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Abstract

Bovine mastitis is an economic burden for dairy farmers and preventive control measures are crucial for the sustainability of any dairy business. The identification of etiological agents is necessary in controlling the disease, reducing risk of chronic infections and targeting antimicrobial therapy. The suitability of a detection method for routine diagnosis depends on several factors, including specificity, sensitivity, cost, time in producing results, and suitability for large-scale sampling of milk. This article focuses on current methodologies for identification of mastitis pathogens and for detection of inflammation, as well as the advantages and disadvantages of different methods. Emerging technologies, such as transcriptome and proteome analyses and nano- and microfabrication of portable devices, offer promising, sensitive methods for advanced detection of mastitis pathogens and biomarkers of inflammation. The demand for alternative, fast, and reliable diagnostic procedures is rising as farms become bigger. Several examples of technological and scientific advances are summarized which have given rise to more sensitive, reliable and faster diagnostic results.

Keywords

Bovine mastitis diagnostic methods technological advances

Introduction

Bovine mastitis is an economic burden for farmers because of decreased milk yield, premature culling, cost of veterinary treatments, and other factors. Mastitis leads to changes in milk composition, which is dependent on the inflammatory response.⁴⁰

The most frequent standard in measuring inflammation is cytological investigation, including milk somatic cell count (SCC). The evaluation of milk quality for premium value or penalties applied to milk prices is generally assessed by SCC. Intramammary infections (IMI) are detected more frequently through milk culturing; however, pathogen isolation is not necessarily associated with inflammation. The identification of mastitis pathogens is of major importance in order for adequate control measures to be taken, risk of appearance of chronic infections reduced, and antimicrobial therapy targeted. This work aims to discuss the advantages and disadvantages of methods used in the diagnosis of bovine mastitis and technological advances that have the potential to offer a “cow-side” use.

Identification of pathogens causing intramammary infection

Phenotypic methods

Bacterial culture has for some time served as the gold standard for the examination of phenotypic characteristics. Appropriate use of culture-enhancement methods can significantly increase sensitivity in the detection of mastitis-associated organisms in milk, and targeted use of selective media may offer significant improvements in sensitivity in composite cow samples and bulk tank milk culture.¹⁰

Phenotypic identification is based on an evaluation of morphology, growth characteristics, ability to metabolize substrates, antimicrobial resistance, and other features that result from DNA expression.⁸¹ Commercial manufacturers of diagnostic tests have developed a number of identification methods based on phenotypic traits, including test systems^a–d and other combinations of biochemical tests.⁸¹ Some advantages of phenotypic methods are that such methods rely on biochemical characteristics that are common and associated with bacterial species, are usually easy to perform, have good market availability, and have a relatively low cost. An inherent weakness in phenotypic methods is that there is variability in expression of characteristics by isolates belonging to the same species,²³ and their interpretation may be subjective.⁹ The reproducibility of these tests is limited by the variability in expression and interpretation of phenotypic characteristics. In addition to reproducibility, the typeability (proportion of isolates that are assigned a type by a typing system)⁶² is imperfect either at the species or at the strain level. Microbiological culture methods are also considered to be laborious and time consuming.²¹

On-farm culture systems are increasingly used, as they offer economic benefits to farmers by reducing therapy costs and the amount of discarded milk.⁴² Studies on the diagnostic validity of on-farm culture systems showed acceptable performance, although the specificity recorded was relatively low.⁵² Previous researchers^42,52 compared 2 on-farm culture systems^e,f for clinical mastitis pathogen identification. One system^e consisted of a culture plate with 2 different media, one allowing the growth of both Gram-positive and Gram-negative bacteria, and one selective for the growth of Gram-negative bacteria. The other on-farm culture system^f included 2 media, one allowing the development of aerobic bacteria and another allowing the growth of coliform bacteria. Both methods were able to successfully categorize isolates of clinical cases of mastitis based on their ability to differentiate between Gram-positive and Gram-negative organisms, with sensitivities of 97.9%^e and 93.8%,^f and specificities of 68.6%^e and 70.1%,^f respectively, but neither had the ability to determine if a sample was contaminated.

Mass spectroscopy using the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF MS) can also be performed to determine bacterial species^3,61 and bacterial strains⁸ within a few minutes.⁵³ It is a reliable, easy to use, and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification, reaching sensitivity and specificity values of 100%.⁵ Nevertheless, the ability of MALDI-TOF MS in identification is limited to specific spectra databases of the existing bacterial protein profiles,⁵ and this technology is still too costly to be widespread in diagnostic laboratories.

Genotypic methods

Genotypic methods use DNA as the basis for identification and are used for species identification and strain typing.⁸¹ The genomic sequences of a number of mastitis-causing pathogens are now available and have been used to develop nucleic acid–based testing methods, such as polymerase chain reaction (PCR), which has become one of the most popular methods for direct detection of nucleic acids from infectious agents.⁴⁸ The high sensitivity of PCR, which is capable of detecting a single molecule of DNA, may be seen as an advantage for microbiological diagnostic purposes. Up to 30% of clinical mastitis samples yield no growth in bacterial culture,⁷⁴ but PCR analysis is sensitive enough to detect growth-inhibited and nonviable bacteria. This leads to a possible decrease in the rate of false-negative results. In addition, short throughput times and the potential for objective and user-independent identification⁷⁵ are other arguments in support of PCR assays. Identification of nonviable bacteria has the potential to enable integration of IMI diagnostics with SCC determination in dairy herd improvement programs through the use of bronopol-preserved samples.⁴¹ A previous study⁷⁴ showed, through a PCR assay, that microbiologically negative samples often contained several common mastitis pathogens, some of which displayed high bacterial counts. Use of PCR-based tests may also be of interest for IMI diagnosis when culturing only detects minor pathogens.⁴ Traditional PCR has advanced from detection at the reaction end-point, to detection while the reaction is occurring. This improvement was necessary because end-point collection of PCR products is not quantitative. In contrast, real-time PCR is quantitative. Disadvantages of traditional PCR are the use of agarose gels in the detection of DNA amplification, in which resolution is very poor (~10-fold), while real-time PCR can detect as little as a 2-fold resolution variation.⁷⁹ Another improvement in traditional PCR has been the multiplex PCR, which can simultaneously detect different genes making it potentially faster and cheaper, as all species can be amplified in a single reaction.¹¹ However, multi-target analysis by PCR has 2 main constraints. First, multiplex PCR is limited in the number of targets that can be consistently amplified simultaneously because of uncontrollable primer–primer interactions. Second, identifying solution-phase multiplex PCR amplicons typically requires a secondary method for the separation of size or sequence verification prior to analysis and data interpretation,¹⁸ which may increase direct costs. A previous study⁴¹ asserts that the development of a PCR test capable of complementing or replacing conventional methods in IMI diagnosis presents a challenge because of the large number of pathogens responsible for IMI, many of which are closely related genetically. Furthermore, milk contains PCR-inhibiting substances, and an assay designed for use in mastitic milk must include dedicated DNA extraction protocols and reagents to obtain results.

Molecular diagnostic methods, in general, may also help identify particularly virulent strains of an organism or distinguish between clonal and nonclonal infection outbreaks. In a clonal outbreak, the predominance of a single strain could indicate contagious transmission of the organism or exposure of multiple cows to a particular environmental point source.⁵⁴ Other molecular typing methods used for bovine mastitis pathogen identification include amplified fragment length polymorphism (AFLP) analysis at a species level⁷³; restriction fragment length polymorphism (RFLP) analysis at a strain level⁶⁴; multiple locus variable-number tandem repeat analysis (MLVA) at a strain level⁵⁸; ribotyping at a species level¹²; transfer DNA intergenic spacer length polymorphism analysis at a species²⁸ and strain level⁶⁹; pulsed-field gel electrophoresis (PFGE) typing at a strain level¹⁶; and DNA sequencing of housekeeping genes at a species²⁷ and strain level.²⁸ From these 8 methods, only 3 (AFLP,⁷³ RFLP,⁶⁴ and PFGE¹⁶) have been performed directly from milk samples but all required prior isolate recovery by microbiological culture. There appears to be higher reproducibility, resolution, and sensitivity to AFLP, but both this technique and RFLP have a similar response time and cost efficiency.⁵⁵ According to a previous study,¹² automated ribotyping is a reproducible method, easy to perform, and operator-independent. However, when performed manually, it is time-consuming and technically demanding, requiring highly skilled personnel. The more recent typing methods provide a higher degree of reproducibility, such as MLVA,⁵⁸ triggered by the independent development of a large range of protocols by many different laboratories leading to several different typing schemes for each organism. This led to interlaboratory comparisons and is one of the main limiting factors in currently available genotyping techniques.⁵⁸ Nevertheless, MLVA has a strong discriminatory capacity, high robustness, portability, objectivity, and throughput^34,75 but low versatility, as most protocols are species or serotype specific. In comparison, PFGE, the current gold standard method for molecular subtyping, has a strong discriminatory capacity and versatility, but is less robust and portable, and has lower objectivity and throughput.³⁴

Other technological advances of genotypic methods for the identification of bovine mastitis pathogens include microarray technology, which is capable of detecting 7 common species of mastitis-causing pathogens within 6 hours, with an observed sensitivity of 94.1% and specificity of 100%.⁴⁵ The platform used was based on PCR technology where pathogen-specific targets of DNA were amplified and transferred to react and hybridize with specific probes that were pre-spotted on the biochip. At the end of the process, colorimetric techniques were used to identify pathogen patterns present in the sample. The detection limit of this method was 10³–10⁵ CFU/mL. A previous study¹⁴ also described a DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze pathogens directly from milk samples. These bacterial groups were identified based on the 16S rRNA gene. Results demonstrated high specificity with sensitivity as low as 6 fmol per volume unit.

Another study⁷¹ identified coagulase negative staphylococci isolates with an updated transfer (t)DNA-PCR, which resulted in 91.0% typeability and 99.2% accuracy. The study also showed that the updated tDNA-PCR associated with capillary electrophoresis was almost as accurate as gene sequencing but faster (increased automation) and cheaper (only $3 per isolate).

Immunoassays

Immunological methods are often used because of their speed, simplicity, relatively low cost, and the availability of commercial kits.²⁴ The detection limits of enzyme-linked immunosorbent assays (ELISAs) have been shown to range between 8 × 10⁻⁴ and 8 × 10⁻³ μg of antibody/mL.⁴⁷ Despite these features, ELISAs are not able to detect some antigens that are present at low concentrations.⁴⁸

ELISAs exist for Staphylococcus aureus detection in cases of bovine mastitis,⁹ but the antibody titer does not correlate well with the amount of infecting bacteria.⁶² Other ELISAs have been developed to screen milk for natural contamination with S. aureus and Listeria sp. organisms.⁶² A previous study⁸⁰ developed a magnetic bead–based ELISA employing monoclonal antibodies for the detection of staphylococci in milk. This method detected between 10⁴ and 10⁵ organisms/mL and took 3 hr. An earlier study⁵¹ investigated a milk antibody test^g that detected S. aureus antibodies in milk samples. The ELISA results were scored both visually and by means of an optical density plate reader and compared against positive controls. This test had several potential points of contention with microbiological tests: a cow in the early stages of infection could be culture positive but antibody negative, and a cow could be antibody positive but culture negative because of the intermittent shedding pattern of cows with chronic S. aureus mastitis, or because milk from a single infected quarter was not included (or was diluted) in composite milk samples. If cows were ≤30 days in milk or producing ≤13.6 kg of milk per day, the test was also not considered to be accurate. The sensitivity of this antibody test^g has been reported to range between 69% and 90%, while specificity values were 61–97%.²⁹ Despite this test being available for several years, its use seems to be limited.

Another study⁸² for S. aureus identification in milk samples based on immunoagglutination compares 6 commercially available slide agglutination tests, which are currently used in human medicine. The highest sensitivity (86.7%) and specificity (90.1%) was obtained for a test consisting of latex particles coated with human fibrinogen and immunoglobulin G.^h

Mastitis detection

Cell counting

Somatic cell count has been used as the gold standard for decades to diagnose subclinical mastitis and is an important parameter for the dairy industry as it affects the price of milk paid to the farmer. The mononuclear leukocytes, monocytes, and lymphocytes, along with the neutrophils, are often the only cells taken into account.⁵⁷ SCCs do not always correlate with infection of the udder, and they may be affected by other factors (e.g., lactation number, stage of lactation, milk production level, stress, season, and breed).⁶⁶ Suggested cutoff values for SCC in mastitis diagnosis differ between publications because different conventions are used in different countries as well as various types of milk samples.³² The most frequently used cutoff value to define subclinical mastitis is a SCC ≥200,000 cells/mL.^60,66 Cell counts below this threshold in composite milk samples indicate that a mammary gland is likely to be free of IMI,¹⁵ but this threshold is based on the assumption that the culture test is perfect, which does not take into account the chances of false-negative results in a SCC ≤200,000 cells/mL. Therefore, a study⁷⁷ determining the accuracy of both SCC and culture to detect IMI proposes a lower threshold of 150,000 cells/mL, which can account for misclassification of the culture. The authors of that study suggest this is a more accurate SCC cutoff, providing information about the prevalence of subclinical mastitis corresponding to test sensitivity and specificity maximization. Even so, on an individual quarter basis, a SCC cutoff point of 100,000 cells/mL may be more appropriate⁵⁷ if using differential cell counting as an alternative method in defining the presence of mastitis.

Somatic cell count can be measured by means of direct or indirect methods. Direct methods use either portable automatic cell counters, which are practical for field use, or automatic counters in a laboratory setting.ⁱ There are a number of portable cell counters available. Some counters use an esterase-catalyzed enzymatic reaction^j and others^k count somatic cells optically by staining cell nuclei with a DNA-specific fluorescent reagent (propidium iodine). The advantages of these portable cell counters include cost-effectiveness, speed of use, and user friendliness, but they are generally considered to have poor sensitivity at low SCC.⁷⁶ The laboratory cell counterⁱ operates on the principle of optical fluorescence as in the portable assay^k mentioned previously, but in this case the fluorescent reagent employed is ethidium bromide. The fluorescent signal generated is used to estimate the SCC in milk.⁷⁶ When comparing both methods,^i,k the automatic method has a higher repeatability.²³ The advantage with an automatic cell counter is that it is objective and accurate. Disadvantages are that it is time consuming because samples need to be sent to a laboratory, and the initial investment is high as the equipment is very expensive.

Another direct detection method is differential cell count (DCC), which shows changes in relative cell proportions and can be used to differentiate healthy glands from inflamed glands. DCCs are performed on quarter milk samples by cytometry⁵⁶ and have been proposed as a valid tool for the identification of inflammatory processes in cases with low SCC.⁶³ Recent studies⁵⁷ have shown that DCC can reveal inflammatory mastitis processes with a sensitivity and specificity of 97.3% and 92.3%, respectively, even in milk with an SCC of 1,000 cells/mL.⁵⁶

The California Mastitis Test (CMT) is a common indirect method for measurement of SCC. The test is performed by adding a detergent to a milk sample with a high cell count, which promotes cell lysis, nucleic acid release, and formation of a “gel-like” matrix. When the cell count is above a certain threshold, the sample viscosity interpretation is subjective, and might result in false positives or negatives.⁷⁶ A sensitivity of 66.7% and specificity of 54.8% using the CMT to detect IMI has been reported in fresh cows.⁶⁵ The main advantages of CMT are that it is quick, cheap, simple, and can be used as a “cow-side” test.

The Wisconsin Mastitis Test (WMT) is a laboratory test generally conducted on bulk tank milk samples. The scores can be used to predict the average number of somatic cells. This indirect method uses the same reagent as the CMT; however, the reaction is not estimated but measured by gel height in a tube, providing a more precise result than CMT. The results are generally reported in millimeters. The WMT is usually used as a screening test on producer’s milk because of its simplicity and objectivity, and also provides a convenient method of monitoring udder health on a herd basis.

In 2010, an indirect method to assess SCC and fat content in milk samples was published.¹⁹ The low-cost portable microfluidic sedimentation cytometer has a 15-min response time and shows a lower detection limit of 5 × 10⁴ cells/mL.

Ion variation: milk conductivity

An effect of mastitis is changes in ion concentrations caused by increased vascular permeability³⁷ leading to modifications in electrical conductivity of milk. Electrical conductivity can be measured by rising conductance in milk caused by an increase in levels of sodium, potassium, calcium, magnesium, and chloride. To date, measurement of electrical conductivity is the most widespread automated detection method for mastitis in milking robots. In such systems, mastitis detection is generally performed through a combination of human inspection of animals, by electrodes in the milking system to detect changes in milk electrical conductivity, and by data analysis in herd management software to detect changes in milk yield and milking frequency. Although milk conductivity change might be useful in detecting mastitis, it is not a reliable or sensitive parameter for conclusive diagnosis³³ on its own because of the high number of false positives.

Biomarker evaluation

A biomarker is a characteristic that can be measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions.⁶ To be considered a “good” biomarker, the indicator must be specific for a disease and should remain unchanged by unrelated disorders. Likewise, reliable and reproducible biomarker quantification must be demonstrated.³⁶

As with the aforementioned ions, enzymes are also released into milk as a result of an animal’s immune response against infection and changes in vascular permeability. The enzymes dealing with milk synthesis tend to decrease, while the enzymes related to inflammation tend to increase.⁶⁰ The enzymes originating from phagocytes increase exponentially. Such enzymes include N-acetyl-d-glucosaminidase (NAGase), β-glucuronidase, and catalase. The activity of other enzymes originating in blood also increases, including plasminogen, which is locally activated as plasmin, a proteolytic enzyme that degrades fibrin and casein.⁶⁰ Several enzymes could be used as biomarkers for mastitis diagnosis, including NAGase,³⁸ serum amyloid A (SAA), haptoglobin (Hp), and lactate dehydrogenase (LDH), a cytoplasmic enzyme.³¹ A previous study¹ showed that LDH was the biomarker with the lowest variation between milkings in clinically healthy cows when compared with SAA, Hp, and NAGase. These authors assert that whatever the method used, several measurements over time could be a viable approach as well as information on the normal biomarker variation in clinically healthy udder quarters. Colorimetric and fluorimetric assays have been developed for measuring milk enzyme concentrations, which increase during the early stages of mastitis, including NAGase³⁸ and LDH.⁴³

Milk proteins may be submitted to proteolysis caused by bacteria or endogenous proteases during episodes of mastitis. Peptide biomarkers of milk could thus be used in the diagnosis of mastitis and could discriminate between bacterial causes.⁴⁹ A 2013 study⁴⁹ used capillary electrophoresis, liquid chromatography, and mass spectrometry to reveal a biomarker panel of 47 peptides, with a sensitivity of 75% and specificity of 100%.

Immunoassays have also been used in the diagnosis of bovine mastitis to detect acute phase proteins Hp and SAA, which increase in milk during inflammation,²⁵ but could be ≤1 and ≤0.3 μg/mL, respectively, in healthy milk samples.² An ELISA has been developed for Hp with a detection limit of 0.07 μg/mL in milk and serum,³⁰ and a commercially available solid-phase sandwich ELISA has been developed for SAA.⁷² A previous study⁷⁸ used an automated optical biosensor–based immunoassay to detect NAGase in milk. The limit of detection for the assay was 1 μg/mL. Nevertheless, other researchers⁷ assert that while ELISAs feature accuracy and specificity, antibody-based strategies are restricted by the ability to detect and quantify 1 protein at a time and by a reliance on the availability of species-specific antibodies. ELISAs, therefore, have little application to the discovery of novel inflammatory mediators, as currently only a limited number of bovine-specific antibodies are available.

Discussion

Phenotypic methods continue to be more frequently used than genotypic methods for the identification of mastitis pathogens. However, we are faced with a few problems and challenges. One problem is the large proportion of milk samples submitted to bacteriological analysis from mastitis cases that lead to no-growth results.⁷⁴ These results are generally recognized as false negatives corresponding to detection failure of IMI causative agents.⁴¹ Therefore, a considerable number of infected cows may remain undetected without a concomitant increase in SCC,⁶⁷ and mastitis pathogen control and eradication in herds may be compromised.¹⁴ Consequently, phenotypic identification is being supplemented with genotypic methods. A 2013 study⁶¹ supports the combination of conventional microbiology as first identification for triage and multiplex PCR use for rapid identification of bacteria associated with IMI. Molecular detection assays are a promising avenue in resolving false-negative issues, and several assays have already been developed that can provide solutions to this problem.⁷⁴

On the other hand, phenotypic methods are usually considered less expensive than genotypic methods.⁸¹ Whether or not this is true, depends in part on the number of samples processed per time unit. In some laboratories, phenotypic tests are used with such high frequency that an investment in automation for test reading could become profitable.³⁵ Regardless of sample number, additional testing may be needed to obtain conclusive results from phenotypic methods. The potential increase in cost and turnaround time of phenotypic testing may narrow or eliminate the cost and time differences between phenotypic and genotypic identification.³⁵ In addition, the cost of inaccurate results must also be taken into consideration.⁸¹ Ultimately, several genotypic methods require investment in equipment that is usually very expensive, which limits their use in routine diagnosis.

Future trends

Development prospects for new bovine mastitis diagnosis methodologies point to new biomarkers and technological advances for high sensitivity and specificity, fast and efficient devices that can offer a “cow-side” use. More recently, transcriptome and proteome analyses have been introduced to the biomedical research field. Such tests allow for the identification of biomarkers, gene expression profiles, and the understanding of complex molecular mechanisms in cell physiology and pathology.³⁹ Advances in relevant proteomic techniques such as 2-dimensional gel electrophoresis and mass spectroscopy⁶⁸ have led to the identification of several new proteins involved in mastitis. Progress in microbial proteomics has been achieved with the availability of whole genome sequences for a number of bacterial groups,¹³ including proteome profiles of mastitis-causing pathogens, which, combined with newly available information on toxins, enzymes, and metabolites produced in the udder, could lead to their identification in milk.⁷⁶ Proteomic studies performed for several mastitis pathogens have led to information on protein expression pattern, which can be applied to the discovery of new therapeutic targets⁴⁶ (e.g., bacterial immunogenic proteins for vaccines) and new diagnostic biomarkers.

Biosensors are fast becoming the next generation of tools in analyzing areas such as environmental research, medicine, biodefense, agriculture, and food control.⁴⁴ Biosensors use biological receptor molecules (e.g., antibody, enzyme, and nucleic acid) combined with a transducer to produce a signal that shows a specific biological event (e.g., an antibody–antigen interaction). Nanotechnology-based pathogen detection has created an array of technologies that have advanced detection, diagnosis, and imaging of biomarkers of disease pathogenesis,¹⁷ shortening the time span between sample uptake and results. Portability of biosensors has been explored over the past 15 years though lab-on-chip platforms, incorporating electronics, sampling, and detection modules necessary for a fast, accurate, and low-cost analysis. Several types of these biochips have been demonstrated, using several detection principles: chemical,⁵⁹ mechanical,²⁰ optical,²⁶ electrical,²² and magnetic.⁵⁰

Conclusion

A variety of mastitis diagnostic tests are routinely used to evaluate microbiological milk quality in dairy farms. The successful choice for a test that evaluates milk requires methodological knowledge and diagnostic capabilities for each test currently available. The suitability of a detection method for routine diagnosis depends on its specificity, sensitivity, cost, amount of processing time, and suitability for a large number of milk samples. New technical advances in mastitis diagnosis still require specialized training and experience to interpret results. The personnel responsible should be aware of the strict compliance of each step in the process for good quality control in obtaining reliable data.

PCR and conventional bacteriological culture are the most common tools used for pathogen detection and represent reliable diagnostic methodologies for veterinarians and farmers. The sensitivity of culture tests may be complemented by PCR analysis and are often combined together to yield more robust results. However, to make treatment decisions, this combination does not allow for a timely answer. Proteomic research for reliable biomarkers is viable for the early detection of mastitis and drug efficacy, and to discover potentially novel targets for the development of alternative therapies. However, these innovations are still not possible to use for routine diagnosis. In conclusion, the demands for an alternative, fast, and accurate diagnostic procedure for mastitis is rising as farms increase in size, cows produce more milk, and milking techniques such as automatic milking systems become more common.

Footnotes

Authors’ contributions

All authors contributed to conception and design of the study; contributed to acquisition, analysis, and interpretation of data; and agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CM Duarte drafted the manuscript. R Bexiga critically revised the manuscript. PP Freitas and R Bexiga gave final approval.

a.

API microbial identification kits, BioMérieux Inc., Durham, NC.

b.

Vitek microbial identification kits, BioMérieux Inc., Durham, NC.

c.

Staph-Zym identification kit for staphylococci, Rosco Diagnostica A/S, Taastrup, Denmark.

d.

BBL Crystal identification systems, BD, Franklin Lakes, NJ.

e.

Minnesota Easy Culture system II bi-plate, University of Minnesota Laboratory for Udder Health, St. Paul, MN.

f.

3M Petrifilm plate methods, 3M Microbiology, St. Paul, MN.

g.

ProStaph test, Proscience Corp., Sterling, VA.

h.

Masta-Staph test, Mast Group Ltd., Bootle, Merseyside, UK.

i.

Fossomatic cell counter, FOSS, Hillerød, Denmark.

j.

Portacheck somatic cell counter, Portacheck Inc., Moorestown, NJ.

k.

Cell counter, DeLaval International AB, Tumba, Sweden.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research and authorship of this article. Publication fees are funded by CIISA at FMV (CIISA: UID/CVT/00276/2013).

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Technological advances in bovine mastitis diagnosis