CASE NOTES

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A case of botulism in a dairy herd

Helen Schaefer, South East Local Land Services, Bega

Posted Flock & Herd March 2015

Introduction

The first recorded case of botulism goes back to 1735 when German sausages were blamed for an episode of food poisoning. The term botulism was not employed until 1870 when a German doctor named the disease "botulism" after the Latin word for sausage, botulus. The bacteria was isolated in 1895. The first of the neurotoxins it produces was identified in 1944.

Botulism is caused by the Gram-positive, spore forming anaerobe, Clostridium botulinum, and affects animals, birds and humans. Cl. botulinum spores are present around the world in soil, water and marine sediments, and are resistant to destruction with the potential to survive in the environment for over 30 years. The bacteria can also be found as a normal inhabitant of the intestinal tracts of cattle, horses and poultry.

This case demonstrates the challenge it can be to diagnose a disease which has limited diagnostic aids, particularly when it does not present as classically recognised. It also raises a challenge to the veterinary profession in some areas, to be proactive in disease prevention in livestock industries as the practices of those industries change.

CASE REPORT

History

• Dairy farm, 337 hectares, Bega, Far South Coast NSW
• year round calving
• 260 milkers, predominantly large frame Holstein Friesians with some smaller types and a few cross breds
• averaging 26L milk daily
• Vaccination - 7 in 1 + Salmonella
• Diet:
  • milkers - intensive Partial Mixed Ration (PMR), fed
  • A. via feed pad ( ~120m concrete): 8kg corn silage, 2.8kg biscuit meal, 6kg wheaten hay, 4kg Distillers Condensed Solubles (DCS) -syrup.;
  • B. via dairy feeders: 4kg 18% dairy pellets + 5kg Dried Distillers Grain (DDG).
  • dry + springers PMR, ~ as above, smaller quantity/head + pasture

• Have been operating the farm based on a PMR system for a number of years.
• Changed nutritionist Dec 2013

Timeline:

• January 2014 - commenced converting to the above ration over a 2 month period, initial silage was rye grass
• March - opened Pit A, which consisted of corn sileage
• May - opened Pit B, which was also corn silage; fed 6kg ~50/50 mix until Pit A ran out
• ~May 25 - 100% silage from Pit B, 6kg/hd/day

• Days 1-16 (28/5 -12/6):
• 15 cows became recumbent, 3yrs - 8yrs of age, 46-289 DIM, all big strong cows, always ate very well;
• 8 shot due to failure to get up in spite of treatment with 4-in-1* milk fever injection (* calcium 27.5g/L, glucose 182g, magnesium hypophosphite 4.7g, phosphorus hypophosphite), propylene glycol, anti-inflammatory injection and intensive supportive care
• 7 recovered, with the aid of immediate lifting +/- 1 x 500ml bag, 4-in-1 milk fever injection subcutaneously
• Private vets had run blood samples in-house: cbc + mba + electrolytes from a number of the recumbent cows. Apart from elevated CK and AST (secondary to downer cow syndrome), no significant findings
• Clinical findings on the downer cows: apart from weakness, nad. Normal reflexes, temp nad, tongue and tail tone nad. If did get up with lifting, appeared strong, went straight to feed pad/in for milking
• No change in diet over that time

• Day 16 (12/6):
• Private vet requested district vet assistance. Cow went down 10 days ago; was lifted, all good; she went down again 3 days later, lifted - all good; went down again 3 days ago - not responding to being lifted. Had been maintaining sternal recumbency, bright alert responsive on day 1, went into lateral recumbency last 48 hours; faeces became very soft and malodorous last 36 hours. No other findings on clinical examination. Private vet requested assistance to do PM, work up the case involving all cows
• 12/6 post-mortem on shot cow - no significant findings; liver, kidney, mesenteric lymph nodes, SI all nad on histology, and for salmonella culture (erroneously did not sample rumen contents)

• Day 17 (13/6):
• 1 cow sudden death on farm in the morning - cow ~250DIM. Private vet did the PM later in day - nsf grossly or from lab. Unsure if this was part of the "episode" - not consistent presentation with other affected cows

• Day 18 (14/6):
• DCS - syrup removed from diet by nutritionist (there had been a suggestion that perhaps the high quantity of syrup was interfering with rumen function)

• Day 20 (16/6):
• cow down - up with lifting only, blood sampled for mba - nad; nil recurrence, milked that pm - all good

• Day 21 (17/6):
• cow down - HL's caudal extension - farmer went to get blood tubes - cow up by self - into dairy, milked - all good

• Day 21 (17/6)
• sileage pit examined by nutritionist to sample for mould and pH check by Westons - also checking other ration components. Observed that Pit B had relatively "high" contamination of "castor oil" plant as it was referred to by the farmer. There was leaf in Pit A and ~45 seeds/kg. There was ~ 225 seeds/kg in Pit B
• The "contamination" of the corn silage by this plant had been recognised in previous years but the higher seed content had not been noted. After some research and discussion it was determined that the plant in question was most likely Datura sp. (false castor oil plant), not Ricinus communis (castor oil plant) as initially suggested by use of the term "castor oil plant" by the farmer. Definitive identification of seeds required.

• Day 22 (18/6):
• silage removed from diet; new ration for milkers, on the feedpad: 25.5kg green chop (rye grass dominant), 2.3kg biscuit meal, 6kg oaten hay

• Where we were at Day 22 (18/6/14)
• No deaths since Day 17 (13/6), no recumbencies since Day 21 (17/6).

Chart 1: Deaths and recoveries during the outbreak

Working on 3 "rule out" fronts:

1. Botulism - via silage pH (too high >4.6) + attempt isolation of the toxin from silage + syrup
2. Seed toxicity - confirm the ID. + potentially the levels of toxin
3. Metabolic disease - the mid-lactation hypoCa theory that the farmer was quite keen on was highly unlikely given the blood results so far, however it would be good to get some more "on the spot" samples of a few to be sure...if any more go down.

• Results as at Day 23 (19/6):
• Blood tests on multiple cows: nsf. No evidence of metabolic disease
• Post-Mortems : no significant findings grossly or on laboratory examination/testing
• Mould negative for silage Pits A & B, dairy pellets, DDG, biscuit meal
• pH correct for silage Pits A & B, DCS syrup (3.75-3.94)

Farmer now also considering the possibility of botulism from the silage or the syrup.

To further progress the "rule outs"

1. Botulism:
• silage sample sent to Biosecurity Qld (ref. Joanne Mollinger) via EMAI for attempted toxin isolation; to also send syrup sample for toxin check
• Spoke to DAFWA re: ELISA testing (ref. John Creeper) - looking at doing convalescent samplesi.e. 3-4wks post acute episode. If all negative, is reasonable to rule out botulism; if some positives, is highly suggestive of botulism in a non-endemic area. Won’t necessarily help with exact time of exposure
2. Toxicity:
• Toxicity: source = sileage - Datura sp.
• * Toxic parts: seeds, leaves, flowers
• * Toxins: tropane alkaloids including atropine
• * Toxic dose: can be wide variation in the alkaloid content in batches of the plant especially in the seeds. Doses of 600-900mg/kg/day have been shown to affect cattle. Regulations in Australia stipulate that grain for livestock feed must not contain more than 5 Datura seeds per kilogram. An experiment in Nth America where over 4000 seeds/kg were fed in a total mixed ration induced inappetance,dry muzzles, mydriasis, bloat and tenesmus. The signs then subsided and the cattle started eating again after 2-3 days.
• * Clinical signs of toxicity: as above, plus tachycardia, polydipsia, tremors, with possible convulsions, paralysis, coma and death.
• * While signs observed in these cattle were not consistent with alkaloid toxicity, the seeds in question were sent to the National Herbarium of NSW, Royal Botanic Gardens, for identification. Subject to findings/other developments, there was the option to send the seeds to the ChemCentre in WA (ref. Ken Dods) to determine the alkaloid levels in the seeds.
3. Metabolic:
• have emphasised importance with farmer of getting bloods for MBA at time of going down - only one sampled since then was nad

• Results as at Day 37 (3/7): (no recumbencies since Day 21 (17/6))
• nil toxin recovered from silage or syrup samples
• seeds confirmed as Datura sp. Most likely Datura stramonium

• Plan as at Day 37 (3/7):
• continue withholding sileage and syrup from the diet;
• booked in to take convalescent samples from 9 cows 9^th July = 3 weeks since last cow went down on 17/6, 6 weeks since the first went down on 28/5, and recovered; 2mls serum to Animal Health Laboratories, DAFWA; turn around time ~2weeks
• cost for Datura sp. alkaloid i.d. + quantification ~$2000 will not pursue at this stage
• recommended vaccination against botulism. Even if this is not a botulism case - on this diet - cattle are at risk. Looking into vaccine options.

• Day 43 (9/7):- Farm Visit to sample the recovered cows
• 9 cows sampled

• Day 44 (10/7) : Review of diet by nutritionist
• Advised running out of green chop
• After discussion by all parties, agreed to re-introduce silage from Pit B at 1.5kg/hd/dayi.e. 25% of the feed rate when the cows went down. All on high alert.

• Day 55 (21/7):- Results from Animal Health Laboratories DAFWA, Botulinum antibody ELISA:
• 3/9 positive for antibodies against botulinum C toxin. (ref. table 1)
• Lab comment: "This seroconversion is indicative of herd exposure to botulinum C. The absence of antibodies to botulinum D supports the interpretation that this was a natural exposure rather than a vaccinial response".
• Spoke to the WA pathologist:"These are strong positives. The test is a "herd test" - it is not particularly sensitive but it is highly specific. From these results, the diagnosis is botulism."
• Diagnosis: Botulism

Test Type: Botulism Type C Antibody - ELISA

Test Type: Botulism Type D Antibody - ELISA

Table 1: Animal Health Laboratories Report from 21^st July 2014

• Day 59 (25/7):
• Vaccinated the herd with SingVac 1 year vaccine. "In previously unvaccinated animals, SingVac 1 Year vaccine will generate protective levels of circulating antibodies against Cl.botulinum types C&D within 35 days of vaccination."

• Day 61 (27/7):
• Increased silage from Pit B to 3kg/hd/day. All parties agreed to leave at this level (50% of feed rate when cows went down) until the vaccine takes effect.

• Day 99 (3/9):
• Increased silage from Pit B to 6kg/hd/day, 40 days after vaccination.

Update:

19/1/2015 :- Spoke to farmer: no issues with botulism since 17/6/14. Full rate of silage feeding since 3/9/14. Cows averaging 30L per cow per day. Are having some issues with high somatic cell counts. Have not re-introduced the DCS syrup as yet - were about to when the cell count went up; don’t want to change anything until that is resolved.

Will continue to vaccinate with 7-in-1, for salmonella and botulism (at different sites/sides) and drench, the entire herd, annually.

Discussion

Bovine botulism is endemic in Australia and has most often been associated with extensive enterprises in Queensland, Northern Territory and Western Australia where cattle develop pica associated with phosphorous and/or protein deficiency. This induces them to chew on bones or eat carrion to satisfy their cravings. Over the last decade or so botulism has become more common in intensive systems, with the increase of feedlots involved with beef production and the use of conserved fodder via total or partial mixed rations in dairy herds.

During its vegetative growth state, which is encouraged by warm anaerobic conditions, C. perfringens produces neurotoxins, seven types of which have been identified (A-G). Ideal situations for neurotoxin production include rotting carcasses and decaying vegetation. Disease in cattle most commonly occurs when individuals ingest the preformed toxin. Toxin types C and D are responsible for almost all cases of botulism in cattle in the southern hemisphere. Type B toxin is more common in Europe and Northern America. The toxin is absorbed from the intestinal tract, enters the bloodstream and is transported to peripheral cholinergic nerve terminals. Inhibition of the release of acetylcholine ensues and leads to flaccid paralysis, with death being caused by respiratory paralysis.

Botulism is recognised as being a difficult diagnosis to make, particularly in geographical areas not recognised as having experienced botulism outbreaks previously. The difficulty is increased in the absence of the classical signs associated with botulinum toxicity such as sudden death, tongue paralysis, tongue protrusion, dysphagia, drooling. Diagnosis has generally relied on clinical signs, ruling out other possibilities and the demonstration of toxin, the latter of which can be very challenging.

What can be learned from this case?

Does the case involve-

• Cluster of seriously affected cattle within 1-2 weeks followed by fewer, less severe cases?

A botulism outbreak in an intensive system generally involves the majority of deaths occurring within the first week, followed by a number of clinically affected animals who recover over the next couple of weeks, with intermittent deaths occurring for up to 3 weeks.^11,15 ?

• No clinical signs apart from recumbency/weakness?

The clinical signs of botulism do depend on the dose of the toxin ingested and the immune status of the animal. Sub-lethal botulism in cattle is now recognised as a syndrome, though perhaps it is not as widely recognised as it ought to be, particularly with respect to intensive systems. There have been a number of cases where the only sign observed of botulism is muscle weakness, and many cases where animals have been observed to recover from botulism, especially later in an outbreak.

The absence of any other clinical signs can be useful to rule out other diseases eg.hypomagnesaemia, listeriosis.

• No significant findings on blood tests?

Given that botulism, particularly as the sub-lethal syndrome, can be confused with a number of other diseases, eg. milk fever, pathology can be very useful to rule out differential diagnoses.

• No significant findings on post-mortem examination?

There are no pathognomonic lesions for botulism discernible on post-mortem or laboratory examination of tissues

• Conserved fodder is being used eg. silage or hay, particularly as part of a total or partial mixed ration?

Silage can provide an ideal environment for the botulism bacteria to grow, particularly if a dead animal ends up in the pit, such as a rodent, bird, snake or cat. Hay can also be contaminated by such carcasses. The risk of significant numbers of animals being affected with botulism, if such contamination exists, increases when the silage or hay is put through a mix-all type process and is evenly distributed through the ration. While this process can lead to a lower dose being consumed by individuals, it does promote the ingestion of the toxin by more of the herd.

• A change in the conserved fodder being used has occurred in any way within the previous two - three weeks. The incubation period as stated in the literature, from ingestion of the botulinum toxin to cattle exhibiting clinical signs, varies from 12 hours (extreme event, massive dose of toxin) to up to 17 days (low doses of toxin).¹¹It is recognised as being most commonly 2-7 days.^11,14

...THINK BOTULISM:

• eliminate most likely source as soon as possible,
• continue with the work up/rule outs

• if botulism remains as a differential diagnosis, collect bloods from all recovered cows, 3-4 wks post occurrence of clinical signs. Submit those samples for botulinum antibody ELISA. The only laboratory currently providing this testing in Australia is the Animal Health Laboratory, Department of Agriculture and Food, Western Australia.

Botulism antibody-ELISA

• The ELISA detects antibodies to the C and D botulinum toxins, and some associated proteins.
• Vaccines produce a strong ELISA response, to both botulinum toxins C and D,
• Natural exposure to toxin will produce a weak ELISA reading to type C or D.
• The reference range is based on ELISA units (EU). The cut off values are as follows:
  type C result is <11EU = negative
  type C result is > 11EU= positive
  type D result is < 7EU = negative
  type D result is > 7EU = positive

The cut-off at 11EU and 7EU for types C and D respectively is statistically calculated to exclude "false-positives" at a level of 99% or better. There is therefore, a reasonable chance of "false negatives" occurring.

• The test has high specificity and low sensitivity.

• It is a very useful herd test where low titres (usually <50EU) to either C or D, in animals surviving a suspect botulism outbreak can be considered confirmatory. Failure to demonstrate antibodies does not exclude the diagnosis.
• Botulism antibody ELISA can be an extremely useful tool in supporting the diagnosis of botulism, particularly in a situation where the disease is suspected of having occurred in a non-endemic area, in the absence of the classic clinical signs.^,

• Cattle in a dairy system being fed a mixed ration, or beef cattle in a feedlot system, should be vaccinated against botulism. The potential cost of an outbreak demands that the risk of botulism be minimised. Vaccination is the most important and reliable way to do that.¹⁸ It is vital that producers involved in such enterprises are well informed of the risk of botulism, and advised that there is the option to vaccinate against it.

Acknowledgements

For being vital members of the team effort:

• Dr. Nikki-Lea Esmond, Bega & Cobargo Veterinary Hospitals
• Phil Pittolo, Pittolo Nutrition
• Duncan & Tash Gowing, Bega Valley Dairy Farmers

References

1. www.botulism-symptoms.com
2. Wilkinson JM (1999) Silage and animal health. Nat.Toxins, 7:224
3. George Weston Technologies, Analytical Reports June 2014
4. George Weston Technologies, Analytical Reports June 2014
5. McKenzie, RA (2012) Australia's Poisonous Plants, Fungi and Cyanobacteria.CSIRO Publishing
6. McKenzie, RA (2012) Australia's Poisonous Plants, Fungi and Cyanobacteria.CSIRO Publishing
7. Virbac Animal Health July 2011
8. De Witte K, Taylor L. Managing the risk of botulism in beef and dairy herds. The Australian Cattle Veterinarian
9. Parkinson TJ, Vermunt JJ, Malmo J. Diseases of Cattle in Australasia. A comprehensive textbook. Vetlearn 2010 p320
10. Western Australian Agriculture Authority Botulism in cattle 2013
11. Radostits OM, Gay CC, Hinchcliff KW and Constable PD. Veterinary Medicine. A textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th Edition. Elsevier Ltd 2007 p824-826
12. Jubb TF, Ellis TM and Gregory AR (1993) Diagnosis of botulism in cattle using ELISA to detect antibody to botulinum toxins. Aust. Vet. Journal 70: 226-227
13. Main D and Gregory A (1996) Serological diagnosis of botulism in dairy cattle. Aust. Vet. Journal 73: 77-78
14. De Witte K and Taylor L. Managing the risk of botulism in beef and dairy herds. The Australian Cattle Veterinarian.
15. McKenzie, Ross pers. comm. 18th June 2014
16. Poe, Ian pers. comm. 23rd June 2014
17. Wilkinson JM (1999) Silage and animal health. Nat.Toxins 7:224
18. De Witte K and Taylor L. Managing the risk of botulism in beef and dairy herds. The Australian Cattle Veterinarian.
19. Parkinson TJ, Vermunt JJ and Malmo J. Diseases of Cattle in Australasia. A comprehensive textbook. Vetlearn 2010 pp320-321
20. Sharpe AE, Brady CP, Byrne W, Moriarty J ,O’Neill P and McLaughlin JG. Major outbreak of suspected botulism in a dairy herd in the republic of Ireland. The Veterinary Record March 29, 2008
21. Palmer D. Department of Agriculture and Food, Western Australia pers. comm.
22. Jubb TF, Ellis TM and Gregory AR (1993) Diagnosis of botulism in cattle using ELISA to detect antibody to botulinum toxins. Aust. Vet. Journal 70: 226-227
23. Gregory A, Ellis T, Jubb T, Nickels R and Cousins D (1996) Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigation of botulism in cattle. Australian Veterinary Journal 73: 55-61
24. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine. A textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th Edition. Elsevier Ltd 2007 p826