REAL TIME PCR VALUES FOR MASTITIS PATHOGENS – RELATIONS TO MILK
QUALITY AND HERD CHARACTERISTICS IN DANISH DAIRY HERDS
1
Torben Werner Bennedsgaard1 and Jorgen Katholm2
Department of Animal Bioscience and Health, Aarhus University, Foulum, Denmark,
2
Knowledge Center for Agriculture, Cattle, Aarhus, Denmark
Abstract
In October to December 2009 bulk tank milk samples from all 4258 Danish dairy herds were
tested by real-time PCR. Data on bulk tank somatic cell count (BTSCC), for most herds from
every milk delivery, and total bacteria count (TBC) for the last 3 weeks before to one week after
sampling were available for all herds. Furthermore information on milking system (automatic
milking system (AMS) divided by major brands or non-AMS), herd size and production type
(organic and conventional) was also available. Linear relations between the last BTSCC and the
cycle threshold (Ct)-values for Staphylococcus aureus, Streptococcus uberis, Streptococcus
dysgalactiae and Streptococcus agalactiae were found when the Ct-values were below 33.5,
34.5, 32 and 32 respectively. Linear relations between the average log transformed TBC and the
Ct-values for the same bacteria were also significant with slightly lower cut-off values. The
prevalence of Str. agalactiae increased with herd size in all herd types from 4 percent in the
smallest herds to over 15 percent in herds with more than 250 cows. The prevalence also varied
with production type and milking system with nearly double prevalence for one brand of AMS.
The relation between the Ct-values and milk quality and especially the differences seen for Str.
agalactiae and special herd characteristics are discussed.
Introduction
In Denmark, BMSCC and treatment frequency for mastitis are the two main parameters for
monitoring the overall udder health status, and Danish veterinarians use bacterial culture of
quarter milk samples routinely for monitoring prudent use of antibiotics and as a diagnostic tool
in mastitis management. Bulk tank milk (BTM) culture has until know only been use to monitor
the prevalence of S. agalactiae. In contrast, BTM culture is widely accepted in many other
countries as a tool for evaluating the quality of milk and for monitoring udder health status
(Jayarao and Wolfgang, 2003).
Molecular methods have been suggested to improve the sensitivity of intramammary pathogen
detection. In particular, PCR based methods are now being used increasingly in mastitis
diagnostics. Recently, analytical accuracy was reported for a real-time PCR based reagent kit
capable of detecting 11 important IMI species/species groups and the beta-lactamase gene
(PathoProofTM Mastitis PCR Assay, Thermo Fisher Scientific, Espoo, Finland) based on a large
collection of culture isolates (Koskinen et al., 2009).
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The objective of this study was to evaluate the results of a PCR test of all Danish dairy herds to
describe relations between PCR results and milk quality and to describe the effect of different
herd characteristics on the prevalence of S. agalactiae based on PCR testing.
Material and Methods
BTM samples from all 4258 Danish dairy herds from 2009 were included. The samples were
collected between the 20th October 2009 and 6th January 2010. BTM sampling was carried out
during milk collection and stored on ice. Within 24 hours, the samples were shipped to Eurofins
Laboratory, Holstebro, Denmark. In 2009, the milk samples for culture and PCR testing were
taken after the BTM sample had been through the laboratory for routine testing of SCC, fat,
protein and urea. Culture of S. agalactiae was done by mixing 120 µl of milk on a selective
aesculin agar containing 5% of sterile calf blood, 0.5% of N-solution and 0.3% S. aureus β-toxin
(Agger et al., 1994).
All BTM samples were tested using a PCR based assay (PathoProofTM Mastitis PCR Assay,
Thermo Fisher Scientific, Espoo, Finland). A total of 350 µl of milk was used as a starting
volume for DNA extraction. Cycle threshold (Ct) values were recorded for all samples and for
all bacterial targets. The Ct value represents the number of PCR cycles it takes to obtain the
threshold level. Ct values higher than 39.9 were set as negative (NoCt).
BTSCC was measured at each delivery to the dairy, typically every second day. TBC was
measured once every two weeks on samples collected the same way as samples for PCR. The
TBC was not necessarily measured the same day as the PCR analysis. TBC from up to three
weeks before to one week after the sampling for PCR was used in the analysis. The results of
BTSCC and TBC were stored in the Danish Cattle Database for all herds. Information on herd
size, milking system and production system was available from the Danish Cattle Database.
Herds infected with S. agalactiae are recorded in the Danish B-register on the basis of a positive
individual cow or BTM testing. Free herds found BTM positive are tested again. If the second
sample is positive, the herd is recorded in the B-register. If the second sample is negative, a third
test will settle the status of the herd. Herds can leave the register if four consecutive BTM
samples each more than 30 days apart test negative for S. agalactiae.
The correlation between Ct-values for the different bacterial DNA and BTSCC and Ct values
and the logarithm of TBC analyzed. A generalized additive model was made using PROC GAM
in SAS 9.2 (SAS institute) to explore changes in correlation with changing Ct values. Based on
the GAM a breakpoint was established and Ct values above the breakpoint were included in the
model using a piecewise regression model.
Results
The breakpoints for a correlation between Ct value and BTSCC were decided to be 32 for S.
agalactiae and S. dysgalactiae, 34.5 for S. uberis and 33.5 for S. aureus. The breakpoints for the
correlation between Ct value and the logtransformed TBC were decided to be 32 for S.
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agalactiae and S. dysgalactiae, 34.5 for S. uberis and 30.5 for S. aureus. The results of the
piecewise regression are presented as graphs in Figures 1 and 2.
Figure 1. Correlation between Ct value of the PathoproofTM PCR assay for different mastitis
pathogens and bulk tank somatic cell count in 4298 Danish Dairy herds.
Bulk tank somatic cell count (cells/ml)
Figure 2. Correlation between Ct value of the PathoproofTM PCR assay for different mastitis
pathogens and bulk tank total bacteria count in 4298 Danish Dairy herds.
Total bacteria count (CFU/ml)
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For S. agalactiae the correlation between a positive PCR reaction and herd size, milking system
and organic farming status was analyzed. The correlation of herd size and milking system are
shown in figure3 for herds with conventional milking and the each of the two most common
automatic milking systems. The prevalence of S.agalactiae increased with increasing herd size.
The prevalence of S. agalactiae was higher than conventionally milked herds for one AMS and
lower for the one AMS.
Figure 3. Correlation between positive PCR reaction for Str. agalactiae, herd size and milking
system.
Significantly fewer organic herds were positive for S. agalactiae compared to conventional herds
(2.6 % compared to 8.2%). Only one out of 41 organic herds with AMS had a positive reaction
for S. agalactiae (2.4 %).
Discussion
The analysis showed that low Ct values for specific major mastitis pathogens are correlated with
both higher BTSCC and TBC. The correlation to BTSCC was strongest for S. aureus and S.
uberis. The correlation for TBC was strongest for S. agalactiae and S. uberis. The results
indicate that surveillance of bulk tank milk Ct values for mastitis pathogens might give
information relevant to milk quality.
The prevalence of S. agalactiae varied with both herd size and milking system. The effect of
herd size is not surprising given the infectious nature of the bacteria. Large herds have more
animals to acquire an infection whether it is from buying animals or from other sources of
infection (humans, other animals). When the first cow is infected the bacteria can spread to other
cows. The big differences between different milking systems are expected to be related to the
milking process. In the AMS-type with the high prevalence of S. agalactiae the bacteria has been
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isolated from the cleaning aggregates and surfaces of the robot which come in contact with the
teats during the milking process.
The low prevalence of S. agalactiae in organic herds was surprising. Danish organic dairy herds
have the same size as conventional herds and the use of AMS is comparable to the conventional
herds. One possible explanation could be if organic herds buy fewer adult cows in to the herds
because of more restrictions on introducing conventional cows in the herd.
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