AHEAD IN ANIMAL HEALTH
Validation of an Escherichia coli mastitis model A.G.J. Velthuis, E. van Engelen and Y.H. Schukken Royal GD, Deventer, The Netherlands
Background Escherichia coli (E. coli) infection is one of the most frequent causes of severe clinical mastitis in dairy cows. E. coli mastitis causes acute inflammation and leads to severe loss of animal welfare. Moreover, adequate treatments options are limited. In search for better treatments, for instance with antibiotics, and to acquire more knowledge on this type of mastitis a reproducible animal model is essential. GD animal health validated an intra-mammary challenge model for E. coli mastitis in lactating cattle. Aim The aim was to achieve a robust and reproducible E. coli mastitis model using a standardized inoculum and a good monitoring of clinical and laboratory parameters.
Materials and Methods The E.coli mastitis model was generally based on the model described by Sipka et al. (2013, 2014). Four dairy cows were included, all healthy, with low SCC and negative
bacteriological results in milk samples from each quarter. Prior to inoculation, a well-characterized low virulent E. coli strain was grown in-vitro. From each cow, two quarters were inoculated with 100 cfu E.coli: two cows were inoculated in the hind quarters and two in the front quarters. After inoculation, clinical and laboratory parameters were measured every 3 h for d 0 and 1 post inoculation, every 6 h for d 2 and 3 and every 12 h for the d 4, 5 and 6. Cow level parameters include general health and rectal temperature.
Quarter level parameters were quarter appearance, appearance of milk, numbers of live E. coli per ml of milk, and SCC. In addition, quarter milk production was measured using a sensor in an a milking unit set up for challenge experiments. The quality of this study was guaranteed by a GCP system and ISO 9001 certification. Milk samples were analyzed in an ISO 17025 accredited laboratory. Data collection, processing and analysis was done by trained and competent technicians.
Main results
CFU/ml (log)
6 4 2 0 0
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Fig 1. Number of E. coli after inoculation
LogSCC/ml
8 6 4 2 0 0
1
Rectal temp. °C
Fig 2. Somatic cell count after inoculation
42 40 38 36 0
1
Conclusions It can be concluded that we were able to induce a robust and reproducible E. coli infection. This E.coli mastitis model can, therefore, be used for determination of efficacy of treatments that aim to reduce E.coli mastitis signs and shedding of the bacterium.
Fig 3. Rectal temperature after inoculation
Kg milk per quarter
We were able to inoculate all eight quarters in four dairy cows with a doses of E. coli close to 100 cfu. After inoculation, numbers of E. coli far higher than 100 cfu could be isolated from all inoculated quarters for at least 2 to 3,5 days post inoculation (see figure). All inoculated quarters showed clinical signs of mastitis and the milk had a typical mastitis appearance with flocks, watery and yellowish. All four cows showed some signs of general illness and a typical short high peak in body temperature. Somatic cells could only be estimated in milk samples with a normal appearance: a sharp increase in numbers of somatic cells was observed. Milk production dropped in the first day in inoculated quarters but also in the control quarters, which was restored afterwards only in the control quarters. The onset of the various clinical signs started uniform in the inoculated quarters and across cows. Hence, the induction and progression of the E. coli mastitis was similar and reproducible in all cows. Moreover, measures of symptoms and secretion of E.coli were homogeneous in all cows and inoculated quarters.
1,2 0,8 0,4
Control quarters Inoculated quarters
0,0 0
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Fig 4. Average daily quarter milk production relative to T=0
References Sipka A, Klaessig S, Duhamel GE, Swinkels J, Rainard P, Schukken Y. Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis. PLoS One. 2014 Jan 14;9(1):e85579. Sipka A, Gurjar A, Klaessig S, Duhamel GE, Skidmore A, Swinkels J, Cox P, Schukken Y. Prednisolone and cefapirin act synergistically in resolving experimental Escherichia coli mastitis. J Dairy Sci. 2013 Jul;96(7):4406-18.
a.velthuis@gdanimalhealth.com www.gdanimalheath.com
GD2146/01-20