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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.37 No.2 pp.87-91
DOI : https://doi.org/10.13041/jpvm.2013.37.2.87

Devastating botulism outbreak in cattle, associated with contaminated soil

You-Chan Bae1, †, Jae-Won Byun1, Byeong-Yeal Jung2, Ha-Young Kim1, Kyung-Hyun Lee1, Oun-Kyung Moon3, Soon-Seek Yoon1, So-Young Park4, O-Soo Lee1, Myoung-Heon Lee1
1Animal Disease Diagnostic Division, Animal and Plant Quaratine Agency, Anyang 430-757, Republic of Korea
2Bacteriology Disease Division, Animal and Plant Quaratine Agency, Anyang 430-757, Republic of Korea
3Veterinary Epidemiology Division, Animal and Plant Quaratine Agency, Anyang 430-757, Republic of Korea
4North-Branch, Gyeonggido Northern Livestock & Veterinary Service, Namyangju 472-757, Republic of Korea
Received 9 April 2013, revised 20 June 2013, accepted 22 June 2013.

Abstract

We describe here cases of botulism at two dairy cattle farms where the soil was submerged by flooding from anadjacent river. All cattle at farms 1 and 2 (91 and 56 head, respectively) died or were euthanized with posterior flaccidparalysis and recumbency. Necropsy and histopathological examination showed no specific lesions. Botulinum toxin type Bwas detected in sera of the affected cattle, and type D toxin was found in ruminal contents and soil from cattle pens at bothfarms. A likely source of the botulinum toxin is the soil of the adjacent river, from which toxins were introduced during flooding.To our knowledge, these appear to be the first cases of bovine botulism associated with contaminated pen soil after flooding.

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INTRODUCTION

 Botulism is caused by neurotoxins produced by Clostridium botulinum [14]. Of the seven toxin types (A~F) produced by this bacterium, types A~D can induce poisoning in cattle [14]. A total of 168 suspected botulism outbreaks in cattle were recorded between 2003 and 2009 in the UK [13]. Type A botulism has rarely been reported in cattle [5].

 Type B botulism in cattle is associated with the consumption of forage carrying botulinum toxin, which is inhibited below pH 4.5 [14]. If the forage pH is lower than 4.5, botulinum spores cannot germinate and produce toxin. Forage botulism caused by type B botulinum toxin has been reported in the US and Europe [3, 12]. Type C or D botulism is usually as sociated with the consumption of feed containing a decomposing carcass, such as a cat, dog, or bird [14]. Moreover, poultry litter in cattle bedding or feed has caused type C or D botulism [11, 13]. Type D botulism in cattle has also been traced to feeding with contaminated bakery waste [6]. Evidence that C. botulinum producing type C or D toxins can use plant material as a substrate for growth has been presented [6, 10]. However, defining the source in suspected cases of botulism is often problematic, as it is difficult to detect the toxin and isolate the organism from clinical samples [13].

 We describe here a devastating outbreak of botulism in two herds of dairy cattle, in which the soil of the cattle pens was contaminated by flooding from an adjacent river.

CASES

Farm 1

 All cattle of a 91-head dairy herd died (n=28) or were euthanized (n=63) between September 10, 2011, and November 23, 2011. The clinical presentation was characterized by sudden flaccid paralysis of the hind legs, sternal or lateral recumbency, mild salivation, respiratory distress, and death. The rectal temperature of each animal was slightly lower than normal. Consciousness was retained until death. Some cattle showed sternal recumbency with ‘swan neck’. The affected cattle were dead within two or three days after the onset of clinical signs. The herd was raised in a cowshed, and consisted of milking cows (34 head), dry cows (33 head), grower calves (7 head), and nursery calves (17 head). The age of the affected cattle ranged from 1 to 76 months. Treatment with calcium, antibiotics, and nutrient supplements was attempted without response. As shown in Fig. 1, this farm was 5 m away from the Yeongpyeong River, which was severely flooded by heavy rains and the soil and pen of it were completely submerged in July 2011.

Fig. 1. The locations of farms 1 and 2 relative to the Yeongpyeong River.

 Between September 26 and November 23, 2011, 11 of the affected cattle (3~30 months old), sera, milk, and saliva samples from other affected cattle, samples of the total mixed ration (TMR), and soil samples from the cattle pen and from the adjacent River were submitted to the Animal Disease Diagnostic Division of the Animal, Plant, and Fisheries Quarantine and Inspection Agency (QIA). Serum chemistry tests revealed elevated creatine kinase (CK) in eight head of cattle (205~2,000 U/ℓ: reference range, 46~169 U/ℓ). Aspartate aminotransferase (AST) was elevated in two head (179~214 U/ℓ: reference range, 60~150 U/ℓ). Full necropsy and a histopathological examination of parenchymal tissues, including the brain and spinal cord, were performed on each animal. At necropsy, much fibrous forage was observed in the rumen, and no specific gross lesions were observed in the other organs. No specific histopathological lesions were noted, other than mild-to-moderate eosinophilic perivascular pachymeningitis, which was detected in the spinal cords of 6 head out of 11.

 Routine microbiological cultures of the parenchymal tissues and ruminal and intestinal contents revealed no specific bacteria. Samples of sera, milk, and saliva from the affected cattle, TMR, and of soil from the cattle pen and River were tested for botulinum toxin and cultured for C. botulinum. All samples were stored in containers and transported on ice to the QIA. Toxin testing was carried out on all samples by intraperitoneal inoculation into mice according to the protocol of the Centers for Disease Control and Prevention (CDC) with minor adaptations [2]. Toxin types were confirmed using a mouse neutralization assay. Specific antisera were incubated with positive samples at 37℃ for 30 min and inoculated intraperitoneally. In addition, an anaerobic broth culture was performed in cooked meat medium (BD, Franklin Lakes, USA) after heat shock (80℃, 30 min). The presence of C. botulinum cells or spores was confirmed by the presence of toxins in the broth culture, assayed according to the protocol of the CDC with minor adaptations. The mouse bioassay used was approved by the Institute’s ethical committee. Type B toxin was detected only in the serum of one downed animal.

 Polymerase chain reaction (PCR) was performed to isolate viral agents such as arboviruses (Akabane virus, Aino virus, Chuzan virus, Ibaraki virus, and bovine ephemeral fever virus), bovine viral diarrhea virus, bovine herpesvirus-1, and bovine herpesvirus-5, but no viruses or viral genomes were detected. An assay for organophosphate chemicals was performed using ruminal contents, but none were detected. Additionally, bovine spongiform encephalopathy (BSE) tests using brain stem samples were negative in all cases.

 After the devastating outbreak of botulism, the pen soil at the farm was replaced with new soil. Nine calves, aged 1~7 months, were experimentally introduced to this farm between March 17 and March 31, 2012. Two calves out of nine were injected with botulinum B, C, and D vaccines once or twice; the other calves were not vaccinated. Seven unvaccinated calves died after recumbency or were euthanized due to recumbency. Six affected calves (non-vaccinated), soil from the pen and adjacent River, grain pellets, roughage feed, drinking water, and cattle feces were submitted to the QIA. Clostridium botulinum isolation and mouse inoculation assays were performed. Botulinum type D toxin was detected in an anaerobic broth culture of ruminal contents from three calves and in soil from the pen using a mouse inoculation test, PCR, and sequencing of the PCR products [20]. However, no botulinum toxin was detected in the other specimens.

Farm 2

 Two milking cows died on October 24 and October 25, 2011, after showing similar clinical signs to those of the cattle at farm 1. All cattle of the 56-head dairy herd died or were euthanized between October 24 and November 28, 2011. A total of 7 cattle died and 49 were euthanized. The salivation observed in this herd was more severe than that in the farm 1 herd. Some cattle showed melana. Antibiotics, calcium, and nutrients were given, but no response was noted. The affected herd was raised in a cowshed, and consisted of milking and dry cows (35 head), grower calves (9 head), and nursery calves (12 head). The age of the affected cattle was 1~88 months. This farm was 100 m away from the Yeongpyeong River (Fig. 1). The soil and pen of the farm were completely submerged by the flood, as farm 1 had been. The distance between farms 1 and 2 is 300 m.

 Between November 5 and November 28, 2011, two of the affected cattle (14~24 months old), sera and saliva samples from other affected cattle, samples of the TMR, and soil samples from the cattle pen were submitted to the QIA. Serum chemical analysis revealed that CK was increased in one animal (2,000 U/ℓ: reference range, 46~169 U/ℓ). The results of gross and histopathological examinations were non-specific, as at farm 1. No C. botulinum was isolated from any sample by direct or anaerobic broth culture. Type B botulinum toxin was detected in the sera of two downer cattle in the initial stage using a mouse inoculation assay. The results of virological tests, organophosphate chemical assays, and BSE tests were identical to those of farm 1.

 After all of the cattle had died, the pen soil at the farm was replaced with new soil. Seven calves, aged 1~9 months, were experimentally introduced to the farm between March 21 and March 31, 2012. Three calves out of seven were injected with botulinum B, C, and D vaccines once or twice; the other calves were not vaccinated. Four non-vaccinated calves died after recumbency or were euthanized due to recumbency. Three of the affected calves (non-vaccinated), samples of soil from the pen, grain pellets, roughage feed, drinking water, and feces were submitted to the QIA. Clostridium botulinum isolation and a mouse inoculation assay were attempted. Botulinum type D toxin was detected in an anaerobic broth culture of ruminal contents from the three calves and soil from the pen, but no toxins were detected in the other specimens, as at farm 1.

DISCUSSION

 To our knowledge, these are the first reported cases of bovine botulism associated with soil contaminated by river flooding and subsequent farm submergence. In this study, botulinum type D toxin was detected in the ruminal contents of cattle and in soil from both farms. Toxins from the soil of the adjacent River may have been introduced to the farms during the flood. Alternatively, toxins in the deep soil of the farms may have been exposed to the surface after submergence. Botulinum type B toxin was detected in the sera of downer cattle in the initial stage at both farms. Samples should be taken from cattle during the initial stage of recumbency in cases where botulism is suspected, because the possibility of toxin detection in the initial stage is higher than at a later stage. The origin of type B toxin in these cases is obscure. The feed (TMR, grain pellets, and roughage feed) is unlikely to be the source because these feed items were supplied to other farms. We assume that the toxin was introduced to the farms by the same route as the type D toxin. These cases suggest that particular attention should be paid to farms that have been submerged by flooding. Heavy rains and warm weather, as were seen in the summer and fall of 2011, provide favorable conditions for botulinum spores in soil or feed to germinate, and for vegetative cells to multiply rapidly, producing a highly lethal toxin [14].

 Clostridium botulinum types B~E have been isolated from the soils of lakes or rivers in the UK, and in the soil of a former cattle market in London [17-19]. Clostridium botulinum types B and E were isolated from river soil in Japan, and the genome of C. botulinum type B was detected in lake soil from Korea [16, 22]. Thus, epidemiological surveys for C. botulinum types B-D in the soils of lakes or rivers near cattle farms would be useful in the future.

 After the outbreak of botulism described in this report, the soil in the cattle pens was exchanged for new soil at both farms. However, a recurrence was observed in experimentally introduced calves in both cases. This means that complete removal of soil contaminated by botulinum toxin is difficult, and thorough vaccination should be applied to newly introduced cattle.

 The clinical duration of most previously reported botulinum type C or D intoxications was 8~23 days [8, 10, 15]. In this study, it was 2.5 months and 1 month, at farms 1 and 2, respectively. The clinical duration at farm 2 was shorter than at farm 1. Therefore, we assume that the amount of toxin at farm 1 was much higher than at farm 2. In a previous report, in a herd of milking cows, the final outbreak was observed more than 7 months after the first outbreak [7]. This phenomenon may be understood if the amount of toxin in the soil or feed was small or if the toxin was distributed unevenly [11]. Moreover, toxicoinfectious botulism has been proposed to explain the long clinical duration of botulism, although for a type of botulism not yet reported in cattle. When a small amount of toxin is ingested initially, it induces intestinal stasis and creates an environment for C. botulinum spores to proliferate and to produce toxin in vivo [9].

 The clinical signs in this study were similar to those of typical botulism cases [14]. As reported previously, no specific changes in serum chemistry were observed [14]. The elevations in serum CK or AST were attributed to muscle damage caused by prolonged recumbency [6]. Gross and histopathological examinations revealed no specific lesions, consistent with typical botulism [6]. The eosinophilic perivascular pachymeningitis in the spinal cord noted in this study may have been caused by cerebrospinal setariasis [21]. However, the lesion was clearly differentiated from that of setariasis because it was confined to the spinal cord, and no larvae of Setaria spp. were seen in the lesion. Moreover, eosinophilic pachymeningitis of the spinal cord was also observed in healthy slaughtered cattle. Therefore, we concluded that this lesion was not specific and was seen incidentally.

 To make a differential diagnosis, hypocalcemia, hypokalemia, organophosphate poisoning, arbovirus infection (e.g., Akabane virus), and BSE were considered; however, these diseases were ruled out by laboratory tests [13, 14]. In particular, the flaccid paralysis due to botulism is very similar to that caused by hypocalcemia [13]. Based on our epidemiological data, clinical signs, detection of C. botulinum types B and D toxins, and the absence of pathological lesions, these cases were confirmed as botulism.

 Clostridium botulinum poisoning in cattle occurs sporadically worldwide. It induces significant economic loss, with mortality as high as 35~60% [1, 4]. Farms that have not experienced botulism previously appear to have a higher risk of further incidents following a first diagnosis, as many farms experience repeated outbreaks [13]. Making a definitive diagnosis remains difficult because the detection rate of botulinum toxin from clinical samples in cattle is about 50% [13]. Therefore, farmers and field veterinarians should remain alert to the potential problems caused by botulism.

ACKNOWLEDGEMENTS

 The authors thank Drs. In-Soon Roh, Woo-Hee Park, Moon-Young Rhyoo, Mr. Jong-Hyeong Lee, and Mr. Jung-Won Park for their assistance with the necropsies. The authors also thank Dr. Albert Byungyun Jeon for bacteriological test and Dr. Jae-Ku Oem for virological tests. The authors also thank Dr. Choi-Kyu Park for assisting with the sampling and Dr. Mee-Kyung Kim for analyzing the samples for organophosphorus compounds. This research was supported by a grant from the Animal, Plant, and Fisheries Quarantine and Inspection Agency; Ministry for Food, Agriculture, Forestry, and Fisheries; Anyang; Republic of Korea.

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