involvement in the pathogenesis of proliferative ileitis is questionable. In this study hamsters were inoculated with infective ileal homogenates prepared from ilea which were extensively washed to remove the ileal contents before grinding. The ilea from hamsters inoculated with this homogenate were also washed before being ground and used to experimentally inoculate a second group of hamsters. Of the 20 hamsters from this second group, 12 developed lesions typical of proliferative ileitis. Extensive microbiologic cultures from these hamsters were negative for C. jejuni. Immunofluorescence studies with a C. jejuni-specific monoclonal antibody were also negative. The use of a Campylobacter genus-specific monoclonal antibody, however, revealed numerous campylobacter-type organisms within the ileal epithelial cells of the crypts and villi. The presence of C. jejuni is therefore apparently not necessary for the production of proliferative ileitis in hamsters, and the intracellular campylobacter-type organism present in the ileal epithelial cells of infected hamsters is probably not C. jejuni.

Leukocyte migration inhibition (LMI) assays were performed using 3 M KCI extracts to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of swine melanoma. Positive LMI reactivity was 70% or greater in melanoma-bearing swine compared to 10% reactivity or less in normal swine tested with extracts of either fresh or tissue-cultured swine melanoma cells. Positive LMI reactivity was 10% or less in both melanoma and normal swine tested with extracts of normal fetal and adult swine tissues. Extracts of tissue-cultured swine melanoma and human melanoma cells were equally capable of stimulating positive LMI reactivity in melanoma swine and human melanoma patients; normal swine and human donors demonstrated 10% or less positive LMI reactivity to these extracts. Positive LMI reactivity was not stimulated in swine melanoma or human melanoma donors by extracts of tissue-cultured human breast tumor cells or fresh and tissue-cultured mouse B16 melanoma cells. Evaluation of these results indicate that melanoma-bearing swine develop cell-mediated immune reactivity toward TAA on swine melanomas and, inasmuch as LMI assays were performed with allogeneic extracts, suggests the presence of common TAA on swine melanomas. Furthermore, these data suggest that swine and human melanoma cells may share some common TAA and that this common TAA is not expressed on mouse B16 melanoma cells.