ddY Strain Research Articles

Studies on Kochiae Fructus. I. Antipruritogenic effect of 70% ethanol extract from kochiae fructus and its active component

The antipruritogenic effect of the 70% ethanol extract obtained from Kochiae Fructus (fruits of Kochia scoparia) and its active components were investigated on a compound 48/80-induced pruritogenic model in male ddY strain mice. The extract (200, 500 mg/kg, p.o.) inhibited the scratching behavior as a pruritogenic indicator. Oleanolic acid oligoglycoside, momordin Ic, isolated from the extract also exhibited the inhibition. These results suggest that Kochiae Fructus could be used as an antipruritogenic agent and its inhibitory effect may be partially attributed to momordin Ic.

寒冷馴化過程のマウスにおけるadenosineおよびadenine摂取の影響

The effects of adenosine (ADO) and adenine (ADE) on cold acclimation were studied in mice of the ddY strain. At 5 weeks of age, mice were maintained at 4 degrees C for 4 weeks. Test groups of mice were supplied with a 0.05% (w/v) solution of ADO or ADE as drinking water in addition to water. We measured changes in body weight, intake of water and food, rectal temperature upon acute exposure to -20 degrees C, weight of interscapular brown adipose tissue (BAT) and the levels of glucose, nonesterified fatty acids (NEFA) and 3H-butyrate in the blood prior to and after exposure to the lower temperature of -20 degrees C. Chronic exposure to cold (4 degrees C) reduced body-weight gain for the first two weeks but weight gain recovered within the next two weeks. ADO partially but selectively inhibited the gain in body weight. This effect was marked in mice maintained at room temperature. Such an inhibitory effect was the case of ADE in the room temperature. Exposure to cold stimulated the daily consumption of food. ADO further and selectively enhanced food intake, but ADE enhanced that in room temperature. Cold increased in daily intake of water. Both ADO and ADE accelerated the intake of water. This effect was marked in mice maintained at 4 degrees C and the effect of ADE was considerable. Mice chose to drink the water that contained ADO, while the choice of water that contained ADE was apparent only during exposure to cold. Exposure to cold rendered mice resistant to severe cold (-20 degrees C). ADO and ADE partially reduced this resistance. Exposure to cold increased the weight of BAT but ADO selectively prevented this increase. Cold did not change the levels of the various compounds measured in the blood. In mice exposed to 4 degrees C for 4 weeks, acute exposure to severe cold decreased the glucose level and increased the levels of NEFA and 3H-butyrate. ADO selectively prevented the changes in the levels of NEFA and 3H-butyrate. It remains to be confirmed whether the growth of brown adipocytes is always necessary when mice acclimate to cold.

Effect of aging on the expression of iNOS and cell death in the mouse cochlear spiral ganglion.

The age-related induction of inducible nitric synthase (iNOS) and apoptotic cell death in spiral ganglion cells (SGCs) of ddy strain mice were studied with immunohistochemical method and TdT-mediated dUTP-biotin nick end labeling (TUNEL), respectively. A large amount of iNOS was expressed in SGCs of 18- to 24-month-old mice, but not in those of the mice less than 12 months of age. Moreover, these mice were accompanied by a great rise in auditory brainstem response threshold as well as a great decrease in the number of SGCs that seemed to be due to preceding cell death of the cells. However, we were unable to find apoptotic TUNEL-positive cells in the spiral ganglions. This was assumed to be due to a very short clearance time of the dead cell bodies of less than one hour. As has been known, NO produced by iNOS can implicate in causes for either protection of cells from peroxidation and cell death. In the present study, therefore, the steep augmentation of iNOS in the SGCs of senescent mice implies that the iNOS initially induced to protect SGCs from the cytotoxicity of cellular peroxidation eventually contribute to the cell death of SGCs themselves.

Selective breeding for high serum IgA levels from noninbred ddY mice: isolation of a strain with an early onset of glomerular IgA deposition.

An outbred mouse strain known as ddY has been reported to spontaneously develop, late in life, mesangioproliferative glomerulonephritis with a severe glomerular immunoglobulin A (IgA) deposition that mimics human IgA nephropathy. However, the incidence of the disease in this strain is not very high, probably due to its heterogeneous genetic background. Therefore, we attempted to isolate a strain with a high incidence and an early onset of the disease through selection for high serum IgA from the outbred ddY mice. The selection procedure was successful in increasing the serum IgA level of the selected line and proved effective both in increasing the incidence and in accelerating the onset of the disease. We propose to designate this line of mice 'HIGA', denoting a line with high serum IgA levels. More than half of the mice from the HIGA strain showed a moderate to severe glomerular IgA deposition as early as 25 weeks of age. The severe deposition observed was comparable to that occasionally seen in the original nonselected ddY strain after 40 weeks of age. Thus, we have succeeded in generating a mouse model of IgA nephropathy with a high incidence and an early onset of glomerular IgA deposition. Using light microscopy, progressive and marked mesangial matrix accumulation was shown to develop in HIGA mice. However, they showed only mild proteinuria (100-300 mg/dl) and did not show hematuria.

Role of nitric oxide and prostaglandins in lipopolysaccharide-induced increase in vascular permeability in mouse skin

To examine the possible role of increased vascular permeability in the circulatory shock induced by endotoxin (lipopolysaccharide), we examined whether lipopolysaccharide elicits plasma extravasation in the skin of ddY strain mice. We also studied whether nitric oxide (NO) and prostaglandins may mediate the lipopolysaccharide-induced increase in vascular permeability. Subcutaneous injection of lipopolysaccharide (100–400 μg/site) induced a dose-related and delayed increase in vascular permeability at the injection site as determined by the leakage of pontamine sky blue. Concurrent administration of aminoguanidine (a putative inducible NO synthase inhibitor) (10 mg/kg, i.v.) inhibited the lipopolysaccharide (400 μ/site)-induced dye leakage by 71%. N G-Nitro- l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (10 and 20 mg/kg, i.v.) inhibited the lipopolysaccharide-induced dye leakage by 36% and 54%, respectively, whereas the inactive enantiomer, N G-nitro- d-arginine methyl ester (10 mg/kg, i.v.), had no effect. Pretreatment with an intraperitoneal injection of dexamethasone (500 μg/kg) or indomethacin (a cyclooxygenase-1 and −2 inhibitor) (5 mg/kg) almost completely inhibited the response induced by lipopolysaccharide, by 96% and 84%, respectively. [ N-(2-Cyclohexyloxy-4-nitrophenyl)methanesulphonamide (a cyclooxygenase-2-specific inhibitor) (0.01–1 mg/kg, i.p.) also induced a dose-related inhibition of dye leakage elicited by lipopolysaccharide: 38% and 80% suppression at the doses of 0.1 and 1 mg/kg, respectively. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the effect of lipopolysaccharide by 74%. These results suggest that the increase in vascular permeability induced by lipopolysaccharide is mediated by both NO and prostaglandins and that synthesis of inducible NO synthase and cyclooxygenase-2 may be involved in this effect of lipopolysaccharide.

Decreased capacity of aged mice to produce interferon-gamma in Legionella pneumophila infection

We investigated the difference in natural resistance to Legionella pneumophila infection between aged (18–20-month-old) and young (3-month-old) mice of ddY strain. Aged mice were more susceptible to the bacterial infection than young mice; 50% lethal doses of L. pneumophila for aged and young mice were 2.2 × 10 7 and 8.5 × 10 7 colony forming units (CFU), respectively, after intraperitoneal injection of the bacteria. The bacterial burden in the livers was larger in aged than young mice after a challenge with a sublethal dose of L. pneumophila. However, peritoneal macrophages of aged mice paradoxically had a greater capacity to kill intracellular L. pneumophila than those of young mice. Interferon-gamma (IFN-γ) production from naive spleen cells was compared after an in vitro stimulation with formalin-killed L. pneunophila. Spleen cells of aged mice produced significantly less IFN-γ than those of young mice. When anti-murine IFN-γ monoclonal antibody was administered before the bacterial infection, the subsequent bacterial burden in the livers significantly increased in young but not in aged mice. These data suggest that, in aged mice, IFN-γ production is depressed at an early phase of L. pneumophila infection and it renders aged mice more susceptible to the infection.

EFFECT OF DIFFERENT WAVELENGTHS OF LOW LEVEL LASER THERAPY ON WOUND HEALING IN MICE

Two bilateral 6.0 mm round skin defects were made on 60 DDY strain mice. The mice were divided into four groups and treated with low incident doses of laser energy at different wavelengths for 15 days: (a) Group I- Control; (b) Group II - 830 nm wavelength; (c) Group III - 680 nm wavelength; and (d) Group IV - 632.8 nm wavelength. For the treatment groups, only the right skin defect was irradiated. The rate of healing was assessed by examination through a monitor microscope, measurement of skin defect area and histological examination. No significant difference was observed between the four groups by macroscopic examination. The area of skin defect of the treated groups was significantly smaller than that of the control groups (p < 0.05) from the 1st-13th day after irradiation. For both irradiated and non-irradiated skin defects of treatment groups, Group III was smaller than Group II (p < 0.05) and Group IV was smaller than Group II (p < 0.05) Histological examination of the tissue skin defect showed greater proliferation of connective tissue and blood vessels in the dermis in treated groups than in control.

Possible dysfunction of ionotropic glutamate receptors in cerebellum of epileptic E1 mouse brain

Binding of [ 3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) to an ion channel domain on the N -methyl- d-aspartate (NMDA)-sensitive subclass of brain glutamate (Glu) receptors was highest in the hippocampus of the hereditary epileptogenic mutant E1 as well as its parent ddY strain mice, when determined before and at equilibrium in the presence of 3 different agonists at the respective domains on the NMDA receptor complex, including Glu, glycine (Gly) and spermidine (SPD). Cerebellar [ 3H]MK-801 binding before equilibrium was significantly lower in E1 mice than in ddY mice, while the binding was not significantly different from each other in other brain structures of both strains of mice. Kinetic analysis revealed that the association rate was significantly lower with [ 3H]MK-801 binding in the cerebellum of E1 mice than of ddY mice. In contrast to ddY mice, furthermore, Gly failed to potentiate cerebellar [ 3H]MK-801 binding before equilibrium in E1 mice, with SPD being active in significantly inhibiting the binding. However, saturation analysis revealed that the affinity and density of cerebellar [ 3H]MK-801 binding at equilibrium in E1 mice were not significantly different from those in ddY mice. In addition, epileptogenic E1 mice had significantly higher levels of [ 3H]SPD binding in all brain structures examined than ddY mice, whereas [ 3H] dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding was significantly lower in the cerebellum of E1 mice than of ddY mice. These results suggest that dysfunction of cerebellar Glu receptors may be at least in part responsible for a variety of abnormal symptoms observed in epileptic E1 mice.

Strain- and age-associated changes in cytokine release from endotoxin-treated mouse monocytes

Levels of cytokines released from endotoxin-mediated monocytes were investigated in mice of DBA/2, BALB/c, C57BL/6, C3H/He, ddY and ICR strains and in ICR mice of various ages (4-52 weeks old). Interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) levels from monocytes were low in DBA/2 and BALB/c mice, and high in ddY and ICR strains. In addition, interleukin-6 (IL-6) levels were low in DBA/2, and high in ddY and ICR strains. Therefore, monocytes obtained from ddY and ICR strain mice released large amounts of cytokines as compared to those from the DBA/2 strain. The levels of IL-1 alpha, IL-1 beta and IL-6 released from monocytes of ICR mice were constant at 4-18 weeks old, and low at 26 and 52 weeks old. In contrast, the TNF alpha level was low at 18 weeks and high at 26 and 52 weeks. These results indicate that age-associated changes in cytokine release from monocytes correlate well with the cytokine levels in bone marrow.

Axonal sprouting at the node of Ranvier of the peripheral nerve disconnected with the cell body

In order to examine whether the axonal sprouting at the node of Ranvier is a local event reactive to the lesion independent of influences from the cell body, the sciatic nerve of mouse (ddY strain) was freeze-injured at the mid-thigh and at the same time transected at the level 2 cm proximal to the frozen site. Within 5 h after freeze-transection, sprouts were formed at some nodes of Ranvier located within 0.5 mm proximal to the frozen site. These sprouts had almost the same ultrastructural features as those found in ordinary freeze-injury, showing a tendency to grow toward the overlying Schwann cell basal lamina. However, the sprouts thus formed ceased to grow and became atrophic within 10 h after freeze-transection. This fact indicates that the axon has a property of sprouting at the node of Ranvier in the absence of the cell body. However, nutritional as well as constitutive substances from the cell body are needed to maintain the growth of the sprout.

Comparative study on factors regulating the level of oxidative stress toward pulmonary nuclei in two different strains of mice

1. 1. The levels of active oxygen formed and thiobarbituric acid reactive substances (TEARS) in pulmonary nuclei of A/J strain were higher than those in the nuclei of ddY strain, while nuclear α-tocopherol level in the lungs of the former strain was lower than the the level in the latter strain. 2. 2. Unsaturation index (UI) of nuclear fatty acids and oxidative damage to DNA in the lungs of A/J strain were higher than those in the lungs of ddY strain. 3. 3. These results suggest that the capacity of nuclear active oxygen formation, nuclear a-tocopherol level and UI of nuclear fatty acids in the lungs can be main factors to regulate oxidative stress toward the pulmonary nuclei in the two strains of mice.

Lack of strain differences in spatial learning among seizure-prone and seizure-resistant mice.

Learning rates were examined in the following inbred mice strains: DBA/2, C3H/He, C57B1/6J, E1, and ddY. DBA/2 mice become susceptible to audiogenic seizures after 2-3 weeks of age and E1 mice have generalized seizures in response to handling after 3 months of age, but the remaining three strains do not develop seizures. In this study, mice from all five strains underwent 32 training trials in a Morris water maze at 7-9 weeks of age. The seizure-prone DBA/2 and E1 mice, along with the nonepileptic ddY and C57B1/6J mice, exhibited learning at similar rates, but the nonepileptic C3H/He mice were unable to learn the water maze task, probably due to visual difficulties. In the C57B1/6J strain only, female mice learned the task significantly faster than males. There was no difference in the learning rate between the E1 strain and its parent ddY strain, or any correlation between spatial learning ability and kindling rates in these strains.

Sex hormone-related control of hepatic epoxide hydrolase activities in mice.

Sex-related differences in hepatic epoxide hydrolase (EH) activities towards 7-(2',3'-epoxy)propoxycoumarin (7-glycidoxycoumarin, GOC) were investigated, mainly in mice but also in rats. Hepatic subcellular EH activities in the ddY mouse were higher in microsomes than in the soluble and mitochondrial fractions and sex-related differences were noted in all the subcellular fractions where males had significantly higher activities than females. Sex differences in the hepatic microsomal and soluble activities similar to those in the ddY strain were also observed in two other strains of mice, A/J and C3H/He, and in Wistar rats. In the ddY strain, castration of the males caused decreases in microsomal and soluble EH activities, while no alteration in the activities in those fractions was found following castration of females. Treatment of the male castrates with testosterone led to recovery of the activities in microsomal and soluble fractions while hormone treatment of female castrates caused a rise only in microsomal activity. Estradiol treatments of both sexes did not cause any changes in the hepatic subcellular activities. In intact ddY mice, testosterone treatment did not affect the male microsomal and soluble EH activities, but resulted in stimulation of both subcellular enzyme activities in females. In contrast, estradiol treatment showed a suppressive effect on both subcellular activities in males, but had no effect on female activities. These results show that hepatic EH activities towards GOC are mainly under androgenic stimulatory control in mice.

Learning dysfunction model in ddY mouse and IgG

Mice of the ddy strain having once received an electric shock in the dark chamber of a passive avoidance response unit were divided into two groups according to their behavior pattern. It is suggested that learning function of the mice which did not avoid the dark chamber (negative-avoidance mice: N-mice) was disturbed. After an injection of IgG obtained from the positive-avoidance mice (P-mice) into the N-mice, these prior N-mice displayed the behavior pattern similar to that of P-mice following an application of electric shock. It was found that the sugar chain structure of N-mouse IgG was different from that of P-mouse IgG. This result indicates that the sugar chain structure is related to the learning ability of ddy mice.

A deficiency in the mechanism for p34 cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34 cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34 cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated froms of p34 cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34 cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34 cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34 cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34 cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34 cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4′,6′-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34 cdc2 to induce mitotic events but the protein remains in a latent form.

Age-related changes in NMDA-induced [3H]acetylcholine release from brain slices of senescene-accelerated mouse

From the brain slices of normal mice (ddY strain, subcloned from dd strain in National Institute of Health in Japan), N-methyl-d-aspartic acid (NMDA) at 0.01~1 mM evoked [3H]acetylcholine (ACh) release in a concentration dependent manner. [3H]ACh release evoked by 1 mM NMDA was significantly inhibited by 2-amino-5-phosphonovaleric acid (APV), phencyclidine (PCP) and 5-methyl-10,11-dihydroxy-5H-dibenzo[a,d]cyclo-hepten-5,10-imine maleate (MK-801). The effects of NMDA were not seen in the Ca2+ free medium and were inhibited by physiological concentration (0.83 mM) of Mg2+. NMDA seems to cause ACh release from nerve terminals through the receptorion channel mediated mechanism in the mouse brain. Based upon these results, we determined the activity of a high K+- or NMDA-evoked [3H] ACh release using prone/8 strain of senescence-accelerated mouse (SAM-P/8) (a murine model of accelerated aging and memory dysfunction) and SAM-resistance/1 strain (SAM-R/1) (normal aging mice as the control) and these release activities were compared between both strains and during aging. [3H]ACh release evoked by 30 mM KCl was significantly lower than that of age-matched SAM-R/1 at 9 and 12 months. NMDA evoked the [3H]ACh release at 2, 6, 10 and 14 months in R/1 mice. In SAM-P/8 mice the activity of NMDA-evoked release was seen at 2 months, but markedly decreased afterwards. Nonsignificant difference was observed on the uptake of [3H]choline and on the spontaneous release of [3H]ACh between SAM-P/8 and SAM-R/1 strains, and during aging. These results suggest: 1.(1) NMDA receptor-ion channels are involved in the ACh release from nerve terminals in the CNS;2.(2) the NMDA-evoked ACh release is decreased after 6 months in SAM-P/8 mice but not in SAM-R/1 mice; and3.(3) the high K+-evoked ACh release is decreased at an earlier age in SAM-P/8 than it is in SAM-R/1. These results suggest that the NMDA- and cholinergic-system in the CNS seems to be deficient in SAM-P/8 and that this deterioration may be a possible cause for their dysfunction of learning and memory.

Effects of Total Asphyxia on the Development of Synaptic Junctions in the Brains of Mice

Using ethanolic phosphotungstic acid staining, we studied the development of synaptic junctions in the brains of mice subjected to total asphyxia. One-day-old mice, Std:ddY strain, were put into a chamber continuously flushed with 100% CO2-gas for 35 min. The surviving mice (28.1%) were used as experimental models, while unasphyxiated littermates served as controls. The synaptic junctions in the frontal motor cortex and hippocampal cortex were studied at 20 and 60 days of age using E-PTA staining, and the numbers of synaptic junctions in both areas were counted. In comparison with the control animals, the frontal motor cortex of the subject mice showed a smaller increase in the number of synaptic junctions, both at 20 and at 60 days of age. In the hippocampal cortex, the number of synaptic junctions seen in the experimental mice brains was similar to that in the controls at 20 days of age; however, the number of synaptic junctions in the treated mice brains showed a slower rate of increase than in the controls at 60 days of age.

Egg production of Clonorchis sinensis in different strains of inbred mice.

In order to compare the intraspecific variation in host-parasite relationship of Clonorchis sinensis, six strains of inbred mice, ICR, DDY, GPC, BALB/c, nude and DS, were infected orally with 20 metacercariae of C. sinensis. The biologic incubation period of C. sinensis was the shortest in DDY mice, 21.2 days in average, followed by GPC 21.4, BALB/c and DS 23.2, ICR and nude 23.4 days, respectively. The fertile period of the fluke was also the longest in the DDY strain, 164 days on average, followed by GPC 132, BALB/c 97, nude 37, DS 32 and ICR 28 days. The egg-laying capacity of the fluke in DDY and GPC was relatively high and stable compared with the other four strains of mice. It was found that there are intraspecific variations in biologic incubation period, fertile period, and fecundity of C. sinensis. The DDY mouse is likely to be the most suitable experimental animal among the six strains of the mice tested.

Deficiency of Osteoclasts in Osteopetrotic Mice Is Due to a Defect in the Local Microenvironment Provided by Osteoblastic Cells*

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Susceptibility of Newly Established Mouse Strain MPS to Mycoplasma pneumoniae Infection

A newly established mouse strain, MPS, which is more sensitive to Mycoplasma pulmonis than ICR, ddY and other mouse strains was examined for its susceptibility to Mycoplasma pneumoniae. In experimental infections with M. pneumoniae, it was observed that M. pneumoniae attached to tracheas of MPS mice, and M. pneumoniae cells were isolated from tracheas and lungs of MPS mice even after four weeks of infection, while no mycoplasmas were isolated from ICR and ddY mice after one week of infection. Specific antibodies against M. pneumoniae were also observed by the Western blotting in the sera of MPS mice infected with M. pneumoniae. Although any lung lesion could not be observed in this work, this newly established mouse strain MPS may be useful for experiments of M. pneumoniae infection, especially for the analysis of strain differences in susceptibility to M. pneumoniae infection.