5.4 Epidemiological insight in Clostridium species on dairy farms
5.4.2 Occurrence of C. perfringens types
Previously, some studies were carried out on the general occurrence of C. perfringens types in cattle and a few of them in dairy (NIILO et AVERY 1963;
VANCE 1967; GURJAR et al. 2008). Most studies on the occurring toxin types in animals were carried out on field isolates, often originating from animals with enteric diseases, making it impossible to use data obtained from those studies for a general overview due to selection bias.
Different types of C. perfringens are confirmed to cause severe enteric disease in cattle, mostly in calves (UZAL et al. 2010). In addition, for some clinical disorders a role of C. perfringens was assumed, but not confirmed so far. These include a subacute lethal syndrome called “haemorrhagic bowel syndrome” or “jejunal hemorrhage syndrome”, for that a causative role was attributed to type A strains (with or without the beta-2 toxin) (DENNISON et al. 2002; CECI et al. 2006). Also an enterotoxaemia syndrome in calves was associated with C. perfringens (MANTECA
et al. 2001).
Within our study for the occurrence of the different types of C. perfringens no statistically significant differences were observed between case and control farms or the animal groups within the particular categories. Therefore, also this data can be used to get an insight in the occurring types on German dairy farms.
Over 99% of all isolates were assigned to type A that is ubiquitously present in the environment and intestinal tract of animals (PETIT et al. 1999). Solely two (0.3%) type D isolates were cultured. The study conducted in Southern Germany parallel to our investigations found also mainly type A (96.6%) and one isolate (1.8%) of each type D and E (DIETSCHE 2015). These findings of mainly type A correspond to results of other studies on fecal samples or isolates of cattle not suffering from acute enteric diseases (NIILO et AVERY 1963; VANCE 1967).
Beta-2 toxin producing strains of C. perfringens are reported to be associated with necrotic enteritis in piglets (BUESCHEL et al. 2003). Additionally, they were previously suspected to have a role in enteric diseases of cattle (DENNISON et al.
2005; LEBRUN et al. 2007). Lebrun et al. found an association between the expression of cpb2 in bovine isolates and enterotoxaemia cases in cattle.
Compared to the 45.7% (n = 202) cpb2 positive isolates cultured from feces within this project, a study previously conducted in Pennsylvania, on fecal samples of seven dairy farms, found a higher amount of 64% (GURJAR et al. 2008). A rate of 46%
(similar to ours) was found in fecal samples of healthy calves (LEBRUN et al. 2007).
Another study, conducted in North America, screened for cpb2 in field isolates obtained from clinical laboratories and found the gene in 12.8% of all isolates from cattle (including animals with and without enteric disease) (BUESCHEL et al. 2003).
Isolates of adult cattle and calves with enteric disorders were more frequently positive with 21.4% and 47.3%, respectively. The high amount in calves with enteric disease was found mainly due to type E isolates that in 97.3% carried cpb2. No comparison to this finding can be made as in the present study no type E isolates were obtained. A study conducted on Belgian blue calves detected cpb2 positive isolates in around 60% of controls as well as in calves that had died from
enterotoxaemia (MANTECA et al. 2002). Looking at this broad variation between the different studies, the presence of cpb2 in C. perfringens isolates from cattle seems to differ widely. Within the present study a distinction was made between the two occurring allelic variants of the cpb2 gene (atypical and consensus). Previously it was reported, that the consensus variant is more frequently present in isolates from pigs, especially piglets (JOST et al. 2005). In contrast, in isolates of non-porcine origin the atypical variant was detected more frequently. Most of the previous studies on the occurrence of C. perfringens in cattle made no distinction between both variants. The present investigation found the atypical cpb2 gene almost twice as often as the consensus variant as well in fecal isolates (31.2% compared to 14.5%) as in those from rumen content (26.1% compared to 14.5%). The first study making an distinction between both variants in bovine isolates found that seven of ten type A, both type C, and 12 of 12 type E isolates that were positive for cpb2 harbored the atypical variant (JOST et al. 2005). Kircanski et al. investigated 11 isolates from cattle and found the atypical and consensus variant in 5 and 1 of these, respectively (KIRCANSKI et al. 2012). A study that investigated the occurrence of both variants in healthy calves and those suffering from diarrhea detected in the enterotoxaemic group only the consensus variant (LEBRUN et al. 2007). They found also in the group of healthy calves more frequently the consensus cpb2 (67.5% compared to 32.5%). One investigation was carried out on a larger number (n = 218) of bovine isolates (SCHLEGEL et al. 2012). Deviating from our results, no isolate was positive for the consensus variant and the atypical allele was detected in only 13% of all isolates. Nonetheless, our results and those of previous studies show that both allelic variants occur frequently in cattle, whereby the atypical variant seems to dominate.
The sources of CPE producing strains that are involved in human food poisoning are still not clear (LINDSTROM et al. 2011). Their general occurrence is estimated to be around 5% of all C. perfringens (MESSELHAEUSER 2013). In this study, cpe positive strains were just detected on four farms (2.9%) confirming that dairy cattle and products of these seem to be no primary source for enterotoxin gene posivite
C. perfringens. Gurjar et al. found a rate of 4.6% cpe positive strains in Pennsylvania obtained from fecal samples from seven different cattle farms (GURJAR et al. 2008).
A much higher finding of 26% was reported by Miva et al. who investigated feces of slaughter cattle in Japan with a nested PCR assay (MIWA et al. 1997). It was suggested that maybe unspecific amplification lead to the finding of high rates (LINDSTROM et al. 2011). In contrast to the study conducted by Miwa et al. (1997), who isolated DNA directly from enrichment broth, within this study we tested isolates already confirmed to be C. perfringens based on their 16S rRNA gene sequence.
Thus, this study confirms that dairy cattle seems to be no primary source for potentially human pathogenic enterotoxin gene positive C. perfringens.
However, also regional differences may be the reason for the different detection frequencies of cpb2 and cpe carrying C. perfringens. This was previously at least suggested for cpb2 positive strains (VAN ASTEN et al. 2010).
Conclusion
Within the present study no significant differences regarding the detection of C. botulinum neurotoxin genes were detected between case and control farms as well as apparently healthy and chronically diseased animals. Moreover, it was shown that viable cells were present in the animals’ intestines without causing disease.
Thus, the postulated role of C. botulinum in chronic disease in dairy herds could not be confirmed.
The cultured isolates of C. perfringens mainly belonged to type A. Both variants of the cpb2 gene were frequently present, whereby the atypical allele dominated. A few C. perfringens isolates carried the cpe gene. This confirms that dairy cattle seem to be no primary source for human pathogenic enterotoxin gene positive C. perfringens.
For both species, Clostridium botulinum and C. perfringens, this study gives an important further insight in their occurrence on dairy farms as data, especially for C. botulinum, in cattle is rare.
6 Summary
Occurrence of Clostridium botulinum neurotoxin genes and toxin-genotypes of Clostridium perfringens in dairy cattle
Svenja Fohler
In the end of the 1990s a new syndrome affecting dairy cattle was proposed. On supposedly affected farms, many animals suffered from a chronic lingering illness.
The described clinical picture comprised a wide variety of symptoms. These included signs of a generalized disease, as well as effects on the locomotor or nervous system, and intestinal disorders. Additionally, emaciation and a loss in milk yield should be characteristic. Detection of C. botulinum or its toxins was the only consistent finding on affected farms, thus the disease was named “visceral botulism”.
This group of bacteria is able to cause an acute disease due to the production of highly neurotoxic proteins (BoNTs). After their formation in the environment (ex vivo) these are classically taken up with food or feed. BoNTs are then resorbed into the blood stream, inhibit the release of acetylcholin at the neuromuscular junction and lead to an, in most cases, acute flaccid paralysis. Death often occurs due to respiratory failure. In contrast, for the “visceral botulism” another pathogenesis was assumed. After ingestion of cells or spores, a colonization of the intestine with subsequent production of low amounts of neurotoxins within the intestine (in vivo) was postulated. These should then cause a generalized chronic disease.
To evaluate the possible causative role of C. botulinum in the proposed syndrome, a case-control study was carried out. Feces and rumen content of 1389 animals from 139 farms as well as feed (n = 411) and water samples (n = 139) were collected. All samples were tested by real time-PCR for the presence of C. botulinum types A to F after enrichment at 37°C in RCM (Reinforced Clostridial Medium). The fecal samples were also enriched at the Friedrich Loeffler-Institut (Institute of Bacterial Infections and Zoonoses, Jena, Germany) in MCM (Modified Cooked Meat Medium) at 30°C and tested with a conventional PCR. In addition, an attempt was made to directly isolate DNA from fecal samples without the inclusion of an enrichment step.
In animal samples from 25 (17.99%), 3 (2.16%), 0 (0.0%), 2 (1.44%), 1 (0.72%), and 3 (2.16%) farms BoNT A, B, C, D, E, and F genes were detected, respectively.
Eleven of all feed samples were BoNT A gene positive. C. botulinum was detected without significant differences in feces and rumen content from chronically diseased and clinically healthy animals from case as well as control farms. Moreover, it was shown that viable bacteria were present in the animals’ intestines without causing disease. Thus, a role of C. botulinum in the so called “visceral botulism” could not be confirmed.
In a second study part, 662 isolates of C. perfringens were cultivated from rumen content, feces and feed. This pathogen is the causative agent of several diseases in animals and humans, including histotoxic infections and enteric diseases. Also the pathogenicity of C. perfringens is attributed to the production of different toxins. To gain more insight in the occurring types on dairy farms a further toxin genotyping was carried out.
Additionally to the mainly detected type A (n = 660), two type D isolates were cultured. The consensus and atypical variants of the cpb2 gene were present in 64 (14.5%) and 138 (31.2%) of all isolates from feces, and 30 (14.5%) and 54 (26.1%) of all isolates from rumen content, respectively. Thus, both allele variants of the cpb2 gene were frequently present in the cultured isolates, whereby the atypical variant dominated. Only 5 (0.8%) of all isolates were positive for the enterotoxin gene. This confirms that dairy cattle seem to be no primary source for potentially human pathogenic enterotoxin gene positive C. perfringens.
The outcome of the studies presented here support the general opinion that both bacteria are ubiquitous and can be either occasionally (C. botulinum) or frequently (C. perfringens) found in the intestine of dairy cows.
7 Zusammenfassung
Vorkommen von Clostridium botulinum Neurotoxingenen und Toxin-Genotypen von Clostridium perfringens in Milchkühen
Svenja Fohler
Ende der 1990er Jahre wurde ein neues Krankheitsbild in Milchviehherden beschrieben. Viele der Tiere auf den betroffenen Betrieben zeigten Anzeichen einer chronischen, fortschreitenden Erkrankung. Das beschriebene klinische Bild schloss vielfältige Symptome ein. Zu diesen gehörten sowohl Anzeichen einer Allgemeinerkrankung als auch Beeinträchtigungen des Bewegungsapparates und Symptome intestinaler Erkrankungen. Außerdem wurden Abmagerung und ein Rückgang der Milchleistung beobachtet. Nach eingehenden Untersuchungen war die einzige Gemeinsamkeit auf allen Betrieben der Nachweis von C. botulinum oder entsprechenden Toxinen. Die von dieser Gruppe von Bakterien in der Umwelt (ex vivo) produzierten Neurotoxine (BoNT) verursachen im klassischen Fall des
„futtermittelassoziierten Botulismus“ eine akute Erkrankung, die durch die Blockade der Acetylcholinfreisetzung an der motorischen Endplatte zu Lähmungs-erscheinungen führt. Der Tod betroffener Individuen tritt schließlich durch Versagen der Atmung ein. Für den sogenannten „viszeralen Botulismus“ wurde eine andere Pathogenese postuliert. Die Bakterien selbst sollten aufgenommen werden, den Darm besiedeln und durch eine andauernde Produktion sublethaler Mengen Neurotoxins (in vivo) zu einer generalisierten chronischen Erkrankung führen.
Um einen möglichen Kausalzusammenhang zwischen dem Vorkommen von C. botulinum und dem beschriebenen chronischen Krankheitsbild zu prüfen, wurde eine Fall-Kontroll-Studie durchgeführt. Es wurden auf 139 Milchviehbetrieben Faeces und Pansensaft von 1389 Tieren sowie Futtermittel- (n = 411) und Wasserproben (n = 139) genommen. Diese wurden nach Anreicherung in RCM (Reinforced Clostridial Medium) bei 37°C auf das Vorhandensein der Neurotoxingene (bont) A – F untersucht. Ergänzend wurden im Friedrich Loeffler-Institut (Institut für bakterielle Infektionen und Zoonosen, Jena) alle Kotproben bei 30°C in MCM (Modified Cooked
Meat Medium) angereichert und ebenso auf des Vorhandensein von bont A – F getestet. Zusätzlich wurde dort eine direkte DNA Isolierung aus Kotproben getestet.
In Probenmaterial von 25 (17,99%), 3 (2,16%), 0 (0,0%), 2 (1,44%), 1 (0,72%), und 3 (2,16%) Betrieben wurden Neurotoxingene der Typen A, B, C, D, E und F nachgewiesen. In elf Futtermittelproben wurde bont A nachgewiesen. Alle Wasserproben waren negativ. C. botulinum wurde ohne signifikante Unterschiede sowohl in Kot als auch in Pansensaftproben von chronisch kranken und klinisch gesunden Tieren von Kontroll- und Fall-Betrieben nachgewiesen. Es konnte gezeigt werden, dass vermehrungsfähige C. botulinum im Intestinaltrakt von klinisch gesunden Kühen vorkommen können. Die Ergebnisse dieser Studie können daher einen möglichen Kausalzusammenhang zwischen dem Vorkommen von C. botulinum und dem beschriebenen chronischen Krankheitsbild nicht bestätigen.
In einem zweiten Projektteil wurden 662 Isolate von C. perfringens aus Probenmaterial derselben Tiere kultiviert. Dieser Keim ist ursächlich für verschiedene Krankheitsbilder, einschließlich histotoxischer und intestinaler Erkrankungen, bei Menschen und Tieren. Auch die verschiedenen mit C. perfringens assoziierten Erkrankungen werden durch die Wirkung von Toxinen verursacht. Um einen Einblick in die in deutschen Milchviehherden vorkommenden Toxintypen zu bekommen, wurden alle Isolate anhand ihrer Toxingene weiter molekularbiologisch Typisiert.
Die meisten Isolate gehörten zum C. perfringens Typ A (n = 660). Des Weiteren wurden zwei dem Typ D zugeordnet. Von allen Isolaten von Faeces wurde bei insgesamt 64 (14.5%) die „consensus“ Variante und bei 138 (31.2%) die „atypische“
Variante des cpb-2 Gens nachgewiesen. Ebenso wurden beide Varianten bei 30 (14.5%) bzw. 54 (26.1%) der von Pansensaft kultivierten Isolate gefunden. Fünf (0.8%) aller Isolate waren positiv für das Enterotoxin-Gen. Dies bestätigt, dass Milchkühe keine primäre Quelle für potentiell humanpathogene Enterotoxingen positive C. perfringens darstellen.
Die Ergebnisse dieser Studie bestätigen die allgemeine wissenschaftliche Ansicht, dass beide Clostridium Spezies ubiquitär vorkommen und entweder gelegentlich (C. botulinum) oder häufig (C. perfringens) im Magen-Darm-Trakt von Milchkühen gefunden werden können.
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