Guidelines for Sanitary Inspection

“Guidelines for Sanitary Inspection”:National Institute of Health, Masami Kitaoka

Published by Kyodo Igaku Sho Shuppansha, October 1950 (2nd edition)

Serological diagnosis have developed, and identification tests were performed in addition to diagnosis by Negri body and animal experiments.

The following process was established to determine rabies.

▶ Postmortem Diagnosis

◎Method for Negri body detection

Negri bodies were detected by staining smears of Ammon’s horn on glass slides.

The Sellers staining method, which uses basic fuchsin and methylene blue to stain the Negri bodies reddish purple to dark red, is now newly used. This method was the best because it did not require fixation of the test specimens and staining could be done in seconds.

Eight slides prepared by the Sellers staining method were examined to determine if they were positive for Negri bodies.

◎Animal Test

It is performed by intracerebral inoculation of mice with high susceptibility and short incubation period.

An emulsion is prepared by taking small pieces from the Ammon’s horn, brainstem, and cerebellum, and inoculated intracerebrally into five or more mice.

If the animal dies within four days of inoculation, it is not rabid. (However, it is desirable to determine the cause of death.)

Three mice each are inoculated with brain emulsion from mice that died after day 5.

Street virus usually develops in 7 to 11 days, so if no symptoms develop during this period, kill one and inoculate three with brain emulsion.

When rabies develops, the following symptoms occur.

First, the hair stands up. When pinched with tweezers, the animal shows swimming movements, and when released on a table, the ears are horizontal, the neck is extended, and the moves are peristaltic. Then comes drooling, paralysis, and death.

If any of these symptoms occur during the observation period, the animal should be killed immediately, some of it preserved in glycerin, and the remainder used for identification tests. If there is trouble determining the animal test, inoculate again from a glycerin specimen or brain emulsion of dead mice.

◎Identification Tests

A standard immunized serum is necessary to identify rabies. There are various methods for preparing immunized sera, but the following methods allow the complement binding reaction to be unobstructed by normal mouse brain antibodies.

Inoculate guinea pig brains with an emulsion of street or fixed virus-infected mouse brains and prepare a 20% emulsion of infected guinea pig brains in 1% phenol-added sterilized saline solution.

Store in a cool, dark place for 5–7 days and shake several times daily. Inoculate guinea pigs intraperitoneally with about 3 mL of a 2-fold dilution of the emulsion three times every 7 days. the third time, the phenol-free brain emulsion could be used.

On the 28th day after the start of immunization, whole blood samples are taken and antibody titers are measured.

◎Neutralization test

Using rabies-positive and rabies-negative sera, make a 10-fold step dilution of 20% infected mouse brain emulsion, and react each with an equal volume of serum on ice for a certain time. Mice are then inoculated intracerebrally. Each dilution is inoculated into 3 to 5 mice at a time, observed for at least 4 weeks, and the LD50 is calculated.

<Complement binding reaction>

Test serum, brain from rabies-infected mice as antigen, guinea pig serum as a complement, and sheep red blood cells are used to test for the presence of anti-rabies antibodies in the samples.

The samples were tested at least twice at 7–10-day intervals to see if antibody titers increased.

<Cross-infection protection experiment>

A vaccine is prepared from the brains of mice infected with rabies and mice inoculated with the specimens using 1% phenol saline solution. This is then inoculated intraperitoneally into the mice twice at 1-week intervals. On day 21 after the start of immunization, a brain emulsion of the mice used to make the vaccine will be inoculated intramuscularly into the immunized mice, and the lethality rates of each group will be compared.

▶ Clinical Diagnosis

◎Typical Rabies

1.Incubatino Period:Very indefinite, ranging from 6–9 days to years, but generally 2–6 weeks. Young animals tend to have shorter incubation periods.

2. Prodromal Phase

Animals generally show signs of anxiety, resist owner commands, or run away. Reflexes are heightened, and even minor stimuli can excite the animal to bite.

Appetite is normal, but the animal may exhibit abnormal behavior such as eating foreign objects. This period lasts from 0.5 to 2 days. In many cases, the virus can already be detected in the saliva during this period.

3. Manic phase:Lasts from 2 to 4 days and is characterized by symptoms specific to rabies.

Infected animals become ferocity, biting their owners, animals, plants, and inanimate objects, and even biting themselves. The eyesight becomes strange, the animal stares at the front corner (exotropia), and the animal hallucinates and bites the air as if it were feeding on a fly in the air.

The voice changes completely, becoming hoarse or silent and drooling. After a mania, the animal stops for a while and then goes into another mania. The frequency of the depression gradually increases.

4. Paralytic phase:In this stage, the animal is depressed, and paralysis affects the mandible, eyes, and hindquarters, leading to death.

◎Atypical rabies

In addition to the typical cases, there are other types of rabies that begin in the prodromal phase and quickly progress to the paralytic phase, or that begin with paralytic symptoms and die within a short period of time, or that follow a long course after the onset of the disease and in rare cases may be cured.