Misdiagnosis of Milk Sample Cultures
The infection status of mammary quarters is determined by microbiological
culture of aseptically obtained milk samples and interpretation of the
culture results. As in all biological data, diagnosing intramammary
infection is subject to error. Culture of milk samples generally results in one of three events: A frequent assumption is that the recovery of the contagious pathogens
Staphylococcus aureus or Streptococcus agalactiae from
a single milk sample is evidence of intramammary infection. However,
F(+) samples can occur with all pathogens including Staph. aureus
and Strep. agalactiae, and the frequency of F(+) samples will
increase as the number of truly infected quarters in a herd increases.
False-positive samples associated with the environmental pathogens likely
will increase as environmental contamination increases. Attempts to reduce the number of F(-) samples by using enrichment techniques
or a period of preliminary incubation should be avoided. Plating larger
volumes of milk (0.1 ml per half-plate) will help reduce the number
of F(-) samples but may increase the number of contaminated samples
if aseptic sampling technique is poor. Clinical quarters are generally
assumed to be infected. Two degrees of contamination are generally recognized: The second type of contamination is "gross contamination".
Three or more colony types are present on the milk streak, often in
relatively heavy growth. Such samples should be declared contaminated
and no attempt should be made to identify potential pathogens within
the mix of microbial growth. When gross contamination is observed, the
quarter should be resampled. Common contamination sources include dirty teat ends, milk touching
fingers before entering the tube, nonsterile tubes or inoculating needles,
streaking milk samples on contaminated media, excess alcohol on teats
or hands, contaminated cotton swab container, and the container lid
not sealed tightly resulting in alcohol evaporation from cotton swabs. Source: National Mastitis Council publication "Laboratory Handbook
on Bovine Mastitis" (1999) p. 35
1) no bacterial growth
2) growth of a pure culture
3) growth of multiple colony types
Any of the three outcomes may not represent the true infection status
of the quarter. Therefore, strict adherence to aseptic sampling technique
and proper storage and handling of milk samples are absolutely essential.
In addition, diagnosis of intramammary infection status based on multiple
samples is more reliable than diagnosis based on a single sample.
False-positive samples [F(+)] result when a pathogen is isolated in
pure culture but the quarter is truly not infected. Such samples occur
as a result of contamination at some point during sample collection and/or
processing. When intramammary infection status is based on culture of
a single sample, F(+) samples get interpreted as an infected quarter.
False-negative samples [F(-)] result when no microbial growth is detected
following microbiological culture but the quarter is truly infected. Reasons
for such samples include:
1) the colony-forming units of the organism in the milk are below the
detection limit of the assay
2) special media or growth conditions are required
3) inhibitors in the milk sample, such as antibiotics, have interfered
with the growth of the pathogen
4) the sample was mishandled during storage, resulting in death of the
pathogen.
False-negative samples are more likely to occur with coliform and Staph.
aureus infections than infections caused by Strep. agalactiae.
A common finding is that 20-30% of samples from clinical quarters will
result in no microbial growth. Clinical signs may be present but the
pathogen has been eliminated by the cow’s immune system.
When a quarter milk sample results in the culture of three or more
dissimilar colony types, the milk sample is most likely contaminated and
the sample should be recorded as such. All mastitis pathogens present
in milk samples can be a result of contamination, including Staph.
aureus and Strep. agalactiae.
The first is "low level" contamination in which dissimilar
colonies are present. These may be the only colonies on the streak or
they may be present in the streak of an otherwise pure culture of a
pathogen. The "low level" contamination should be recorded
together with the pathogen.