Background
Shiga toxin producing
Escherichia coli (STEC) are an emerging issue for veterinary public health. Their zoonotic relevance is related to the severity of caused diseases and the low dose of ten to 100 bacteria [
1]. The highly cytotoxic Shiga toxin combined with other virulence factors causes gastroenteritis and severe sequelae in children with a low, but steady incidence. Most research to date has concentrated on the STEC-serovar O157:H7, but in Germany and other European countries human infections by non-O157:H7 serovars have also been frequently assessed [
2‐
5]. The only consistent difference of these STEC-serovars to apathogenic
Escherichia coli (
E. coli) is the possession of Shiga toxin (
stx) genes. Potentially pathogenic STEC for humans, causing hemorrhagic colitis and mostly possessing the genes for
stx2,
EHEC-hly
A
and
eae, encoding intimin, a factor of adherence, are so-called enterohemorrhagic
E. coli (EHEC), but also other STEC can induce serious disease in humans [
2,
3,
6]. Ruminants, and especially cattle, are considered as a natural reservoir of STEC. Extended risks originate in domesticated cattle kept in close contact to humans. Furthermore, cattle-derived foods can be contaminated and represent an important source of infection as well [
7,
8]. Bovine carriers of STEC show no sign of clinical disease whilst shedding this pathogen.
Risk factors for fecal shedding by dairy cattle are varied, including different factors of management such as hygiene, the kind of diet, husbandry and changes in housing or structure of the herd [
9‐
11]. Moreover, at least for the serovar O157:H7, the individual characteristics of each cow also seem to have an influence [
12]. Based on data from Synge et al., Matthews et al. formed a model simulating the excretion- and transmission-dynamics of STEC O157:H7 in a cattle herd [
13‐
15]. The model fit best to the real data if a small proportion of cows had a 50-fold higher excretion of STEC O157:H7 compared to the other animals [
14,
15]. Up to 80.0% of the transmissions of STEC O157:H7 to uninfected cows originated in 20.0% of the most infectious cows [
14]. These high-level STEC shedding cows were defined as Super-Shedders [
12,
15]. A high-level excretion of STEC as shown by these Super-Shedders is associated with the colonization of the recto-anal junction of their gastrointestinal tract [
12,
15,
16].
According to the definition of Chase-Topping et al., all cows that shed at least 10
4 cfu (colony forming units) STEC O157:H7 per gram feces were characterized as Super-Shedders [
12]. In this definition, the longitudinal component of Super-Shedding was totally neglected. However, the concentration of STEC O157:H7 was shown to be associated with the duration of shedding [
17]. Hence, in the present study, as in the investigations of Lim et al. and Carlson et al., the longitudinal part of the definition was used [
18,
19]. In both studies, cows were defined as Super-Shedders, or alternatively as Persistent-Shedders, if they shed STEC O157:H7 for more than three consecutive months. In this study, the definition of a Super-Shedder was extended as follows: cows were classified as Super-Shedders if at least half of their samples and equal or more than four consecutive samples were
stx- positive.
The aim of this study was to investigate risk factors in milk production for STEC shedding in general by dairy cattle. Furthermore, the occurrence of possible Super-Shedding cows, shedding not only STEC O157:H7 but also other STEC-serovars, was examined by longitudinal and qualitative sampling over the period of twelve months.
Results
In 24.7% (407) of all samples, stx genes were detected by the screening PCR. Within the herds, the mean stx prevalence varied between 11.1% and 32.3%.
Of all cows sampled for more than six months (n = 140), only 12.9% (18 cows) were detected as constantly
stx-negative. Most of the animals excreted
stx at least once (45.5%, 55 cows), and 17.1% (24 cows) were classed as
stx- positive in more than 50% of their samples. In 40.7% (57 cows), the maximal duration of
stx shedding was one sampling, whereas 29 of these cows were identified as intermittent shedders. They were detected in one to three additional but not consecutive samplings. For 32.9% (46 cows) that shed
stx in two to three consecutive samplings, these samplings in most of the cases (35 cows) were followed by other short periods of
stx shedding. Seventeen cows (12.1%) were determined to be
stx-positive in the screening PCR in four or five consecutive samplings. In total, twelve of these cows had additional periods of shedding intermittently or for short periods. Two cows (1.6%) were characterized by a great number of consecutive
stx-positive samplings: one cow was determined to be
stx-positive in seven consecutive samplings, the other even over eight consecutive samplings (Table
1). In total, 14 cows (10.0%) were identified as Super-Shedders according to the definition with positive
stx-detection in the screening PCR on at least four consecutive samplings and in equal to or more than half of their samples. In the sampling period, STEC-colonies were isolated resulting in 1,105 STEC-isolates from 144 of the 407
stx-positive fecal samples. Dominating combinations of virulence genes were
stx2 and
EHEC-hly
A
(434 isolates, 39.3%), and
stx1,
stx2 and
EHEC-hly
A
(311 isolates, 28.1%) (Table
2).
Table 1
Maximal number of consecutive stx-detection in the feces of all cows, investigated for more than six months (n = 140)
0 | 18 | 12.9% |
1 | 57 | 40.7% |
2-3 | 46 | 32.9% |
4-5 | 17 | 12.1% |
7-8 | 2 | 1.6% |
Table 2
Prevalences of virulence factors from isolated STEC (n = 1,105 isolates)
- | + | - | + | 434 | (39.3%) |
+ | + | - | + | 311 | (28.1%) |
- | + | - | - | 145 | (13.1%) |
+ | + | - | - | 56 | (5.1%) |
- | + | + | + | 43 | (3.9%) |
+ | - | - | - | 35 | (3.2%) |
+ | - | - | + | 33 | (3.0%) |
+ | - | + | + | 24 | (2.2%) |
+ | + | + | + | 17 | (1.5%) |
- | + | + | - | 4 | (0.4%) |
+ | + | + | - | 2 | (0.2%) |
+ | - | + | - | 1 | (0.1%) |
From 14 Super-Shedding animals, 380 isolates were identified. From these isolates, 61 STEC-isolates of six Super-Shedders were selected for serotyping based on their virulence pattern, the date of sampling and the cow of origin. To represent each of the four farms with Super-Shedding cows, STEC-isolates from one Super Shedder per farm were chosen. Assuming that on one farm different serovars might be present, STEC-isolates from two additional cows on one of the farms were serotyped. The characterized STEC-isolates belonged to 24 sorbitol-fermenting non-O157:H7 STEC serovars (Table
3). The two most predominant serovars were serovar O113:NM (16 isolates, 26.2%) and O22:H8 (10 isolates, 16.4%), the most frequent virulence patterns of these STEC were
stx2 and
EHEC-hly
A
(39 isolates, 63.9%).
Table 3
Serotypes and virulence patterns of tested STEC-isolates (n = 61) from six selected Super-Shedders
O113:NM | 16 | 13 | 2 | 1 (stx
2
+) | cattle, human (D) |
O22:H8 | 10 | 4 | 5 | 1 (stx1+, EHEC-
hlyA
+) | cattle, human (D, HUS) |
Ont:H25 | 7 | 5 | 1 | 1 (stx2+, eae+) | cattle, human (D) |
O130:H11 | 6 | 2 | 4 | | cattle |
O8:H19 | 2 | 2 | | | cattle, human (D, HUS) |
O18:H8 | 2 | 1 | | 1 (stx1+, EHEC-
hlyA
+) | - |
O113:H21 | 2 | 2 | | | cattle, human (D, HUS, TTP) |
O138:H34 | 2 | 1 | | 1 (stx2+) | cattle |
O5:H7 | 1 | | 1 | | cattle |
O8:H21 | 1 | | | 1 (stx1+) | human (HUS) |
O28:H31 | 1 | 1 | | | - |
O80:H45 | 1 | 1 | | | - |
O91:H7 | 1 | 1 | | | cattle |
O150:H8 | 1 | | 1 | | cattle |
Ont:H7 | 1 | | | 1 (stx2+) | cattle, human (D) |
Ont:H19 | 1 | 1 | | | cattle, human (D) |
Ont:H21 | 1 | 1 | | | cattle, human (D) |
Ont:H39 | 1 | 1 | | | - |
Ont:H42 | 1 | 1 | | | cattle |
Orough:NM | 1 | 1 | | | cattle, human (D, HUS) |
Orough:H2 | 1 | | | 1 (stx1+, eae+, EHEC-
hlyA
+)
1
| cattle, human |
Orough:Hnt | 1 | 1 | | | cattle, human (D) |
In one cow, the serovar O22:H8 was isolated on each sampling date from July to November. In September, this cow began to shed additionally the serovar O113:NM for the next five consecutive samplings until January. On the same farm, another cow shed the serovar O113:NM over nearly the same period from November to December.
Several risk factors were identified in this study as significant influences on the detection of
stx in bovine feces: days in milk (DIM) and the number of lactations, the somatic cell count, the content of protein and urea in milk and the nutritional condition. Furthermore, the presence of Super-Shedding cows in the herd and the month of sampling represented significant risk factors. Depending on the DIM class, dry cows, and cows with 50 to 150 days as well as cows with more than 350 days in milk showed a significantly higher risk or a higher tendency to shed STEC (Table
4). First-calving cows, cows with a somatic cell count lower than 100,000 cells/ml milk, a protein content in milk higher than 3.0%, respectively a body condition score higher than 3.50 had a significantly or tendentially increased risk to be identified as STEC-shedders by the screening PCR, whereas cows with an urea content lower than 150 mg/L milk had a decreased risk (Table
4).
Table 4
Significant risk factors for shedding of STEC by dairy cattle
days in milk | | | | | | | |
51.-100.d | 175 | 44 | 25.1% | 1.73 | 0.98-3.04 | 1.-50.d |
t
|
101.-150.d | 192 | 52 | 27.1% | 1.93 | 1.11-3.35 | 1.-50.d | * |
>350.d | 114 | 36 | 31.6% | 2.06 | 1.12-3.79 | 1.-50.d | * |
dry cows | 182 | 50 | 27.5% | 1.67 | 0.95-2.91 | 1.-50.d |
t
|
number of lactation | | | | | | | |
first calvers | 293 | 93 | 31.7% | 1.74 | 1.22-2.47 | >3 completed lactations | ** |
first calvers | 293 | 93 | 31.7% | 1.51 | 1.07-2.11 | 2-3 completed lactations | * |
somatic cell count (/ml milk) | | | | | | | |
<100,000 | 1028 | 279 | 27.1% | 1.57 | 1.18-2.10 | >100,000 | ** |
protein content in milk (%) | | | | | | | |
3.00-3.80 | 906 | 220 | 24.3% | 1.80 | 1.06-3.08 | <3.00 | * |
>3.80 | 198 | 54 | 27.3% | 2.30 | 1.18-4.48 | <3.00 | * |
content in urea in milk (in mg/l) | | | | | | | |
<150 | 580 | 144 | 24.8% | 0.69 | 0.45-1.06 | 150-300 |
t
|
BCS | | | | | | | |
3.50-5.00 | 318 | 81 | 25.5% | 1.92 | 1.22-3.04 | 1.00-3.25 | * |
presence of a Super-Shedder | | | | | | | |
yes | 1071 | 323 | 30.2% | 2.63 | 1.90-6.64 | no | *** |
Additionally, a cow kept in a herd with Super-Shedders had a significant, more than two-fold risk of being positive in the
stx-detection by PCR compared to a cow in a herd with no Super-Shedder (Table
4).
The
stx prevalence was highest in the late summer months (August, September, and October) with 27.9%, 28.8% and 35.0% in contrast to the spring months with lower prevalence (February: 20.5%, March: 17.6%, April: 16.3%). In consequence, cows showed a significant higher risk for detection as
stx-positive in summer, autumn and winter compared to that in spring. Furthermore, cows had a significantly higher risk in autumn for shedding
stx than in winter (Table
5).
Table 5
Seasonality of stx-detection in feces of examined cattle expressed in Odds Ratios
spring | X | 0.61* | 0.43*** | 0.63* |
summer | 1.64* | X | 0.70t | 1.02 |
autumn | 2.35*** | 1.43t | X | 1.47* |
winter | 1.60* | 0.98 | 0.68* | X |
Discussion
Many studies have examined the epidemiology of STEC O157:H7 in cattle populations, but there have been only a few investigations into the relevance and the transferability of these results to other serovars, for instance O26, O91 or O113. These serovars have been detected in cattle herds and are also relevant for human diseases [
2,
3,
20]. Therefore, this study considered STEC in general, not only O157:H7.
Following an evaluation of the shedding patterns of all investigated cows, the strict definition of Super-Shedders with at least four consecutive
stx-positive samples in the screening PCR and at least half of their samples deemed
stx-positive was necessary to distinguish intermittent-shedding and persistent-shedding cows. A strictly longitudinal definition of a Super-Shedder as in the present study was also used in the investigations of Lim et al. and Carlson et al. [
18,
19]. Carlson et al. reasoned this definition by findings of Woerner et al. that the persistence of shedding of O157:H7 is much more important than the STEC count in the feces, as suggested by Chase-Topping et al. in their definition of a Super-Shedder [
12,
19,
21].
Due to the methodological approach which included a step of high dilution (10
-5-10
-6) to plate the bacteria for the colony-hybridization, an efficient isolation of STEC gave a hint of high numbers of STEC in the fecal sample. The isolation of STEC in this study mainly was possible in the samples of Super-Shedders, which is congruent with the results mentioned by Chase-Topping et al. who defined Super-Shedders basically about the number of shed STEC [
12].
To our knowledge, the study presented here is the first showing Super-Shedding of serovars other than O157:H7 by Super-Shedder dairy cattle.
Compared to the study of Low et al. with 3.9% of all cows identified as Super-Shedders for the serovar O157:H7, the proportion of analyzed Super-Shedders for non-O157:H7 serovars in all investigated herds in this study was high with 10.0% (14 cows) [
22]. This is possibly due to the higher colonization of cattle with non-0157:H7-serovars [
23,
24]. In general, a high proportion of Super-Shedders is associated with a high proportion of low-level and intermittent shedders due to the higher possibility of transmission [
22,
25]. As previously described for STEC strains in cattle, most of the isolated strains do not possess the
eae gene [
23,
26]. However, the mechanisms of adherence for bovine non-O157:H7 STEC-isolates are not restricted to
eae but also include local or diffuse adherence on HEp2-cells [
27]. Further virulence factors of adherence for non-O157:H7 STEC, also associated with human diseases, could comprise Saa (an autoagglutinating adhesion), Iha (adherence-conferring protein similar to
Vibrio cholerae IrgA), Efa1 (
E. colifactor for adherence) and LPF (long polar fimbriae; closely related to LPF of
Salmonella enterica serovar Typhimurium) [
28‐
32]. Concerning Efa1, Stevens et al. proved in
in vivo experiments the relevance of this virulence factor for the colonization of intestinal mucosa in cattle [
29]. As we currently do not know whether Super-Shedding of non-O157:H7 STEC is also caused by enhanced colonization of the terminal rectum, as it has been shown for O157:H7 colonization, future studies clearly should address these questions [
33].
Congruent to other literature, a STEC prevalence of 24.7% was detected in healthy dairy cows in Northern Germany in this study [
23,
26]. In other studies in Germany, values from 18.0% up to 86% were assessed [
23,
34‐
36]. In the other parts of Europe, the prevalence was supposed to be slightly lower with percentages of 11.0% to 21.0% [
37]. Differences in the obtained prevalence can be related to different methods of detection on the one hand and the sampling method and the sample size on the other hand. The methodology used in the present study and also in the study by Geue et al. with an enrichment step before the PCR and the colony-hybridization resulted in the hitherto highest prevalence of STEC in beef cattle and heifers in Germany [
23]. The most sensitive sampling method, at least for STEC O157:H7, seemed to be the rectal swab [
38,
39]. This is probably due to the colonisation of the recto-anal junction of the intestinal mucosa, which is directly sampled by the swab [
16,
22].
Only 18 animals (12.9%) of 140 sampled cows were stx-negative over the whole period of sampling. Most of the cows were defined as intermittent shedders; they were stx-positive in the PCR for short periods of consecutive samplings or for one sampling, but did not reach the number of consecutive stx-positive samplings necessary to fulfill the definition of a Super-Shedder.
With regard to the virulence gene patterns, the assessed dominance of
stx2 and
stx1 +
stx2-positive strains in cattle in the present study is in accordance with other authors [
23,
26]. This high proportion of
stx2, known as the most virulent Shiga toxin for humans, in bovine STEC strains poses an important risk for humans in contact with cattle and cattle-derived foods. This holds especially true because several cases of STEC infections in humans have been associated with STEC strains without the
eae gene, but with
stx2 and other factors for adhesion [
1,
30,
40‐
42].
Based on the technical and methodological efforts of serotyping, only 61 isolates originating in the Super-Shedding cows were examined. These serovars belonged to 24 non-O157:H7 STEC-serovars, partly with previous references of description in healthy cattle but also in healthy and diseased human [
43]. This diversity of serovars is typical for cattle; Blanco et al. isolated in 328 cattle 66 STEC that belong to 25 serovars [
44]. Three of the most frequent serogroups in the present study were also identified in the investigation of Blanco et al. [
44]. Eight of the serovars isolated in the present study have already been associated with diseases in humans [
43]. Especially the serovars O8:H19, O8:H21, O22:H8, O113:H21 and Orough:NM were also detected in diseased humans with hemolytic-uremic syndrome (HUS) and thrombotic-thrombocytopenic purpura (TTP), seven other isolated serovars (see Table
3) were isolated in cases of humans with diarrhea [
43]. The most predominant serovars O113:NM and O22:H8 induced diarrhea respectively diarrhea and hemolytic-uremic syndrome (HUS) in some cases in humans [
43]. The serovar Orough:H2 was not associated with the induction of severe human disease but was characterized by the possession of
stx
2
, EHEC-hly
A
and
eae genes, which is the typical virulence patterns for the human pathogen EHEC [
43].
In the present study, one single cow hosted two to four different serovars. However, this number is limited by the number of colonies taken from the plate. In the study of Blanco et al., up to three different serovars were isolated by some cows [
44].
The serotyping of the small proportion of all isolates was sufficient to confirm the hypothesis of Super-Shedders of non-O157:H7 STEC by at least two cows shedding continuously O113:NM and 022:H8. This indicates that also non-O157:H7 serovars are able to colonize the bovine intestinal mucosa, resulting in persistent Super-Shedding. Further studies under defined experimental conditions are needed to investigate the precise colonization and shedding in detail. Regarding the other risk factors, the seasonality for the shedding strains serogroup O157 showed highest prevalence in summer, those for STEC in general a prevalence peak in autumn [
45,
46]. In accordance with both studies, the highest prevalences in this study were also detected in the late summer months. Thus, saisonality is clearly associated with STEC shedding, regardless of which serotype is tested for.
The higher risk for heifers and for cows with 50 to 150 days in milk can be explained by the metabolic and emotional stress of these animals and actually was shown for serovar O157:H7 [
25,
47]. The relevance of age for the occurrence of pathogenic
E. coli has already been presented by Wieler et al. [
48]. Regarding O157:H7, only few studies have dealt with the connection of milk content and the shedding of this serovar [
11]. These authors clearly showed the impact of milk content, probably related to diet, health and stress, on the shedding of non-O157:H7 STEC as well. Moreover, the presence of a Super-Shedder in the herd as a persistent source of infection was shown to be a risk factor for the shedding of serogroup O157 and was confirmed for non-O157:H7 STEC [
12].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AM carried out all samplings and the bacteriological analyses and draft the manuscript. LW made substantial contributions to the interpretation of the results. KH and TS provided support in the validation of results. LW, KH and TS critically revised the manuscript. AF managed the serotyping of the STEC-isolates. NK designed and coordinated the study and was involved in drafting the manuscript.
All authors read and approved the final manuscript.