Hisamasa Joshima

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.

Transmission experiments of cilia-associated respiratory bacillus in mice, rabbits and guineapigs

Transmission experiments of cilia-associated respiratory (CAR) bacillus were performed in mice in order to clarify the principal route of the infection, and in rabbits and guineapigs in order to examine their susceptibility. Determination of the infection was evaluated serologically by the indirect immunofluorescence assay (IFA) technique and histologically by the presence of CAR bacillus in the airways. BALB/c mice were intranasally inoculated with the SMR strain of CAR bacillus. The IFA antibody to the bacteria in these mice rose to more than 1 : 160 at 4 weeks postinoculation (PI) and the mice were utilized as transmitters for the following experiments. One out of 15 uninfected mice kept in intracage contact with infected mice became infected from 4 weeks after contact. Incidence of contact infection increased thereafter. On the other hand, there was no evidence of infection in the uninfected mice housed in the separate cages from the cage in which infected mice were housed throughout the 12-week observation period. The primary method of CAR bacillus transmission seems to be direct contact with infected mice or fomites contaminated by infected mice; airborne transmission appears to be of little importance. Rabbits and guineapigs were also intranasally inoculated with the SMR strain of CAR bacillus. IFA antibodies were positively detected by 4 weeks PI, but no CAR bacillus nor histological changes relating to the infection were observed in the airways of either species. It is suggested that rat origin CAR bacillus can transmit to rabbits and guineapigs, and that the infection can spread to other species of rodents and rabbits.

Pathology of rats intranasally inoculated with the cilia-associated respiratory bacillus

Five-week-old Wistar/Ms rats were inoculated intranasally with a lung homogenate containing a strain of cilia-associated respiratory (CAR) bacillus and were examined on days 4, 7, 14, 21, 28 and 56 postinoculation (PI). Some rats showed clinical signs with wheezing and considerable body weight loss from day 21 PI. Gross lesions, including enlargement of lungs with focal atelectasis, bronchiectasis and emphysema, were observed from day 21 PI. Histologically, round cell infiltration was first present in the lamina propria of the nasal respiratory mucosa on day 7 PI. From day 14 PI, colonization of the CAR bacillus (4-8 µm in length), associated with round cell infiltration in the lamina propria and the peripheral regions, was observed in the ciliated mucosa of the bronchioles, bronchi, trachea and nasal cavities. Generally, the lesions progressed and expanded from upper to lower airways with time. Sporadic mucopurulent bronchopneumonia was observed from day 21 PI in some rats. The CAR bacilli (0·2-0·25 µm in diameter) were also demonstrated electron-microscopically in the ciliated epithelium of the intrapulmonary airways. The CAR bacillus antigen was demonstrated on the ciliated mucosa of the affected airways by the indirect immunofluorescence assay technique. Microbiological examination revealed that the rats used in this study were free from other known respiratory pathogens throughout the experimental period. Thus, it is suggested that the CAR bacillus alone can produce a murine respiratory disease. Fourteen days were needed for pathological lesions to develop.

CHANGES OF BLOOD PLASMA ELEMENT CONTENTS IN X-RAY IRRADIATED MICE BY PIXE ANALYSIS

The quantitative changes of elements in blood plasma were observed with the passage of time after X-ray whole body irradiation with 12 Gy on C57BL/6J mice by PIXE method. From 4 days after irradiation, dead mouse was found and all mice died by 8 days. Hematocrit (Ht) values indicated a decrease from the 1st day, but on days 3 and 4 there was a small rise. Finally the values became 64 % of that of non-irradiated control on day 7, it was just before death. By analysis with PIXE method, 15 elements were observable in blood plasma of control mice. The elements such as P, S, Cl, K, Ca and Cr were abundant and Fe and Br followed. As trace elements, the peaks of Zn, Cu and Ni were clearly observed. After irradiation, K and Ca decreased on day 1st, afterwards increased gradually. On the contrary, the elements, S, Cl, were rather stable. While Fe decreased from 1st day, Cu increased from the day 2. Zn and Ni showed intensely down and rise in amount, and decreased on day 7. The results of possible measurement of the changes in amount of these elements of blood plasma suggest PIXE method is an easy and useful way for diagnosis.

PIXE STUDIES ON POTASSIUM AND CALCIUM IN MOUSE BLOOD PLASMA AFTER TRANSPLANTATION OF EL-4 TUMOR CELLS

The quantitative changes in the elements, amounts of Cl, K, Ca, in blood plasma were measured by PIXE method. The samples were obtained at appropriate intervals after transplantation of EL-4 tumor cells in three strains of mice, C57BL/6J (H-2b), C57BL/10J (abbreviation: B10; H-2b) and A/J (H-2a). Transplanted EL-4 tumor cells proliferated in both strains of C57BL/6J and B10. In A/J mice, transplanted EL-4 cells proliferated about 10 days and then were rejected completely by the immunological reaction according to the difference of major histocompatibility antigens. The amounts of Cl in plasma remained at similar level in the time course in any strains, but K fluctuated in C57BL/6J and B10, and less in A/J. On the other hand, Ca showed always higher values in C57BL/6J than other two strains of mice. In B10 mice, Ca increased just before death, but in A/J it decreased at the time of healing by rejection. These changes of Ca in the three strains of mice were related quantitatively 10 the hematocrit values of these strains of mice after transplantation of EL-4 cells.

POSSIBLE APPLICATION OF PIXE METHOD TO INTESTINAL PERMEABILITY MEASUREMENT

We measured intestinal permeability of mice by PIXE method. A mixture of a rubidium chloride solution and a manganese chloride solution was administered orally into mice. The blood was sampled successively after the administration, and the element contents in the blood were measured by PIXE method. The Rb content in the plasma of Rb+Mn administered group reaches maximum 5 min after the administration and decreases subsequently. The value in the whole blood of Rb+Mn administered group increases slightly for first 30 min after the administration and remarkably from 3 hrs. This increase continues for 48 hrs. That in control group is very little. Rb is probably transferred into plasma from intestinal mucosa at first, and subsequently moves into blood cells. The Mn content in control group is little. The values in the whole blood and the plasma of Rb+Mn administered group reach maximum 5 min after the administration and then decrease, and turn to decline to control level at 24 hrs. Mn is probably transferred into blood from intestinal mucosa throughout, and washed out more rapidly than Rb. The element contents of Rb and Mn in the plasma of Rb+Mn administered mice are found to increase to the peak level 5 min after the administration. Rb and Mn are useful as a tracer to study of intestinal disease with measurement by PIXE method. With this manner by PIXE method, intestinal permeability can be measured more briefly than existing methods. In order to assess intestinal permeability, we propose an indicator which is obtained by comparing the element amounts of Rb and/or Mn in blood sampled just after the administration.

Use of rubidium, manganese, and zinc as tracers to measure intestinal permeability by PIXE analysis: basal study in an experimental enteritis model.

Intestinal permeability has been suggested to be closely linked with the etiology or activity of Crohn’s disease. However, current methods for measurement of intestinal permeability are too laborious for routine examination, as they require urine collection and/or use of radioisotopes. The present study was performed to develop a more convenient and safer method for assessing intestinal permeability using blood samples rather than urine. Rats with indomethacin-induced enteritis were orally administered Rb, Mn, and Zn as tracers. Intestinal permeability was determined by assaying the levels of Rb, Mn, and Zn in blood samples by particle-induced X-ray emission (PIXE). The distributions of Rb, Mn, and Zn in the small intestine after administration were analyzed by micro-PIXE. The conventional PIXE analysis showed that the levels of Rb and Zn in the blood in the enteritis group were correlated with the grade of enteritis. The micro-PIXE analysis showed that Rb, Mn, and Zn were translocated into the wall of the proximal small intestine 5 min after administration, and this effect was more conspicuous in the enteritis group than in controls. Analysis of blood or small intestine tissue samples using the PIXE allows determination of both intestinal permeability and the route of permeation.