Pinworm detection in mice with immunodeficient (NOD SCID) and immunocompetent (CD-1 and Swiss) soiled bedding sentinels in individually ventilated cage systems.

Sentinel exposure to soiled bedding is frequently used for health monitoring of mice housed in individually ventilated cage systems (IVCS). Despite its advantages, the use of soiled bedding sentinels (SBSs) is far for being a reliable method. Two studies were conducted to evaluate the sensitivity of immunodeficient SBSs NOD.CB17-Prkdc(scid)/NCrHsd (NOD SCID) against two immunocompetent outbred strains, Hsd:ICR (CD-1) and RjOr1:Swiss (Swiss) to pinworm detection in IVCS-housing. Four different diagnostic methods were used: perianal tape test, fecal flotation, plate method and histology. Positivity was considered if at least one of the techniques used was positive. In the first study NOD SCID were more sensitive than CD-1 SBSs (P < 0.05), and except for the fecal flotation test performed at week 6, all the diagnostic methods were more sensitive with NOD SCID mice (P < 0.05). In the second study differences between the Swiss and NOD SCID mice were less obvious (P = 0.08). When compared separately, the different diagnostic methods, except for the fecal flotation test, were all more sensitive in the NOD SCID mice (P < 0.05). In addition, the anal tape test in the Swiss SBSs was more sensitive at week 7 than at week 15 (P < 0.05). In conclusion, combining various diagnostic techniques and samplings at week 7 post-exposure with non-invasive methods increases the rate of pinworm detection. Immunodeficient SBSs showed higher sensitivity than immunocompetent ones. Thus, use of immunodeficient SBSs is highly recommended in health control protocols.

Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice.

Pasteurella pneumotropica is an opportunistic pathogen in rodents. Natural infection in immunodeficient animals suggests that immunodeficiency is a major factor in P. pneumotropica pathogenesis. To understand this process, we performed clinical, pathological and bacteriological studies of immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice experimentally infected with P. pneumotropica ATCC 35149. From 14 days postinoculation, some of P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of weight loss. Three of 10 P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of depression, ruffled coat, and weight loss and died at 27, 34, and 59 days postinoculation. At 35 days postinoculation, almost all P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice had lung abscesses. The bacteria were isolated from the upper and lower respiratory tracts, including the lungs, and blood. In contrast, P. pneumotropica-infected ICR mice exhibited no clinical signs or lesions. The bacteria were isolated from the upper, but not the lower respiratory tracts. We developed an animal model for understanding host interactions with P. pneumotropica.

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.

Dual infection with Pneumocystis carinii and Pasteurella pneumotropica in B cell-deficient mice: diagnosis and therapy.

BACKGROUND AND PURPOSE: The clinical presentation, diagnosis, histopathologic findings, and elimination of dual respiratory tract infection with Pasteurella pneumotropica and Pneumocystis carinii were studied in 100 adult barrier-reared C.B17 and MRL- lpr mice homozygous for a targeted mutation of the JH region of the immunoglobulin heavy chain. METHODS: Necropsy, aerobic bacteriologic culture of hematogenous and pulmonary tissues, histochemical staining of pulmonary tissues, polymerase chain reaction analysis of pulmonary tissues and feces, and viral serologic testing were performed on 19 clinically affected mice and 8 clinically normal mice, then later on antibiotic-treated and caesarian re-derived mice. Therapeutic strategies included sequential administration of trimethoprim/ sulfamethoxazole and enrofloxacin or enrofloxacin administration and caesarian rederivation. RESULTS: Clinically affected mice had diffuse, nonsuppurative, interstitial pneumonia with superimposed pyogranulomatous lobar pneumonia that was detected microscopically. Affected lung tissue yielded pure culture of P. pneumotropica. Aged-matched, clinically normal mice of both genotypes had interstitial histiocytic pneumonia without lobar pneumonia, and P. pneumotropica was not isolated. Histochemical staining of lung tissues from normal and clinically affected mice revealed scattered cysts consistent with P. carinii, principally in the interstitium. Treatment with sulfamethoxazole/trimethoprim and enrofloxacin eliminated bacteriologic detection of P. pneumotropica, decreased mortality from 50% to 6%, and improved breeding performance. CONCLUSION: A successful antibiotic therapy and rederivation approach, incorporating enrofloxacin, cesarian section, and isolator rearing, was developed for B cell-deficient mice with opportunistic infections.

Tumor necrosis factor alpha and gamma interferon are required for the development of protective immunity to secondary Corynebacterium pseudotuberculosis infection in mice.

The production and role of endogenous cytokines during the course of secondary Corynebacterium (C.) pseudotuberculosis infection were investigated in mice. When immunized mice were challenged on day 28 after primary infection, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were found to appear at 3 hr and to reach the maximum at 24 hr after challenge. Spleen cells of mice primarily infected from 2 to 8 weeks before produced a significant amount of TNF-alpha and IFN-gamma when stimulated with formalin-killed bacteria. However, they could not produce detectable amounts of IL-4. The administration of anti-TNF-alpha monoclonal antibody (MAb) and IFN-gamma MAb increased bacterial proliferation in the organs of immune mice and exacerbated the secondary infection. Injection of anti-CD4 MAb alone or anti-CD4 plus anti-CD8 MAbs resulted in significantly increased mortality and a marked suppression of bacterial elimination as well as cytokine production of secondarily infected mice, while the treatment with anti-CD8 MAb alone showed no effect on either the resistance or cytokine production of mice. These results suggest that CD4, probably Th1 T cells, play an important role for establishment of protective immunity against secondary C. pseudotuberculosis infection by secreting TNF-alpha and IFN-gamma.

Mapping the extracellular topography of the alpha-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain.

The extracellular surface of the alpha-chain of Torpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptides alpha 1-16, alpha 89-104, alpha 158-174, alpha 262-276, and alpha 388-408. Lower, but significant, levels of reactivity were obtained with antibodies against peptides alpha 67-82, alpha 78-93, alpha 100-115, and alpha 111-126. On the other hand, free AChR bound high levels of antibodies against peptides alpha 34-49, alpha 78-93, alpha 134-150, alpha 170-186, and alpha 194-210. It also bound moderate levels of antibodies against peptides alpha 262-276 and alpha 388-408. Low, yet significant, levels of binding were exhibited by antibodies against peptides alpha 45-60, alpha 111-126, and alpha 122-138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).

The role of cytotoxic macrophages in non-obese diabetic mice: cytotoxicity against murine mastocytoma and beta-cell lines.

The cytotoxicity of macrophages from non-obese diabetic (NOD) mice against murine mastocytoma (P-815), and murine beta-cell lines having the NOD gene background (MIN6N-9a), were examined. Peritoneal exudate cells from 20-week-old mice showed higher cytotoxicity, measured as inhibition of thymidine uptake into P-815, than those from 12-week-old mice (p < 0.01). In cyclophosphamide-injected mice, cytotoxicity of peritoneal exudate cells had increased at 8 days post-injection, at which time the mice were not diabetic. To confirm macrophage cytotoxicity against pancreatic cells and examine its cytolytic mechanism, the cytotoxicity of peritoneal exudate cells from cyclophosphamide-injected NOD mice against MIN6N-9a cells was measured by the chromium release assay. These peritoneal exudate cells showed higher cytotoxicity as compared to those of saline-injected mice (p < 0.001). Macrophages were demonstrated to be the major component of peritoneal exudate cells (50%) by flowcytometric analyses. Cytotoxicity increased with macrophage enrichment by adhesion (p < 0.01). Furthermore, a macrophage toxin, silica, completely blocked the cytotoxicity (p < 0.001). Cytokines (interleukin 1 and tumour necrosis factor) and a nitric-oxide-producing vasodilator, sodium nitroprusside, were cytotoxic to MIN6N-9a cells but only sodium nitroprusside showed cytotoxicity when incubated for the same period as peritoneal exudate cells. Thus, macrophages play an important role in beta-cell destruction and soluble factors other than cytokines (e.g. nitric oxide) may be mediators of this early cytolytic process.

Induction of anti-Escherichia coli activity in mice by phenothiazines, benzo[a]phenothiazines and benz[c]acridines.

The abilities of 14 phenothiazines, 8 benzo[a]phenothiazines and 12 benz[c]acridines to induce anti-Escherichia coli activity in mice were compared. Pretreatment with several benzo[a]phenothiazines or benz[c]acridines protected mice from lethal infection of Escherichia coli in a dose-dependent manner, whereas most of the phenothiazines induced much weaker anti-Escherichia coli activity. However, direct contact of Escherichia coli with these compounds or their administration just after bacterial inoculation were ineffective. These data suggest the immunopotentiation activity of benzo[a]phenothiazines and benz[c]acridines.

Immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice.

The development of antibody against lactic dehydrogenase virus in five strains of mice (NZB x NZWF1, BALB/c, C.B-17, ICR and C.B-17 scid or SCID mice) was examined by indirect immunofluorescence (IIF) of infected liver sections. IIF antibody appeared 1 to 3 weeks and rose progressively 2 to 4 weeks after infection in four strains of mice (NZB x NZWF1, BALB/c, C.B-17 and ICR mice). SCID mice did not develop antibody. These results suggest that IIF may be applicable for detecting LDV infection in many other ordinary strains of mice.

Influence of lead on the host's defence mechanisms (I)--Influence of lead on antibody production.

To clarify the influence of lead on the host's defense mechanisms, antibody production in mice pretreated with lead was tested using the hemagglutination titer against SRBC (Sheep Red Blood Cells) and HRBC (Hamster Red Blood Cells) as indicator and the following results were obtained. 1. When mice were pretreated intraperitoneally with lead one day or six days before immunization and then immunized with SRBC, which is known as a strong antigen, antibody developed smoothly in the first immunization showing the same tendency as that of the control group. After the booster immunization, antibody production was markedly suppressed in the group of mice pretreated with lead six days before the immunization. When mice were immunized with HRBC, which is known as a weak antigen, antibody production was very poor in the first immunization, but after the booster immunization, the antibody titers rose rapidly in the group pretreated one day before the immunization. However, the titers of the group pretreated with lead 6 days before the immunization was considerably suppressed, showing the same tendency as that of mice immunized with SRBC. 2. When mice received intraperitoneally three or six doses of lead before immunization with SRBC or HRBC, antibody titer of these groups were somewhat lower than that of the control group. 3. When mice were pretreated intravenously with lead one day or six days before immunization with SRBC or HRBC, the antibody production ability was not remarkably damaged, showing almost the same titer as in the control group.

Antitumor action of an acidic glycoprotein (SAGP) from Streptococcus pyogenes in mice.

We have shown that an acidic glycoprotein (SAGP) isolated from a cell-free extract of Streptococcus pyogenes (Su strain) prolonged the life-span of Ehrlich ascites carcinoma (EAC)-bearing mice. The present study shows that the life-span prolonging effect of SAGP in EAC-bearing mice was reduced by whole body X-irradiation before EAC inoculation. SAGP (500 micrograms protein/mouse/day X 4, i.p.) also showed a life-span prolonging effect (T/C (%) = 169) on Meth A fibrosarcoma (Meth A)-bearing mice, but the effect of SAGP was abrogated by an i.p. pretreatment of the host with carrageenan, an antimacrophage agent. The spleen cells from the Meth A-inoculated and SAGP-treated mice were found to have a considerable cytostatic activity by a 3H-thymidine incorporation assay. But the activity disappeared in the presence of carrageenan. These results suggest that the in vivo antitumor effects of SAGP are mediated through its immunomodulating action.

Studies on route of immunization with a mixture of killed parasites and adjuvants against Babesia rodhaini infection in mice.

A series of experiments were undertaken to determine the most effective route of immunization with a mixture of killed Babesia rodhaini antigen (S antigen) and formalin-fixed Corynebacterium parvum (Propionibacterium acnes) bacterin (CPB) against challenge infection with B. rodhaini 3 weeks later. The mice pretreated with S antigen and CPB mixture intraperitoneally, but not intramuscularly, were significantly resistant to intraperitoneal (IP) or intravenous (IV) challenge with 10(6) organisms. The survival rates were 70.0 (IP challenge) and 60.0% (IV challenge) respectively. Fairly protective activities were equally produced in mice intravenously pretreated with S antigen and CPB with survival rates of 60.0% against IV challenge, but 30% against IP. These results indicated that the IP injection of S antigen and CPB mixture is desirable route for immunization against subsequent IP or IV challenge with B. rodhaini. On the other hand, lower protective effect was reconfirmed in the mice treated with S antigen and Freund's Complete adjuvant, regardless of immunization routes in the additional experiment. The survival rates were 33.3, 14.3 and 11.8% in the intraperitoneally, intramuscularly and subcutaneously-treated mice respectively against IP challenge with 10(6) organisms.

In vitro and in vivo interferon production in NOD mice.

The production of interferon-alpha/beta (INF-alpha/beta) and interferon gamma (IFN-gamma) in NOD and ICR mice was studied in vitro and in vivo. The in vitro IFN-alpha/beta production in the spleen cells of NOD mice, which were stimulated with either Newcastle disease virus (NDV), Sendai virus, poly(I:C) or lipopolysaccharide (LPS), was very similar to the IFN-alpha/beta production in the spleen cells of ICR mice. Contrastingly, the in vitro IFN-gamma production in the spleen cells of NOD mice, which were stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM), was greater than the IFN-gamma production in spleen cells of ICR mice. The in vivo IFN-alpha/beta production in NOD mice induced by NDV was also very similar to that in ICR mice, whereas the in vivo IFN-gamma production in the BCG-sensitized NOD mice, which was induced by purified protein derivative (PPD), was greater than that in the ICR mice. These results may indicate that NOD mice have abnormalities on the IFN-gamma production.

Resistance of mice to genital infection with Neisseria gonorrhoeae.

Five strains of mice (C3H, CBA, BALB/c, TO and ICR) were inoculated intra-vaginally with Neisseria gonorrhoeae in an attempt to produce an animal model of gonorrhoea. Of a total of 68 mice inoculated, only three (4.4%) were culture-positive after 3 days. Histological examination of both the genital mucosa of inoculated animals, and the mucosa of genital tract organ cultures inoculated in vitro failed to show any evidence of gonococcal adherence or colonisation. Mice of these strains, therefore, appear resistant to gonococcal infection of the genital tract.

Successful oral rabies vaccination of raccoons with raccoon poxvirus recombinants expressing rabies virus glycoprotein.

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.

Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses.

Recent studies indicate that Ig class and IgG subclass induction varies for different proteins and further that some Ig subclasses, like IgG2a, are more efficient in important biologic processes such as antibody-dependant cell-mediated cytotoxicity (ADCC). Many proteins of herpes simplex virus type 1 (HSV-1) are immunogenic and induce immunoglobulin responses. To determine the distribution of immunoglobulins induced by HSV-1 proteins, we studied immune mouse serum using an Ig isotype specific Elisa assay for antiviral activity. We found by endpoint analysis that the antiviral titer was 1:12,903 for IgG1, 1:5141 for IgG2a, 1:2140 for IgG2b and 1:229 for IgG3. To identify which isotypes were induced by individual glycoproteins and other viral proteins, Western blots containing HSV-1 proteins were probed with immune serum and isotype specific second antibodies. gB, gC, gD and the 42/44KDa nucleocapsid complex induced strong IgG1, IgG2a, IgG2b responses. IgG3 reactivity with viral proteins appeared weaker. Among the IgG3 reactivities detected on immunoblots, gB and gC were the most intense. Other proteins which elicited IgG1, IgG2a and IgG2b responses were 170KDa, 154KDa and gE. IgA responses were induced by 154KDa, gC, gB, gE and gD. Prominent IgM responses included gB, gC, gD and the 42KDa protein. These results indicate that HSV-1 glycoproteins induce prominent responses in all IgG isotypes except IgG3. The biologic implications of the data are discussed.

Early events in B-cell activation: anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells.

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent and allele-specific manner. Antibodies to other B-cell surface molecules such as Ia did not induce a PI response. The effect of anti-Lyb2 mab was always less in magnitude than that induced by anti-IgM, but the effects of the two antibody preparations were most comparable in larger, presumptively preactivated cells. To explore the issue that Lyb2 may represent a receptor for a growth factor, possibly the early-acting B-cell growth factor BSF-1, we studied the PI response to BSF-1 and the effect of BSF-1 on Lyb2-induced PI turnover. BSF-1 neither induced a PI response nor inhibited competitively the response induced by anti-Lyb2 mab.

Immune responses in Mycoplasma pneumoniae infection of infant mouse and of man.

Infant mice (2 to 4 days old) were exposed to living Mycoplasma pneumoniae. The organisms were isolated in the order of 10(3) to 10(4) colony-forming units from the lungs of mice for 2 weeks after infection. Mononuclear cell infiltration was present in the lungs of infected mice. The specific IgG antibodies to membrane proteins of M. pneumoniae in the sera of infected mice were detected by enzyme-linked immunosorbent assay for about 800 days. In immunoblotting analysis, a 160-kilodalton (kDa) protein strongly reacted with the infected mouse sera. The dams immunized with membrane proteins conferred passive immunity on their offspring via colostrum. Specific IgG antibody appeared in the serum of infant mice that were given mouse anti-M. pneumoniae serum or convalescent human patient serum orally. These mice were also protected from challenge with M. pneumoniae. The immunoblotting patterns of patient sera were similar to those of infected mouse sera. The infant mouse model may be useful to investigate the host immune responses to M. pneumoniae.