Apr. 12, 1924 



Development of American Foulbrood 



135 



that of larvse at the age when European foulbrood makes its attack, comprises 

 nearly 45 per cent of the dried substance, or nearly 14 per cent of the fresh 

 substance. From these facts it may be assumed that, because of the great 

 amount of food given the larva at this age, there must be present in the larval 

 intestine, at all times during the active feeding period, considerable amounts 

 of this food rich in sugar unassimilated, up to and even after active feeding 

 ceases. A number of larval and prepupal intestines were dissected from 

 healthy larvse and tested roughly with Benedict's qualitative solution {34) 

 for the presence of reducing sugars. The results indicated the presence of 

 relatively large amounts of reducing sugar in the intestines of larvse just prior 

 to sealing. Little or no reducing sugar could be demonstrated in the intestines 

 of sealed larvse or prepupoe. 



It may therefore be assumed that certain of the organisms associated with the 

 early stages of European foulbrood are able to grow in the presence of a high 

 sugar concentration. Experiments were devised in 

 which a medium containing 10 per cent dextrose was 

 used. It was found that while a few types of organ- 

 isms, such as one resembling Streptococcus apis {S8), 

 could be grown in varying numbers, an organism sim- 

 ilar to that described by Maassen (S9), resembling the 

 larger'forms of Bacillus pluton, called Bacillus lanceolatus, 

 could be isolated and grown from over 50 per cent 

 of the samples cultured. As described by the writer 

 (.4'^), "This organism was found to grow best on a 

 10 per cent dextrose yeast extract agar with a reac- 

 tion slightly acid. It is differentiated from Bacillus 

 pluton and Streptococcus apis in being gram-negative, 

 and does not grow readily if at all in media with- 

 out sugar." From these studies it was suggested 

 that possibly this comparatively high sugar content of 

 the unassimilated food in the larval intestine may 

 have an influence on the germination of the spores and growth of Bacillus 

 larvae and that a change may occur when the sugar content is suflBciently re- 

 duced by assimilation in the larval intestine after it has been capped and when 

 the intestines have been emptied by the opening of the ventriculus into the 

 end gut. Therefore, with these preliminary observations as a basis, experi- 

 nental work on this subject was begun during the spring of 1922. 



GROWTH OF BACILLUS LARVAE IN CULTURES IN RELATION TO 

 VARIATION IN SUGAR CONCENTRATION 



The first step in the substantiation of this theoretical assumption is to de- 

 termine whether there is a correlation between germination of the spores of 

 Bacillus larvae and vigor of vegetative growth and variations in concentration of 

 reducing sugars in culture media. Ordinary culture media are unsuitable for 

 the growth and isolation of Bacillus larvae; in fact, one of the confirmatory tests 

 for this organism in laboratory diagnosis of American foulbrood (31) is the 

 absence of growth on plain beef infusion agar plates, since the spores wiU not 

 germinate thereon. There are rarely any secondary invaders associated with 

 Bacillus larvae in the decayed material, and these plates practically never show 

 growth. 



GROWTH EBQUIEEMENTS OF BACILLUS LARVAE 



Various special culture media have been devised which answer more or less 

 satisfactorily the requirements for the ordinary growth of the organism. The 



Fig. 6.— Healthy coilea larva 

 at age of maximum intestinal 

 sugar content and approxi- 

 mately the age when the ma- 

 jority die from European foul- 

 brood. (White (iff)) 



