Outbreak of Type C Botulism in Birds and Mammals in the Emilia Romagna Region, Northern Italy

Avian botulism, mainly caused by ingestion of type C toxins produced by Clostridium botulinum, is a common cause of death for waterbirds worldwide (Na-Ri et al., 2010). We describe an outbreak of botulism that occurred in August 2011 along Crostolo stream, which flows through Reggio Emilia (44°67′64″N, 10°62′57″E) in the Emilia Romagna region, Italy. In the summer, stream flow rate is usually reduced, and as a consequence, water stagnation is observed. The highest average monthly temperature (27.3 C) and the lowest precipitation (0.00 mm) for 2011 were recorded in August; the biochemical oxygen demand was 6 mg/L, and the chemical oxygen demand was 16 mg/L.

The Crostolo stream is populated commonly with waterbirds. In the late summer of 2008, a botulism outbreak occurred involving a large number of birds. On 8 August 2011, several Mallards (Anas platyrhynchos) were found dead in the Crostolo stream. Over the following days, mortality extended to Hooded Crows (Corvus corone cornix), Little Egrets (Egretta garzetta), Common Moorhens (Gallinula chloropus), Yellow Wagtails (Motacilla flava), and several coypus (Myocastor coypus). The dead animals and the decaying carcasses were collected and destroyed promptly. From the first report of bird mortality on 8 August 2011 until the end of the phenomenon, which lasted 1 wk, 80 dead birds and 16 coypus were collected by the Provincial Veterinary Service along the banks or in the water of the Crostolo stream.

Thirty-five live, sick Mallards that showed clinical signs characterized by weakness of legs and wings, inability to sustain flight, and flaccid paralysis were collected. These were sent to a wildlife rehabilitation center and given fluid and antibiotic therapy. Thirty of 35 Mallards sent to the wildlife rehabilitation center recovered completely.

Twenty-eight Mallards, two Hooded Crows, and three coypus found moribund on the Crostolo stream bank were collected by the Provincial Veterinary Service and transported to Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Laboratory of Reggio Emilia, for diagnostic testing. None of the animals showed specific gross lesions. Maggots (third-stage larvae of Lucilia caesar [Szpila, 2009]) were observed in the stomach content of 20 of 28 dead Mallards. The alimentary canal was empty in the remaining birds. During postmortem examination, blood clots were collected from the hearts of all animals and maggots (if any) from their stomachs. Blood clots were centrifuged to obtain serum. Mallard sera were pooled into seven pools of four samples each to obtain sufficient material for the mouse bioassay. Sera from crows and coypus were tested individually. Maggots collected from 20 Mallard stomachs were also pooled into five pools, four samples each. Maggots were carefully washed to remove external contamination, homogenized, and submitted to overnight cold extraction (4 C) in gelatin diluent (0.2% gelatin, 0.4% Na₂PO₄; pH 6.4, 1∶1 [w/v]). Serum samples and maggot extracts were used for mouse bioassays carried out according to Centers for Disease Control and Prevention (1998) using type E and C antitoxins. Samples of intestine, heart, kidney, liver, and brain were collected and tested for viral infectious diseases using different PCR methods: avian influenza (Spackman et al., 2002), Flavivirus genus (Manarolla et al., 2009), Usutu virus (Scaramozzino et al., 2001), and West Nile virus (Tang et al., 2006). Samples of intestine and liver were tested for Salmonella spp. using standardized methods. Liver also was cultured directly on blood agar, serum agar, and Hektoen enteric agar. All pools of sera and maggots were positive by mouse bioassay, showing typical signs of botulism during the first 12 hr after inoculation. Mice were subsequently protected by C. botulinum type C antitoxin. Virologic and bacteriologic tests were negative.

The first avian botulism outbreak in Italy, reported in the late summer of 1973 in the Emilia Romagna region, involved Mallards and other ducks. Since its first description, 14 avian botulism outbreaks have been reported in seven Italian regions: Emilia Romagna, Sardinia, Lazio, Tuscany, Piedmont, Veneto, and Liguria (Table 1 and Fig. 1; Annibali et al., 2011). All of these outbreaks were characterized by the presence of conditions favorable for the growth of C. botulinum. These factors include optimal environmental conditions for spore germination and bacterial growth, suitable material or substrates that provide energy for bacterial replication, and a means of toxin transfer to birds. All of these conditions influenced the occurrence of outbreaks; however, some conditions in places (lakes, streams, and rivers) where outbreaks had occurred were significantly different compared with those of places in which no botulism outbreak occurred (Rocke, 2006).

In the outbreak area in the late summer (August/September) environmental conditions were suitable for growth of C. botulinum: shallow water, increase in air and water temperature, increase in the sediment temperature, and decaying organic matter. Temperature plays a critical role in the multiplication of C. botulinum and toxin production. Temperatures at the time of the outbreak were 24.2–27.2 C, satisfying the conditions required for C. botulinum multiplication and toxigenesis (Segner et al., 1971). Additionally, the absence of precipitation in August could have enhanced the deterioration of the aquatic conditions, increasing spore germination, bacterial growth, and toxin production by C. botulinum.

The source of the botulinum toxin cannot be identified; however, it is likely that the toxin was released into the food chain because of several contributory factors. Decaying vertebrate carcasses may have played a major role by providing a suitable substrate for growth of C. botulinum and production of type C toxins; decaying carcasses provide a protein-rich and anaerobic environment. The role of migratory birds in the origin of botulism outbreaks is a topic of debate. Birds can act as mechanical carriers of C. botulinum spores and can disperse them to adjacent or distant water reservoirs, where they can germinate and cause avian botulism (Zdenek, 2004). Some outbreaks of avian botulism have been linked to bird migration routes (Zdenek, 2004).

Every year several species of wild birds, in particular the Mallard, migrate from other European countries to Italy. Italy hosts Mallards originating from a wide geographic area: Central Europe (Germany, Austria, Poland, Czech Republic, Switzerland, French Camargue, and southern Germany), northern Europe (Denmark, Sweden, and the UK), and eastern Europe. The movement of migratory birds from these countries to Italy starts at the end of August, increases in November, and reaches a peak from December until March (Spina and Volponi, 2008). Spores of C. botulinum transported mechanically from contaminated areas may trigger outbreaks even several months after the occurrence of the environmental contamination when the conditions became optimal for spore germination and bacterial growth.

The detection of C. botulinum type C toxins in dead larvae collected in the alimentary canal of the carcasses suggests that consumption of toxin-bearing fly larvae was a likely source of intoxication in waterfowl (Shayegani et al., 1984). Fly larvae and other invertebrates are unaffected by the toxin but, as they feed on decaying matter, effectively concentrate the toxin. In this way, botulism outbreaks in waterfowl often become self-perpetuating (Duncan and Jensen, 1976; Wobeser, 1976).

For these reasons, the removal of bird carcasses has been suggested as a tool to manage C. botulinum outbreaks (Evelsizer et al., 2010) since a reduction in density of toxin-laden maggots produced within bird carcasses is assumed to improve the survival rate of healthy birds.

Our demonstration of C. botulinum type C intoxication in the strictly vegetarian coypu implicates water and plant contamination by infected carcasses and maggots as a source of the toxin. Further investigations are needed to gain a better understanding of the epidemiologic role of migratory birds, the effect of environmental factors, and the role of invertebrates as vectors of toxins in botulism outbreaks.

This study was partially funded by Emilia-Romagna Region, Italy, COMM.07/001.