Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland

Abstract

Background: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk.

Results: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland.

Conclusions: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Keywords: Escherichia coli; Staphylococcus aureus; Streptococcus agalactiae; Streptococcus uberis; Bacterial identification; Epidemiological data; Mastitis; Polish cattle.

Fig. 1

The percentage of bacterial species identified by using culture methods in bovine milk from cows with clinical mastitis (A) and from cows from the control group (B). Legend: SA – Staphylococcus aureus, EC – Escherichia coli, GBS – Streptococcus agalactiae, SU – Streptococcus uberis, KP – Klebsiella pneumoniae, SE – Staphylococcus epidermidis, CS – Corynebacterium spp., SV – Viridians Group Streptococcus, NASM – Non-aureus staphylococci and mammaliicocci (exc. Staphylococcus epidermidis)

Fig. 2

The results of a comparison between the two approaches of isolating the bacterial DNA from CM milk samples. Legend: without TSB – milk samples subjected to direct DNA isolation; with TSB – milk samples subjected to pre-incubation in TSB before further DNA isolation procedures; SA – Staphylococcus aureus, EC – Escherichia coli, GBS – Streptococcus agalactiae, SU – Streptococcus uberis

Fig. 3

The percentage of bacterial species identified by molecular methods in bovine milk from 100 cows with clinical mastitis (A) and from 103 cows from the control group (B). Legend: SA – Staphylococcus aureus, EC – Escherichia coli, GBS – Streptococcus agalactiae, SU – Streptococcus uberis

Fig. 4

The percentage of simultaneous co-occurrence of species studied determined by the PCR method and in culture methods determined for all (N = 203) milk

Fig. 5

The combination of co-occurrence of the particular bacterial species of mastitis determined in the PCR method and in the culture method. Legend: SA – S. aureus, EC – E. coli, GBS – S. agalactiae, SU – S. uberis

Fig. 6

Map of Poland with marked regions and cities from which the tested milk samples came. Legend: Region 1 – the north-east, Region 2 – the south, Region 3 – the south-west

Fig. 7

The distribution of S. aureus (SA), E. coli (EC), S. agalactiae (GBS), and S. uberis (SU) in the three regions of Poland: the north-east (Region 1), the south (Region 2) and the south-east (Region 3) determined by molecular (A) and culture (B) method