Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Abstract

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n = 67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrosprayionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Application of Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry for Identification of Coagulase-negative Staphylococci Isolated from Milk of Cows with Subclinical Mastitis

“…In six cases, the authors were unable to identify species of CNS causing subclinical mastistis using MALDI-TOF MS (log value less than 2.0). Similar results were obtained by Perreten et al (2013), who were unable to determine the primary etiological factor of mastitis in 18% of disease cases using the PCR/ESI-MS technique. Other studies (Tomazi et al 2014) demonstrated that the sensitivity of MALDI-TOF MS for the identification of CNS isolated from cow udders is 95.4% in comparison to PCR-RFLP.…”

Molecular biological tools applied for identification of mastitis causing pathogens

“…In the same way, it is also possible to design a more complex form in which variable number of forward primers is added to face less number of reverse primers in the reaction. This reduction in the number of primers in the reactions minimizes possible faulty annealing or dimer building [3] . Another variant is the combination of different assays together such as performing Multiple PCR followed by electrospray-ionization mass spectrometry (PCR/ESI-MS) in order to enable rapid detection of nearly all known pathogens.…”

“…Mastitis pathogens are usually bacteria, however, molds, yeast, and prototheca may also induce mastitis. It is important to identify the mastitis prompting organism in order to (1) properly treat and select the suitable antibiotics, (2) understand their route of spread and evaluate the contagiousness of the case, (3) calculate their public health impact, (4) to judge the prognosis of the affected quarter/cow considering early culling decision, (5) select the suitable hygienic and preventive measures, and finally for (6) choosing the proper mastitis vaccine programs to be applied in the farm [2] , [3] .…”

“…Field diagnosis of clinical mastitis is usually based on udder examination, changes in the physical properties of the milk and the increase in somatic cell count and even the use of ultrasonography [3] , [4] while the diagnosis of subclinical mastitis is more difficult and depends on indirect techniques such as California Mastitis Test (CMT), electrical conductivity, or the detection of body enzymes released due to tissue damage (e.g. LDH, and NAGase) [2] .…”

Food Pathogens

“…With broad-range amplification followed by detection of mixed amplicons, the process can identify genetic proof of known and unknown pathogens. Perreten and colleagues demonstrated that PCR-ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease [78]. Identification by PCR-ESI-MS using oligonucleotides is more specific for bacterial groups than for particular species, even if changeable regions are amplified between species and strains.…”