Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria

Abstract

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Antimicrobial Resistance of Escherichia Coli Isolated From Milk, Beef and Chicken Meat in Bangladesh

“…Relatively higher numbers of E. coli isolates were obtained in conventional method (37.87%) than molecular technique (35.94%). Similar finding was also reported by Koskinen et al (2010) who reported culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis and dissimilar with Salauddin (2015) who reported all isolates of E. coli were 16S rRNA positive. Multi-drug resistant E. coli isolates were found for 10 commonly used and market available antibiotics.…”

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

“…However, this technology failed to identify some of the primary mastitis pathogens in 17.9% of the samples which may also be related to the prolonged transport time. Negative results of up to 12% were also reported for other PCRbased assays compared with culture (Koskinen et al, 2010). Otherwise, primary pathogens, such as Strep.…”

“…Similar ranges were obtained when PCR-based molecular methods were compared with culture (Paradis et al, 2012;Spittel and Hoedemaker, 2012). However, molecular methods have been shown to higher detection potential than culture (Koskinen et al, 2009;Koskinen et al, 2010). We also show that the rapid detection of barely cultivatable and slow-growing mastitis pathogens, such as M. bovis, by PCR/ESI-MS represents a major advantage for veterinarians in preventing the spread of the disease within respective herds (Aebi et al, 2012).…”

Phenotypical and Mass Spectral Assessment Methods for Identification of Some Contagious Mastitis Pathogens

“…Although the culture of milk is considered as a gold standard test for mastitis diagnosis, there are several disadvantages associated with bacterial culture, including no bacterial yielding from truly subclinically infected cows due to presence of high number of leukocytes or presence of preservatives or residual therapeutic antibiotics in submitted samples. A further recognized disadvantage of culture is that about 25 to 45% of all milk samples yield no bacterial growth even after two days of incubation (Koskinen et al, 2010). Moreover, microbiological culture of milk is time consuming and species identification by standard biochemical methods requires more than 48 h to be completed.…”

“…Moreover, microbiological culture of milk is time consuming and species identification by standard biochemical methods requires more than 48 h to be completed. Due to the above-mentioned limitations of cultural methods, Polymerase Chain Reaction (PCR) has been developed to identify various mastitis pathogens (Koskinen et al, 2010). The progress of PCR based techniques supplies a talented option for the rapid detection of different microorganisms.…”