Anaerobe 27 (2014) 100e105
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Pathogenesis and toxins
Relationship between gastrointestinal dysbiosis and Clostridium botulinum in dairy cows Monika Krüger a, Awad A. Shehata*, a, b, Anke Grosse-Herrenthey a, Norman Ständer a, Wieland Schrödl a a b
Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2013 Received in revised form 25 March 2014 Accepted 27 March 2014 Available online 16 April 2014
The gastrointestinal tract is a balanced ecosystem that can get out of balance and predisposed to clostridial diseases or other pathological conditions. The objective of the present study was to evaluate the gut microbiota in dairy cows suffering from chronic botulism. Cows were investigated for Clostridium (C.) botulinum in faeces and rumen fluids. In order to study the relationship between botulism and gastrointestinal microbiota, faeces and rumen fluid were tested for bacterial composition using conventional microbiological culture techniques and fluorescence in situ hybridization (FISH). Protozoa were analyzed in rumen fluid microscopically. The presence of C. botulinum was associated with specific changes in the faecal microbiota, especially a significant reduction of total aerobic bacteria, total anaerobic bacteria, enterococci, Clostridium perfringens and yeast and fungi. Also C. botulinum positive rumen fluid had significantly more Bacteroides spp., C. histolyticum group, Alfa- proteobacteria, Gammaproteobacteria, and sulfate-reducing bacteria; as well as significantly fewer Euryaracheota, and the protozoa Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. In conclusion, C. botulinum is common in dairy cows in Germany but the incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Bacteria, yeast and protozoa appear to be crucial in the colonization process; however, the chronology of these events and role of each microbial group needs further evaluation. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: C. botulinum Dysbiosis Gut microbiota BoNT Protozoa
1. Introduction The bacterium Clostridium botulinum is widespread in the environment. It can be found in soil, dust, manure, slaughter-house wastes, and residue of biogas plants, bio-compost and mud. The gastrointestinal tract of humans and animals is also a habitat for this agent. So far, seven serologically different botulinum neurotoxin (BoNT) types (AeG) are known that block the release of acetylcholine at the neuromuscular junctions. The BoNT may be taken up orally if preformed in food or feed (intoxication) [1e5]. In addition to the feed-borne diseases in domestic animals, another form of botulism is reported in cattle, called “visceral botulism” or “chronic botulism”. This form of botulism is thought to be caused by colonization of the lower intestine with C. botulinum and the subsequent production of BoNTs [2,5]. The presumed cause of clostridial colonization in the intestinal tract is microbial imbalance in * Corresponding author. Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany. Tel.: þ49 03419738183; fax: þ49 03419738199. E-mail address: [email protected] (A.A. Shehata). http://dx.doi.org/10.1016/j.anaerobe.2014.03.013 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.
the digestive tract accompanied by replication and toxin production as part of a multifactorial disease. Gut microbiota have continuous communication with host cells and form long-lasting, interactive associations with their host. These associations play a critical role in conservation of mucosal immune function, epithelial barrier integrity, motility, and nutrient absorption [6e8]. Under normal conditions, gut microbiota display a symbiotic relationship with the host to contribute to its intestinal health; however, a disturbance in normal microbiota of the intestinal tract can lead to an imbalance of hoste microbe relationships, known as “dysbiosis” [9]. Disturbances within this system by feed, heavy metals, toxic substances, bacterial toxins antibiotics, etc. can lead to localized inflammation, extensive infection or intoxication [10e12]. Protozoa are an important part of the rumen microbiota. They serve to reduce bacterial populations in the rumen and any changes in rumen bacteria influence their population [13]. Rumen protozoa represent an appreciable proportion of the rumen biomass [14] and have much longer generation time than bacteria (8e36 h vs. about 20 min). They depend on rumen bacterial support for vitamin B complex and vitamin K.
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Little is known about the role of immunological homeostasis in the gastrointestinal tract of cattle. It is postulated that under dysbiotic conditions, facultative pathogenic bacteria are able to induce disease because otherwise antagonistic bacterial populations fail to suppress them [15]. Thus, we hypothesize that colonisation and neurotoxin generation by C. botulinum in the gastrointestinal tract of cows also requires this precondition. In the last 15 years, C. botulinum associated chronic diseases have increased in Germany without known causes. The objective of this study was to shed light on the distribution of C. botulinum in dairy cows and its relationship to the microbial (bacteria, protozoa) composition of gut and rumen fluid.
with 4.5 mL) in reinforced clostridial medium (RCM; SIFIN, Berlin, Germany), vigorously mixing, heating at 80 C for 10 min and incubating at 37 C for 7 d in anaerobic chamber (MACS anaerobic Workstation, Don Whitley Scientific Limited, West Yorkshire, England) before subsequent storage at 25 C until analysed. After thawing, the culture samples were centrifuged at 7000 g for 15 min and the clear supernatant was analysed for type specific BoNTs/AeE with polyclonal antibodies using ELISA [16]. The relative units (RUs) of BoNTs were calculated with the measured optical densities (OD) values as follow: (sample OD minus twice the value of the control OD [BoNT negative sample of bovine faeces]) multiplied by dilution factors [17].
2. Materials and methods
2.4. Bacterial enumeration in faeces and rumen fluid
2.1. Animals
2.4.1. Bacterial cultivation A total of 237 faecal samples collected from the selected 5 farms (GH, R, E, GZ, and K) were investigated by conventional microbiological culture techniques. Faecal specimens (0.5 g in 4.5 mL PBS) were serially diluted in PBS for quantitative bacterial investigations. Dilutions were tested for total aerobic cell numbers developing on sheep blood agar (Oxoid, Germany), Gram negative cell numbers on Gassner agar (SIFIN, Berlin), enterococci on Citrate azide tween carbonate agar (CATC agar, SIFIN, Berlin), total anaerobe cell numbers on sheep blood agar (OXOID, Germany), Lactobacilli on deMan, Rogosa and Sharpe Lactobacillus agar (MRSA agar, SIFIN, Berlin), Bacteroides spp., on sheep blood agar supplemented with vitamin K, Clostridium perfringens on sheep blood agar containing polymyxin B and neomycin, and yeasts and fungi on Sabouraud agar (SIFIN, Berlin). The total aerobic cell numbers, Gram negative cell numbers and enterococci were cultured aerobically at 37 C for 24 h. The total anaerobic cell numbers, Lactobacilli and Bacteriodes were cultured at 37 C for 48 h in anaerobic chamber. To differentiate the total anaerobic cells number from aerobic cells, samples were cultivated on sheep blood agar in which one plate is cultivated aerobically and the second is incubated anaerobically. Yeasts and fungi were cultured aerobically at 37 C for 5 days.
Cows at 16 farms, located in Saxony, Thuringia, SaxonyeAnhalt and Schleswig Holstein, Germany were investigated to update the epidemiological situation of C. botulinum during the period 2008e 2010. Cows of fourteen farms showed clinical signs of botulism while cows in 2 farms (designated GZ and K) were apparently healthy. 2.2. Samples A total of 484 faecal specimens collected from the 16 farms were tested for C. botulinum. Faecal specimens (about 100 g per animal) were taken from Ampulla recti. All specimens were collected within three to 14 days after giving birth. Five farms (GH, R and E were diseased and had the highest percentages of botulinum positive cows; GZ and K were apparently healthy and had the lowest detection rate of botulinum positive cows) from the 16 farms were selected for further investigation (Table 1). Rumen fluids, taken orally using a pumped stomach tube, were tested for protozoa and C. botulinum. Faeces and rumen fluid samples from the selected 5 farms were further investigated by conventional microbiological culture techniques and fluorescence in situ hybridization (FISH), respectively. 2.3. Indirect detection of C. botulinum The presence of the BoNT upon incubation of faecal and rumen specimens was considered predictive of the presence of the bacterium C. botulinum. Faecal specimens and rumen fluids were tested for C. botulinum by diluting samples 1:10 (0.5 g or 0.5 ml
2.4.2. Fluorescence in situ hybridization (FISH) of rumen fluid A total of 182 rumen fluid samples were investigated using FISH test. Briefly, samples were fixed in ice-cold ethanol (1:1, v/v), methanol (1:1, v/v) and fresh paraformaldehyde (1:3, v/v) as described previously [18,19] and hybridized using fluorophore (indocarbocyanin Cy3) labelled 16S/23S rRNA-targeted oligonucleotide probes on silanized microscope slides [20]. The following probes were used in this study: bacteria (Eub338) [21], Archea
Table 1 Clostridium botulinum types in faeces and rumen fluid of cows at five German dairy farms. Farm
Animal number
Health status
GH
700
Diseased
59
25
R
380
Diseased
89
88
E
550
Diseased
25
57
GZ
652
Healthy
30
30
K
280
Healthy
35
25
238
225
Total
Sampling Faeces
C. botulinum in ruminal fluid
C. botulinum
C. botulinum faeces A
B
C
D
E
A
B
C
D
E
Positive Suspected Positive Suspected Positive Suspected Positive Suspected Positive Suspected Positive Suspected
1 4 6 12 0 3 0 7 0 7 7 (2.9% 33 (13.9%)
0 0 0 0 0 0 0 0 0 0 0 0
2 2 1 5 0 0 0 1 1 2 4 (1.6%) 10 4.2%
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 1 1 23 2 0 0 24 (10.1%) 3 (1.2%)
2 5 13 22 1 0 0 0 0 5 16 (7.1%) 32 (14.2%)
0 0 2 1 1 0 0 0 0 0 3 (1.3%) 1 (0.4%)
1 0 1 0 1 0 0 1 0 0 3 (1.3%) 1 (0.4%)
0 0 0 1 2 0 0 0 0 0 2 (0.8%) 1 (0.4%)
0 0 0 0 0 0 0 0 0 0 0 0
Rumen fluid
Positive (BoNT concentration in enrichment culture of faeces or rumen fluids 10 RU/g or mL). Suspected (BoNT concentration in enrichment culture of faeces or rumen fluids < 10 RUg or mL).
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(Arch915) [22], Euryarcheota (Eury496) [23], BacteroidesePrevotella-group (Bac 303) [24], CytophagaeFlavobactereBacteroides (Cfb286) [25], Bacteroides fragilis group (Bfra602) [24], Bacteroides distasonis (Bdis656) [26], C. histolyticum group (Chis150) [24], C. lituseburense group (Clit135) [24], C. leptum group (Clept) [24], Eubacterium rectale group (Erec) [24], Lactobacillus (Lab158) [24], sulfate-reducing bacteria (Srb385) [27], Alphaproteobacteria (Alf1b) [28], and Gammaproteobacteria (Gam42a) [28]. To increase permeability of the cell envelope for Chis, Lab, Erec, Eub and Srb probes, samples were treated with 10 ml lysozyme buffer (100 mM TriseHCl at pH 8.0, 50 mM EDTA, and 130,000 U lysozyme, Boehringer, Mannheim, Germany) and incubated at 37 C for 30 min in a moist chamber. If more stringent conditions were needed, formamide was added to the hybridization buffer from 5 to 40% (v/v). Cells were counterstained with 40 , 6-diamidino-2-phenylindole and counted visually using a Zeiss Axioskopepi fluorescence microscope (Axioskop, Carl Zeiss, Germany).
were botulinum positive, 36 (15.1%) botulinum suspected and 171 (81.1%) botulinum negative. Some samples were positive for more than one type of C. botulinum. The percentages of positive C. botulinum in diseased farms were 2.9%, 0%, 1.6%, 0% and 10.1%, for types A, B, C, D and E, respectively, (Table 1), however, the percentages of suspected animals were 13.9%, 0%, 4.2%, 0% and 1.2%, respectively. Surprisingly, C. botulinum was also detected in the apparently healthy farms (GZ and K) (Table 1). C. botulinum was also detected in the rumen fluid samples collected from the selected five farms (R, E, GH, GZ and K). Out of 225 rumen fluid samples, 21 (9.3%) were botulinum positive (some samples were positive for more than one type of C. botulinum) and 35 (15.5%) were suspected. The dominant C. botulinum in rumen fluids was type A. The percentages of positive C. botulinum types A, B, C, D and E were 7.1%, 1.3%, 1.3% and 0.8%, and 0%, respectively, while the percentages of suspected positive animals were 14.2%, 0.4%, 0.4% and 0.4% and 0%, respectively.
2.5. Enumeration of protozoa in rumen fluids
3.2. Bacterial counts in faeces in relation to C. botulinum detection
Aliquots of rumen fluids were diluted 1:2 with methyl green formalin NaCl solution (0.6 g methyl green, 100 mL formalin, 8.0 g NaCl, 900 mL distilled water) and subsequently stored at 8 C until counting and microscopic differentiation. The fixed rumen fluid specimens were counted with a Sedgewick-Rafter counting chamber using a light microscope. Phenotypic criteria were used according to Irle [28] und Lynn [29]. 2.6. Statistical analysis The statistical analysis was carried out with GraphPad Prism 4 (GaphPad Software, La Jolla, USA). Two-way analysis of variance followed by unpaired Student t-test was used to identify significant differences between means. 3. Results 3.1. C. botulinum detection in dairy cows German cows at 16 farms were investigated for visceral botulism. Cows at fourteen farms showed clinical signs of botulism including peripartual indigestion (constipation alternating with diarrhea), postnatal downer cows, acute laminitis, ataxia and stiff stilted gait, paralysis (unable to get up), apathy, engorged veins, positive venous pulse, oedema in legs, udder and dew-lap, retracted abdomen, forced respiration and sudden death. The cows demonstrated clinical symptoms such as mobility disorders, swollen joints, suppurations of skin wounds, viscous saliva, and droopy heads and tails. The behaviour of diseased animals was very listless, without agility and curiosity. On marginally diseased farms, symptoms were milder but there were some downer cows and reduction of milk production. Cows at GZ and K, were apparently healthy. Of the 14 diseased farms and the 2 apparently healthy farms tested for the presence of C. botulinum, 74 of the 484 faeces (15.3%) were definitely positive (BoNT concentration in enrichment culture of faeces 10 RU g1) and 123 (25.4%) were suspected positive (BoNT concentration in enrichment culture of faeces < 10 RU g1) for one of the C. botulinum types A, B, C, D and E based on the detected BoNT type. In this study, the dominant C. botulinum in bovine faeces was type E. The percentages of positive C. botulinum types A, B, C, D and E were 2.5%, 0.4%, 1.8%, 1.2% and 15.3%, respectively. Five farms (R, E, GH, GZ and K) were selected for further investigations (Table 1). Out of 238 faecal samples, 31 (13%) samples
The detection of C. botulinum in faeces was associated with changes in the faecal microbiota (Fig. 1). There was a significant reduction of total aerobic bacteria (p < 0.0001), total anaerobic bacteria (p < 0.02), yeast and fungi (p < 0.001), Enterococci (p < 0.04) and C. perfringens (p < 0.03) in botulinum positive cows (N ¼ 31). Botulinum suspected animals (N ¼ 36) had also significantly lower total aerobic bacteria (P < 0.01), yeast and fungi (p < 0.007) and C. perfringens (p < 0.01). On the other hand, there were no significant differences in total Gram negative bacteria, Bacteriodes spp. and Lactobacillus spp. between botulinum positive and botulinum negative (N ¼ 171) animals. 3.3. Bacterial counts in rumen fluids in relation to C. botulinum detection Also the detection of C. botulinum in rumen fluids was associated with changes in the rumen fluids microbiota (Fig. 1 and Table 2). C. botulinum positive rumen fluid samples had a significantly higher population of Archea (p < 0.003), BacteroidesePrevotella-group (p < 0.0001), B. fragilis group (p < 0.0001), B. distasonis (p < 0.005), C. histolyticum group (p < 0.001), Lactobacillus (p < 0.0001), sulfatereducing-bacteria (p < 0.0004), Alphaproteobacteria (p < 0.0001), Gammaproteobacteria (p < 0.0003), along with a significant decrease of Euryarcheota (p < 0.003). There were no significant changes of CytophagaeFlavobactereBacteroides, C. lituseburense group, C. leptum group and Eubacterium rectale group in botulinum positive (N ¼ 21) and botulinum negative (N ¼ 64) rumen fluids (Table 2). 3.4. Protozoa in rumen fluids The Entotodinium spp., Epidinium spp., Isotricha spp., Dasytricha spp., Diplodiniinae spp., and Ophryoscolex spp. were detected in rumen fluids (Fig. 2). In botulinum positive rumen fluids (N ¼ 20), there was a significant reduction of Epidinium spp. (p < 0.001), Dasytricha spp. (p < 0.02), Diplodiniinae spp. (p < 0.01) and Ophryoscolex spp. (p < 0.0001) compared with botulinum negative samples (N ¼ 140). Also botulinum suspected rumen fluids (N ¼ 32) had a significant reduction of Epidinium spp. (p < 0.01), Dasytricha spp. (p < 0.01), Diplodiniinae spp. (p < 0.01) and Ophryoscolex spp. (p < 0.0001) compared with botulinum negative samples (Fig. 3). However, there were no significant changes of Entotodinium spp. and Isotricha spp. between botulinum positive and botulinum suspected rumen fluids.
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Fig. 1. Distribution of bacteria and yeast in faeces of botulinum positive (N ¼ 31) botulinum negative (N ¼ 171) and botulinum suspected cows (N ¼ 36).
4. Discussion There has been a clear increase in cases of botulism on dairy farms without a known reason since the mid-1990s. Two forms of the disease are known in cattle: food-born botulism from ingestion of preformed neurotoxins and visceral or chronic botulism with ingestion of vegetative forms and/or spores of C. botulinum [2,16]. The source of the spores is the environment and ingested spores may germinate in the intestinal tract of animals which lack a protective indigenous bacterial flora [16]. The present study was initiated to update the distribution of C. botulinum in German and to study the relationship between botulism and members of the intestinal microbiota. In our study, the dominant C. botulinum in bovine faeces in Germany was type E based on the detected BoNT type. In contrast, Table 2 FISH test results of C. botulinum positive (N ¼ 21) and negative (N ¼ 64) rumen fluids of cows. BoNTþ
Eub Arch Eury Bac Cfb Bfra Bdis Chis Clit Clept Erec Lab Srb Alf1b Gam42a
BoNT
P value
Mean
SD
Mean
SD
2,Eþ08 2,Eþ07 7,Eþ06 3,Eþ07 2,Eþ05 3,Eþ06 1,Eþ07 4,Eþ05 1,Eþ05 1,Eþ07 1,Eþ07 2,Eþ07 9,Eþ06 7,Eþ06 5,Eþ06
3,Eþ08 1,Eþ07 7,Eþ06 4,Eþ07 1,Eþ05 3,Eþ06 1,Eþ07 6,Eþ05 4,Eþ03 1,Eþ07 8,Eþ06 2,Eþ07 9,Eþ06 1,Eþ07 5,Eþ06
3,Eþ08 8,Eþ06 2,Eþ07 2,Eþ06 4,Eþ05 5,Eþ05 7,Eþ06 2,Eþ05 2,Eþ05 2,Eþ07 1,Eþ07 4,Eþ06 3,Eþ06 4,Eþ05 2,Eþ06
3,Eþ08 1,Eþ07 2,Eþ07 5,Eþ06 7,Eþ05 9,Eþ05 7,Eþ06 2,Eþ05 4,Eþ05 2,Eþ07 1,Eþ07 5,Eþ06 6,Eþ06 1,Eþ06 3,Eþ06
No significant 0.003 0.03 0.0001 No significant 0.0001 0.005 0.001 No significant No significant No significant 0.0001 0.0004 0.0001 0.0003
Eub ¼ total bacteria, Arch ¼ Archea, Eury ¼ Euryarcheota, Bac ¼ BacteroidesePrevotella-group, Cfb ¼ CytophagaeFlavobactereBacteroides, Bfra ¼ Bacteroidesfragilis group, Bdis ¼ Bacteroidesdistasonis, Chis ¼ C. histolyticum group, Clit ¼ C. lituseburense group, Clept ¼ C. leptum group, Erec ¼ Eubacteriumrectale, Lab ¼ Lactobacillus, Srb ¼ sulfate-reducing bacteria, Alf1b ¼ Alpha proteobacteria, and Gam42a ¼ Gamma proteobacteria.
the dominant C. botulinum in rumen fluids was type A. Widespread exposure of C. botulinum in Germany farms was found. Even on the apparently healthy farms (GZ and K). C. botulinum types C and E were also detected in faeces (Table 1). Although human cases are mostly caused by types A, B, or E and animal diseases are mostly caused by types C and D [30,2], dairy cows are considered a carrier for all types. In order to investigate the link between dysbiosis and botulism, faeces and rumen fluid samples were tested for different bacteria and protozoa. The enumeration of bacteria in faeces and rumen fluid showed significant dysbiosis in these substrates. Populations of Enterococci and C. perfringens and yeast and fungi were significantly lower in C. botulinum positive and suspected cows. Enterococci are important bacterial antagonists to C. botulinum in the gastrointestinal tract [31,32]. This could be due to a bacteriocin effect [32], hence bacteriocin produced from lactic acid bacteria mediated inhibition of C. botulinum. Sullivan and coworkers [33] thought that the inhibition of C. botulinum spores or vegetative cells indicated that the normal flora of healthy individuals had a bacteriostatic, rather than a bactericidal effect on the growth of C. botulinum and concluded that infant botulism may result, in part, from the absence of inhibitory organisms in the normal flora of the infant intestine. On the other hand, C. botulinum positive rumen fluid samples had significantly higher population of Archea, BacteroidesePrevotella-group, B. fragilis group, B. distasonis, C. histolyticum group, Lactobacillus, sulfur reducing bacteria (SRB), Alphaproteobacteria, Gammaproteobacteria, along with a significant decrease of Euryarcheota. The release of sulfate from mucine degradation by Clostridia, Bifidobacteria and B. fragilis to produce disulfide results from utilization of H2 accumulating in rumen due to fermentation. Clostridia and Enterobacteria produce H2 by reduction of pyruvate [15,31,34]. Consequently, SRB use H2 to produce H2S, a very toxic gas that damages the epithelium of the gastrointestinal tract. In other studies, Clostridium difficile infected patients had a high population of facultative anaerobes and low levels of Bifidobacterium and Bacteriodes [35,36]. More recently, C. difficile infections have been associated with changes in the gut microbiota which allows C. difficile to multiply and colonize the gut [37e39]. Fallani
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Fig. 2. Micromorphology of important rumen protozoa; 1 ¼ Total protozoa, 2 ¼ Entotodinium spp., 3 ¼ Epidinium spp., 4 ¼ Isotricha spp., 5 ¼ Dasytricha spp., 6 ¼ Diplodiniinae, and 7 ¼ Ophryoscolex spp.
and coworkers [40] observed that infants with detectable proportions of C. difficile had a lower percentage of Bifidobacteria and higher proportions of Bacteriodes spp. Also, the C. histolyticum subgroup in rumen fluid of C. botulinum positive cows was significantly higher demonstrating an increase of pathogenic Clostridia. Vijay and co-workers [41] found an association between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota in animals with nosocomial diarrhea, including C. difficile infection.
The rumen of ruminants contains a mixed population of ciliate protozoal species. In our study, Entotodinium spp., Epidinium spp., Isotricha spp., Dasytricha spp., Diplodiniinae spp, and Ophryoscolex spp. were detected in rumen fluids. Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. were significantly lower in C. botulinum positive rumen fluid. A typical protozoal population is able to breakdown w17% of available rumen bacteria every h [13]. However, the amount of bacterial degraded by protozoa depends on the protozoal group. Diplodiniinae spp. and Holotriches
Fig. 3. Distribution of protozoa in rumen fluid of botulinum positive (N ¼ 20), botulinum suspected (N ¼ 32) and botulinum negative (N ¼ 140) cows.
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(Dasytricha spp. and Isotricha spp.) have the greatest and least activity, respectively. Newbold and coworkers [42] reported that bacterial degradation occurs only in intact rumen ecosystems. For example, Listeria monocytogens was degraded 25% in three hours. In contrast, no degradation of pathogenic bacteria occurred in protozoa free rumen fluid and the microbiota were dominated by the C. botulinum cluster. Rumen fluids with intact microbiota have a large capacity for proteolytic inactivation of BoNT/C [43]. Ozutsumi and coworkers [44] showed that rumen protozoa influence the population and composition of its microbiota. The causes of this dysbiosis could be rumen acidosis but also could be due to contamination with antimicrobially active substances in their feed. Oral antibiotic treatment in dairy cows is very rare; however, glyphosate, the active substance of the systematic herbicide “RoundupÒ” is a common contaminant in feed and a potent antimicrobial agent that affects different bacteria differentially. Enterococci, Lactobacillus and Bifidobacteria are highly susceptible to glyphosate while C. botulinum is highly resistant [15,31]. High concentrations of this selective antimicrobial in feed occurred about the same time as its herbicidal use increased for genetically engineered herbicide-tolerant crops and as a crop desiccant. Further investigation of the impact of this herbicide and other dysbiotic agents on GI tract microbiota are necessary. C. botulinum is detectable in faeces and rumen fluids of dairy cows in Germany. The incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Such dysbiotic conditions in the gastrointestinal tract could be responsible for colonization by C. botulinum bacteria. However the chronology of these events and role of each microbial group needs further evaluation.
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Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 1992;15:593e600. [23] Jurgens G, Glöckner FO, Amann R, Saano A, Montonen L, Likolammi M, et al. Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization. FEMS Microbiol Ecol 2000;34:45e56. [24] Franks AH, Harmsen HJ, Raangs GC, Jansen GJ, Schut F, Welling GW. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA targeted oligonucleotide probes. Appl Environ Microbiol 1998;64:3336e45. [25] Weller R, Glöckner FO, Amann R. 165 rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum CytophagaFlavobacterium-Bacteroides system. Appl Microbiol 2000;23:107e14. [26] Suau A, Rochet V, Sghir A, Gramet G, Brewaeys S, Sutren M, et al. Fusobacterium prausnitzii and related species represent a dominant group within the human fecal flora. Syst Appl Microbiol 2001;24:139e45. [27] Amann R, Stromley J, Devereux R, Key R, Stahl DA. Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl Environ Microbiol 1992;58:614e23. [28] Irle A. Untersuchungen zum Einfluss von Clostridiengaben bei Grassilagen mit auffällig niedrigen Reineiweißanteilen auf die Pansenfermentation (in vitro). Dissertation (Hannover); 2011. [29] Lynn DH. The ciliated protozoa. Characterization, classification and guide to the literature. 3rd ed. Netherlands: Kluwer Academic Publishers; 2008. [30] Foran PG, Mohammed N, Lisk GO, Nagwaney S, Lawrence GW, Johnson E. Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E, and F compared with the long lasting type A. Biol Chem 2003;278:1363e71. [31] Shehata A, Schrödl W, Neuhaus J, Krüger M. Antagonistic effect of different bacteria on Clostridium botulinum types A, B, D and E in vitro. Veterin Rec 2013;172(2):47. [32] Okereke JD, Montville PR. Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures. Appl Environ Microbiol 1991;57:3423e8. [33] Sullivan NM, Mills DC, Riepmann HP, Arnon SS. Inhibition of growth of Clostridium botulinum by intestinal microflora isolated from healthy infants. Microb Ecol Health Dis 1988;1:179e92. [34] Gibson GR, Macfarlane GT, Cummings JH. Sulphate reducing bacteria and hydrogen metabolism in the human large intestine. Gut 1993;34:437e9. [35] Zinkevich VV, Beech IB. Screening of sulfate-reducing bacteria in colonoscopy samples from healthy and colitic human gut mucosa. FEMS Microbiol Ecol 2000;34:147e55. [36] Hopkins MJ, Macfarlane GT. Changes in predominant bacterial populations in human faeces with age and with Clostridium difficile infection. J Med Microbiol 2002;51:448e54. [37] Wilcox M, Minton J. Role of antibody response in outcome of antibiotic e associated diarrhea. Lancet 2001;357:158e9. [38] Rousseau C, Levenez F, Fouqueray C, Doré J, Collignon A, Lepage P. Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition. J Clin Microbiol 2011;49(3):858e65. [39] Antharam VC, Li EC, Ishmael A, Sharma A, Mai V, Rand KH, et al. Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J Clin Microbiol 2013;51(9):2884e92. [40] Fallani M, Rigottier-Gois L, Aguilera M, Bridonneau C, Collignon A, Edwards CA, et al. Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods 2006;67:150e61. [41] Vijay C, Antharam EC, Li AI, Anuj S, Volker M, Rand H, et al. Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial Diarrhea. J Clin Microbiol; 2013:2884e92. [42] Newbold CJ, Stewart CS, Wallace RJ. Developments in rumen fermentation e the scientist’s view. In: Garnsworthy PC, Wiseman J, editors. Recent advances in animal nutrition. Nottingham: Nottingham University Press; 2001. pp. 251e79. [43] Allison MJ, Maloy SE, Matson RR. Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep. Appl Environ Microbiol 1976;32: 685e8. [44] Ozutsumi Y, Tajima K, Takenaka A, Itabashi H. The effect of protozoa on the composition of rumen bacteria in cattle using 16S rRNA gene clone libraries. Biosci Biotechnol Biochem 2005;69:499e506.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and toxins
Relationship between gastrointestinal dysbiosis and Clostridium botulinum in dairy cows Monika Krüger a, Awad A. Shehata*, a, b, Anke Grosse-Herrenthey a, Norman Ständer a, Wieland Schrödl a a b
Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2013 Received in revised form 25 March 2014 Accepted 27 March 2014 Available online 16 April 2014
The gastrointestinal tract is a balanced ecosystem that can get out of balance and predisposed to clostridial diseases or other pathological conditions. The objective of the present study was to evaluate the gut microbiota in dairy cows suffering from chronic botulism. Cows were investigated for Clostridium (C.) botulinum in faeces and rumen fluids. In order to study the relationship between botulism and gastrointestinal microbiota, faeces and rumen fluid were tested for bacterial composition using conventional microbiological culture techniques and fluorescence in situ hybridization (FISH). Protozoa were analyzed in rumen fluid microscopically. The presence of C. botulinum was associated with specific changes in the faecal microbiota, especially a significant reduction of total aerobic bacteria, total anaerobic bacteria, enterococci, Clostridium perfringens and yeast and fungi. Also C. botulinum positive rumen fluid had significantly more Bacteroides spp., C. histolyticum group, Alfa- proteobacteria, Gammaproteobacteria, and sulfate-reducing bacteria; as well as significantly fewer Euryaracheota, and the protozoa Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. In conclusion, C. botulinum is common in dairy cows in Germany but the incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Bacteria, yeast and protozoa appear to be crucial in the colonization process; however, the chronology of these events and role of each microbial group needs further evaluation. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: C. botulinum Dysbiosis Gut microbiota BoNT Protozoa
1. Introduction The bacterium Clostridium botulinum is widespread in the environment. It can be found in soil, dust, manure, slaughter-house wastes, and residue of biogas plants, bio-compost and mud. The gastrointestinal tract of humans and animals is also a habitat for this agent. So far, seven serologically different botulinum neurotoxin (BoNT) types (AeG) are known that block the release of acetylcholine at the neuromuscular junctions. The BoNT may be taken up orally if preformed in food or feed (intoxication) [1e5]. In addition to the feed-borne diseases in domestic animals, another form of botulism is reported in cattle, called “visceral botulism” or “chronic botulism”. This form of botulism is thought to be caused by colonization of the lower intestine with C. botulinum and the subsequent production of BoNTs [2,5]. The presumed cause of clostridial colonization in the intestinal tract is microbial imbalance in * Corresponding author. Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103 Leipzig, Germany. Tel.: þ49 03419738183; fax: þ49 03419738199. E-mail address: [email protected] (A.A. Shehata). http://dx.doi.org/10.1016/j.anaerobe.2014.03.013 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.
the digestive tract accompanied by replication and toxin production as part of a multifactorial disease. Gut microbiota have continuous communication with host cells and form long-lasting, interactive associations with their host. These associations play a critical role in conservation of mucosal immune function, epithelial barrier integrity, motility, and nutrient absorption [6e8]. Under normal conditions, gut microbiota display a symbiotic relationship with the host to contribute to its intestinal health; however, a disturbance in normal microbiota of the intestinal tract can lead to an imbalance of hoste microbe relationships, known as “dysbiosis” [9]. Disturbances within this system by feed, heavy metals, toxic substances, bacterial toxins antibiotics, etc. can lead to localized inflammation, extensive infection or intoxication [10e12]. Protozoa are an important part of the rumen microbiota. They serve to reduce bacterial populations in the rumen and any changes in rumen bacteria influence their population [13]. Rumen protozoa represent an appreciable proportion of the rumen biomass [14] and have much longer generation time than bacteria (8e36 h vs. about 20 min). They depend on rumen bacterial support for vitamin B complex and vitamin K.
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Little is known about the role of immunological homeostasis in the gastrointestinal tract of cattle. It is postulated that under dysbiotic conditions, facultative pathogenic bacteria are able to induce disease because otherwise antagonistic bacterial populations fail to suppress them [15]. Thus, we hypothesize that colonisation and neurotoxin generation by C. botulinum in the gastrointestinal tract of cows also requires this precondition. In the last 15 years, C. botulinum associated chronic diseases have increased in Germany without known causes. The objective of this study was to shed light on the distribution of C. botulinum in dairy cows and its relationship to the microbial (bacteria, protozoa) composition of gut and rumen fluid.
with 4.5 mL) in reinforced clostridial medium (RCM; SIFIN, Berlin, Germany), vigorously mixing, heating at 80 C for 10 min and incubating at 37 C for 7 d in anaerobic chamber (MACS anaerobic Workstation, Don Whitley Scientific Limited, West Yorkshire, England) before subsequent storage at 25 C until analysed. After thawing, the culture samples were centrifuged at 7000 g for 15 min and the clear supernatant was analysed for type specific BoNTs/AeE with polyclonal antibodies using ELISA [16]. The relative units (RUs) of BoNTs were calculated with the measured optical densities (OD) values as follow: (sample OD minus twice the value of the control OD [BoNT negative sample of bovine faeces]) multiplied by dilution factors [17].
2. Materials and methods
2.4. Bacterial enumeration in faeces and rumen fluid
2.1. Animals
2.4.1. Bacterial cultivation A total of 237 faecal samples collected from the selected 5 farms (GH, R, E, GZ, and K) were investigated by conventional microbiological culture techniques. Faecal specimens (0.5 g in 4.5 mL PBS) were serially diluted in PBS for quantitative bacterial investigations. Dilutions were tested for total aerobic cell numbers developing on sheep blood agar (Oxoid, Germany), Gram negative cell numbers on Gassner agar (SIFIN, Berlin), enterococci on Citrate azide tween carbonate agar (CATC agar, SIFIN, Berlin), total anaerobe cell numbers on sheep blood agar (OXOID, Germany), Lactobacilli on deMan, Rogosa and Sharpe Lactobacillus agar (MRSA agar, SIFIN, Berlin), Bacteroides spp., on sheep blood agar supplemented with vitamin K, Clostridium perfringens on sheep blood agar containing polymyxin B and neomycin, and yeasts and fungi on Sabouraud agar (SIFIN, Berlin). The total aerobic cell numbers, Gram negative cell numbers and enterococci were cultured aerobically at 37 C for 24 h. The total anaerobic cell numbers, Lactobacilli and Bacteriodes were cultured at 37 C for 48 h in anaerobic chamber. To differentiate the total anaerobic cells number from aerobic cells, samples were cultivated on sheep blood agar in which one plate is cultivated aerobically and the second is incubated anaerobically. Yeasts and fungi were cultured aerobically at 37 C for 5 days.
Cows at 16 farms, located in Saxony, Thuringia, SaxonyeAnhalt and Schleswig Holstein, Germany were investigated to update the epidemiological situation of C. botulinum during the period 2008e 2010. Cows of fourteen farms showed clinical signs of botulism while cows in 2 farms (designated GZ and K) were apparently healthy. 2.2. Samples A total of 484 faecal specimens collected from the 16 farms were tested for C. botulinum. Faecal specimens (about 100 g per animal) were taken from Ampulla recti. All specimens were collected within three to 14 days after giving birth. Five farms (GH, R and E were diseased and had the highest percentages of botulinum positive cows; GZ and K were apparently healthy and had the lowest detection rate of botulinum positive cows) from the 16 farms were selected for further investigation (Table 1). Rumen fluids, taken orally using a pumped stomach tube, were tested for protozoa and C. botulinum. Faeces and rumen fluid samples from the selected 5 farms were further investigated by conventional microbiological culture techniques and fluorescence in situ hybridization (FISH), respectively. 2.3. Indirect detection of C. botulinum The presence of the BoNT upon incubation of faecal and rumen specimens was considered predictive of the presence of the bacterium C. botulinum. Faecal specimens and rumen fluids were tested for C. botulinum by diluting samples 1:10 (0.5 g or 0.5 ml
2.4.2. Fluorescence in situ hybridization (FISH) of rumen fluid A total of 182 rumen fluid samples were investigated using FISH test. Briefly, samples were fixed in ice-cold ethanol (1:1, v/v), methanol (1:1, v/v) and fresh paraformaldehyde (1:3, v/v) as described previously [18,19] and hybridized using fluorophore (indocarbocyanin Cy3) labelled 16S/23S rRNA-targeted oligonucleotide probes on silanized microscope slides [20]. The following probes were used in this study: bacteria (Eub338) [21], Archea
Table 1 Clostridium botulinum types in faeces and rumen fluid of cows at five German dairy farms. Farm
Animal number
Health status
GH
700
Diseased
59
25
R
380
Diseased
89
88
E
550
Diseased
25
57
GZ
652
Healthy
30
30
K
280
Healthy
35
25
238
225
Total
Sampling Faeces
C. botulinum in ruminal fluid
C. botulinum
C. botulinum faeces A
B
C
D
E
A
B
C
D
E
Positive Suspected Positive Suspected Positive Suspected Positive Suspected Positive Suspected Positive Suspected
1 4 6 12 0 3 0 7 0 7 7 (2.9% 33 (13.9%)
0 0 0 0 0 0 0 0 0 0 0 0
2 2 1 5 0 0 0 1 1 2 4 (1.6%) 10 4.2%
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 1 1 23 2 0 0 24 (10.1%) 3 (1.2%)
2 5 13 22 1 0 0 0 0 5 16 (7.1%) 32 (14.2%)
0 0 2 1 1 0 0 0 0 0 3 (1.3%) 1 (0.4%)
1 0 1 0 1 0 0 1 0 0 3 (1.3%) 1 (0.4%)
0 0 0 1 2 0 0 0 0 0 2 (0.8%) 1 (0.4%)
0 0 0 0 0 0 0 0 0 0 0 0
Rumen fluid
Positive (BoNT concentration in enrichment culture of faeces or rumen fluids 10 RU/g or mL). Suspected (BoNT concentration in enrichment culture of faeces or rumen fluids < 10 RUg or mL).
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(Arch915) [22], Euryarcheota (Eury496) [23], BacteroidesePrevotella-group (Bac 303) [24], CytophagaeFlavobactereBacteroides (Cfb286) [25], Bacteroides fragilis group (Bfra602) [24], Bacteroides distasonis (Bdis656) [26], C. histolyticum group (Chis150) [24], C. lituseburense group (Clit135) [24], C. leptum group (Clept) [24], Eubacterium rectale group (Erec) [24], Lactobacillus (Lab158) [24], sulfate-reducing bacteria (Srb385) [27], Alphaproteobacteria (Alf1b) [28], and Gammaproteobacteria (Gam42a) [28]. To increase permeability of the cell envelope for Chis, Lab, Erec, Eub and Srb probes, samples were treated with 10 ml lysozyme buffer (100 mM TriseHCl at pH 8.0, 50 mM EDTA, and 130,000 U lysozyme, Boehringer, Mannheim, Germany) and incubated at 37 C for 30 min in a moist chamber. If more stringent conditions were needed, formamide was added to the hybridization buffer from 5 to 40% (v/v). Cells were counterstained with 40 , 6-diamidino-2-phenylindole and counted visually using a Zeiss Axioskopepi fluorescence microscope (Axioskop, Carl Zeiss, Germany).
were botulinum positive, 36 (15.1%) botulinum suspected and 171 (81.1%) botulinum negative. Some samples were positive for more than one type of C. botulinum. The percentages of positive C. botulinum in diseased farms were 2.9%, 0%, 1.6%, 0% and 10.1%, for types A, B, C, D and E, respectively, (Table 1), however, the percentages of suspected animals were 13.9%, 0%, 4.2%, 0% and 1.2%, respectively. Surprisingly, C. botulinum was also detected in the apparently healthy farms (GZ and K) (Table 1). C. botulinum was also detected in the rumen fluid samples collected from the selected five farms (R, E, GH, GZ and K). Out of 225 rumen fluid samples, 21 (9.3%) were botulinum positive (some samples were positive for more than one type of C. botulinum) and 35 (15.5%) were suspected. The dominant C. botulinum in rumen fluids was type A. The percentages of positive C. botulinum types A, B, C, D and E were 7.1%, 1.3%, 1.3% and 0.8%, and 0%, respectively, while the percentages of suspected positive animals were 14.2%, 0.4%, 0.4% and 0.4% and 0%, respectively.
2.5. Enumeration of protozoa in rumen fluids
3.2. Bacterial counts in faeces in relation to C. botulinum detection
Aliquots of rumen fluids were diluted 1:2 with methyl green formalin NaCl solution (0.6 g methyl green, 100 mL formalin, 8.0 g NaCl, 900 mL distilled water) and subsequently stored at 8 C until counting and microscopic differentiation. The fixed rumen fluid specimens were counted with a Sedgewick-Rafter counting chamber using a light microscope. Phenotypic criteria were used according to Irle [28] und Lynn [29]. 2.6. Statistical analysis The statistical analysis was carried out with GraphPad Prism 4 (GaphPad Software, La Jolla, USA). Two-way analysis of variance followed by unpaired Student t-test was used to identify significant differences between means. 3. Results 3.1. C. botulinum detection in dairy cows German cows at 16 farms were investigated for visceral botulism. Cows at fourteen farms showed clinical signs of botulism including peripartual indigestion (constipation alternating with diarrhea), postnatal downer cows, acute laminitis, ataxia and stiff stilted gait, paralysis (unable to get up), apathy, engorged veins, positive venous pulse, oedema in legs, udder and dew-lap, retracted abdomen, forced respiration and sudden death. The cows demonstrated clinical symptoms such as mobility disorders, swollen joints, suppurations of skin wounds, viscous saliva, and droopy heads and tails. The behaviour of diseased animals was very listless, without agility and curiosity. On marginally diseased farms, symptoms were milder but there were some downer cows and reduction of milk production. Cows at GZ and K, were apparently healthy. Of the 14 diseased farms and the 2 apparently healthy farms tested for the presence of C. botulinum, 74 of the 484 faeces (15.3%) were definitely positive (BoNT concentration in enrichment culture of faeces 10 RU g1) and 123 (25.4%) were suspected positive (BoNT concentration in enrichment culture of faeces < 10 RU g1) for one of the C. botulinum types A, B, C, D and E based on the detected BoNT type. In this study, the dominant C. botulinum in bovine faeces was type E. The percentages of positive C. botulinum types A, B, C, D and E were 2.5%, 0.4%, 1.8%, 1.2% and 15.3%, respectively. Five farms (R, E, GH, GZ and K) were selected for further investigations (Table 1). Out of 238 faecal samples, 31 (13%) samples
The detection of C. botulinum in faeces was associated with changes in the faecal microbiota (Fig. 1). There was a significant reduction of total aerobic bacteria (p < 0.0001), total anaerobic bacteria (p < 0.02), yeast and fungi (p < 0.001), Enterococci (p < 0.04) and C. perfringens (p < 0.03) in botulinum positive cows (N ¼ 31). Botulinum suspected animals (N ¼ 36) had also significantly lower total aerobic bacteria (P < 0.01), yeast and fungi (p < 0.007) and C. perfringens (p < 0.01). On the other hand, there were no significant differences in total Gram negative bacteria, Bacteriodes spp. and Lactobacillus spp. between botulinum positive and botulinum negative (N ¼ 171) animals. 3.3. Bacterial counts in rumen fluids in relation to C. botulinum detection Also the detection of C. botulinum in rumen fluids was associated with changes in the rumen fluids microbiota (Fig. 1 and Table 2). C. botulinum positive rumen fluid samples had a significantly higher population of Archea (p < 0.003), BacteroidesePrevotella-group (p < 0.0001), B. fragilis group (p < 0.0001), B. distasonis (p < 0.005), C. histolyticum group (p < 0.001), Lactobacillus (p < 0.0001), sulfatereducing-bacteria (p < 0.0004), Alphaproteobacteria (p < 0.0001), Gammaproteobacteria (p < 0.0003), along with a significant decrease of Euryarcheota (p < 0.003). There were no significant changes of CytophagaeFlavobactereBacteroides, C. lituseburense group, C. leptum group and Eubacterium rectale group in botulinum positive (N ¼ 21) and botulinum negative (N ¼ 64) rumen fluids (Table 2). 3.4. Protozoa in rumen fluids The Entotodinium spp., Epidinium spp., Isotricha spp., Dasytricha spp., Diplodiniinae spp., and Ophryoscolex spp. were detected in rumen fluids (Fig. 2). In botulinum positive rumen fluids (N ¼ 20), there was a significant reduction of Epidinium spp. (p < 0.001), Dasytricha spp. (p < 0.02), Diplodiniinae spp. (p < 0.01) and Ophryoscolex spp. (p < 0.0001) compared with botulinum negative samples (N ¼ 140). Also botulinum suspected rumen fluids (N ¼ 32) had a significant reduction of Epidinium spp. (p < 0.01), Dasytricha spp. (p < 0.01), Diplodiniinae spp. (p < 0.01) and Ophryoscolex spp. (p < 0.0001) compared with botulinum negative samples (Fig. 3). However, there were no significant changes of Entotodinium spp. and Isotricha spp. between botulinum positive and botulinum suspected rumen fluids.
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Fig. 1. Distribution of bacteria and yeast in faeces of botulinum positive (N ¼ 31) botulinum negative (N ¼ 171) and botulinum suspected cows (N ¼ 36).
4. Discussion There has been a clear increase in cases of botulism on dairy farms without a known reason since the mid-1990s. Two forms of the disease are known in cattle: food-born botulism from ingestion of preformed neurotoxins and visceral or chronic botulism with ingestion of vegetative forms and/or spores of C. botulinum [2,16]. The source of the spores is the environment and ingested spores may germinate in the intestinal tract of animals which lack a protective indigenous bacterial flora [16]. The present study was initiated to update the distribution of C. botulinum in German and to study the relationship between botulism and members of the intestinal microbiota. In our study, the dominant C. botulinum in bovine faeces in Germany was type E based on the detected BoNT type. In contrast, Table 2 FISH test results of C. botulinum positive (N ¼ 21) and negative (N ¼ 64) rumen fluids of cows. BoNTþ
Eub Arch Eury Bac Cfb Bfra Bdis Chis Clit Clept Erec Lab Srb Alf1b Gam42a
BoNT
P value
Mean
SD
Mean
SD
2,Eþ08 2,Eþ07 7,Eþ06 3,Eþ07 2,Eþ05 3,Eþ06 1,Eþ07 4,Eþ05 1,Eþ05 1,Eþ07 1,Eþ07 2,Eþ07 9,Eþ06 7,Eþ06 5,Eþ06
3,Eþ08 1,Eþ07 7,Eþ06 4,Eþ07 1,Eþ05 3,Eþ06 1,Eþ07 6,Eþ05 4,Eþ03 1,Eþ07 8,Eþ06 2,Eþ07 9,Eþ06 1,Eþ07 5,Eþ06
3,Eþ08 8,Eþ06 2,Eþ07 2,Eþ06 4,Eþ05 5,Eþ05 7,Eþ06 2,Eþ05 2,Eþ05 2,Eþ07 1,Eþ07 4,Eþ06 3,Eþ06 4,Eþ05 2,Eþ06
3,Eþ08 1,Eþ07 2,Eþ07 5,Eþ06 7,Eþ05 9,Eþ05 7,Eþ06 2,Eþ05 4,Eþ05 2,Eþ07 1,Eþ07 5,Eþ06 6,Eþ06 1,Eþ06 3,Eþ06
No significant 0.003 0.03 0.0001 No significant 0.0001 0.005 0.001 No significant No significant No significant 0.0001 0.0004 0.0001 0.0003
Eub ¼ total bacteria, Arch ¼ Archea, Eury ¼ Euryarcheota, Bac ¼ BacteroidesePrevotella-group, Cfb ¼ CytophagaeFlavobactereBacteroides, Bfra ¼ Bacteroidesfragilis group, Bdis ¼ Bacteroidesdistasonis, Chis ¼ C. histolyticum group, Clit ¼ C. lituseburense group, Clept ¼ C. leptum group, Erec ¼ Eubacteriumrectale, Lab ¼ Lactobacillus, Srb ¼ sulfate-reducing bacteria, Alf1b ¼ Alpha proteobacteria, and Gam42a ¼ Gamma proteobacteria.
the dominant C. botulinum in rumen fluids was type A. Widespread exposure of C. botulinum in Germany farms was found. Even on the apparently healthy farms (GZ and K). C. botulinum types C and E were also detected in faeces (Table 1). Although human cases are mostly caused by types A, B, or E and animal diseases are mostly caused by types C and D [30,2], dairy cows are considered a carrier for all types. In order to investigate the link between dysbiosis and botulism, faeces and rumen fluid samples were tested for different bacteria and protozoa. The enumeration of bacteria in faeces and rumen fluid showed significant dysbiosis in these substrates. Populations of Enterococci and C. perfringens and yeast and fungi were significantly lower in C. botulinum positive and suspected cows. Enterococci are important bacterial antagonists to C. botulinum in the gastrointestinal tract [31,32]. This could be due to a bacteriocin effect [32], hence bacteriocin produced from lactic acid bacteria mediated inhibition of C. botulinum. Sullivan and coworkers [33] thought that the inhibition of C. botulinum spores or vegetative cells indicated that the normal flora of healthy individuals had a bacteriostatic, rather than a bactericidal effect on the growth of C. botulinum and concluded that infant botulism may result, in part, from the absence of inhibitory organisms in the normal flora of the infant intestine. On the other hand, C. botulinum positive rumen fluid samples had significantly higher population of Archea, BacteroidesePrevotella-group, B. fragilis group, B. distasonis, C. histolyticum group, Lactobacillus, sulfur reducing bacteria (SRB), Alphaproteobacteria, Gammaproteobacteria, along with a significant decrease of Euryarcheota. The release of sulfate from mucine degradation by Clostridia, Bifidobacteria and B. fragilis to produce disulfide results from utilization of H2 accumulating in rumen due to fermentation. Clostridia and Enterobacteria produce H2 by reduction of pyruvate [15,31,34]. Consequently, SRB use H2 to produce H2S, a very toxic gas that damages the epithelium of the gastrointestinal tract. In other studies, Clostridium difficile infected patients had a high population of facultative anaerobes and low levels of Bifidobacterium and Bacteriodes [35,36]. More recently, C. difficile infections have been associated with changes in the gut microbiota which allows C. difficile to multiply and colonize the gut [37e39]. Fallani
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Fig. 2. Micromorphology of important rumen protozoa; 1 ¼ Total protozoa, 2 ¼ Entotodinium spp., 3 ¼ Epidinium spp., 4 ¼ Isotricha spp., 5 ¼ Dasytricha spp., 6 ¼ Diplodiniinae, and 7 ¼ Ophryoscolex spp.
and coworkers [40] observed that infants with detectable proportions of C. difficile had a lower percentage of Bifidobacteria and higher proportions of Bacteriodes spp. Also, the C. histolyticum subgroup in rumen fluid of C. botulinum positive cows was significantly higher demonstrating an increase of pathogenic Clostridia. Vijay and co-workers [41] found an association between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota in animals with nosocomial diarrhea, including C. difficile infection.
The rumen of ruminants contains a mixed population of ciliate protozoal species. In our study, Entotodinium spp., Epidinium spp., Isotricha spp., Dasytricha spp., Diplodiniinae spp, and Ophryoscolex spp. were detected in rumen fluids. Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. were significantly lower in C. botulinum positive rumen fluid. A typical protozoal population is able to breakdown w17% of available rumen bacteria every h [13]. However, the amount of bacterial degraded by protozoa depends on the protozoal group. Diplodiniinae spp. and Holotriches
Fig. 3. Distribution of protozoa in rumen fluid of botulinum positive (N ¼ 20), botulinum suspected (N ¼ 32) and botulinum negative (N ¼ 140) cows.
M. Krüger et al. / Anaerobe 27 (2014) 100e105
(Dasytricha spp. and Isotricha spp.) have the greatest and least activity, respectively. Newbold and coworkers [42] reported that bacterial degradation occurs only in intact rumen ecosystems. For example, Listeria monocytogens was degraded 25% in three hours. In contrast, no degradation of pathogenic bacteria occurred in protozoa free rumen fluid and the microbiota were dominated by the C. botulinum cluster. Rumen fluids with intact microbiota have a large capacity for proteolytic inactivation of BoNT/C [43]. Ozutsumi and coworkers [44] showed that rumen protozoa influence the population and composition of its microbiota. The causes of this dysbiosis could be rumen acidosis but also could be due to contamination with antimicrobially active substances in their feed. Oral antibiotic treatment in dairy cows is very rare; however, glyphosate, the active substance of the systematic herbicide “RoundupÒ” is a common contaminant in feed and a potent antimicrobial agent that affects different bacteria differentially. Enterococci, Lactobacillus and Bifidobacteria are highly susceptible to glyphosate while C. botulinum is highly resistant [15,31]. High concentrations of this selective antimicrobial in feed occurred about the same time as its herbicidal use increased for genetically engineered herbicide-tolerant crops and as a crop desiccant. Further investigation of the impact of this herbicide and other dysbiotic agents on GI tract microbiota are necessary. C. botulinum is detectable in faeces and rumen fluids of dairy cows in Germany. The incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Such dysbiotic conditions in the gastrointestinal tract could be responsible for colonization by C. botulinum bacteria. However the chronology of these events and role of each microbial group needs further evaluation.
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