The aim of this study was to determine the occurrence of pathogens in selected group of ewes and the relationship between somatic cell count (SCC) and the presence of pathogens. The experiment was carried out on a dairy farm, where predominantly breed was a Tsigai. Sampling was carried out in monthly intervals as part of the milk recording test day from February to July 2019. A total of 303 ewes were included in the survey, during the milk recording test day. The ewes with SCC ≥1000 × 103 cells.mL-1 were selected for further sampling at half udder level. Based on SCC the ewes were divided into five groups: <200 ×103; ≥200 <400 × 103; ≥400 <600 × 103; ≥600 >1000 × 103; ≥1000 × 103 cells.mL-1. The first group of SCC contained 33.9% of milk samples, the second 14.1% of samples, the third 5.7% of samples, the fourth 6.2% and the fifth 40.1% of samples. The most common pathogens were coagulase negative staphylococci (CNS). The most frequent CNS was
Mastitis is big healthy, economic and welfare problem in dairy animals. The main cause of increase SCC in milk of ewes is intramammary infection (
The hypothesis of this article is that high SCC in milk is caused by presence of mastitis pathogens. The aim of this study was the evaluation of occurrence of pathogens in milk of ewes and the possible relation of pathogens with SCC.
The experiment was carried out on farm with Tsigai ewes as dominantly kept breed, together with Lacaune and Improved Valachian (303 dairy ewes in farm). The ewes were on pasture during the day and received concentrates in amounts of 200 g per day during milking.
The milk sampling was performed once a month during morning milking as a part of regular milk recording test day from February to July 2019 (February, March, May, June, July). SCC was determined using a Somacount 150 (Bentley Czech, USA). The ewes with SCC ≥1000 × 103 cells.mL-1 at any time during the regular sampling period were selected for further sampling at half udder level three days later. All these ewes were sampled again always on third day after further regular recording test days during whole lactation period even if they had low SCC at regular sampling. The milk samples were collected at half udder level and analysed on SCC and presence of pathogens. Thus 95 ewes (407 milk samples) without symptoms of clinical mastitis were selected into study.
For the bacteriological cultivation and the presence of pathogens the milk samples were colltableected by discarding first squirts of milk and subsequently cleaning of the teat end with 70% alcohol and approximately 5 mL of milk from each udder halves was taken in sterile tube. The inoculum of each sample of milk was inoculated onto blood agar (Oxoid LTD, Hamshire, UK). All plates were incubated aerobically at 37 °C and evaluated after 24 hours. The all plates were re-evaluated after another 24 hours incubation. Colonies were identified on basis of cells morphology, Gram staining, type of hemolysis, the activities of catalase (3% H2O2, Merck, Darmstadt, Germany) esculin hydrolysis and cytochrome oxidase C (Bactident Oxidase, Merck). Presumptive
The identification to species level by applying MALDITOF MS (Bruker Daltonics, Bremen, Germany). Briefly, fresh colony material was spotted by direct transfer method on to MALDI-TOF MS target plate, allowed to dry at room temperature and overlaid with 1 μL of matrix solution (saturated solution α-cyano-4-hydrosy-cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) and allowed to dry at room temperature. Before the matrix solution was added 1 μL of 70% formic acid to the bacterial spot and allowed to dry for direct transfer-formic acid method. A loopful of bacterial colony was suspended in 300 μL distilled water and 900 μL ethanol was added for the protein extraction. The supernatant was discarded after centrifugation of cell suspension at 17,000 × g for 2 min. The centrifugation was repeated and the remaining ethanol was discarded. After dried the pellet was resuspended in 5 to 50 μL formic acid-water (70:30) in depending on the size of pellet and an equal volume of acetonitrile was added finally. 1 μL of the supernatant was transferred to the MALDI-TOF MS target plate after centrifugation at 17,000 × g for 2 min and allowed to dry before applying 1 μL of matrix solution. The MALDI Biotyper software version 2.0 (Bruker Daltonics) was used for bacterial identification.
Samples on the basis of SCC at half udder level were divided into following five SCC groups for evalutation of the distribution of milk samples: first <200 × 103 cells.mL-1; second ≥200 <400 × 103 cells.mL-1; third ≥400 <600 × 103 cells.mL-1; fourth ≥600 <1000 × 103 cells.mL-1; fifth ≥1000 × 103 cells.mL-1 (Using Excel, Microsoft, USA). The distribution of samples according SCC group was done by Microsoft Excel. Somatic cell score was used for statistical evaluation (SCS) and SCS was calculated according formula:
For statistical evaluation the data were divided according month of sampling where five groups of samples were created: Feburary, March, May, Juni and July. The samples also were divided into 9 pathogens group differed by presence of pathogens (1st group – major (
y – was the measurements for somatic cell counts β – the fixed effects of months, pathogens e – random error, assuming e ~ N (0, I δ2e) X, Z – incidence matrices for fixed effects and random cow effect, resp.
When evaluating the entire observation period tested ewes on udder half level the first group of SCC contained 33.9% of samples, the second 14.1% of samples, the third 5.7% of samples, the fourth 6.2% and the fifth 40.1% of samples (Figure
Frequency of distribution of milk samples in SCC groups.
Total 407 examined milk samples tested on presence of mastitis pathogens were as negative classified 63.1% of milk samples, out of these samples 75.9% had SCC below 500 × 103 cells.mL-1. 36.1% of samples were classified as bacteriological positive and 0.7% of milk samples were classified as contaminated. Only 7.5% of bacteriological positive samples had SCC below 500 × 103 cells.mL-1. Two pathogens were identified in 2.7% of bacteriological positive samples. In 67.9% of ewes with bacteriological positive samples there were repeated detected the presence of pathogens during tested period. Thus pathogens could persist in udder throughout whole lactation. Also up to 21.1% from these ewes had infected both udder halves pathogens repeatedly.
Important group of samples are those in fifth group – with very high SCC (Figure
From major pathogens only
The occurrence of pathogens in months of observation.
Pathogen | February | March | May | Jun | July |
---|---|---|---|---|---|
|
|||||
2 | 1 | 3 | - | - | |
- | - | - | - | 1 | |
1 | 2 | 4 | 1 | - | |
1 | 2 | - | - | - | |
2 | 3 | 2 | 1 | 20 | |
- | - | - | - | 1 | |
3 | 7 | 3 | 3 | 5 | |
- | - | 1 | - | - | |
1 | - | - | - | - | |
5 | 4 | 1 | - | - | |
- | - | - | - | 1 | |
- | - | - | 4 | - | |
1 | - | - | - | - | |
- | - | 8 | 21 | - | |
7 | 6 | 15 | 3 | 2 | |
- | - | 1 | - | - | |
1 | 1 | - | - | - |
CNS were the most common group of pathogens in milk followed by increased SCC in milk.
This work was supported by the Ministry of Education Science Research and Sports of the Slovak Republic/the Slovak Research and Development Agency (Projects No. APVV-15-0072) and KEGA 039SPU-4/2019 “Modernization of practical education of hygiene and prevention in animal production”.