Identification of botulism C/D in 2 dogs via polymerase chain reaction

Abstract

Two dogs from the same household were referred to the emergency service of the Veterinary Teaching Hospital of the École Nationale Vétérinaire de Toulouse for evaluation of acute onset of flaccid tetraparesis. At the time of presentation, the dogs were recumbent. Neurological examination revealed lower motor neuron deficits in all 4 limbs. Treatment consisted of supportive care. The 2 dogs recovered completely within 1 mo. Epidemiology, history, clinical signs, and clinical progression were suggestive of botulism. As Clostridium botulinum C/D was identified in the feces of both dogs using PCR, botulism was the most likely diagnosis. The source of contamination could not be determined; however, a sheep carcass in the dogs’ environment was suspected. This case report details clinical signs, neurological examination findings, diagnostic test results, treatment, and outcome of 2 dogs with PCR-confirmed C. botulinum C/D infection. To the authors’ knowledge, this is the first documented case of botulism C/D confirmed with PCR in dogs.

Key clinical message:

Testing with PCR should be incorporated into the diagnostic workups of dogs suspected of having botulism.

Botulism is a neuroparalytic disease induced by inhibited transmission of nerve signals due to the action of botulinum neurotoxins (BoNTs). Botulinum neurotoxins attach irreversibly to synaptic nerve endings and trigger the enzymatic cleavage of syntaxin, a cholinergic vesicle docking protein. As a result, the release of acetylcholine into the synaptic gap is blocked, leading to flaccid paralysis (1,2). Botulinum neurotoxins are produced mainly by Clostridium botulinum (C. botulinum), an anaerobic, ubiquitous, spore-forming, Gram-positive bacterium. Dogs and other carnivores are considered resistant to botulism toxins (1,3); however, several publications described botulism intoxications in dogs (1,3–6). Overall prognosis is guarded (1,5), though respiratory failure and death have been reported (2). A variety of methods to diagnose botulism in dogs, including specific BoNT antibody titer, microcolumn chromatography, C. botulinum identification, electrophysiological methods, and mouse bioassays (MBA), have been reported (3,5,7). After recent improvements, PCR testing for C. botulinum on fecal and gastric content seems a reliable method to diagnose botulism in avian, equine, and bovine species, with the potential to serve as an alternative to the laboratory diagnosis of botulism in dogs (8–10).

CASE DESCRIPTIONS

Two Beauceron dogs from the same household were referred because of acute onset of flaccid tetraparesis with a 2-day duration. According to the history provided, both dogs had ingested a decomposed lamb carcass 2 d before the onset of clinical signs. Mild ataxia had rapidly progressed to tetraparesis in both individuals. Both dogs were in lateral recumbency at admission. Neurological examination showed lower motor neuron deficits in all 4 limbs.

Case 1

A 7-month-old male Beauceron dog weighing 25 kg was referred to the emergency service of the Veterinary Teaching Hospital of the École Nationale Vétérinaire de Toulouse for evaluation of ascending weakness progressing to tetraparesis over a 2-day period. The dog lived on a sheep farm where he was allowed to roam freely and had possible access to carrion, especially lamb carcasses. On physical examination, the dog had a heart rate of 76 bpm, a respiratory rate of 20 brpm, and a rectal temperature of 38.0°C. Abdominal palpation was unremarkable. No peripheral lymphadenopathy was noted. Physical and neurological examination revealed tetraparesis. No cranial nerve abnormalities were identified. The patellar and cranial tibial reflexes were absent in the hind limbs. Withdrawal reflexes were weak in all 4 limbs. Pain perception was present. The dog was unable to urinate. Serum biochemical abnormalities included mild increases in AST [43 U/L; reference interval (RI): 1 to 37 U/L] and ALT (62 U/L; RI: 3 to 50 U/L). Complete blood (cell) count was unremarkable, as were thoracic radiographs.

Based on the history and neurological examination findings, the differential diagnosis for the flaccid tetraparesis included acute polyradiculoneuritis, botulism, organophosphate intoxication, ionophore intoxication, polymyositis, acute fulminant myasthenia gravis, and tick paralysis (2). As the vector of tick paralysis has never been reported in Europe, tick paralysis was ruled out. Other diseases considered typically affect single animals, with the notable exceptions of organophosphate and ionophore intoxications, which can occur as outbreaks. These substances are primarily employed as coccidiostats in poultry farming or as agricultural pesticides and were not used on the premises, according to the farmer. Intoxication with either of these compounds was therefore unlikely.

Fecal samples were collected and analyzed for the presence of C. botulinum. The samples were diluted 10-fold in fortified cooked meat medium, incubated at 71°C for 10 min, and further incubated under anaerobic conditions at 37°C for 18 h. The DNA was extracted from 1 mL of broth, and the presence of genes encoding BoNT/C, D, C/D, and D/C was evaluated using real-time PCR as previously described (11). Clostridium botulinum C/D was identified in the fecal samples using the method described. Treatment consisted of supportive therapy, including intravenous fluids, intermittent catheterization of the urinary bladder, nursing care, and physical therapy. Botulinum antitoxin is not available for veterinary use in Europe. Therefore, specific treatment for botulism could not be administered. Respiratory function was monitored closely using pulse oximetry. Positive pressure ventilation was not required. During hospitalization, the dog’s neurological status improved gradually. At the time of discharge, 6 d after admission, he was able to maintain sternal recumbency. At 1 wk after discharge, the dog was able to walk but was still ataxic. At 30 d after initial presentation, the dog had recovered completely.

Case 2

A 4-year-old female Beauceron dog weighing 50 kg from the same household as the dog in Case 1 was referred to the emergency service of the Veterinary Teaching Hospital of the École Nationale Vétérinaire de Toulouse for evaluation of acute onset of flaccid tetraparesis with a 2-day duration. Physical examination revealed a heart rate of 80 bpm, a respiratory rate of 14 brpm, and a rectal temperature of 38.3°C. Abdominal palpation was unremarkable. Peripheral lymph nodes were within normal limits. Neurological examination findings were as described for Case 1. Serum biochemical abnormalities included moderate increases in AST (145 U/L; RI: 1 to 37 U/L), ALT (62 U/L; RI: 3 to 50 U/L), and ALP (157 U/L; RI: 20 to 155 U/L) associated with a marked increase in CK (2723 U/L; RI: 25 to 467 U/L). Complete blood (cell) count was unremarkable, as were thoracic radiographs.

Clostridium botulinum C/D was detected in fecal samples from this dog using the same method as described for Case 1. The dog was hospitalized and received the same treatment as the dog in Case 1. This dog improved more slowly than the first, possibly due to concomitant orthopedic problems. During hospitalization, the dog appeared to be in pain on palpation of the thoracic limbs. Radiographs of the forelimbs did not reveal abnormalities, and the dog was prescribed meloxicam [0.1 mg/kg (0.045 mg/lb), PO, q24h] for a duration of 5 d. The dog showed little neurological improvement during hospitalization and was unable to maintain sternal recumbency at the time of discharge, 6 d after admission to the hospital. Like the dog in Case 1, this dog had recovered completely by 30 d after initial presentation.

Epidemiological investigations

The farm animal population in the dogs’ environment consisted of 1000 sheep raised for dairy production and lamb meat. The dogs described in this report were the only other animals present on the farm. Wild animals likely inhabited the surrounding area and may have come into contact with the dogs. No poultry were present on the farm. Samples were collected on the farm for testing to try to identify the source of contamination. Swabs were used to sample the housing of the livestock and the carcass container, and water and liquid collected in the rendering bin (Figure 1). Evaluation of the samples for C. botulinum C/D was as previously described (11). No bacteria were detected in the samples evaluated. Due to the owner’s refusal, it was not possible to examine fecal samples from the farm’s sheep.

FIGURE 1.

Rendering bin from the farm where the 2 dogs lived. The bin was used to store carcasses before their removal.

DISCUSSION

This case report details clinical findings, treatment, and outcomes of 2 dogs with suspected botulism, as well as the presence of C. botulinum C/D in the feces of both dogs, identified using PCR. The history, clinical presentation, and neurological examination findings of the 2 dogs were suggestive of botulism. The evolution of clinical signs and the recovery times were similar to those described in previous case reports (1,5). Of note, most conditions included in the differential diagnosis of botulism generally affect only a single individual, except for organophosphate and ionophore intoxications, which were ruled out in these cases. Given the very low prevalence of both myasthenia gravis and acute polyradiculoneuritis in dogs, the likelihood of observing 2 cases in animals from the same household is exceedingly low. To the authors’ knowledge, no clusters of these 2 diseases have been reported in the veterinary literature to date. This context further underscores the increasing importance of accurate botulism diagnosis, particularly when other causes of lower motor neuron disease can be excluded based on clinical history and environmental investigation. Although botulism is often suspected based on clinical presentation, confirmation remains challenging.

Many different methods have been used to confirm canine botulism in the laboratory. These include MBA for BoNT detection, detection of specific anti-BoNT antibodies in dog serum, microcolumn chromatography, and detection of C. botulinum using the MBA (Table 1) (1,5–7).

TABLE 1.

Characteristics of the main methods for diagnosis of botulism.

Method	Targeted element	Sample type	Result time
Chromatography	Toxin	Serum	24 h
Mouse bioassay	Toxin	Serum	15 d
ELISA	AntST	Serum	21 d
PCR	Bacterial DNA	Feces/gastric content	48 h

AntST — Specific toxin antibody, according to (3–5,8).

Mouse bioassay has been the gold standard for BoNT detection; however, ethical concerns require its replacement by alternative methods in line with European regulations. For the detection of BoNTs in sera, samples must be collected at the onset of clinical signs, when the toxin is still present in the blood. Due to the time delay between onset of clinical signs and presentation, it may be difficult to identify BoNT in the serum. Due to the progressive decrease in the amount of BoNT in the bloodstream and the potent nature of BoNT, highly sensitive methods are required for its detection. In human botulism cases, the detection of BoNT in the serum of patients with clinical symptoms of food-borne botulism is reported to be ~70%, with a range of 56 to 82% (12). Farrow et al reported an amount of 25 LD₅₀ per mL in a serum collected from a dog with severe clinical signs 4 d after exposure, illustrating the challenge of detecting BoNT (6).

Although detection of BoNT in samples collected from symptomatic animals is the standard approach for diagnosing botulism, detection of BoNT-producing clostridia has also been reported (13). Identification of the BoNT-producing bacteria instead of BoNT is a method that allows short turnover time and addresses ethical concerns (Table 2). Use of PCR testing has been of particular interest for the diagnosis of botulism in cattle and horses, for which BoNT detection in serum is difficult (14). Therefore, PCR assays are now widely used to diagnose botulism in avian species, cattle, and horses. These tests are typically run on fecal samples or gastric contents, following an enrichment step to enhance sensitivity. In avian, equine, and bovine botulism, PCR has demonstrated diagnostic performance comparable to the MBA (Table 3) (8–10,14). Notably, Johnson et al reported sensitivities of 89, 86, and 96% for Types A, B, and C, respectively, with specificities ranging from 99 to 100%, surpassing the MBA with respect to both accuracy and turnaround time (10). In the case reported here, laboratory confirmation was obtained using PCR to detect C. botulinum, after an enrichment step, as currently recommended in France for the diagnosis of avian or cattle botulism (9,15).

TABLE 2.

Advantages and disadvantages of diagnostic methods.

Test	Advantages	Disadvantages
Mouse bioassay	High specificity, available Gold standard	Low sensitivity, expensive, long processing time, ethical concerns
Serology	High specificity	Seroconversion delay, limited availability
PCR	Inexpensive, widely available, fast results	Possible asymptomatic carriage
Electromyography/electroneurography	Fast results, differentiates PR-MG-PM	Limited availability Not specific

MG — Myasthenia gravis; PM — Polymyositis; PR — Polyradiculoneuritis.

TABLE 3.

Sensitivities and specificities related to PCR testing among species.

Species	Serotype(s)	Se/Sp (%)	Source
Avian	C, D, C/D, D/C, E	95/100	(9)
Equine	A	86/99 to 100	(10)
Equine	B	89/99 to 100	(10)
Equine	C	91/99 to 100	(10)
Bovine	D	100/not reported	(8)

Se — Sensitivity; Sp — Specificity.

Detection of the bacterium, rather than the toxin, may raise concerns about false-positive results, reduced specificity, and asymptomatic carriage. However, available evidence indicates that this method does not commonly yield false-positive results in avian, bovine, or equine species (8,9,16,17). Although Clostridium perfringens and Clostridium difficile are commonly identified in the gut microbiota of healthy dogs, C. botulinum has not been reported in such populations (18). Detection has been limited to dogs exhibiting clinical signs consistent with botulism (6). The lack of studies on asymptomatic carriage limits interpretation of positive PCR findings in dogs.

In the case reported herein, PCR allowed us to identify mosaic serotype C/D. Currently, 9 types of BoNT (i.e., A to H plus X) and > 40 subtypes, including mosaic BoNT/C/D and D/C, first described in 1996, have been identified (19,20). Botulinum neurotoxin C/D is the most common BoNT type involved in avian botulism outbreaks in Europe (21–25). Most reported canine cases of botulism were induced by BoNT/C, and sporadically by BoNT/A, BoNT/B, and BoNT/D (5,26,27). Discrimination between BoNT/C and C/D is not systematically made during laboratory analysis, especially when MBA is used. It is therefore unknown whether previous Type C outbreaks described in the literature were indeed Type C or were actually Type C/D outbreaks (3,6,28).

Contaminated carcasses have been widely reported as the source of botulism in dogs (1–3,29). The 2 dogs reported herein were allowed to eat lamb carcasses, which may have been the source of contamination; however, none of the samples collected on the farm was positive for C. botulinum C/D. Clostridium botulinum C/D is typically involved in avian botulism outbreaks. To our knowledge, no outbreaks of avian botulism were reported in the neighborhood or in wild birds in the vicinity of the farm.

In conclusion, to the authors’ knowledge this is the first case report describing the use of PCR testing to identify C. botulinum C/D for the diagnosis of botulism in dogs. In vitro diagnostic methods for botulism depend on high-quality reference materials and specific reagents, such as antibodies, which are currently difficult to obtain. No validated commercial diagnostic kits are currently available, limiting the broader application of these techniques in routine veterinary diagnostic testing. Compared to other diagnostic methods, PCR testing offers advantages, such as rapid availability of results, which may affect an animal’s medical management. Botulism has typically been diagnosed with reliance on history, clinical signs, epidemiology, and progression of the disease. Use of PCR testing for BoNT-producing clostridia may help to establish a definitive diagnosis. Further studies on the prevalence of asymptomatic carriage of C. botulinum in dogs may strengthen the diagnostic utility of PCR testing.