Mastitis Test

In this paper comparison of efficiency of commercially available antibiotic susceptibility test MASTiK^® with traditionally used Kirby-Bauer disk diffusion method in cow mastitis cases was described. Six samples of examined milk were chosen on a randomly basis. In the samples presence of following microorganisms was determined: Staphylococcus aureus, Escherichia coli, coagulase-negative Staphylococcus sp. and Streptococcus uberis. Based on the result of the trial it was determined that MASTiK^® test is completely comparable to the results obtained by classical disk diffusion method and can be of great help on a field in fast selection of antibiotics, while results of bacteriological identifications are being awaited.

Key words: mastitis, antibiotics susceptibility, MASTiK^®, Kirby-Bauer disc method.

Mastitis in the World as well as in our Country represents one of economically most important diseases of dairy cows. Losses that occur in affected cows raises not only from lowered milk production but and from discarded milk due to withdrawal period, loss of stimulation, cost of treatment and negative impact of mastitis on reproduction.

Early diagnosis is of great importance since cost of mastitis and losses are higher with delayed treatment. European Union regulation (Regulation EC No 1662/2006) (3) states that milk used for human consumption must originate from healthy animals.

Classical methods of mastitis detection include: determination of somatic cell count (SCC), concentration of inflammation markers (i.e. enzymes N-acetil-β-D-glucosaminidase and lactate dehidrogenase), electric conductance measuring and finally isolation of causative agent and antibiograme. Isolation and determination of causative agent is still a gold standard, but partly due to geographical distances between farms and laboratories, mastitis cases are often not sent to laboratories, and most of mastitis cases are treated according to experience. That all can lead to low therapy success and increased risk to human health due to uncontrolled use of antibiotics.

Aim of our trial was to compare efficiency of  MASTiK^® test, commercial fast test for in vitro determination of bacterial susceptibility to antibiotics (MASTiK^® ImmuCell Corp., Portland, ME, USA) and classically used Kirby-Bauer disk diffusion test.

MASTiK^® is fast test for determination of antibioticsusceptibility of mastitis causing organisms (bacteria) that have the ability to ferment lactose. Most of common mastitis causes has the ability to ferment lactose, some of them are: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus disgalactiae, Streptococcus uberis, Escherichia coli, Klebsiella and Arcanobacterium pyogenes. Bacteria that can cause mastitis in cows, that pose ability to ferment lactose but are seldom met are: Streptococcus faecalis, Streptococcus zooepidermicus, Streptococcus of G group, Coagulase negative Staphylococcus, Enterobacter and Citrobacter. Mastitis causing organisms that do not have ability to ferment lactosis and can not be cured with antibiotics regularly used in mastitis treatment or antibiotics in general are: Mycoplasma, Prothoteca and Pseudomonas (5).

Investigation was conducted on 6 milk samples, which were sent to laboratory in order to conduct bacteriological search and antibiograme. Five out of six samples were of changed consistency and colour.

All samples of milk were by standard microbiology technique introduced to blood MacConkey and Sabouraud dextrose agar and incubated at 37ºC. Antibiotic sensitivity survey of isolates was investigated by disk diffusion method. Simultaneously to inoculation to agars MASTiK^® test was performed according to manufactures specification: by 1 ml of milk sample vas inoculated in reagent bottle. Contest was homogenised and incubated on 37ºC in a period of two hours. After incubation contest was stirred with sterile pipette, by two s of mixture were inoculated into each basin of 16 basins on a test plate. Plates were covered with adhesive foil and incubated on 37ºC in a period of 18 hours. Afterwards reading of MASTiK^® test result was done and susceptibility to antibiotics was estimated according to colour change (Purple colour - no growth of bacteria, causative agent is sensitive on antibiotic; Yellow colour - bacteria are growing, causative agent is not sensitive on investigated antibiotic), obtained result were recorder on a MASTiK^® test card.

With standard bacteriological techniques from examined samples following microorganisms were isolated:

Sample No. 1. Staphylococcus aureus.

Sample No. 2. Escherichia coli.

Sample No. 3. Streptococcus dysgalactiae in vast number.

Sample No. 4. Coagulase negative Staphylococcus spp.

Sample No. 5  Streptococcus uberis.

Sample No. 6. Mouldiness from Aspergillum genus.

Results of investigation of susceptibility of bacteria on antibiotics with standard disk diffusion test and MASTiK^® test are shown in tables 1 and 2.

Table 1. Results of antibiotic susceptibility survey - Kirby Bauer disc test

Although great effort is done in mastitis prevention and education of farmers and veterinarians, mastitis treatment often does not have appropriate success since it is done without previous isolation of microorganisms', without sensitivity testing (2). Geographical distances between farms and labs as well as habits of the farmers have greatly influenced this condition. Those were the basic motives to investigate possibility of use of fast tests in selection of antibiotics in mastitis treatment and to do comparison of MASTiK^® test usability compared Kirby-Bauer disk diffusion test.

When in milk samples causative agent is present as solely one species MASTiK^® test has shown to be useful and reliable. That highlights the need that milk samples should be collected under maximally aseptic conditions which is often not conducted in such manner on a filed and these samples often arrive in laboratory with certain degree of contamination. Proper sampling will determine the success of mastitis treatment.

MASTiK^® test has reveals antibiotic susceptibility of following bacteria causative agents of mastitis: Staphylococcus aureus, Escherichia coli, coagulase negative Staphylococcus sp. and Streptococcus uberis and is completely comparable with the results of investigation of isolates sensitivity by standard disc diffusion test for same antibiotics. This finding is in compliance with the findings of other authors that have dealt with same problems (4, 5).

MASTiK^® test inoculated with sample No. 3 was not readable after incubation of 18 hours, due to vast number of bacteria in the sample (isolated Streptococcus dysgalactiae) which highlights the necessity to check the MASTiK^® panels every 4 hour (according to user manual). This sample of milk (No. 3) has changed colour after period of pre incubation (2 hours) and that was also due to vast bacteria and has shown that the statement made by manufacturer that samples of milk with high number of bacteria give result much faster. We did not have anamnesis data's and therefore could not skip this step of pre incubation (according to user manual). Samples were chosen on a random basis and therefore in sample No. 6 mould from genus Aspergillum was isolated. Result of MASTiK^® test has ruled out necessity of antibiotic use in this case of mastitis and can therefore provide more rational use of antibiotics.

Taking in concern simplicity of conductance we have found MASTiK^® test to be useful for field practice as help in choice of antibiotics while results of bacteriological investigation are being awaited, therefore it is desirable to do simultaneous classical microbiological investigation with MASTiK^® test due to:

-   Identification of bacterial cause of mastitis;

-   Identification of causative agents such as: Pseudomonas aeruginosa, yeast, achorofile algae (Prototheca zopfii) and moulds;

-   Possible contamination of samples (which is very often) which can be ruled out by classical microbiological techniques;

The fact that mastitis in dairy cows causes economic losses is well known but its influence goes even further from solely influencing milk production. Clinical mastitis can have significantly negative influence on reproduction since it is estimated that cows which get mastitis in first 45 days after calving have significantly lower conception rate  (1). Having in mind all mentioned, need for formers to pay even more attention in programmes of prevention and control and veterinarians in treatment of mastitis since proper and quick treatment can have positive impact on mastitis therapy success, reproduction therefore on profit and on health of human population.

1.   Obtained results have confirmed that MASTiK^® test is applicative in fast determination of bacterial sensitivity on selected antibiotics.

2.   Considering simplicity in conduction and reliability, test is useful for filed application in selection of antibiotics while results of bacteriological identification are being conducted.

3.  Although MASTiK^® test is not designed for hepl in choosing of the treatment in cases of mastitis caused by moulds, based on the results of this trial, it has shown that antibiotics should not be used in treatment. This data can be of great health and since it has shown that this case is not caused by bacteria and therefore use of antibiotics in not desirable, instead of that it is advisable to do classical microbiology identification technique. This approach can lower irrational use of antimicrobial drugs and lower risk of antibiotic resistance for human population.

1.   Ahmadzadeh, A., Frago, F., Shafii, B., Dalton, J.C., Price, W.J., McGuire, M.A., Effect of clinical mastitis and other diseases on reproductive performance of Holstein cows, Animal Reproduction Science 2009,112, 273-282.

2.   Bradley, A.J., Bovine mastitis: an evolving disease. Vet J 2002, 164, 116-128.

3.   Commission Regulation (EC) No 1662/2006, Health requirements for raw milk and colostrum production, Official Journal of the European Union L 320/4

4.    Hoblet, K.H., Queen, G.W., Howard, R.R., Antibiotic Susceptibility Test for Mastitis: A Comparison of a Commercial Test Kit with Kirby-Bauer, Ag Prac 1993,14, 7, 45-51.

5.   Saperstein, G., Clinical applications of antibiotic susceptibility test for mastitis, Ag Prac 1993, 14, 4, 25-28.