All mammals are susceptible to mastitis (the inflammation of the mammary tissue due to a bacterial or viral

infection). Subclinical mastitis does not display mammary tissue swelling or milk abnormalities; therefore,
it often proceeds undetected. Mastitis infections have degenerative properties, which prohibit the mammal
from producing the quantity and quality of milk that it produced prior to the infection. Therefore, Mastitis is
declared the most economically detrimental infection in the dairy industry (Ruegg 2002). This projects
objective was to determine the most effective and accurate method for diagnosing subclinical mastitis.
Currently there are multiple methods for early detection of mastitis or milk qualities typically associated
with mastitis (conductivity, California Mastitis Test, and temperature). The aforementioned parameters as
well as (turbidity, phosphates, pH, microorganism detection through incubation with methyl blue, and milk
component break downs) were measured from samples of bovine lactation to determine their accuracy of
detecting mastitis as diagnosed by the owners. Furthermore, a control sample was created in which E. Coli
was added to milk and cultured while white blood cells were added to simulate a mastitis infection. Each
parameter was measured throughout the culture to create a curve to help determine the stage in which the
infected animal lies. Specific conductance, pH, and turbidity correlated with mastitis detection in the
control creating a parabola for each test. The accurate and early detection of mastitis will minimize the
scarring of the mammary tissue resulting in higher lifetime milk yield averages and more efficient dairy
farming.

DETECTION OF SUBCLINICAL
MASTITIS IN DAIRY CATTLE
To determine the most cost and time effective method of diagnosing mastitis in dairy cattle.

Obtain permission from the dairy farm(s) to sample their cows directly and
contact a veterinarian about syringes, needles, and Heparin and EDTA tubes.
Collect farm samples and measure parameters such as teat temperature and
milk temperature directly after sampling.
Place samples on ice and test the remaining parameters at a later time in a more
sanitary environment. (milk conductivity, turbidity, pH, phosphates, CMT,
microorganisms, and milk composition).
Create control sample by collecting whole blood samples, separating and
collecting the white blood cells and adding the WBC to milk samples at 37C,
which contain Escherichia coli.

Figure 16: Transporting Blood Samples into anticoagulant filled

tubes for control samples.

Analyze data using t-tests and r^2 values. Create trend lines to predict mastitis
infection.

Figure13: Thermal image of a cows udder to display the greatest

heat loss and where the samples were measured from (Vegricht et.
al. 2007).

Specific Conductivity correlated best with the diagnosis of mastitis.

pH and Turbidity also displayed a correlation with mastitis.
Phosphates displayed the weakest correlation with mastitis diagnosis.
Cows that are going dry have a significantly higher milk temperature.
Cows suspected to have mastitis had significantly lower teat temperatures.
The pH of milk from a cow suspected to have mastitis or a cow that is going
dry was significantly higher than clean cows.
Cows suspected to have mastitis had significantly lower turbidity values.
Cows that are going dry or are suspected to have mastitis have significantly
lower phosphate values.
The correct diagnosis of mastitis in a dairy cow through the CMT was
insignificant.
The component of milk (Ca3(PO4)2) is significantly reduced in cows with
mastitis.
No methyl-blue micro organism test turned entirely white.

Figure 15: Hlubiks Dairy Farm, where all samples for

the purpose of this project were collected-free stall
milking barn.

Mastitis is a common disease in the dairy industry, and occurs in two forms.
Clinical mastitis is the easier form to diagnose because there are visible signs of
mastitis in the milk and throughout the udder. Subclinical mastitis, however, has
no visible signs; such as, inflammation of the mammary glands detectable by the
human hand nor milk abnormalities (Questions about Milk 2013). Subclinical
mastitis is also the most economically important form of mastitis because it is
often reoccurring, undetected, reduces overall milk yields, and it is highly
contagious (Ruegg 2002). Persistent and chronic subclinical mastitis infections
permanently damage the milk secreting cells of the cow, reducing the overall milk
yield of the cow (Ruegg 2002). As of 2009, mastitis cost the dairy industry 1.7-2
billion dollars annually (11 percent of the total U.S. milk production), and the
average dairy farm 171 dollars per cow annually (Jones 2009). Mastitis, or the
inflammation of mammary cells, is caused by the release of leukocytes due to the
invasion of bacteria into the teat canal (Jones 2009). Leukocytes are also referred
to as somatic cells or white blood cells. High concentrations of leukocytes result
in reduced milk production and altered milk composition (Jones 2009). High
levels of somatic cells are unfavorable to distributing companies because they
often reduce the grade of the product. Farmers that supply milk with high somatic
cell counts are often fined furthering the deteriorating economic effect of mastitis.
There are multiple parameters that can be used independently to test for common
abnormalities associated with mastitis. The best current method for cow-side
testing is the California Mastitis Test, which is a qualitative measurement of
somatic cells. The test requires a strip sample of milk and an equivalent amount of
solution, which disturbs the cell membrane of any cells present in the milk
sample, allowing the DNA in the sample to react with the test reagent forming a
gel (White et al. 2005). The reaction is complete within 15 seconds and is a
slightly darker purple area that can quickly disappear. The CMT also has a wide
range for somatic cell count and is qualitative data (see Table 1) (Ruegg 2002).
Other methods of testing for somatic cells are geared towards the bulk tank and
the quality of the entirety of the milk (The Value and Use). However, sampling
from the bulk tank does not pinpoint which cows are infected; thus, disease can
spread rampantly through a farm without alerting the farmer until the damage is
irreversible. Therefore, the best way to prevent mastitis and increase milk quality
is early individual detection.

Figure 1: Change in conductivity as the E. Colis population within the

control samples increased.

Figure 5: The milk turbidity of samples collected from the farm compared
to the farmers diagnosis of mastitis within the cow.

Figure 2: Milk samples measured for mastitis with 1.5 being positive,
0.0 being negative, and values between representing trace amounts.

Figure 6: The turbidity of the milk samples as the population of the E.

coli within the samples increased.

Figure 9: Milk temperature directly from cow. Range shown displays the Figure 10: Temperature of the middle segment of the teat compared to
average body temperature of a dairy cow.
the range shown of a healthy cow body temperature.

Table 1: Typical scoring of the California Mastitis Test that was not
used for this experiment (Ruegg 2002).

Figure14: Diagram of a quarter of the

udder and the teat (Mammary System
2015).

Figure 18: 10mL blood samples in heparin blood thinner

that were centrifuged at 1500-2000rpm*g to separate out
the plasma, white blood cells, and red blood cells.
Figure 17: Top image: progressive amounts of microorganisms. Bottom image:
first sample is a positive trace, middle is negative, right is positive.

Figure 3: pH of the milk control samples as the population of the E. coli

within the samples increased.

Figure 4: pH of the milk samples relative to the farmers diagnosis of

mastitis within the cow.

Figure 7: Change in phosphate levels in parts per million relative to the Figure 8: Phosphate levels within the milk controls compared to
farmers diagnosis of mastitis within the cow.
length of E. coli incubation.

Figure 11: Calcium Phosphate percentages of milk samples from clean

cows and cows with mastitis.

Figure 12: Qualitative levels of microorganisms in the control samples

based on a scale of 3.

Figure 19: A culture after data was collected from the control samples to determine whether or not E. Coli was growing within the
milk, there is a second colony of another bacteria displayed in the third dish.

There were two phases of this project, creating

control trend lines from E. coli laden samples and
sampling lactating bovine of which 2/9 of the
samples were from cows suspected to have mastitis
and 1/9 were going dry and thus have higher
somatic cells and signs of mastitis. Specific
Conductance of the control sample most closely
correlated to the progression of E. Coli growth and
thus the simulated mastitis. Control sample 350 A
most closely correlated to a parabolic graph with an
R^2 value of 0.9991. The parabola formed from the
graph of all three control samples is positive;

however, two of the three samples form a trend line with a negative parabola
and the points associated with those trend lines display a higher R^2 value.
Therefore, the conductivity of the milk samples will eventually plateau rather
indefinitely increase. The pH of the control did not reflect the data collected
for mastitic cows and clean cows, which is likely due to the fermentation
process occurring with the lactic acid in the milk. The milk actually becomes
more basic as mastitis develops. It becomes more basic because the
inflammation causes reduced secretion of milk and higher permeability of the
mammary epithelial cells, which can lead to transfer of components of the
blood into the milk such as citrates, bicarbonates, and Na and Cl ions (Ogola
et. al 2007). Therefore, the trend line developed from the sampled data and
not the control is more accurate. Unlike the hypothesis predicted, the milk
becomes less turbid as mastitis progresses, this is because the milk curdles
within the udder and is released in chunks of the protein casein and milk fat
surrounded by a liquid composed primarily of water, calcium, vitamins, and
sugars such as lactate and glucose. Therefore, the curdles within the sample
quickly settle to the bottom removing their original turbidity from the sensor
within the colorimeter. The cow within the samples that was going dry had
really high turbidity readings because the udder is cleansing itself and
reducing the quantity; thus not releasing as much water into the milk. The
milk temperature of the cow steadily increased with likelihood of mastitis
because the milk temperature accurately represents internal body
temperature. The temperature of the milk of the cow that was going dry was
significantly higher than all other data points because it is a natural process
as the cow goes dry, she cleanses the teats less often; therefore, flushing
the bacteria within her less frequently. So to prevent infection the udder is
flooded with somatic cells and the temperature is increased. The teat
temperature of the cows were measured in the center of the teat; however
for the first 23 data points, a thermocouple was used to collect the
temperature which required the wire to be pressed against the cow causing
it discomfort and thus readings that do not reflect the readings that were
later recorded with a laser temperature reading gun. Phosphates decreased
as the likelihood of mastitis increased; however, the control samples all
displayed a drop in phosphates then an increase all below the average. This
could be attributed to the fact that the phosphates covalently bind to the
protein casein, which then forms ionic bonds to calcium ions forming a
micelle, the casein and triglycerides form the curdles observed; therefore, it
is possible that some of the phosphates were captured in the precipitate. As
the samples became more separated and curdled they were stirred and the
curdles were broken possibly re-releasing the trapped phosphates. Finally,
the most accepted method of diagnosing mastitis, the California Mastitis
Test, produced no significant correct data; thus, supporting my hypothesis
that the California Mastitis Test is not the best cow-side determinant of
mastitis. The methyl-blue test produced no samples that completely turned
white, which is absurd considering the concentration of E. coli within the
control samples. However, it is likely that the E. Coli was primarily
surrounding the fat and protein curdles, which were white. Finally, finding the
percentage of the main components of the milk was removed from this study
because of the time necessary to separate each component that would be
inefficient in a cow-side machine. However, there was a significant difference
between clean milk and milk from a cow with mastitis in the component of
Ca3(PO4)2.

The best single method found in these experiments to diagnose mastitis was pH
according to its correlation with the simulated mastitis for the control samples,
and its correlation with suspected mastitis through samples collected. However,
there is error associated with the pH values and the best way to get around this
error is to add additional tests that correlate well with the progression of mastitis.
Therefore, the tests that would produce the most accurate diagnoses in the most
feasible manner are (in order) pH, turbidity, conductivity, teat temperature,
phosphates, mass of calcium within the milk (milk composition), CMT, and milk
temperature. Since 2 cows suffer through mastitis on average in every dairy farm
of 60 head, it would be beneficial for the farmer to monitor every one of his cattle
every milking to observe trends towards mastitis and detect, prevent, and treat
early. A farmer of 50 head can expect to make more than $13226.45 per month in
optimum conditions; however, mastitis will cost him $539.03 a month. A single
purchase of equipment to test for the four most accurate and cheapest tests can
insure mastitis infections to diminish possibly saving the farmer $539.03 per
month or $6,468.36 a year of which he would only need to spend between
$199.68 and $542.94 (depending on the quality of the equipment) and the cost of
repairs. This is only for a small farm of 50 head of cows. This technique will help
small farmers hold together in the ever changing market and help to prevent
monopolies.

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