Journal of Wildlife Diseases, 46(3), 2010, pp. 951–955 # Wildlife Disease Association 2010

Outbreak of Botulism (Clostridium botulinum type C) in Wild Waterfowl: , Korea

Gye-Hyeong Woo,1,4 Ha-Young ,1 You-Chan Bae,1 Young Hwa Jean,1 Soon-Seek Yoon,1 Eun- Jung Bak,2 Eui Kyung Hwang,3 and Yi-Seok Joo11Laboratory of Pathology, Animal Disease Diagnostic Center, National Veterinary Research and Quarantine Service, Anyang, Gyeonggi-do 430-824, Republic of Korea; 2 Department of Oral Biology, College of Dentistry, , Seoul 120-752, Republic of Korea; 3

Department of Animal Science, College of Life Science and Natural Resources, Sangji University, Wusan-dong, Downloaded from http://meridian.allenpress.com/jwd/article-pdf/46/3/951/2240923/0090-3558-46_3_951.pdf by guest on 30 September 2021 Wonju, Kwangwon-do 220-702, Republic of Korea; 4 Corresponding author (email: [email protected])

ABSTRACT: Over a 6-day period beginning on (Anseriformes; Forrester et al., 1980; 15 October 2008, 93 dead or sick wild Wobeser et al., 1983; Shayegani et al., waterfowl, including Mallards, Spotbills, and 1984), shorebirds (Charadriiformes; For- teal species, were found along the shore of a branch stream of the Hangang River, which rester et al., 1980), gulls (Larus argentatus; flows through Seoul, Korea, and were submit- Neimanis et al., 2007), chickens (Gallus ted to the National Veterinary Research and gallus; Dohms et al., 1982), turkeys (Melea- Quarantine Service (NVRQS) for diagnosis. gris gallapavo; Smart et al., 1983), cattle Clinically, the affected birds showed flaccid (Bos primigenius; Wobeser et al., 1997), paralysis of the legs and wings and paralysis of the neck. Grossly, no bird showed any lesions, and horses (Equus ferus; Bernard et al. but all had almost empty stomachs. Histopath- 1987). In this study, we describe botulism ologic findings included mild lymphocytic in wild ducks caused by C. botulinum toxin hepatitis and mild lymphocytic interstitial type C near Seoul, Korea. nephritis. Clostridium botulinum type C toxin On October 15, 2008, three dead Spot- was identified in sera collected from the birds bills (Anas poecilorhyncha) were found using a mouse bioassay for botulinum toxins; however, no bacteria were isolated from any of in the Anyangcheon (37u339130N, the affected birds. In addition, a low-pathogen- 126u529400E), a small branch stream of ic avian influenza virus was isolated from two the Hangang River (37u339130N, Spotbills, and pesticides such as diazinon and 126u529400E), which flows through Seoul, phorate, were detected in seven Mallards. The Korea. The Anyangcheon rises from Ui- cause of this outbreak is not clear, but an increase in organic materials from sewage due wang (37u20923.840N, 126u59916.670E) to drought, increased temperatures, and an south of Seoul and then flows through increased number of aquatic carcasses resulting Anyang (37u23921.980N, 126u56929.210E) from pesticide contamination may have in- into the Hangang (Fig. 1). The stream is creased the replication of C. botulinum, con- used as a stopover by migratory birds, but tributing to the release of botulinum toxins into the waterfowl food chain. no previous mass deaths were reported Key words: Botulism, flaccid paralysis, prior to this outbreak. Over the 6 days after Mallard, pesticide, Spotbill, Teal. the discovery of dead birds, 93 migratory wild birds were found paralyzed (Spotbills, Botulism is a fatal paralytic disease of n52) or dead (n591) on the shore or in the birds and some mammals caused by the water of the same stream. Approximately ingestion of toxins produced by Clostrid- 51% of the affected waterfowl were Mal- ium botulinum. Although seven types of lards (Anas platyrhynchos, n548). Other toxin have been demonstrated, type C affected species included Spotbills (n521), botulism is the one that typically causes Green-winged Teals (Anas crecca, n518), disease and mortality in wild birds (For- Baikal Teals (Anas formosa, n54), Shovel- rester et al., 1980; Wobeser, 1997) and has ers (Anas clypeata, n51), and Pintails (Anas been reported worldwide in waterfowl acuta acuta, n51). All wild ducks were

951 952 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 3, JULY 2010

bacterial isolation. For viral examination, samples of trachea, kidney, and cecal tonsil were inoculated in chicken embryos. Gross- ly, stomachs were typically almost empty and contained only a small amount of sand and a few seeds. No gross lesions were seen in any of the birds examined. For the mouse inoculations, pooled sera and filtered supernatants of intestinal

contents and maggots were prepared by Downloaded from http://meridian.allenpress.com/jwd/article-pdf/46/3/951/2240923/0090-3558-46_3_951.pdf by guest on 30 September 2021 submitted date and species. For each assay, two mice were injected intraperitoneally with 0.2 ml of the sample (sera and filtered supernatants of the intestinal contents from sick animals, and sera and filtered super- natants of the intestinal contents and/or maggots from dead animals) and observed FIGURE 1. Map of sites of two botulism out- for up to 4 days for characteristic signs of breaks near Seoul, Korea. The first outbreak (solid line) occurred along the Tancheon stream; the botulism, including a ‘‘wasp-waist’’ appear- second (dotted line) occurred along the Anyang- ance. Then, the botulism type was con- cheon. firmed by mouse neutralization tests using antibotulinum toxin types A, B, C, and E submitted to the National Veterinary Re- (National Institute for Biological Standards search and Quarantine Service (NVRQS). and Control, UK). Type C botulinum toxin The two Spotbills affected showed depres- was identified in serum samples collected sion, flaccid paralysis of the legs and wings, from Spotbills (four of 21), Mallards (18 of and paralysis of the neck. Maggots were 48), and Baikal Teals (four of four). found rarely in some carcasses. Upon However, no botulinum toxin was detected necropsy, blood or blood clots, intestinal in intestinal contents or maggot samples. In contents, and maggots were collected for addition, no histopathologic lesions were use in a mouse bioassay for botulinum observed in any of the mice showing toxins; in addition, portions of the gastroin- characteristic signs of botulism or the mice testinal contents were analyzed for pesti- injected with toxin-neutralized serum. cides by gas chromatography (HP6890N, No bacteria were cultured from the GC/NPD, FPD; Hewlett Packard, Palo intestinal contents or livers. A low patho- Alto, California, USA) and gas chromatog- genic avian influenza virus (H3) was raphy/mass spectrometry (HP6890N and isolated from two Spotbills. Histopatho- HP5973N; Hewlett Packard). The limit of logically, a few lymphocytes and macro- detection was about 0.2 ppm, although it phages had infiltrated the livers and differed among the pesticides. Various kidneys in most birds. Although parasites organs and tissues (e.g., brain, lungs, heart, were not classified, they were observed in liver, spleen, kidneys, trachea, proventricu- blood vessels and the bile duct of the liver lus, gizzard, and intestine from birds or in one Spotbill and one Mallard. In brain, spinal cord, lungs, heart, liver, spleen, organophosphate residue tests of gastro- kidneys, and intestine from mice) were fixed intestinal contents, diazinon and phorate in 10% buffered formalin, embedded in were detected in seven Mallards with type paraffin, sectioned at 4 mm, and stained with C botulinum toxin. The concentration of hematoxylin and eosin for histopathology. diazinon in the birds ranged from 0.34 to Intestine and liver samples were cultured 0.57 mg/kg, while that of phorate ranged on blood agar and MacConkey agar for from 0.51 to 0.79 mg/kg. SHORT COMMUNICATIONS 953

During the outbreak, the weather in October was warmer than normal (mean temperature for this time of year for the past 36 years); the monthly average maximum temperature was 22.4 C, 1.7 C higher than normal, and the minimum temperature was 10.7 C, 1.9 C higher than normal. The highest daily temperature in October was 28.3 C in Seoul (37u349180N,

126u579590E; Fig. 2). Further, the month- Downloaded from http://meridian.allenpress.com/jwd/article-pdf/46/3/951/2240923/0090-3558-46_3_951.pdf by guest on 30 September 2021 ly precipitation amounted to 32.7 mm in Korea, only 57% of that recorded in previous years (Table 1). In particular, there was no rainfall in the beginning or middle of October, suggesting that the proportion of sediments may have in- creased due to an influx of sewage and FIGURE 2. Daily minimum and maximum tem- decreased precipitation. The optimal tem- peratures in Seoul before and after the botulism perature for the multiplication of C. outbreak (thin arrow denotes start of outbreak; thick botulinum is between 25 and 40 C (Rocke arrow denotes end of outbreak). The highest daily temperature during the outbreak period was 28.3 C. and Bollinger, 2007). However, Botulinum type C toxin is produced at temperatures as low as 15.6 and 12.8 C (Segner, 1971). Namdonggongdan reservoir may have Botulism occurs frequently in the summer been similar to those in wetlands (i.e., and fall because ambient conditions are shallow water, a decrease in the amount of near the optimal temperature for bacterial dissolved oxygen, an increase in the growth (Dohms et al., 1982; Wobeser et sediment temperature, and decaying or- al., 1983; Rocke and Bollinger, 2007). ganic matter; Rocke and Bollinger, 2007). In Korea, three outbreaks of botulism in Clostridium botulinum is an anaerobic wild waterfowl, including the present organism found in soils, the intestines of outbreak, have been reported: the first animals, and aquatic environments botulism event occurred in October of throughout the world, and it multiplies in 2007 in the Tancheon (37u33912.170N, decaying organic materials, where toxins 126u52940.690E; Fig. 1), a small branch are released. Invertebrates play an impor- stream of the Hangang. Species affected tant role in mediating the transfer of toxins by the Tancheon stream event were from organic materials to wild ducks similar to those affected by the Any- (Rocke and Bollinger, 2007). The car- angcheon stream event and included 20 cass-and-maggot cycle is a well-known species of migratory and resident wild scenario through which botulism out- birds, such as Spotbills, Teal species, breaks occur in waterfowl (Wobeser, Mallards, Pintails, and Mergansers (Kang 1997; Rocke and Bollinger, 2007). Botuli- et al., 2008). The second botulism event num toxin has also been identified in occurred in the Namdonggongdan reser- extracts of maggots collected from car- voir (37u23928.400N, 126u40935.760E) in casses that died of botulism (Shayegani et October and November of 2008. About al., 1984). However, in this outbreak, no 1,200 wild birds were sickened and died, botulinum toxin was detected in maggots. including waterfowl and shorebirds (In- Although the rate of toxin detection was chon Metropolitan Health and Environ- low in this outbreak, type C botulinum mental Research Institute, unpubl. data). toxin was confirmed by mouse inoculation The causes of the botulism outbreak in the from 26 affected birds. Some studies 954 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 3, JULY 2010

TABLE 1. Precipitation, maximum (Max.) and minimum (Min.) temperatures for Seoul, Korea for the fall of 2007 and 2008.

2007 2008

September October November September October November

Precipitation (mm) 411.7 60.4 13.7 63 32.7 23.5 Max. temperature (C) 25.4 21 13.6 27.1 22.4 13.8 Min. temperature (C) 18.3 10.5 1.9 17.3 10.7 3.1 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/46/3/951/2240923/0090-3558-46_3_951.pdf by guest on 30 September 2021 reported that the toxin was not detected paralysis and the detection of C. botuli- via the mouse assay in some birds affected num toxin in sera from wild waterfowl. by botulism (Thomas, 1991; Neimanis et Although the source of the toxin was not al., 2007). However, a mouse assay was determined, an influx of pesticides may used for diagnosis because of the irrevers- kill aquatic animals including vertebrate ible reaction of botulinum toxin at neuro- and invertebrate, and the decaying car- muscular junctions (Swerczek, 1980; Ros- casses would provide increased substrate setto and Montecucco, 2003). for bacterial growth, multiplication, and In this study, low-pathogenic avian toxin production. Furthermore, the con- influenza viruses and organophosphates centration of raw sewage may have been such as diazinon and phorate were detect- increased due to drought. As a result, the ed in two Spotbills and seven Mallards, water became shallow, the current was respectively. Although avian influenza was slow, and sediment deposition was in- isolated from two birds, lack of lesions and creased, thereby decreasing dissolved the isolated nature of this incident do not oxygen in the water. The botulinum toxin implicate this virus in this epidemic. thus produced would then have been The acute oral LD50 of diazinon is released into the food chain, resulting in 2.7 mg/kg in Mallard ducklings, while that an outbreak of botulism. of phorate is 0.62 mg/kg in Mallards We thank Jung-Won Park and Jong- (Tomlin, 2006). Diazinon was present at Hyeong Lee, Laboratory of Pathology, Ani- a lower concentration than would cause mal Disease Diagnostic Center, NVRQS, acute toxicity, whereas the detected con- for technical assistance. centration of phorate was above the threshold for acute oral toxicity in Mallard LITERATURE CITED ducks, suggesting that the death of Mal- BERNARD, W., T. J. DIVERS,R.H.WHITLOCK,J. lards was caused by phorate. Although not MESSICK, AND E. TULLENERS. 1987. Botulism as a all of poisoned birds show clinical signs sequel to open castration in a horse. Journal of the American Veterinary Medical Association and lesions associated with organophos- 191: 73–74. phate toxicity, tracheobronchial secretion, DOHMS, J. E., P. H. ALLEN, AND J. K. ROSENBERGER. lung edema, and cyanosis are observed in 1982. Cases of type C botulism in broiler affected animals (Nettles, 1976), and their chickens. Avian Diseases 26: 204–210. crops and gizzards are filled with feed FORRESTER, D. J., K. C. WENNER,F.H.WHITE,E.C. GREINER,W.R.MARION,J.E.THUL, AND G. A. (Quick, 1982; Reece and Handson, 1982). BERKHOFF. 1980. An epizootic of avian botulism However, there were no gross and histo- in a phosphate mine settling pond in northern pathologic findings associated with organ- Florida. Journal of Wildlife Diseases 16: 323– ophosphate toxicity in this study, suggest- 327. ing that botulinum toxin may have been KANG, M. S., A. KIM,B.Y.JUNG,M.J.KIM,S.J.JOH, Y. J. LEE,Y.H.BONG,N.R.SHIN,C.B.YANG, the main cause of death. AND J. H. KWON. 2008. An outbreak of botulism We identified botulism based on the in wild ducks in the Tancheon. In Proceedings of characteristic clinical signs of flaccid the 2008 KSVS Conference and General Meet- SHORT COMMUNICATIONS 955

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