Enumeration of probiotic bacilli spores in animal feed: interlaboratory study.

Fourteen out of 17 laboratories completed an interlaboratory study comparing 2 pretreatment protocols of feed samples containing authorized probiotic bacilli spores. Both methods used tryptone soy agar for enumeration. Pretreatment A involved preparation of a suspension of the feed sample in 50% ethanol. For pretreatment B, the sample was suspended in peptone salt solution and heated at 80 degrees C for 10 min. Each laboratory analyzed 12 samples (6 per pretreatment), which represented duplicates of a high (10(9) colony-forming units [CFU]/g) and low (10(5) CFU/g) level of bacilli spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSD(r)) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSD(r) values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards.

Bacteriological investigations of clinical mastitis in heifers in Sweden.

Bacterial analyses were carried out of 2069 udder secretions, isolated from 1481 heifers with mastitis in eight veterinary districts in Sweden. Streptococci, e.g. Streptococcus dysgalactiae and Str. uberis, dominated the bacterial flora, being isolated from 34.4 and 19.5% respectively of heifers with clinical mastitis occurring from puberty up to 14 d post partum. Bacterial species generally regarded as important pathogens in the summer mastitis complex, e.g. Actinomyces pyogenes, Stuart-Schwan coccus and strictly anaerobic bacteria such as Peptostreptococcus indolicus, Fusobacterium necrophorum and Bacteroides melaninogenicus were isolated at low frequencies (13.2, 6.3, 9.4, 3.8 and 1.3% respectively). When the cases of mastitis studied were restricted to those appearing in heifers pre partum, May 15 to October 14 (summer mastitis), these bacterial species were isolated at higher percentages (27.1, 14.4, 21.4, 13.5 and 5.2% respectively). These figures were, nevertheless, still lower than those published in reports from other countries. Whether considered up to 14 d post partum or only pre partum, there were no significant differences in the frequencies of A. pyogenes isolated at different seasons. There were geographical differences in bacterial incidence, e.g. Staphylococcus aureus was isolated significantly more often in northern regions whereas Str. dysgalactiae was more common in the south. This and other Swedish investigations support the theory that A. pyogenes and strictly anaerobic bacteria are 'secondary invaders' that depend on Str. dysgalactiae to cause a primary infection. It is stressed that the udders of all heifers should be examined daily so that cases of mastitis can be treated immediately.

[Limulus test--a rapid and simple method for the detection of endotoxins produced by gram-negative bacteria in mastitis milk]. / Limulustestet--en snabb och enkel metod att påvisa endotoxin från gramnegativa bakterier i mjölk vid mastit.

According to the present study the limulus amebocyte lysate test (LAL) seems to be a convenient test to detect endotoxin in milk from udder quarters with and without inflammation. The correlation between endotoxin concentration and the results from the bacteriological investigation of 79 milk samples was good (Table I). Determination of endotoxin in 20 milk samples from cases of acute clinical mastitis with high cell count and a negative bacteriological culture showed that all but one had an endotoxin concentration of greater than 1.0 ng/ml milk (Table II). By using a micromethod of the LAL it is possible to detect cases of mastitis caused by gram-negative bacteria about one hour after the sample has reached the laboratory. In a preliminary field study milk from 13 cases of acute clinical mastitis were tested by a modified limulus amebocyte lysate (LAL) test ("cowshed test"). A 100% correlation to bacteriological findings was observed (Table IV). By using the LAL test to detect mastitis cases caused by gram-negative bacteria great economic advantages and less risk for resistance problems can be achieved by using proper antibiotics. This is the fact in Sweden where the frequency of acute clinical mastitis caused by streptococci (100% of strains sensitive for penicillin) and Staphylococcus aureus (about 90% of strains sensitive for penicillin) is high (70-80%) and about 20% are caused by gram-negative bacteria, mostly E. coli.