Genetic diversity of Listeria monocytogenes strains from a high-prevalence dairy farm

Abstract

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm ("farm A") revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm ("farm B"). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.

FIG. 1.

Percentage of isolates from each sample source according to serotype. Environmental strains not obtained from commodity feeds, silage, or individual fecal samples are listed as “Env.”

FIG. 2.

Dendrogram showing PFGE subtype (AscI REDP) diversity of L. monocytogenes samples from farm A at two collection time points (TP 1 = 2002 collection, TP 2 = 2003 collection). Numbers listed in the first column on the left are the LMB (Listeria monocytogenes Bank) isolate identification numbers. Unique pulsovars are designated using the LMB number of the first strain to be identified with that pulsovar. Source codes are as follows: F (feed), S (silage), C (cow fecal), W (water tank), T (tractor blade), MS (milk sock/filter), B (bovine brain), CYD (commodity yard dust), and O (other). The serotypes associated with each pulsovar are listed in the third column; however, pulsovar/sample ID 0836 includes strains of two serotypes (1/2a and 1/2b). Numbers listed in the two columns on the right indicate the number of samples positive for a particular PFGE type at each time point. The UPGMA dendrogram was constructed from Dice coefficients with 1.5% band matching tolerance.

FIG. 3.

PFGE subtypes and serotypes of L. monocytogenes isolates from the cow 153 fecal sample. (A) AscI REDP; (B) ApaI REDP.

FIG. 4.

PFGE REDP of bovine clinical isolates (LMB 468 and 113) and closely related fecal strains present on farm A. The upper REDP cluster shows ApaI REDP, and the lower cluster shows AscI REDP. The numbers in the left column are LMB strain numbers. The letters in the center column indicate the source (FA, farm A; FB, farm B; CDC, PFGE reference strain obtained from the Centers for Disease Control and Prevention). The sample sources are listed in the right column (C, cow feces; B, bovine brain).

FIG. 5.

UPGMA representation of genetic relationships between 18 L. monocytogenes isolates based on hybridization data derived from 606 microarray probes. Bootstrap values are indicated next to the appropriate nodes (only values >60 are indicated), and phylogenetic divisions are indicated as Div I (Division I), Div II, and Div III. Strains are coded as follows: letter(s) to the left of the LMB number indicate the strain serotype (F, 4b; A, 1/2a; B, 1/2b; FED, 4e/d), and letters immediately following the three- to four-digit LMB number indicate the farm where the isolate was obtained from (FA, farm A; FB, farm B). If no letters are present the isolate was obtained from a farm source unassociated with farm A or farm B. Letter(s) after the period indicate the sample type from which the isolate was obtained from (S, silage; C, cow feces; B, bovine brain; BM, bulk milk; G, goat).