Development and testing of a set of chromogenic media for rapid diagnosis of bovine mastitis

INTRODUCTION

Bovine mastitis, an inflammatory condition of the mammary gland, ranks among the most widespread and economically impactful diseases in dairy production [1-3]. Transmission occurs due to multiple factors, including poor milking hygiene, suboptimal housing conditions, improper milking techniques, weakened animal immunity and inadequate preventive measures [4-6]. The disease presents in both clinical form – characterized by visible symptoms such as udder swelling, redness, and pain – and subclinical form, which lacks overt inflammation but results in reduced milk quality [7].

The etiology of mastitis comprises two primary causative groups: mechanical and infectious. Mechanical causes involve udder injuries resulting from inappropriate milking techniques, defective milking equipment or traumas during grazing. These injuries establish favorable conditions for microbial invasion, potentially leading to inflammatory development [8]. Nevertheless, pathogenic microorganisms constitute the principal factor in mastitis occurrence [9].

The most common pathogens of mastitis are Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and others [10, 11]. S. aureus is one of the most dangerous pathogens, as it can induce chronic forms of mastitis that are resistant to treatment [12-15]. S. agalactiae is transmitted mainly through milking equipment and can persist in cows for a long time [16]. E. coli often causes acute forms of mastitis, accompanied by severe symptoms [17].

To diagnose infectious bovine mastitis, veterinarians have multiple diagnostic tools at their disposal, each tool having its distinct advantages and limitations [18-24]. Among these, bacteriological milk testing remains one of the most widely used methods [25][26]. This procedure involves aseptic milk sample collection followed by inoculation onto culture media. After thermostat incubation, microorganisms are identified based on their morphological, biochemical and cultural properties. While this method enables precise pathogen identification and facilitates targeted treatment selection, it requires specialized equipment and has a relatively long turnaround time (2–3 days) [27]. Chromogenic media can be used to speed up the diagnosis of infectious mastitis. These specialized media contain substrates that undergo color changes when acted upon by pathogen-specific enzymes, allowing for etiological agent identification within 24 hours post-inoculation. Current rapid test options include Compact Dry (R-Biopharm AG, Germany) and RIDA® COUNT (Chisso Corporation, Japan) test plates [28, 29]. These test systems feature various specialized assays for determining S. aureus, Enterobacteriaceae, Salmonella, total microbial count, E. coli, as well as yeast and mold contamination.

The Laboratory for Diagnostics and Control of Antibiotic Resistance of Pathogens of the Most Clinically Significant Infectious Animal Diseases, Federal Scientific Centre VIEV has developed its own formulation of chromogenic media for differentiating mastitis pathogens without requiring lengthy laboratory studies. The set consists of three distinct chromogenic media that, when used together, enable identification of the mastitis pathogen spectrum in each specific case. This approach facilitates determination of the pathogenic spectrum, thereby influencing subsequent therapeutic decisions.

The study aims to evaluate the efficacy and diagnostic quality of these chromogenic media for bovine mastitis diagnosis.

MATERIALS AND METHODS

Chromogenic media. Three chromogenic nutrient media were prepared.

Medium I is intended for the determination and differentiation of the most frequently encountered microorganisms of the Enterobacteriaceae family.

Medium II is intended for the determination and differentiation of microorganisms of the genus Staphylococcus.

Medium III is intended for the determination and differentiation of microorganisms of the genus Streptococcus (in particular, S. agalactiae).

For ease of use, the three media were placed in one Petri dish with sectors.

The efficacy of chromogenic nutrient media was determined according to the following criteria: sensitivity, specificity, cultural properties of microorganism control strains, differentiating and inhibitory properties. A commercially available medium, Columbia blood agar (HiMedia Laboratories Pvt Ltd., India) supplemented with 5% defibrinated ram blood served as control.

Control strains. The control strains comprised 22 microbial cultures from the collection of pathogenic and vaccine strains maintained at the Federal Scientific Center VIEV, including: E. coli ATCC 25922, S. agalactiae ATCC 8057, S. aureus ATCC 12600, Klebsiella pneumoniae B-1392, Proteus mirabilis B-1382, Pseudomonas aeruginosa B-1366, Salmonella typhimurium B-1025, Enterococcus faecalis B-1399, Enterococcus faecium 1921, Acinetobacter baumannii 2516, Enterobacter cloacae 1322, Staphylococcus hominis 1377, Staphylococcus equorum 2511, Staphylococcus haemolyticus 2505, Staphylococcus pseudintermedius B-1849, Morganella morganii 1418, Streptococcus uberis 2114, Streptococcus dysgalactiae 2432, Streptococcus pyogenes 1972, Aerococcus viridans 2320, Streptococcus canis 2326, Streptococcus suis 2383.

Preparation of bacterial suspension dilutions. Initial bacterial suspensions were prepared at concentrations of 1×10⁸ to 1×10⁹ CFU/mL using the pharmacopoeial reference standard (PhRS 3.1.00085). To achieve the required seeding densities, serial ten-fold dilutions of the initial suspensions were performed.

Determination of sensitivity. S. agalactiae, S. aureus and E. coli strains were inoculated onto the studied chromogenic and control media in 1 mL at different concentrations: 1 × 10⁰, 1 × 10¹, 1 × 10² CFU/mL. After (24 ± 2) hours of incubation at 37 °C, the number of grown colonies in all the inoculations was compared. The experiment was performed in triplicate. To compare the mean values of the groups and determine statistically significant differences between them, the Student’s test (t-criterion) was used: the differences are considered statistically significant if p-value < 0.05.

Specificity assessment. Specificity was determined for each chromogenic medium separately. The growth and nature of changes in colonies of different bacterial strains on the same experimental medium were compared and the presence of similarities or differences was noted.

Evaluation of differentiating properties. To determine the differentiating properties, changes in control strains inoculated in chromogenic and control media (structure, color of colonies, color of the medium around the colonies) were compared.

Evaluation of inhibitory properties. Inhibitory properties were determined by the presence or absence of growth of cultures on chromogenic media in comparison with the presence of growth on a control medium.

Testing of media with mastitis milk samples. Eight milk samples (10 mL each) were collected from cows with mastitis confirmed by the Kenotest somatic cell test (CID Lines, Belgium). Samples were collected in sterile biological specimen containers and processed within 2 hours of collection, with storage maintained at +4 to +8 °C. For inoculation, a sterile cotton swab was immersed in each milk sample, excess moisture was removed by touching the container walls, and then streaked in a lawn pattern onto three chromogenic media. The cultures were incubated at 37 °C for 24 hours before result interpretation.

RESULTS AND DISCUSSION

To determine the sensitivity of chromogenic media, three target strains (S. agalactiae, S. aureus, and E. coli) inoculated in 1 mL at three different concentrations (1 × 10⁰, 1 × 10¹, 1 × 10² CFU/mL) were used. After culturing for 24 hours at 37 °C, the number of colony-forming units for all media was counted. The results are presented in Table 1.

To identify statistically significant differences or similarities, the Student’s t-test was used, the results of which are presented in Table 2.

Based on the obtained data, chromogenic media demonstrate sensitivity comparable to the control medium, as confirmed by statistical analysis (Student’s t-test, p > 0.05). The observed differences between chromogenic media and the control medium showed no statistical significance across all tested strains and concentrations. Thus, these results indicate that chromogenic media effectively support growth of target microorganisms even at minimal inoculum levels.

The specificity, differentiating properties and inhibitory characteristics of the chromogenic media were evaluated concurrently using 22 microbial strains representing diverse species. The results are presented in Table 3.

Medium I was found to be highly specific: most of the tested bacteria formed colonies with unique colors. For example, E. coli formed burgundy colonies, S. aureus – golden, P. aeruginosa – gray-green, and S. equorum – violet-pink. However, some microorganisms, such as E. cloacae and K. pneumoniae, had similar colony colors (violet-blue), which may make it difficult to distinguish them visually. Inhibitory properties were weak: all studied strains of microorganisms demonstrated growth within 24 hours. Nevertheless, medium I ensured effective differentiation of control strains by colony color, which allows visually distinguishing microorganisms already at early stages.

The inhibitory properties of Medium II are pronounced: the growth of most bacteria was absent, with the exception of the target microorganisms – Staphylococcus spp. It is worth noting that the specificity of the medium is low – most staphylococci were stained blue-green. However, the same color was predominantly saprophytic microorganisms, while potentially pathogenic staphylococci (S. aureus and S. haemolyticus) differed in color. For example, S. aureus formed purple colonies, and S. hominis and S. equorum formed blue-green ones, which made it possible to visually distinguish them. Medium II as compared with the control one, provided a differentiation of staphylococci by color.

Medium III demonstrated good inhibitory properties, effectively suppressing the growth of most microorganisms, with the exception of gram-positive cocci and some representatives of the Enterobacteriaceae family. The differentiating and specific properties of the medium were weakly expressed and manifested mainly for enterococci, which were stained blue-green, and for S. agalactiae, which formed blue colonies.

The use of all three chromogenic media in combination provides a comprehensive approach to mastitis diagnosis, demonstrating high sensitivity, specificity and differentiating properties. This method allows for a wide range of microorganisms to be covered, selectively isolating target bacterial groups such as staphylococci, streptococci and enterococci.

For testing in the field, milk samples were collected and then inoculated onto three chromogenic media. The results are shown in the Figure.

The simultaneous use of three media for milk sample inoculation enables nearly species-level differentiation of cultures. In Figure (a) it is evident that Enterococcus sp. grew on Media I and III (supposedly E. faecalis, as E. faecium typically exhibits a darker green coloration). Single colonies on Medium II consist of Staphylococcus sp., while the presence of S. aureus can be ruled out, as it would appear purple on Medium II. Additionally, burgundy colonies on Medium I indicate the presence of E. coli in the sample. In Figure (b), the sample microbiome consists almost exclusively of Enterococcus sp. White and green colonies on Media I and II, respectively, are formed by Staphylococcus sp. microorganisms, excluding S. aureus. Figure (c) reveals a monoculture of Enterococcus sp., most likely E. faecium. The fourth sample shown in Figure (d), contained only filamentous fungi. Another milk sample yielded results similar to (b), while three other cultures showed no growth.

CONCLUSION

The developed chromogenic media demonstrate high efficiency in bovine mastitis diagnosis. Medium I, with its high sensitivity and differentiating properties, enables primary screening and detection of a broad spectrum of microorganisms, including members of the Enterobacteriaceae family. Medium II, due to its strong inhibitory properties, selectively isolates staphylococci, which is a critical feature for identifying pathogenic species such as S. aureus. Medium III, while having more limited differentiating capabilities, effectively supports the growth of enterococci and streptococci, including S. agalactiae, making it essential for mastitis diagnosis.

The integrated use of all three media ensures high diagnostic accuracy, enabling not only broad microbial coverage but also selective identification of target bacterial groups. This significantly accelerates pathogen detection and facilitates timely administration of effective therapy. Testing of the media on milk samples from mastitic cows confirmed the media’s practical applicability and effectiveness when used in the field.

During the testing, occasional development of filamentous fungi was observed, which may complicate result interpretation. To address this, further work will focus on optimizing the media composition by evaluating various antifungal preparations at different concentrations. These improvements aim to enhance media specificity by suppressing non-target fungal growth, thereby reducing the risk of false-positive results.

It is worth noting that standardized disposable loops for milk culture make it possible to roughly estimate the number of colony-forming units. While this method lacks high precision, it provides a practical approximation of milk contamination levels, offering valuable preliminary insights into infection severity.

Thus, the developed chromogenic media represent a promising tool for rapid mastitis diagnosis, combining high sensitivity, specificity and differentiating capabilities. Their implementation in veterinary practice could significantly accelerate diagnostic procedures and enhance mastitis treatment efficacy, ultimately improving animal health and dairy herd productivity.