Occurrence of Clostridium botulinum neurotoxin genes and toxin-genotypes of Clostridium perfringens in dairy cattle

University of Veterinary Medicine Hannover

Occurrence of Clostridium botulinum neurotoxin genes and toxin-genotypes of Clostridium perfringens in dairy cattle

INAUGURAL-DISSERTATION

in partial fulfilment of the requirements for the degree Doctor of Veterinary Medicine

-Doctor medicinae veterinariae- (Dr. med. vet.)

submitted by

Svenja Fohler

Castrop-Rauxel

Hannover 2016

Academic supervision: Univ. Prof. Dr. med. vet. Günter Klein Institute of Food Quality and Food Safety Laboratory supervision: PD Dr. med. vet. Amir Abdulmawjood

Institute of Food Quality and Food Safety

1. Referee: Univ. Prof. Dr. med. vet. Günter Klein Institute of Food Quality and Food Safety 2. Referee: Prof. Dr. med. vet. Ralph Goethe

Institute for Microbiology

Day of oral examination: 23.05.2016

The work presented in this thesis was done within the project “Bedeutung von Clostridium botulinum bei chronischem Krankheitsgeschehen” (“Importance of Clostridium botulinum in chronic disease”) that was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office

for Agriculture and Food (BLE).

Grant numbers: 2810HS005 and 2810HS036.

Parts of the laboratory investigations were carried out at the Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany by other contributing

authors of Manuscript #1 included in this thesis.

To my family

Manuscripts included in the doctoral thesis:

Manuscript #1

FOHLER, S., S. DISCHER, E. JORDAN, C. SEYBOLDT, G. KLEIN, H. NEUBAUER, M. HOEDEMAKER, T. SCHEU, A. CAMPE, K. C. JENSEN, A. ABDULMAWJOOD (2016):

Detection of Clostridium botulinum neurotoxin genes (A–F) in dairy farms from Northern Germany using PCR: A case-control study.

Anaerobe 39, 97-104 Manuscript #2

FOHLER, S., G. KLEIN, M. HOEDEMAKER, T. SCHEU, C. SEYBOLDT, A. CAMPE, K. C. JENSEN, A. ABDULMAWJOOD:

Diversity of Clostridium perfringens toxin-genotypes from dairy farms Submitted for publication

The work for the doctoral thesis was conducted within a collaborative project.

Following other publications closely related to the content of this thesis are listed:

Related Articles:

SEYBOLDT, C., S. DISCHER, E. JORDAN, H. NEUBAUER, K. C. JENSEN, A. CAMPE, L. KREIENBROCK, T. SCHEU, A. WICHERN, F. GUNDLING, P. DO DUC, S. FOHLER, A. ABDULMAWJOOD, G. KLEIN, M. HOEDEMAKER (2015):

Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows.

Vet. Microbiol. 177 398-402

ALAJMI, A., G. KLEIN, M. GREINER, N. GRABOWSKI, S. FOHLER, A. CAMPE, T. SCHEU, M. HEODEMAKER, A. ABDULMAWJOOD (2016):

Potential role of real-time PCR for detection of Mycobacterium avium subsp.

paratuberculosis (MAP) in chronically diseased milking cows: a case control study Accepted for publication in Berl. Munch. Tierarztl. Wochensch.

Oral presentations:

FOHLER, S., A. ABDULMAWJOOD, G. KLEIN (2014):

Bedeutung und Nachweis von Clostridium perfringens in der Lebensmittelkette Kolloquien für Tiergesundheit und Lebensmittelqualität im Sommersemester 2014, Hannover, Germany

C. SEYBOLDT, H. NEUBAUER (2014):

C. botulinum-Diagnostik: Nachweis der Toxingene mittels PCR – Ergebnisse und Bedeutung.

Abschluss-Symposium „Bedeutung von Clostridium botulinum bei chronischem Krankheitsgeschehen“, Hannover, Germany

FOHLER, S., A. ABDULMAWJOOD, G. KLEIN (2014):

Vorkommen von Clostridienspezies in Tier und Umgebungsproben aus Fall- und Kontrollbetrieben.

Abschluss-Symposium „Bedeutung von Clostridium botulinum bei chronischem Krankheitsgeschehen“, Hannover, Germany

FOHLER, S., A. ABDULMAWJOOD, G. KLEIN (2015):

Vorkommen und Bedeutung lebensmittelrelevanter Clostridien in Milchviehbetrieben - Untersuchungen und Ergebnisse einer Fall-Kontroll-Studie -

Fachtagung Gissel-Institut 2015, Halle (Westfalen), Germany

FOHLER, S., G. KLEIN, M. HOEDEMAKER, A. WICHERN, K. C. JENSEN, A. CAMPE, J. ROHDE, A. ABDULM AWJOOD (2015):

Clostridien auf Milchviehbetrieben: Vorkommen von Spezies mit Relevanz für Lebensmittelqualität und –sicherheit

56. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene (56th Food Hygiene Congress), Garmisch-Partenkirchen, Germany

Poster:

FOHLER, S., G. KLEIN, M. HOEDEMAKER, T. SCHEU, K. C. JENSEN, A. CAMPE, J. ROHDE, C. SEYBOLDT, A. ABDULMAWJOOD (2015):

Occurrence of pathogenic Clostridium spp. in dairy farms from Northern Germany.

Clostpath 2015, 9th Conference on the Biology and Pathogenesis on the Clostridia, Freiburg, Germany

FOHLER, S., G. KLEIN, M. HOEDEMAKER, A. WICHERN, K. C. JENSEN, A. CAMPE, J. ROHDE, A. ABDULMAWJOOD (2014):

Occurrence of Clostridium spp. in dairy farms from Northern Germany 56. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene (56th Food Hygiene Congress), Garmisch-Partenkirchen, Germany

FOHLER, S., G. KLEIN, M. HOEDEMAKER, T. SCHEU, J. MEENS, K. C. JENSEN, A. CAMPE, T. MAIER, A. ABDULMAWJOOD (2014):

Comparison of 16S rDNA-Sequencing and MALDI-TOF-MS for the identification of different Clostridium spp.

56. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene (56th Food Hygiene Congress), Garmisch-Partenkirchen, Germany

Table of Contents

PUBLICATION LIST... V

TABLE OF CONTENTS ... VII

LIST OF TABLES ... IX

LIST OF FIGURES ... IX

LIST OF ABBREVIATIONS ... XI

1 INTRODUCTION ... 1

2 LITERATURE OVERVIEW ... 3

2.1 Clostridium botulinum ... 3

2.1.1 General ... 3

2.1.2 The toxins of Clostridium botulinum ... 4

2.1.3 Diseases in humans and animals ... 5

2.1.4 Laboratory diagnosis ... 8

2.2 Clostridium perfringens ... 12

2.2.1 General ... 12

2.2.2 The toxins of Clostridium perfringens... 13

2.2.3 Diseases in humans and animals ... 16

2.2.4 Laboratory diagnosis ... 19

2.3 “Visceral botulism” ... 23

3 MATERIAL AND METHODS ... 25

4 MANUSCRIPTS ... 31

4.1 Manuscript # 1: ... 31

Detection of Clostridium botulinum neurotoxin genes (A – F) in dairy farms from Northern Germany using PCR: a case-control study ... 31

4.2 Manuscript # 2: ... 33

Diversity of Clostridium perfringens toxin-genotypes from dairy farms ... 33

5 COMPREHENSIVE DISCUSSION ... 54

5.1 Discussion of study design and used methodology ... 54

5.1.1 Study design ... 54

5.1.2 Laboratory investigations ... 56

5.2 Detection of C. botulinum neurotoxin genes and association to chronic herd health problems ... 65

5.3 Detection of C. perfringens types ... 68

5.4 Epidemiological insight in Clostridium species on dairy farms ... 69

5.4.1 Occurrence of C. botulinum ... 69

5.4.2 Occurrence of C. perfringens types ... 70

6 SUMMARY ... 75

7 ZUSAMMENFASSUNG ... 77

8 LITERATURE ... 80

9 SUPPLEMENTAL MATERIAL ... 104

List of Tables

Table 1 Characteristics of Clostridium botulinum groups I to IV ... 4 Table 2 Clostridium perfringens types and associated diseases ... 13

List of Figures

Figure 1 Study design ... 26 Figure 2 Samples investigated from each of the 139 farms and used methodology for BoNT gene

(bont) detection at the Institute for Food Quality and Food Safety ... 27 Figure 3 Samples investigated from each of the 139 farms and used methodology for BoNT gene

(bont) detection at the Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses ... 28 Figure 4 Samples from each of the 139 farms and used methodology for the cultivation of

Clostridium isolates ... 29 Figure 5 Methods used for toxin genotyping of Clostridium perfringens ... 30

List of Abbreviations

BoNT Clostridium botulinum neurotoxin bont Clostridium botulinum neurotoxin gene

bp Base pair

C. botulinum Clostridium botulinum C. perfringens Clostridium perfringens

CPA Clostridium perfringens alpha toxin cpa Clostridium perfringens alpha toxin gene CPB Clostridium perfringens beta toxin cpb Clostridium perfringens beta toxin gene CPB-2 Clostridium perfringens beta-2 toxin cpb2 Clostridium perfringens beta-2 toxin gene

cpb2con Clostridium perfringens beta-2 toxin gene “consensus” allele cpb2aty Clostridium perfringens beta-2 toxin gene “atypical” allele CPE Clostridium perfringens enterotoxin

cpe Clostridium perfringens enterotoxin gene

DNA Deoxyribonucleic acid

ELISA Enzyme-linked Immunosorbent Assay ETX Clostridium perfringens epsilon toxin etx Clostridium perfringens epsilon toxin gene ITX Clostridium perfringens iota toxin

iap Clostridium perfringens iota toxin gene (enzyme component) ibp Clostridium perfringens iota toxin gene (binding component)

MCM Modified Cooked Meat Medium

PCR Polymerase chain reaction RCM Reinforced Clostridial Medium rDNA Ribosomal deoxyribonucleic acid

RNA Ribonucleic acid

1 Introduction

In 1999, a new syndrome affecting dairy cows was proposed (BOEHNEL et GESSLER 2000; SCHWAGERICK et al. 2000). It has been characterized as a chronically lingering illness affecting many animals per herd, thus leading to significant economic losses. The described clinical picture included a wide variety of symptoms. Signs of a general illness as well as circulatory disorders, effects on the locomotor or nervous system, and the intestine were described. As this combination of symptoms could not be assigned to known diseases in cattle, broad investigations were carried out. The only reported consistent finding in supposedly affected dairy farms was the detection of Clostridium (C.) botulinum and/or its toxins in sample material from living animals or animal carcasses. Therefore, authors of the conducted studies proposed a substantial causative role of C. botulinum and named the disease

“chronical” or “visceral” botulism.

However, most of the published work on this syndrome was just based on case reports or did not include a sufficient number of farms to reliably prove the causal relations within this newly described disease. Additionally, nearly no epidemiological data on the general occurrence of C. botulinum in healthy cattle was available. Most of the previously conducted studies investigated outbreaks of “classical foodborne botulism”. This made it impossible to evaluate the finding of C. botulinum in diseased animals. Due to the lack of scientific proof, the reported great economic losses, and the negative effects on animal welfare, further research was strongly recommended (BFR 2004, 2010; BECHTER 2014). Therefore, an extensive collaborative case control study was designed to clarify the potential causative role of C. botulinum. A case definition was elaborated based on previously published work. Detailed clinical examination of lactating cows, herd screenings for animal health parameters, investigation of feed and water, and interviews with the farmers regarding the farm management were carried out. Laboratory investigations to verify the role of C. botulinum and its toxins included three different methodologies: First, the detection of the neurotoxin genes of C. botulinum using PCR to detect cells or spores of the bacterium, whereby different enrichment procedures were applied. Second, the

sample material was incubated under favorable conditions for clostridia, trying to isolate C. botulinum and other Clostridium species, which could be possibly important in the disease development (as partly also mixed clostridial infections were considered as a potential cause). Third, the gold standard methodology for C. botulinum neurotoxin detection, the mouse bioassay, was performed (SEYBOLDT et al. 2015).

Within the first part of this thesis, the results of the testing of feces, rumen content, feed and water for the presence of C. botulinum by neurotoxin gene detection are presented. The second part focuses on C. perfringens isolated within the collaborative project. The aim of this study part was to analyze the diversity of occurring C. perfringens isolates.

2 Literature Overview 2.1 Clostridium botulinum 2.1.1 General

Botulism, the disease caused by Clostridium (C.) botulinum, is already known for centuries (ERBGUTH 2008). The first cultivation of the organism itself was done by van Ermengem in the end of the 19^th century. He gave the bacterium its first name, Bacillus botulinus (“botulus”, latin = sausage), and described its toxins (ERMENGEM 1897).

C. botulinum is a Gram-positive, spore-forming, obligate anaerobic bacterium that can be found ubiquitously in the environment (LINDSTROM et al. 2010). Several bacterial strains were isolated and assigned to this species because of their ability to produce botulinum neurotoxins (BoNTs). Although they caused similar clinical pictures in affected patients, it was observed that the antigenic properties between the toxins differed as well as the primary affected species. This lead to the differentiation of BoNTs into seven types, A to G, that can be further divided into several subtypes (ROSSETTO et al. 2014). An eighth serotype H has been recently described, but still remains to be confirmed (BARASH et ARNON 2014). Types A, B, E and F are mainly associated with human botulism cases, while types C, D, and their mosaic types, C/D and D/C, are often found in outbreaks in cattle, birds, and some other animals (LINDSTROM et KORKEALA 2006; WOUDSTRA et al. 2012).

Based on phenotypic characteristics C. botulinum is classified into four different groups (Table 1). Group I contains type A, and proteolytic strains of types B and F altogether sharing an optimal growth temperature of 37 - 40°C (LINDSTROM et KORKEALA 2006). They form spores with a high heat resistance that are therefore a hazard for the production of canned food (ESTY et MEYER 1922). C. sporogenes shows close genetic relation to the neurotoxic members of this group (WEIGAND et al. 2015). Group II consists of type E and the non-proteolytic strains of types B and F.

This group has its growth optimum at 30°C or less. Types C, D and their mosaic types are assigned to group III that finds optimal growth conditions at 37 – 45°C.

C. novyi and C. haemolyticum are non-BoNT producing species that are genetically closely related to other members of group III (SKARIN et SEGERMAN 2014).

C. argentinense is the only member of group IV. Some strains of this species are able to produce BoNT G. It grows best at 37°C. Also some other clostridia are able to produce BoNTs. Particularly, these are strains of Clostridium butyricum and Clostridium baratii that produce BoNT E and F, respectively (HALL et al. 1985;

MCCROSKEY et al. 1986).

Table 1 Characteristics of Clostridium botulinum groups I to IV

(based on LINDSTROM et al. 2010, GRENDA et al. 2014 and ROSSETTO et al. 2014)

Groups of C. botulinum

I II III IV

C. botulinum types

A,

proteolytic type B and F

non-proteolytic type B and F,

E

C and D, mosaic forms (C/D and D/C)

C. argentinense

Subtypes A1, A2, A3, A4,

A5, A6, A7, A8, A9, A10;

B1, B2, B3, B5, B6, B7;

A(B); Ab; Af; Af84;

Bf; F1, F2, F3, F4, F5

B4;

E1, E2, E3, E6, E7, E8, E9, E10, E11;

F6

C; D; CD; DC G

Optimal growth temperature

37 – 40°C < 30°C 37 – 45°C 37°C

Non-neurotoxic members

C. sporogenes C. novyi C. subterminale

2.1.2 The toxins of Clostridium botulinum

Primary, BoNTs are synthesized as ~150 kDA proteins. These compose of a 50 kDA light chain (LC), and a 100 kDA heavy chain (HC) that consists of a translocation and a binding domain (TURTON et al. 2002). Additionally to the neurotoxins, C. botulinum also produces a non-toxic-non-heamagglutinating protein (NTNHA). It protects the neurotoxins that are relatively easily biodegraded by environmental proteases and

gastric acids (OHISHI et al. 1977; ROSSETTO et al. 2014). BoNT and NTNHA form, together with several haemagglutinin proteins, the so called progenitor toxin complex (PTC). The heamagglutinins are responsible for the trans-epithelial absorption of the toxin in the intestine after oral uptake (K. LEE et al. 2013). When reaching the bloodstream BoNTs can remain for several days in the circulation (FAGAN et al.

2009). The binding domain of the HC highly specific anchors the toxin to receptor structures on cholinergic nerve terminals (ROSSETTO et al. 2014). Following, the toxin is internalized by endocytosis. Then, the HC translocation domain forms a channel into the vesicular membrane and the LC is released into the cytosol. The LC is a metalloprotease with specific cleavage-activity towards SNARE (soluble N- ethylamide-sensitive factor attachment protein receptor) -proteins that are essential for neurotransmitter exocytosis (PANTANO et MONTECUCCO 2014). Thus, acetylcholine cannot be released in the postsynaptic cleft and the neuromuscular transfer of stimuli is blocked, leading to a flaccid paralysis.

2.1.3 Diseases in humans and animals Sources

Human and animal botulism classically occur after the uptake of preformed neurotoxin with food or feed (ANNIBALLI et al. 2013). Typically contaminated matrices are home preserved food for humans. In cattle, outbreaks often result from the incorporation of carcasses in anaerobic fermented feed like silage. An association with the uptake of poultry litter was also reported (SMART et al. 1987;

MCLOGHLIN et al. 1988; MCLAUCHLIN et al. 2006; PAYNE et al. 2011; LECLAIR et al. 2013). In phosphorus deficient areas botulism can be associated with the uptake of parts of bones of dead animals (SCHOCKEN-ITURRINO et al. 1990). Bovine botulism cases due to type C and D mainly originate from these sources. Outbreaks due to type B are reported to occur after toxin formation in decaying plant material (“forage poisoning”) when for example a protective pH below 4.5 is not reached during the ensiling process (WILSON et al. 1995; KELCH et al. 2000). The preformed toxin is ingested together with the contaminated material and resorbed from the

intestine into the blood stream. It enters peripheral motor-neurons and inhibits, as described above, the release of acetylcholine contained in intracellular vesicles.

Clinical symptoms of “classical foodborne botulism”

In humans, the first signs of botulism are often difficulties with swallowing, articulation disorders and blurred vision (DLABOLA et al. 2016). Also cattle shows difficulties with swallowing and the uptake of feed (BRAUN 2006). The generalized paralysis is whether descending, like in humans, or it starts from the hind hand (KELCH et al.

2000; BRAUN 2006). For the latter, it leads at first to posterior ataxia and difficulties in getting up. In many cases a decrease in tail tone is reported (MARTIN 2003).

Because of the paralysis, the exploration of the mouth is often unusually easy to perform, the tongue tone is reduced, and a typical biphasic inspiration or abdominal breathing can be observed (GALEY et al. 2000; BRAUN 2006; STÖBER 2006).

Partly recorded changes in biochemical blood parameters are increased hematocrit, CK (creatine kinase) and ASAT (aspartate aminotransferase) (JEAN et al. 1995;

BRAUN 2006). Post mortem investigations of bovines normally show no pathologic- anatomic anomalies. Therefore, they can be helpful to exclude differential diagnosis (KELCH et al. 2000; KUMMEL et al. 2012; ANNIBALLI et al. 2013). The disease progresses in both, animals and humans, and most often, finally leads to death due to respiratory failure (LINDSTROM et al. 2010). In cattle, the disease course can differ from sudden death to prolonged durations over several days even in the same outbreak situation (GALEY et al. 2000). Also the incubation period in cattle varies from just a few hours up to more than 14 days (YERUHAM et al. 2003;

MYLLYKOSKI et al. 2009). Often, many animals per herd are affected at once (ORTOLANI et al. 1997; GALEY et al. 2000; STEINMAN et al. 2006).

Therapy and prevention

For all, animals and humans, only a symptomatic therapy can be tried (including mechanical ventilation and supportive care). Additionally, antitoxin can be administered, but it is of use only as long as free toxin is still circulating (ANNIBALLI et al. 2013). It has no impact on toxin that is already bound to the nerve endings. The

antitoxins are expensive and therefore mostly no option for treatment of animal botulism, especially not in livestock (DLABOLA et al. 2016). Additionally, at the moment none of them is approved for the application in animals for food production in Germany, precluding the use for treatment of cattle. Often, euthanasia of affected animals is an act of humanity and should be carried out, at least in recumbent cases (BRAUN 2006). To prevent additional cases, feeding of supposedly contaminated feed to any animals on the farm should be stopped immediately (BRAUN 2006). In many outbreaks an association can be made with a recent change of one part of the fed ration, occasionally in association with the finding of enclosed animal carcasses (YERUHAM et al. 2003; MYLLYKOSKI et al. 2009).

Vaccines are available to prevent disease in cattle (ANNIBALLI et al. 2013).

However, in Germany none of these is commercially licensed. Thus, a governmental permission is needed if a veterinarian wants to administer one of the available type C and D vaccines. In other countries, bovines are vaccinated regularly against botulism. In Israel for example, this is now done since the late 1970´s (STEINMAN et al. 2006).

Other forms of botulism

More rarely, three other types of botulism occur in humans. The first is “infant botulism” in children under one year of age (ROSOW et STROBER 2015). It is suspected to be a cause of sudden infant death (ARNON et al. 1978). The pathogenesis is different from that described above. C. botulinum itself is ingested, and as the intestinal flora of the infant is not entirely developed it colonizes the gut and locally produces toxin. An frequently reported source of the bacterium in infant botulism cases is honey (GRABOWSKI et KLEIN 2015). Often observed symptoms include hypotonia, bulbar weakness, and a flaccid paralysis of skeletal muscles, like in “classical foodborne botulism” cases (ROSOW et STROBER 2015). Another form of botulism can develop after the contamination of wounds with bacteria or spores (“wound botulism”) (MERSON et DOWELL 1973). In these cases, the toxin is produced by the bacteria within the wound, it enters the blood stream and causes the muscular paralysis. In recent years, this form most frequently occurred in drug users

that accidently injected C. botulinum (MACDONALD et al. 2013). The bacterium finds ideal anaerobic conditions deep in the tissue where it is able to grow and produce its toxins. Inadvertently, botulism can be initiated by incorrect administration of BoNTs as therapeutic agents or in plastic surgery (commonly known as Botox^®), when it is tried to take advantage of its ability to block the transmission of neuronal signals (MEZAKI et al. 1996; BAKHEIT et al. 1997).

Cases of botulism in Germany

In Germany, animal botulism is not an obligatory notifiable disease as in other European countries (e.g. France). Therefore, no secure data exist on the frequency of botulism cases or outbreaks in cattle, but they are generally considered to occur only rarely (DLABOLA et al. 2016). For other European countries, in the past decades a rise in animal botulism cases was reported (ANNIBALLI et al. 2013).

Whereby, many of these were outbreaks in poultry, but also a rise in cattle outbreaks was seen. A survey conducted in England and Wales revealed a number of 168 incidents in cattle between 2003 and 2009, with a change of the primary causative type from C to D (PAYNE et al. 2011). Bovine botulism cases in Europe are today mainly caused by type D or D/C (WOUDSTRA et al. 2012). In contrast, for the United States of America it was reported that botulism outbreaks due to feed contaminated with BoNT type B are more common (HOGG et al. 2008).

Unlike animal botulism outbreaks, human cases have to be reported in Germany.

During the last decade, not more than ten human cases were reported annually (RKI 2015).

2.1.4 Laboratory diagnosis

A clinical tentative diagnosis is easily made in big outbreaks in cattle. Then, often a high morbidity, with many animals showing specific disease symptoms, and mortality is observed (HOGG et al. 2008). The secure diagnosis of botulism in smaller outbreaks or situations when just single individuals are affected is often difficult. The only reliable proof of a causative role of BoNTs is their detection in serum or feces of

affected patients. This can be complemented by additional testing of remains of supposedly contaminated food or feed (LINDSTROM et KORKEALA 2006). But, the toxin is often already bound at the neuromuscular junction and free toxin in non- sterile sample material could be rapidly degraded by proteolytic microorganisms (ALLISON et al. 1976). Therefore, its detection often fails and only a tentative diagnosis based on the observed symptoms can be formulated.

Toxin detection

The standard method for C. botulinum neurotoxin (BoNT) detection is the mouse bioassay (DORNER et al. 2013). For this assay, mice are injected intraperitoneally with a suspension suspected to contain biologically active toxin. This could be serum of patients or feces mixed with gelatin diluent, but also a culture supernatant after enrichment. If biologically active toxin is present, the injected mice show characteristic symptoms beginning with ruffled fur and labored abdominal breathing (wasp waist). In most instances, the mice die after 6 to 24 hours (CDC 1998). For detection of BoNTs produced by group II strains it is necessary to trypsinate the suspension before its injection into mice (DUFF et al. 1956). This needs to be done if enzymatic cleavage that activates these toxins in the natural pathway has not taken place. The mouse bioassay is very sensitive as it detects already 0.01 ng/ml of toxin, but it also has some drawbacks (WICTOME et al. 1999). To figure out which type of BoNT lead to the symptoms observed in the laboratory animals, additional mouse lethality assays using specific antibodies need to be conducted. For this retesting, antibodies for all types of BoNTs are required. These are then each injected into mice together with the suspension presumptively containing botulinum neurotoxin.

Only those mice protected with the specific antibodies for the BoNT type present within the sample will stay unaffected, whereas all others will show symptoms of botulism (CDC 1998). This laboratory method is not only critical because of the need of the lethality assay using laboratory animals. It is also a time consuming and an elaborative technique, with the need of experienced staff and animal housing facilities. Another problem revealed by the FLI (Friedrich-Loeffler-Institute, Jena, Germany), which conducted an inter-laboratory comparison study, was that results,

obtained by the different participating laboratories, were not concordant. In some laboratories a significant need for improvement was observed (SEYBOLDT et NEUBAUER 2013).

To replace the mouse bioassay, different other assays also targeting biological active toxin were developed. One in vivo assay, called toe spread reflex model, tests for local muscle paralysis after intramuscular injection in mice (WILDER-KOFIE et al.

2011). Also ex vivo assays like the hemidiaphragm assay that uses electrical stimulation measurement of an isolated phrenic nerve from rats or mice were proven suitable for detection of biological active toxin (BURGEN et al. 1949; RASETTI- ESCARGUEIL et al. 2009). More recently, cellular based assays were developed, i.e.

a Cellular-Based Potency Assay (CPBA) that was approved by the American Food and Drug Administration (USA), and a neuronal cell based assay (NCB) that shows a higher sensitivity compared to the mouse bioassay (FERNÁNDEZ-SALAS et al.

2012; BASAVANNA et al. 2013). But, to apply the latter assay on samples like food or feces the elaborative isolation and purification of BoNT is required (PELLETT 2013). Also a lot of immunological methods were developed for BoNT detection, whereby ELISAs are the most common and some of them are commercially available (DEZFULIAN et al. 1984; STANKER et al. 2008; BROOKS et al. 2010). The problem is that also non-active toxin can be detected by these methods. The most recently published endopeptidase ELISAs got over this drawback as they only detect biologically active toxin (GRENDA et al. 2014). Thus, it may be a promising technique to replace the mouse bioassay in the future.

Detection of Clostridium botulinum

Although in general, the detection of C. botulinum cells or spores cannot replace methods targeting the toxin to confirm botulism cases, because of its ubiquitous occurrence and the fact that “classical foodborne botulism” cases are caused by the uptake of preformed toxin, in some instances it can be useful to target the organism itself (DLABOLA et al. 2016). These include epidemiological studies on its occurrence, the clarification of infant botulism cases, or the detection of cells or spores in materials suspected to contain toxin producing C. botulinum. For the latter

it can be either done to confirm it as the outbreak source or to achieve at least a tentative diagnosis if toxin detection was not successful in patients and/or suspected materials. However, the cultivation of C. botulinum is difficult and often fails, in particular because sample materials like feces or feed often contain a high competitive microflora (LINDSTROM et KORKEALA 2006). Therefore, many PCR based assays were developed, to detect C. botulinum cells or spores. The majority of these assays focus on the detection of the genes coding for the neurotoxins (bont).

As for most other pathogens, at first conventional gel-based PCR assays were developed (TAKESHI et al. 1996; LINDSTROM et al. 2001). With the general progress in molecular detection methods, also faster and less elaborative procedures, like real time-PCR assays (MESSELHÄUSSER et al. 2007; FACH et al.

2009; KIRCHNER et al. 2010), were published. As the NTNH gene (ntnh) locus is associated to the bont locus, Raphael and Andreadis developed an assay targeting this gene (RAPHAEL et ANDREADIS 2007). Further work was done to establish assays for the determination of the expression level of bont by reverse transcriptase real time-PCR (LOVENKLEV et al. 2004; SHIN et al. 2006). Generally, the use of amplification controls is recommended to verify PCR results, because the investigated materials like intestinal content and environmental samples often contain PCR inhibiting substances (SCHRADER et al. 2012). Before DNA isolation and PCR assays can be performed, an enrichment step (two to five days) should be conducted, to increase the amount of bacteria and thereby the DNA copies present for amplification. This is necessary, as the number of cells or spores present in natural samples is often very low (DAHLENBORG et al. 2003; LINDSTROM et KORKEALA 2006).

2.2 Clostridium perfringens 2.2.1 General

C. perfringens causes a broad range of diseases in animals and humans, but it is also one of the most widespread bacteria (PETIT et al. 1999). This species was first isolated during a human necropsy in 1892 by Welch and Nutall who initially assigned the name Bacillus aerogenes capsulatus that was later changed to Bacillus welchii.

Finally, the bacterium was renamed to C. perfringens (MESSELHAEUSER 2013).

C. perfringens is classified into five types, A to E, depending on the production of four major toxins (alpha, beta, epsilon and iota toxin) (UZAL et al. 2014). Each of these five toxinotypes is associated with various diseases (PETIT et al. 1999). Additionally to the major toxins, up to 12 minor toxins can be produced (UZAL et al. 2010).

Clinical importance is to date assigned to three of them: the enterotoxin, the netB toxin and the beta-2 toxin (SKJELKVÅLE et UEMURA 1977; BUESCHEL et al. 2003;

KEYBURN et al. 2008).

Following, first a short introduction is given on the four major and two minor toxins (enterotoxin and beta-2 toxin) possibly involved in diseases in cattle, including their pathogenicity mechanisms. This is followed by a brief introduction in diseases caused by the different types of C. perfringens. The main focus lies on diseases in cattle, as the investigations of this thesis were done on dairy farms. Also human diseases are introduced, because a dissemination of pathogenic strains along the food chain could pose a risk to consumer health. For a general overview, also including diseases in other species, see table 2.

Table 2 Clostridium perfringens types and associated diseases (based on Petit et al. 1999, Uzal et al. 2014)

Type Toxins Diseases in

Humans Cattle Other animals

A α gas gangrene,

food poisoning^e, antibiotic-associated diarrhea^e, sporadic diarrhea^e

enterotoxaemia, histotoxic infections

necrotic enteritis of poultryⁿ, canine gastrointestinal disease^e, necrotizing enteritis in piglets^b2

B α, β, ε hemorrhagic enteritis

in neonatal calves

lamb dysentery, dysentery in sheep

C α, β necrotic enteritis necrotic enteritis of calves

necrotic enteritis of foals, lambs, piglets, acute enterotoxaemia in adult sheep (“struck”)

D α, ε enterotoxaemia of

calves

hemorrhagic enterocolitis of goats; enterotoxaemia/ pulpy kidney disease in sheep

E α, ι enteric disease in

calves

enteric disease in rabbits and lambs

e = enterotoxin producing strains

n = netB-toxin producing strains

b2= beta-2 toxin producing strains

2.2.2 The toxins of Clostridium perfringens

Mode of action

The alpha toxin (CPA) is the major pathogenicity factor in histotoxic infections due to C. perfringens type A strains (AWAD et al. 1995). Although produced by all members of this species, type A strains are superior to all other types in CPA production (NIILO 1980). This toxin is a zinc-dependent phospholipase that modifies cellular membranes, activates the arachidonic acid cascade, and finally leads to lysis of affected cells (TITBALL et al. 1999). It is hemolytic and reduces capillary perfusion by inducing intravascular platelet aggregation (HICKEY et al. 2008). Thus, the proteolytic anaerobe C. perfringens is able to supply itself with the optimal growth conditions by generating an anaerobic environment containing necessary growth factors.

The second major toxin, the beta toxin (CPB), can be produced by type B and C strains and is easily degraded by trypsin (UZAL et al. 2010). It forms transmembrane pores, thus leading to ion changes in affected cells (NAGAHAMA et al. 2003). This causes swelling of those cells and finally necrosis (effects that can be primarily observed in the jejunum and ileum). The villi in affected intestinal compartments are destroyed and histologically a diffuse necrotizing enteritis can be found (SAYEED et al. 2008). After uptake in the circulatory system, CPB is additionally able to cause lethal effects (UZAL et al. 2010). The mechanisms leading to these effects are until now not completely understood.

The C. perfringens epsilon toxin (ETX) is also a pore forming protein that is produced by type B and D strains (UZAL et al. 2014). Before it can affect host cells it has to be activated by intestinal proteases, in contrast to CPB that is inactivated. After entering the blood stream by a paracellular pathway, accumulation of ETX is mainly found in the kidney and the brain (NAGAHAMA et SAKURAI 1991; POPOFF 2011;

FREEDMAN et al. 2014). Interestingly, it is able to pass the blood-brain barrier, where it causes in high doses perivascular edema leading to peracute death (FINNIE et al. 1999; SOLER-JOVER et al. 2007). When lower doses of ETX are administered, bilaterally symmetrical lesions like necrosis and hemorrhages can be observed (WIOLAND et al. 2013). ETX is able to stimulate the release of the neurotransmitter glutamate (O. MIYAMOTO et al. 2000). This is probably the main reason for the observed excitation in affected animals (POPOFF 2011).

The fourth major toxin, the iota toxin (ITX), consists of two proteins, a binding (IB) and an enzymatic (IA) component (SAKURAI et al. 2009). These protoxins also need to be activated by proteolytic enzymes (GIBERT et al. 2000). The IB binds to host cell receptors, forms pores that lead to ion changes and triggers the internalization of the enzymatic component into the cytosol (UZAL et al. 2014). IA then interacts with ADP-ribosylating actin and therefore, destroys the integrity of the cytoskeleton and leads to disorganization of cellular junctions.

All four major toxins are produced during the exponential growth phase and are secreted actively. The enterotoxin in contrast, is produced within the bacterium

during sporulation, accumulates in the cytoplasm, and is finally released when the mother cell is lysed (DUNCAN 1973). It specifically binds to certain claudin receptors on enterocytes (SHRESTHA et MCCLANE 2013). Together with these claudin receptors it forms a so called “small complex”, that then hexamerizes, forms a prepore complex, and following an active pore in the cellular membrane. This leads to death of enterocytes as result of a strong calcium influx (CHAKRABARTI et MCCLANE 2005). Due to villus shortening and blunting as well as epithelial desquamation, a loss of intestinal integrity is observed that results in electrolyte dysregulation, with diarrhea being the most obvious characteristic (FERNANDEZ MIYAKAWA et al. 2005). This diarrhea sets a massive amount of spores free. Thus, it seems to be an important mechanism for the further distribution of enterotoxic C. perfringens strains.

Several studies suggested a possible role of the beta-2 toxin (CPB-2) in animal diseases, especially in piglets (GIBERT et al. 1997; GARMORY et al. 2000;

BUESCHEL et al. 2003). Compared to the other toxins of C. perfringens, its pathogenic effects are until now only rarely studied. It was demonstrated that it has cytotoxic effects on different cell lines, in animal experiments it was able to induce hemorrhagic enteritis in bovine intestinal loops, and it was lethal to mice after intravenous administration (GIBERT et al. 1997; MANTECA et al. 2002).

Coding genes

Interestingly, solely CPA and in some strains CPE are coded by chromosomal genes.

All other toxins introduced here are coded by mobile genetic elements. The alpha toxin is coded by cpa, the beta toxin by cpb, the epsilon toxin by etx, the two iota toxin components by iap and ibp, the enterotoxin by cpe and the beta-2 toxin by cpb2. cpe as well as cpb2 can be found in every type, A to E (UZAL et al. 2014). Two allelic variants of the cpb2 gene, called “consensus cpb2” and “atypical cpb2”, exist (JOST et al. 2005). The high conservation of cpa in all strains of C. perfringens can be utilized for laboratory confirmation of cultured isolates (PIATTI et al. 2004). An association was found between the location of the cpe and the primary caused syndrome in humans. Chromosomally-coded cpe was more frequently detected in

food poisoning strains, whereas a plasmid coded gene was mostly associated with non-foodpoisoning type A strains (SPARKS et al. 2001). This difference may be explained by the fact that isolates harboring a chromosomally coded cpe were found to be more heat resistant compared to those with an cpe coded on a plasmid (SARKER et al. 2000).

2.2.3 Diseases in humans and animals

Type A of C. perfringens is known to be ubiquitous in the environment and the most common toxin type (J. G. SONGER 1996). It leads to gas gangrene (malignant edema) in humans and animals (UZAL et al. 2014). This disease can also be caused by Clostridium novyi or Clostridium septicum (CHIPP et al. 2009; RIBEIRO et al.

2012). Most often it develops after the contamination of deep wounds. Inside, C. perfringens finds optimal anaerobic growth conditions and actively secretes, among others, the alpha toxin. Observed symptoms include a massively hurting wound and edema with gas production in the surrounding tissue (MACLENNAN 1962). The disease progresses rapidly if no fast surgical intervention is done. In the end, it leads to the patient’s death due to a systemic shock (STEVENS et al. 2012). A special form of this tissue necrosis can affect the udder of sheep or cows, mostly occurring as secondary infection (MAYER 2006; OSMAN et al. 2009). The major pathogenicity factor in gas gangrene is the alpha toxin (AWAD et al. 1995). A second toxin the perfringolysin O (PFO) works synergistically with CPA (AWAD et al. 2001).

Additionally, type A strains are suspected to be associated with clostridial enteritis in calves and a synergistic role of CPA and PFO was demonstrated in an intestinal loop model (MANTECA et al. 2001; G. J. SONGER et MISKIMINS 2005).

About 5% of type A strains are able to produce CPE (MESSELHAEUSER 2013).

These enterotoxic strains can potentially cause three types of intestinal diseases in humans. The first and most common is a mild toxico-infection (food poisoning) that occurs after the ingestion of highly contaminated food (at least 10⁶ cfu/g) (MCLAUCHLIN et GRANT 2007). Matrices often involved in outbreaks are meat and

poultry meat products as well as prepared foods containing these. Due to the high heat resistance of spores and the ability of vegetative cells to grow at temperatures up to 53°C, infections mainly occur after ingestion of pre-prepared food that was undercooked and/or stored under inappropriate conditions (MCCLANE et al. 2013).

Due to the mild symptoms, often only bigger outbreaks or outbreaks in institutionalized settings are reported. The main symptoms of this food poisoning, diarrhea and abdominal pain, typically begin 8 to 24 hours after ingestion of contaminated foods, and end self-limiting after another 12 to 24 hours. The frequency of C. perfringens food poisoning is supposed to be underestimated in annual summary reports on foodborne diseases and the associated pathogens, as most affected persons don’t visit a doctor (MESSELHAEUSER 2013). Within the European Union even no consistent surveillance program concerning this food borne pathogen exists. Nonetheless, for the United States of America it is reported that it is one of the major pathogens causing foodborne diseases (SCALLAN et al. 2011).

The second human intestinal disease caused by CPE producing C. perfringens strains is a diarrhea observed after antibiotic treatment. It was assumed that 5 to 15% of all antibiotic-associated diarrhea cases in men are caused by CPE producing type A strains (CARMAN 1997). Also a sporadic non-foodborne diarrhea can develop after ingestion of CPE producing strains (BRETT et al. 1992). Both diseases have stronger effects on infected patients and last longer than food poisoning.

Type C of C. perfringens causes more severe diseases than type A. Men can suffer from a necrotizing enteritis that was observed in Northern Germany just after the second world war. Following, it was reported to be endemic in some parts of Southeast Asia (MURRELL et WALKER 1991). The symptoms include vomiting, bloody stool and abdominal pain. In severe cases, a toxemia develops leading to a rapid death. Type C strains cause also in animals a hemorrhagic necrotizing enteritis that mostly affects neonates (NIILO et al. 1974). In both, animals and humans, the main pathogenicity factor is the beta toxin (CPB). It affects the intestinal epithelium (mainly of jejunum and ileum) that results in fluid accumulation and hemorrhagic necrosis (UZAL et al. 2014). Then, the toxin is absorbed into the blood stream and is

able to cause generalized symptoms, i.e. due to brain lesions. It was shown that CPB is inactivated by intestinal proteases. Thus, an important role in disease evolution is assigned to trypsin inhibiting substances (NIILO 1986). These are as well present in colostrum, ingested by animal neonates, as in food (sweet potatoes) that is frequently consumed in endemic areas (UZAL et al. 2014).

Type D of C. perfringens causes enterotoxaemia in cattle, although less frequent when compared to sheep and goats (JONES et al. 2015). The epsilon toxin (ETX), produced by these strains within the intestine, less strongly affects the gut while it leads more to damage in the central nervous system, especially in sheep (FINNIE 2003). In peracute cases, just slightly patho-histological changes are observed in the brain (including vasogenic edema). But, if lower amounts of the toxin are circulating in the bloodstream and therefore a longer disease duration takes place, macroscopic visible changes like parenchymal brain necrosis and bilateral focal malacia can be found at necroscopy. Symptoms observed in calves experimentally injected with ETX included neurological signs (opistothonus, convulsion, recumbency, loss of consciousness) and dyspnea (UZAL et al. 2002).

Diseases due to types B and E are until now not so well studied compared to those induced by other types mentioned above. C. perfringens type B strains are able to produce the beta as well as the epsilon toxin and can lead to a fatal hemorrhagic dysentery in sheep lambs (UZAL et SONGER 2008). Type E is the only type that produces the iota toxin. Till now, regarding types B and E no reports of human diseases exist. But, for both it was suggested that each may play a role in hemorrhagic enteritis or enterotoxaemia in calves, respectively (J. G. SONGER 1996). Additionally, Type E was found in cases of acute abomasitis in calves (J. G.

SONGER et MISKIMMINS 2004).

As mentioned above, strains producing CPB-2 are suspected to be involved in enteritis in piglets and also for some bovine enteric diseases a role in their pathogenesis was suggested (BUESCHEL et al. 2003; DENNISON et al. 2005;

LEBRUN et al. 2007).

2.2.4 Laboratory diagnosis

Diagnosis of diseases caused by C. perfringens is generally based on several criteria. In cases of gas gangrene, the clinical findings in combination with a positive wound culture (eventually positive for other clostridia) give a clear diagnosis (WELLS et WILKINS 1996). For the laboratory confirmation of all intestinal disorders one thing must receive attention. It is not possible to base an etiologic diagnosis on the simple finding of the bacterium in sample material, because of the ubiquitous occurrence of type A (VANCE 1967). Thus, in disease cases a quantitative analysis should be carried out and the exact type should be determined. This can be either done by typing of pure cultures or toxin detection in enrichment broth, but also by isolation of the causative toxin from patients´ sample material (feces or serum) and subsequent typing. For food poisoning cases, additionally the contaminated food matrix and the reason for contamination should be figured out (e. g. inappropriate storage conditions). If it is not possible to track back and identify the contaminated food in an outbreak situation, it is at least necessary to detect the same type of C. perfringens or its toxins in samples from more than one of the affected patients to confirm a causal relationship between different cases.

Diagnosis of enteric diseases in farm animals should be based on farm history, clinical signs, and necroscopy findings combined with laboratory confirmation, that includes toxin detection as well as microbiological methods (UZAL et SONGER 2008). All enteric diseases mainly occur when C. perfringens is present in high numbers. Therefore, quantitative methods followed by further typing should be routinely carried out to confirm a causal role of it in clinical cases.

Cultivation of C. perfringens

Compared to other members of the genus Clostridium, it is relatively simple to culture C. perfringens. Reasons for this are among others that it is a less strict anaerobe and in investigated sample materials, like contaminated food in outbreak situations or intestinal content of patients, it should mostly be present in high numbers. Therefore, several culture methods have been developed for the detection and enumeration of C. perfringens. Agars that can be used for cultivation include in example Schaedler-

agar, RCM-agar, egg-yolk-agar, TSC-agar and TSN-agar (HARMON et al. 1971;

STARR et al. 1971). For the horizontal detection and enumeration of C. perfringens in food and feed an ISO (International Organization for Standardization) norm based on colony counts exists (ISO 7937:2004) as well as for the enumeration in water (ISO 14189:2013-11

)

Species confirmation

In contrast to other clostridia, C. perfringens shows some biochemical reactions that in combination enable a clear species identification. This encloses its lecithinase activity, the formation of nitrate and the fermentation of different sugars, while it is negative for lipase, indole and urease tests (ZIEGLER 2013). In addition, C. perfringens is non-motile and shows on blood agar plates typically a double zone of β-hemolysis. Physiological characteristics can be evaluated in example by the use of commercially available tests like the API^® system (MESSELHAEUSER 2013).

The gene coding for the alpha toxin (cpa) is present in all strains of C. perfringens.

Thus, it was recommended and is often used for laboratory confirmation of cultured isolates (PIATTI et al. 2004). This confirmation is highly specific and much faster than classical biochemical tests, especially when for pure cultures just a simple boiling method is used to isolate DNA instead of more complex protocols that include several washing and binding steps.

Molecular identification of cultures could be done by sequencing of the 16S rRNA gene that is present in all prokaryotic cells (CLSI 2008). Species identification based on this gene is considered to be the molecular gold standard and is especially suitable in screening approaches or when bacteria are cultured that cannot be easily distinguished from other species based on phenotypic characteristics.

Another method, leading to an accelerated identification, that was proven to be suitable for C. perfringens identification and is now often used in clinical laboratories is MALDI-TOF-MS (Matrix assisted time-of-flight mass spectrometry (ZIEGLER 2013)). This method detects species specific spectra of ribosomal proteins, thus enabling the identification of a wide variety of pathogens without a restriction to certain targets in advance.

Typing methods

Unfortunately, a distinction between the different types of C. perfringens is not possible solely based on phenotypic characteristics of pure cultures or the above mentioned assays. Therefore, further investigations have to be subsequently conducted after cultivation.

The classical typing methods for C. perfringens are based on tests needing laboratory animals, like toxin neutralization test in mice or guinea pigs (PETIT et al.

1999). Because of the same drawbacks of animal-experiments that were already mentioned above in the section on the mouse bioassay for botulism diagnosis, much simpler and faster methods that get along without the use of laboratory animals were developed. Several serological assays were developed that can be used to directly test patient sample material or enrichment culture supernatant after incubation.

Especially for the detection of CPE in patients’ stool ELISA assays are commercially available (GOLDSTEIN et al. 2012). Also for the detection of other toxins, a lot of ELISA assays were developed and proven suitable, whereby some of them are even more sensitive than the neutralization assays in animals (UZAL et SONGER 2008).

RPLA (reverse passive latex agglutination) kits are offered for enterotoxin detection and could also be used as well as cell based assays (i.e. using vero cells) (MCLAUCHLIN et GRANT 2007).

Mahony et al. demonstrated for the enterotoxin that in vivo and in vitro toxin production can differ widely (MAHONY et al. 1992). Therefore, typing based on toxin detection in enrichment broths or culture based assays may produce false results.

Today, in many laboratories PCR based protocols are used for the typing of clinical isolates of C. perfringens. A lot of molecular assays have been developed. Most of these detect at least the genes of the four major toxins, cpa, cpb, etx, and iap and/or ibp (MEER et SONGER 1997; YOO et al. 1997). In addition, assays for the detection of the genes of the minor toxins with clinical importance, CPE (cpe), NetB (netB), and CPB-2 (cpb2), have been developed (BAUMS et al. 2004; HEIKINHEIMO et KORKEALA 2005; KEYBURN et al. 2008). Jost et al. (2005) found that different variants of the cpb2 gene exist and designed primers to distinguish between the

consensus and the atypical allele (JOST et al. 2005). Because of the possible presence of many different toxin genes in each individual strain it was obvious that multiplex approaches would enable fast and efficient typing. Conventional gel based multiplex assays were developed by several research groups (BAUMS et al. 2004) as well as multiplex real time-PCR assays (MESSELHAUSSER et al. 2007; ALBINI et al. 2008; GURJAR et al. 2008). Molecular methods were also evaluated for quantitative analysis (WU et al. 2011). Additionally, some assays were developed to distinguish between chromosomal and plasmid borne cpe (K. MIYAMOTO et al.

2004).

Further molecular characterization and epidemiological investigations on strains isolated from outbreaks can be carried out using various protocols like PFGE, ribotyping, plasmid profiling, MLVA (multiple-locus variable number tandem repeat analysis) or AFLP (amplified fragment-length polymorphism) (EISGRUBER et al.

1995; RIDELL et al. 1998; SCHALCH et al. 1999; MCLAUCHLIN et al. 2000;

SAWIRES et SONGER 2005).

2.3 “Visceral botulism”

First cases of a potentially new and unknown disease in dairy cows were reported in the end of the 1990s for Northern Germany (SCHWAGERICK et al. 2000). On the first supposedly affected farms, clinical signs were observed in many animals. Cows either died after a few days or showed chronic wasting. A vague case description including manifold disease symptoms was given. Symptoms of a general illness like apathy and depression, but also effects on the intestine like indigestion (constipation or diarrhea) and a retracted abdomen, symptoms typical for circulatory disorders (edemas, engorged veins), and effects on the locomotor or nervous system (ataxia or unsteady gate, difficulties in standing up, recumbency, the head turned to the side, partly salivation, difficulties with swallowing of feed, and paralysis of the tongue or lower jaw) were described. The disease should additionally lead to a decrease in milk yield as well as emaciation (BOEHNEL et GESSLER 2000; SCHWAGERICK et al.

2000; BOEHNEL et al. 2001). Many of these symptoms were previously also reported for classical botulism cases in cattle.

The clinical picture observed on the farms could not be associated with well-known causes for diseases in dairy holdings and conventional therapy of animals failed (e. g. therapy for milk fever for downer cows after giving birth) (SCHWAGERICK et al.

2000). Therefore, further investigations were conducted in many directions to find the potential cause of disease. One outcome was the finding of C. botulinum and / or its neurotoxins in sample material from farms with herd health problems. Based on this and the lack of other outcomes, authors conducting those studies concluded an important role of C. botulinum in the observed herd health problems and named the disease-complex “visceral botulism” (BOEHNEL et al. 2001). The proposed pathogenesis for this “visceral botulism” should be analog to that of human infant botulism. It was suggested, that spores (or cells) are taken up with contaminated feed, then passage the upper intestine, and following colonize the lower intestine (caecum and colon) when favorable conditions (i. e. a gut dysbiosis) are present (SCHWAGERICK et al. 2000; BOEHNEL et al. 2001). The neurotoxins should then, in contrast to “classical foodborne botulism”, be formed by the bacteria within the animals gut, directly affect the nervous system of the intestine, and be resorbed

continuously in small amounts into the blood stream leading to symptoms of a generalized disease. The disease duration should be up to several months, but also sudden unexpected deaths were described.

Further studies were carried out as herd health problems, supposedly caused by this

“visceral” form of botulism, occurred on additional farms leading to great economic losses. These studies supported the assumption of a causative role of C. botulinum and its toxins on the affected farms, as also toxin or bacteria were found (KRUGER et al. 2011; KRUGER et al. 2014a; KRUGER et al. 2014b). However, none of these studies included a representative number of farms and most of them failed to include controls. Although data on the general occurrence of C. botulinum in dairy farms are rare, some studies on its occurrence in bovine sample material exist. In example a previously conducted study on healthy cattle at slaughter revealed a high prevalence of C. botulinum type B in fecal samples (DAHLENBORG et al. 2003). Also other authors found C. botulinum in healthy cattle (KLARMANN 1989; SOUILLARD et al.

2015). Taking this into account, it was impossible to draw secured conclusion from the already available data and it was proven necessary to conduct further investigations (BFR 2010).

Therefore, a case control study was carried out to prove the hypothesis of a causative role of C. botulinum in chronically diseased dairy herds, and to gain a deeper insight in the occurring herd health problems by comparing a statistically valid number of farms that keep animals showing signs of the described syndrome with unaffected controls.

3 Material and Methods

Material and Methods are described in detail in Manuscript #1 and Manuscript #2.

The design of the study and the laboratory investigations were planned in collaboration by the following institutes:

1. Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

2. Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

3. Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany

4. Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

The on farm visits and sampling were conducted by the Clinic for Cattle. Laboratory investigations were carried out at the Institute of Food Quality and Food Safety, and for the investigations presented in Manuscript #1 partly at the Friedrich-Loeffler- Institut. The cultivation of Clostridium isolates was carried out at the Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover. Statistical analysis was done by the Institute of Food Quality and Food Safety together with the Department of Biometry, Epidemiology and Information Processing.

Figure 1 shows the general study design including the criteria defined for the three investigated farm categories (Control, Case-1 and Case-2), while figures 2 and 3 show the work steps of the laboratory investigations presented in Manuskript #1, and figures 4 and 5 those presented in Manuskript #2. All statistical analyses were conducted using SAS (Version 9.3, SAS, Institute, USA).

Figure 1 Study design

from (FOHLER et al. 2016)

Figure 2 Samples investigated from each of the 139 farms and used methodology for BoNT gene (bont) detection at the Institute for Food Quality and Food Safety

Figure 3 Samples investigated from each of the 139 farms and used methodology for BoNT gene (bont) detection at the Friedrich-Loeffler- Institut, Institute of Bacterial Infections and Zoonoses

Figure 4 Samples from each of the 139 farms and used methodology for the cultivation of Clostridium isolates

Figure 5 Methods used for toxin genotyping of Clostridium perfringens

(Capital letters A to E indicate the type of C. perfringens used as positive controls: A: CCUG 1795T, B: CCUG 2035, C: CCUG 2036, D:

CCUG 2037, E: CCUG 44727; Z: bovine type A inhouse collection strain LMQS-BP-8160); X: molecular-weight seize marker; italic letters indicate the names of C. perfringens toxin encoding genes, cpa: alpha toxin gene, cpb: beta toxin gene, etx: epsilon toxin gene, iap: iota toxin gene (enzyme component), cpe: enterotoxin gene, cpb2 and cpb2con:

consensus beta-2 toxin gene, cpb2aty: atypical beta-2 toxin gene; neg:

negative control)

4 Manuscripts

4.1 Manuscript # 1:

Detection of Clostridium botulinum neurotoxin genes (A – F) in dairy farms from Northern Germany using PCR: a case-control study

The results of the laboratory investigations on the occurrence of C. botulinum on the investigated farms using different enrichment procedures and PCR based protocols (detecting the neurotoxin genes) are presented in Manuscript # 1 included in this thesis.

Parts of the Laboratory investigations were carried out at the Friedrich-Loeffler- Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany.

Contribution of the first author to this work:

Svenja Fohler was involved in the design of the study, performed the laboratory investigations that were conducted at the Institute of Food Quality and Food Safety (Sample processing, enrichment in RCM, DNA isolation, real time-PCR assays), conducted the analysis and interpretation of the results, and wrote and revised the manuscript.

Detection of Clostridium botulinum neurotoxin genes (A – F) in dairy farms from Northern Germany using PCR: a case-control study

Published in Anaerobe, June 2016, Volume 39, Pages 97–104

Svenja Fohler^1*, Sabrina Discher^2*, Eva Jordan², Christian Seyboldt², Guenter Klein¹, Heinrich Neubauer², Martina Hoedemaker³, Theresa Scheu³, Amely Campe⁴, Katharina Jensen⁴, Amir Abdulmawjood^1#

1. Institute of Food Quality and Food Safety, Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany

2. Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Straße 96a, 07743 Jena, Germany

3. Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany

4. Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Foundation, Buenteweg 2, 30559 Hannover, Germany

* These authors contributed equally to this work

# Corresponding author

doi:10.1016/j.anaerobe.2016.03.008

http://www.sciencedirect.com/science/article/pii/S1075996416300221