Influence of genetic background on host resistance to experimental murine tularemia.

The host response to experimental murine tularemia was examined in different inbred mouse strains. The kinetics of growth of Francisella tularensis live vaccine strain (LVS) in the livers and spleens of A and C57BL/6 mice were monitored, and it was observed that mice of the A strain were more susceptible to the proliferation of LVS than were C57BL/6 mice. The difference was most marked 5 days following infection, when the number of bacteria isolated from the spleens of A mice was found to exceed that of C57BL/6 mice by 100-fold. In addition, the C57BL/6 strain exhibited a more pronounced splenomegaly 8 days after infection than did the A strain. When the response of other inbred strains was evaluated by determining the splenic count of LVS on day 5 postinfection, several levels of antiularemic resistance were observed. Mice of the AKR, BALB/cBy, C57BL/10, and SJL strains were found to be most resistant, while SM mice were most susceptible to the proliferation of LVS. The DBA/2, CBA, 129, C3H/HeJ, and A strains expressed a resistance phenotype which was intermediate between the two extremes, with A and C3H/HeJ mice being somewhat more susceptible than DBA/2, CBA, or 129 mice. The trait of resistance or susceptibility was analyzed genetically in (C57BL/6 x A)F1 hybrid mice and in F2 generation and recombinant inbred (RI) mouse strains derived from C57BL/6 (resistant) and A (susceptible) strain progenitors. The F1 progeny exhibited a level of resistance to infection which was similar to that of the resistant parent. In both the F2 generation mice and the RI strains, a continuous spectrum of resistance levels was observed. The results of these experiments indicate that the genetic background of the host influences host resistance to experimental murine tularemia and that multiple genetic loci are involved in this response.     

Polyclonal increase in certain IgG subclasses in mice persistently infected with the 87V strain of scrapie

Eight different combinations of seven strains of scrapie agent and the three known Sinc genotypes of mice were screened for changes in the concentration of IgG in serum. A single radial immunodiffusion assay was used to measure IgG throughout the incubation period which in different models ranged from an average of 125 days to longer than the maximum observation period of about 600 days. The only major changes occurred with the 87V strain of scrapie injected intracerebrally (i.c.) or intraperitoneally (i.p.) into mouse strains of the Sinc genotype p7p7. IgG concentration reached 1.5 to 2.0 times the control values in i.c. infected mice, which developed clinical disease after 270 to 320 days and also in i.p. infected mice, which did not develop the disease within the 600-day observation period. At very high IgG concentration, the increase was polyclonal; it involved the IgG 1 subclass more than the others and was accompanied by an increased rate of IgG clearance from serum. It is suggested that some scrapie infections of mice (and sheep) may upset the control of IgG production. The underlying mechanism may involve cell-pathogen interactions which are common to all scrapie infections, but only lead to gross changes in IgG in some combinations of agent strain and host genotype.     

Propagation of ovine prions from "poor" transmitter scrapie isolates in ovine PrP transgenic mice

Ovine prion strains have typically been identified by their transmission properties, which include incubation time and lesion profile, in wild type mice. The existence of scrapie isolates that do not propagate in wild type mice, defined here as "poor" transmitters, are problematic for conventional prion strain typing studies as no incubation time or neuropathology can be recorded. This may arise because of the presence of an ovine prion strain within the original inoculum that does not normally cross the species barrier into wild type mice or the presence of a low dose of an infectious ovine prion strain that does. Here we have used tg59 and tg338 mouse lines, which are transgenic for ovine ARQ or VRQ PrP, respectively, to strain type "poor" transmitter ovine scrapie isolates. ARQ and VRQ homozygous "poor" transmitter scrapie isolates were successfully propagated in both ovine PrP transgenic mouse lines. We have used secondary passage incubation time, PrPSc immunohistochemistry and molecular profile, to show that different prion strains can be isolated from different "poor" transmitter samples during serial passage in ovine PrP transgenic mice. Our observations show that poor or inadequate transmissibility of some classical scrapie isolates in wild type mice is associated with unique ovine prion strains in these particular sheep scrapie samples. In addition, the analysis of the scrapie isolates used here revealed that the tg338 mouse line was more versatile and more robust at strain typing ovine prions than tg59 mice. These novel observations in ovine PrP transgenic mice highlight a new approach to ovine prion strain typing.     

Pathogenesis of scrapie: agent multiplication in brain at the first and second passage of hamster scrapie in mice.

The intracerebral (i.c.) injection of mice with a particular source of hamster passaged scrapie produced disease after an incubation period of 325 +/- 6 days (mean +/- s.e.). The incubation period at the second i.e. passage in mice was reduced to 149 +/- 2 days. Studies were made of the dynamics of agent replication at 1st and 2nd passages in mice. At first passage, there was a 'zero phase' lasting about 175 days, when no infectious agent was detected in brain (or spleen), followed by a period of agent replication which lasted 150 days. At second passage, there was no significant 'zero phase' and agent replication occupied the whole of the incubation period. The occurrence of a 'zero phase' on interspecies passage of scrapie is discussed in relation to other reports of a 'zero phase' in mouse passaged scrapie.     

Interaction of scrapie agent and cells of the lymphoreticular system

Summary The current study focused on the role of lymphoid elements of the lymphoreticular system in scrapie pathogenesis. In the first experiment, adherent and non-adherent splenocytes from mice infected with the 139A scrapie strain were prepared. The level of infectivity on a per cell basis was significantly higher in the adherent cell population. In a second set of experiments, thymocytes, unfractionated splenocytes, T-cell enriched and T-cell depleted fractions of splenocytes were infected in vitro with ME7 scrapie strain. There was no evidence of replication of scrapie in ME7-exposed cells in any of the preparations during the first 5–14 days post-exposure. In assays done 5 days after infection, most of the infectivity was cell-associated. These data suggest that lymphoid cells are not involved in scrapie replication. The level of IgA in the serum of 139A-infected mice was markedly reduced compared to the levels in mice injected with normal mouse brain homogenate or with the ME7 scrapie strain. The reduction in IgA levels in 139A-infected mice was evident at each of the 4 time points tested. The final experiment dealt with the question of scrapie replication in the lymphoreticular organs in mouse strains with different incubation periods for 139A after intraperitoneal injection. The results in this experiment suggest that the difference in incubation periods is related to differences in time of access of infection to the central nervous system rather than to differences in the ability of agent to replicate in spleen.     

Genetic control of mouse cytomegalovirus-induced myocarditis.

Mouse cytomegalovirus (MCMV) infection of mice induced myocarditis, characterized by a mononuclear cell infiltrate with associated necrosis of myofibres. Myocarditis was observed in parallel with viral inclusion-bearing cells in the heart during the acute phase of the infection. Myocarditis also persisted after the acute phase when viral antigens were no longer detectable by immunoperoxidase histochemistry and infectious virus could not be cultivated from various organs. The influence of host genetic factors on the development of cytomegalovirus-induced myocarditis was investigated using H-2 congenic and recombinant inbred mouse strains. Analysis of congenic variants with C57BL/10 and BALB/c backgrounds and the A/J strain revealed that genes linked to the H-2 complex influenced susceptibility to peak levels of MCMV-induced myocarditis seen 7 and 10 days post-infection. In addition, non-H-2 genes of the BALB/c background were important in determining the severity of myocarditis. Analysis of the strain distribution pattern of the CXB recombinant inbred series did not disclose the identity of the BALB/c non-H-2-linked allele conferring susceptibility to MCMV-induced myocarditis. The level of myocarditis seen in the F1 hybrid between the high-responder BALB/c and low-responder C57BL/6 strains suggested dominant inheritance. The amount of viral replication in the major target organs did not correlate with the severity of myocarditis. In conclusion, at least two genes, one mapping to the H-2 complex and another non-H-2-linked gene, influenced the development of myocarditis in MCMV-infected mice.     

Genetics of murine resistance to Trypanosoma cruzi.

Resistance to the protozoan parasite Trypanosoma cruzi is governed by multiple genetic factors, including at least one coded for by a locus in or near the major histocompatibility complex of the mouse. The influence of the H-2 locus on resistance was evident when H-2 congenic mice on a strain background of intermediate resistance were challenged or when the survival of H-2 typed F2 mice was followed. The H-2k haplotype of the susceptible C3H/An strain was associated with higher mortality when compared with the H-2b haplotype of the resistant C57BL/10 strain. Genetic studies showed that resistance was a dominant trait and increased with genetic heterozygosity. F1 mice derived from crosses between resistant and susceptible strains, or even between two susceptible strains, were much more resistant than either parent. Crosses between two resistant strains, C57BL/6J and DBA/2J, led to resistant progeny in the F1 and F2 generations; but when recombinant inbred strains derived from these parental strains were challenged, susceptible strains were identified, indicating that different genes were responsible for resistance in the two strains.     

Serial studies on the development of cerebral amyloidosis and vacuolar degeneration in murine scrapie

The number of amyloid plaques and the severity of vacuolar lesions were traced through the incubation period following an intracerebral injection of scrapie agent with 3 combinations of scrapie agent and mouse strain. Amyloid plaques were the first histological lesions to be seen, appearing as early as 60 days after injection in the case of LM mice with 87A scrapie strain, long before vacuolation or any other changes became apparent. It is therefore concluded that amyloid plaques are not simply secondary to the degenerative vacuolar lesions usually seen in scrapie. For each combination, the first plaques to appear were in areas close to the lateral ventricles, suggesting that early plaque production is related to the localization of components of the inoculum soon after intra-cerebral injection. In areas distant from the ventricles, plaques were only seen during the later part of the incubation period, at a time when vacuolation could also be detected. The majority of plaques throughout most of the incubation period were of the “shadowy” morphological type, containing little amyloid. This suggests that shadowy plaques are formed as such and that the other types of plaques, which are larger and richer in amyloid, may develop from them.     

Host modifier genes affect mouse autoimmunity induced by the lpr gene.

A defect in apoptotic signal transmission through CD95 is an essential genetic mechanism for lymphoproliferation and autoimmunities in lpr or gld mice. However, disease manifestations are largely affected by the host genetic background. To identify and map such host genes modifying lpr gene effect, ie, the lpr modifier (Lprm) genes, 82 MRL/lpr x (MRL/lpr x C3H/lpr) F1 mice were subjected to immunopathological and genetical analyses. High-grade vasculitis and glomerulonephritis among backcross mice were observed in separate groups of mice. Microsatellite analysis revealed that there were two host genes affecting the occurrence of vasculitis, Lprm1 (chromosome 4) and Lprm2 (chromosome 3). A recessive MRL allele at Lprm1 enhanced vasculitis to occur in both sexes, whereas that of Lprm2 inhibited its development selectively in females. Genotype combinations of these two genes explained the severity of vasculitis in crosses of MRL/lpr and C3H/lpr mice and also the vasculitis-prone recombinant inbred strain McH5/lpr. A recessive MRL allele at Lprm3 (chromosome 14) suppressed glomerulonephritis. The weight of the spleen was increased by a recessive MRL allele at Lprm4 (chromosome 5) yielding a logarithm of odds score of 2.02 in a quantitative trait locus analysis. In contrast, the weight of axillary lymph nodes was increased by a recessive MRL allele at a locus on chromosome 2, but its presence was not supported by the quantitative trait locus analysis. The titer of anti-dsDNA autoantibody was controlled by the locus Lprm5 on chromosome 16, which had an logarithm of odds score of 3.41. Possible candidate genes for Lprm genes deduced from their map locations are discussed and compared with the autoimmunity genes reported thus far. In conclusion, autoimmune disease manifestations by the lpr mutation are affected by multiple host genes separately.     

Changes in drinking and feeding habits of mice with experimental scrapie

Several strains of mice, some differing at the sinc locus which controls scrapie incubation period, were injected with one or other of three divergent strains of scrapie agent. A decline of drinking habits occurred in all cases, commencing at a relatively early stage of incubation and long before typical signs of the disease were apparent. In some host-agent strain combinations there was a corresponding increase in feeding which was not present in other combinations.     

Isolation of two distinct prion strains from a scrapie-affected sheep

We performed a transmission study using mice to clarify the characteristics of the most recent case of scrapie in Japan. The mice that were inoculated with the brain homogenate from a scrapie-affected sheep developed progressive neurological disease, and one of the scrapie-affected mice showed unique clinical signs during primary transmission. This mouse developed obesity, polydipsia, and polyuria. In contrast, the other affected mice exhibited weight loss and hypokinesia. In subsequent passages, the mice showed distinct characteristic scrapie phenotypes. This finding may prove that different prion strains coexist in a naturally affected sheep with scrapie.     

Characteristics of a short incubation model of scrapie in the golden hamster.

Repeated passage of the "Chandler" strain of scrapie in female golden hamsters using the intracerebral route of inoculation reduces the minimum incubation period to 60 days, about half of the minimum incubation period so far found in any of the mouse models of scrapie. The infectivity titres in brain in the clinical stage of the disease are considerably higher (greater than 8-0 -log10 LD50 i.c. units/0-05 g) than those found in mouse scrapie. The biological characteristics of this model of hamster scrapie are reported, including the effects on incubation period of route of inoculation, dose of agent, sex of hamster, ambient temperature (hibernation) and splenectomy. Some general and specific applications of this experimental model of scrapie are discussed.     

Experimental murine hyalohyphomycosis with soil-derived isolates of Fusarium solani.

Two strains of soil-borne Fusarium solani, both characterized for their ability to produce cyclosporin A and C, were examined for their pathogenicity in severe combined immunodeficiency (SCID) and BALB/c male mice. Intravenous (i.v.) infections with F. solani conidia were performed. No mortality was observed after infection with 0.3-1.6 x 10(7) cfu per mouse in SCID and BALB/c mice. When mice were infected with 0.8-1.5 x 10(6) cfu per mouse and 2 days later with 1.2-1.9 x 10(6) cfu per mouse, 28.6-85.7% survival occurred over a 25-day period, depending on the F. solani strain and the inbred mouse line used. Death was preceded by renal insufficiency affecting both kidneys. Furthermore, i.v. injection with heat-killed conidia followed 2 days later by injecting viable conidia resulted in renal infection in both breeds of mice. F. solani isolated from infected organs was more virulent than the original isolate, and 3/8 (37.5%) of BALB/c and 4/7 (57.1%) of SCID mice died after receiving a single dose. Dissemination to the brain was found only in SCID mice, but torticollis was observed in both mouse breeds. Soil-borne F. solani isolates possess poor pathogenic potential for mice, but either two successive infective doses or a primary injection with heat-killed conidia followed by a single infective dose breaks through host defenses in normal and immunoincompetent mice. Mouse passage increased the pathogenicity of two soil-derived F. solani strains.     

Host Defenses in Experimental Scrub Typhus: Genetics of Natural Resistance to Infection

Genetic resistance to lethal infection with Rickettsia tsutsugamushi was studied in over 30 inbred strains, inbred hybrids, and outbred stocks of mice. Inbred mice infected intraperitoneally with the Gilliam strain of R. tsutsugamushi showed three patterns of response: susceptible (A/HeJ, C3H/HeDub, C3H/HeJ, C3H/HeN, C3H/St, CBA/J, DBA/1J, DBA/2J, and SJL/J), resistant (AKR/J, BALB/cDub, BALB/cJ, C57BL/6J, C57L/J, and SWR/J), and selectively resistant (A/J). The selectively resistant pattern was characterized by random deaths occurring throughout the titration range and was also observed in three of the six outbred mouse stocks surveyed. No correlation was evident between the H-2 haplotype of inbred mice and their response to Gilliam infection. The progeny from five different Gilliam-resistant by Gilliam-susceptible inbred parental crosses were all resistant. Study of F(1), F(2), and parental backcross generations of BALB/cDub (resistant) and C3H/HeDub (susceptible) hybrids indicated resistance was dominant and was controlled by a single gene or a closely linked cluster of genes that were autosomal and not linked to coat color. The resistance of BALB/cDub mice was not due to an inability of host cells to support rickettsial growth, since C3H/HeDub and BALB/cDub embryo cell cultures supported similar growth of Gilliam organisms. C3H/HeDub mice, although susceptible to intraperitoneal Gilliam infection, were capable of mounting an immune response to Gilliam antigens, since subcutaneous infection was not lethal and did protect animals against subsequent intraperitoneal challenge with either the Gilliam or Karp strains of R. tsutsugamushi.     

Studies of the lymphoreticular system in the pathogenesis of scrapie: the role of spleen and thymus.

It has been established that a high proportion of extraneural replication of scrapie agent occurs in tissues associated with the lymphoreticular system, such as spleen. The question arises as to which components of the system are responsible for scrapie replication and whether such components can be identified by means of physiological manipulations. A series of experiments in mice showed that splenectomy deprived the host of a significant fraction of its capacity to permit ME7 scrapie agent to replicate, resulting in increases (10 to 30 per cent) of incubation period following intraperitoneal infection. This lost capacity remained for at least 60 days of age after neonatal splenectomy, although a smaller fraction of the total replicative capacity was vested in the spleen of the newborn than in that of mice aged 5 or more days; prolongation of incubation period in splenectomized newborn mice was over 9 per cent irrespective of the interval to injection.Experiments involving thymectomy were performed to discover whether the thymus-dependent portion of the lymphoid system was responsible for some of the spleen's ability to permit scrapie agent to replicate. The results failed to show any involvement of the thymus and its dependent lymphoid system in the replication and pathogenesis of scrapie in mice.     

Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis

The pathogenesis of lethal infection by the nonecapsulated, toxigenic Sterne strain of Bacillus anthracis and the genetic basis of resistance were characterized in mice. Lethal doses of Sterne spores produced disease in susceptible mice similar to that caused by toxigenic and encapsulated B. anthracis. At the inoculation site, the mice developed an edematous exudate with large concentrations of bacilli and toxin. In the susceptible A/J strain, lethal infection was accompanied by systemic invasion and serum anthrax toxin levels increased in parallel with systemic bacterial concentrations and with the mortality rate. Host resistance to Sterne infection was associated with the ability to synthesize the complement component 5 (C5). All Sterne-resistant mouse strains had a functional gene (Hc) encoding C5, whereas susceptible mice were deficient in C5. A/J mice could be passively protected from lethal challenge by C5-positive serum but not by serum from C5-negative congenic mice. Also resistance was linked to production of C5 in individual backcross (97%) and F2 (98%) mice. The distribution pattern for recombinant inbred mice was consistent with a major role in host resistance of Hc or a closely linked locus, although other genes probably contribute. This mouse model will be useful in characterizing the pathogenesis of anthrax and testing the safety and efficacy of new anthrax vaccines.     

Evidence for co-infection of ovine prion strains in classical scrapie isolates

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.     

Role of the H-2s haplotype in survival of mice after infection with Trypanosoma cruzi.

In studies of the resistance of inbred mice to infection with Trypanosoma cruzi Peru, mouse strain B10.S was the only strain which survived the infection resulting from the inoculation of 10(3) trypomastigotes. This is the only inbred mouse strain studied to survive infection. To investigate the effect of the H-2 haplotype on survival, C57BL/10 congenic mouse strains bearing H-2S recombinant haplotypes and mouse strains A.SWSn/J and SJL/J were tested for their ability to overcome the T. cruzi infection. None of the recombinant strains tested, including B10.S(7R), B10.S(8R), B10.S(9R), and B10.HTT, survived the infection, indicating that at least two or more regions of the H-2 locus must be H-2S to ensure survival. Strains A.SWSn/J and SJL/J with the H-2S haplotype did not survive, indicating that the genetic background outside the H-2 complex also influences survival. The congenic F1 hybrid (C57BL/10 X B10.S) F1 exhibited intermediate survival levels when compared with the parental strains, indicating that H-2S survival is affected by gene dosage. The F1 hybrid strain [B10.S(7R) X B10.S(8R)]F1, which possesses the complete H-2S haplotype in the trans configuration, did not survive T. cruzi infection, suggesting that H-2S-mediated survival does not operate by trans complementation.     

Adaptation of the agent of scrapie to hamsters

The agent of scrapie (Compton strain) can be transmitted from mice to hamsters; the incubation period of the disease is 5–6 months. Passage of the agent of scrapie through suspensions of brain tissue was repeated 10 times. The scrapie agent was found in the spinal cord and spleen but it could not be found in the liver, kidneys, adrenals, and lungs of the infected animals in the last stage of the disease.Division of General Epidemiology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 2, pp. 199–201, February, 1976.