Investigation of clinical outbreaks of American foulbrood in honey-bee operations in Saskatchewan

Abstract

Four outbreaks of American foulbrood were investigated in honey-bee operations in Saskatchewan during the summer of 2019. Clinical signs were confirmed by the Saskatchewan Provincial Specialist in Apiculture and the causative agent was cultured and identified through matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Evaluation of management practices revealed off-label metaphylactic use of oxytetracycline in 3 of 4 operations and a discontinuation of antibiotic use in the fourth. Recent regulatory changes regarding access to medically important antimicrobials has provided an opportunity for veterinarians to promote evidence-based use of antimicrobials in apiculture while safe-guarding the health of commercial honeybee populations and the economic viability of their producers.

American foulbrood (AFB), caused by Paenibacillus larvae, is a highly infectious, destructive bacterial disease that affects the young brood of honey-bee colonies (1,2). Newly hatched larvae are inadvertently fed bacterial spores, which germinate and replicate in the midgut causing an overwhelming septicemia and producing over 1 billion new spores per infected larva (2,3). As deceased larvae are cleaned from their cells by worker caste bees, spores are picked up and disseminated throughout the hive products including honey and wax, as well as hive equipment including frames and boxes (4–7). Spores are resilient to environmental conditions and chemical treatments and have been reported to maintain infectivity for decades (8). This resiliency poses significant challenges regarding the management and control of disease. If clinical signs consistent with AFB infection are observed, burning the bees and equipment of affected colonies or, if available, subjecting equipment to gamma irradiation or high velocity electron-beam irradiation are the only practical solutions to eliminate spores (9–12).

In Canada, prevention of clinical disease relies heavily on the use of biannual, metaphylactic, antibiotic treatments with oxytetracycline in the spring and fall. Treatments are administered as either a dusting of antibiotic mixed with powdered sugar, or as a direct feed within sugar syrup. If Oxysol-62.5 (DIN 00560189) is used, each dusting treatment consists of mixing 400 g of oxytetracycline HCl (62.5 mg/g) with 3.5 kg of powdered sugar, and dusting 32 g of this mixture into each hive 3 times at 4-to 5-day intervals. Syrup feed is made by dissolving 400 g of oxytetracycline HCl (62.5 mg/g) within 300 kg of a 1:1 mixture of sugar and water and feeding 2.5 kg of this solution to each hive 3 times at 4- to 5-day intervals. If Oxytet-25 (DIN 02231111) is used, then 454 g of oxytetracycline HCl (55 mg/g) is mixed with 3.5 kg of powdered sugar or 300 kg of 1:1 sugar syrup for dusting powder or syrup administration, respectively. Volumes can be scaled down as needed. Antibiotic use can prevent the replication of the vegetative state and suppress clinical manifestation, but it does not affect the spores present in treated colonies (13). This metaphylactic treatment is thought to have contributed to both the development and subsequent spread of antibiotic resistant strains of P. larvae and necessitated the use of alternative antibiotics such as tylosin for control (14–17).

A definitive diagnosis of AFB requires the identification of clinical signs consistent with disease (Figure 1) and isolation of the causative agent through laboratory testing (7). As of the fall of 2019, AFB was added to the list of provincially notifiable diseases in Saskatchewan, and laboratory-confirmed cases must be reported to the Chief Veterinary Officer within 24 h of confirmation. The disease is annually notifiable in Canada.

Figure 1.

Common clinical signs of American foulbrood. A — larval ropiness, representing decaying and macerated pre-pupae (arrow); B — Scattered, ‘shotgun’ brood pattern as a consequence of larval/pupal mortality; C — Perforated cell cappings (arrows) and sunken cell cappings (arrowheads); D — Scale, representing dead, desiccated pre-pupae (arrows)

Following infection, larvae and pupae change from pearly white to tan to brown as they are invaded by large numbers of replicating vegetative bacilli (18). The large number of bacteria create a glue-like consistency to the pre-pupa/pupa that can be macerated and strung out using a probe to test for larval ropiness. If macerated pupal tissue can be drawn out and stretched more than 2 cm from a cell (Figure 1A), it is highly suggestive of AFB infection (10). When worker bees recognize an abnormal larval or pupal cell either before or after capping, they will clean and remove the diseased or dead larva or pupa, which can produce a scattered, “shotgun” appearance to the brood pattern (Figure 1B) (19). Remaining cell cappings of affected larvae may also appear sunken, greasy, or perforated (Figure 1C) (2). Scattered, “shotgun” brood appearance and abnormalities in cell cappings may be suggestive for AFB, but they are not as specific as the presence of ropey brood and firmly attached larval scale (10). As infection advances, dead larvae will desiccate and form dark brown scales that adhere firmly to the ventral lateral wall of the brood cell (Figure 1D) and a strong, unpleasant foul odor (hence the name “foulbrood”) may be noted from affected bee frames (18). Samples of comb that contain evidence of ropey larvae and/or scales can be cultured to isolate the bacterium (6,7,20,21), or tested using molecular techniques such as polymerase chain reaction (PCR) (6,22).

Clinical description

Four suspected outbreaks of AFB were identified in commercial honey-bee operations across central and southeastern Saskatchewan in June, 2019. In this manuscript, outbreaks are defined as an incidence of clinical disease in hives in excess of what is expected. All 4 operations — identified here as Operations A, B, C, and D — varied in their management practices for control of AFB, which are summarized in Table 1. All 4 operations are large honey-producing operations with 2100 to 4000 honey-producing colonies. Given the use of metaphylactic antibiotics in 3 operations and the long duration of freedom from observable disease in the 4th, antibiotic-free operation, the expected incidence of clinical disease in each operation is zero.

Table 1.

Summary of management variables from 4 commercial honey-bee operations in Saskatchewan diagnosed with a clinical outbreak of American foulbrood during the summer of 2019.

	Operation ID
Management variable	A	B	C	D
Total number of honey-producing colonies	4000	2700	2800	2100
Total number of bee yards	125	85	45	50
Number of affected yards	1	1	1	N/Ab
Total number of colonies in affected yard	40	195	44	N/Ab
Number of affected colonies in affected yard	1	1	1	7
Affected colony status	Alive	Alive	Dead	Dead
Metaphylactic antibiotic use	No	Yes	Yes	Yes
Antibiotic used	—	Oxytetracycline	Oxytetracycline	Oxytetracycline
On-label antibiotic use?	—	No	No	Noc
Frequency of brood inspection	Spring and whenever weak	Spring and whenever weak	Overwinter loss, Spring and whenever weak	Spring and whenever weak
Who performs brood inspection	Owner and staff	Owner and staff	Owner and staff	Owner
Proportion of brood frames inspected	Few	Few	Few	All
Percentage of brood frames renewed annually	10%	Over 50%a	10%	Less than 5%
Last suspected case of American foulbrood	25 years ago	3 years ago	3 years ago	1 year ago

^aEstimated frame replacement proportion for new nucleus hives, from which the affected colony originated.

^bAll colonies were overwintered together indoors and individual yard assignments were not tracked for nucleus colonies.

^cFall treatment of oxytetracycline in 2018 was given as 2 applications, not 3.

N/A — Not applicable.

Operation A

Operation A had ceased using metaphylactic antibiotics and had last treated its colonies with oxytetracycline in the fall of 2016. On June 10th, 2019, staff identified a single colony in 1 yard with a scattered, “shotgun” brood pattern, abnormal cell cappings, larval scale, and larval ropiness. The colony was treated with a single application of oxytetracycline administered over the frames in icing sugar, and the Provincial Specialist in Apiculture was contacted to inspect the affected hive. A clinical diagnosis of AFB was confirmed based on the described clinical signs, and the entire hive was destroyed through burning; the remainder of the yard was treated with oxytetracycline as per label directions. Several affected frames were submitted to the laboratory for bacteriological culture.

Operation B

Operation B treats its colonies twice annually with oxytetracycline mixed with powdered sugar in the spring and fall, but only performs a single application of antibiotics per feed treatment instead of the on-label instructions requiring 3 applications given 4 to 5 days apart. Brood frames are inspected frequently throughout the season and, on June 14th, 2019, a single colony within a large yard of nucleus colonies (n = 195) was marked with clinical signs similar to those described in Operation A. Frames and bees from the colony were destroyed by burning, and the remaining colony materials (i.e., super boxes, lids, and bottom) had their surfaces scorched with a torch before being re-used in circulation. The last suspected case of AFB in this operation was identified 3 y ago, and a clinical diagnosis was made by the Provincial Specialist in Apiculture at that time.

Operation C

Unlike operations A and B, which had identified clinical signs in living colonies, Operation C identified a suspect colony during its annual inspection of dead colonies that failed to survive overwintering. Frames contained scattered, abnormal cappings and adhered scale along the ventral aspect of numerous cells. The Provincial Specialist in Apiculture was alerted, and the entire hive was burned. Similar to Operation B, this commercial outfit reported a 3-year gap between this case of AFB and its last suspected case. The previous case was identified by clinical signs observed by the operation owner alone without visual confirmation by the Provincial Specialist. The owner of this operation also noted that a nearby hobbyist had lost their colonies to AFB in 2013 but had left the hives out instead of having them destroyed. There was a high likelihood of these contaminated colonies being robbed out by bees belonging to Operation C, a behavior whereby foraging bees from strong hives will collect (steal) unprotected honey within weak and dead hives and bring it back into their hives. Operation C had therefore been using metaphylactic oxytetracycline, but only once annually in the spring since 2010 instead of the label-prescribed twice annual treatments.

Operation D

Operation D identified a total of 7 deceased nucleus colonies suspected to have succumbed to AFB during examination of its overwinter mortalities. This operation overwinters all of its colonies together indoors and performs rigorous inspection of all brood frames 2 to 3 times each spring. Suspect colonies were initially identified due to a noticeable malodor, and subsequent inspection of frames revealed capping abnormalities and the presence of scale. All identified colonies and frames were destroyed by burning. The operation reported administering oxytetracycline twice annually and adhering to label instructions but noted that the fall treatment in 2018 was only given as 2 applications instead of the label-prescribed 3.

From all operations, only those frames with either visible larval scale or cells identified as positive for ropiness were collected for bacterial culture. Scales were collected from affected frames, vortexed in sterile water, and subjected to heat treatment to eliminate other microbial contaminants. Samples were plated on both Columbia sheep blood agar (CSA) (BD Biosciences, San Jose, California, USA) and MYPGP [prepared according to published protocols (6,7)] and incubated at 37°C with 5% CO₂ for 48 h. Plates were confirmed for lawns of colony growth consistent with the high concentration of spores expected from contaminated larval scale and with colony morphology consistent with P. larvae (7). Two serial sub-cultures were performed from these lawns to generate a pure culture. Colony growth from the second sub-culture was submitted to Prairie Diagnostic Services, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, and tested using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) with a pre-existing library for Paenibacillus larvae. Bacterial isolates from all operations were positively identified as Paenibacillus larvae. An isolate from Operation A was confirmed as Paenibacillus larvae using both MALDI-TOF MS and PCR with primers designed by Dobbelaere et al (22). Independent samples collected by the Provincial Specialist in Apiculture were submitted to the Animal Health Laboratory, (University of Guelph, Guelph, Ontario) for antimicrobial susceptibility testing by Kirby Bauer disk diffusion following Canadian Laboratory Standards Institute (CLSI) VET06 guidelines. Isolates from all operations were confirmed to be susceptible to oxytetracycline.

Follow-up with all 4 operations was performed following routine fall inspections. All operations confirmed that they had detected no additional clinical evidence of AFB. Operation A returned to metaphylactic, on-label use of oxytetracycline and the 3 other operations had adjusted their metaphylactic antibiotic protocols to follow label instructions.

Discussion

The current landscape of AFB in Saskatchewan remains largely unknown due to the long-term masking of disease through antibiotic metaphylaxis; outbreaks are difficult to predict. Suppression of clinical disease can lead to a high build-up of infectious spores within contaminated colonies, unknown to both hobbyists and commercial beekeepers applying treatment (17,23,24). In Saskatchewan, cases of AFB are identified through a combination of random inspections and self-reporting by beekeepers. Four outbreaks of AFB were reported in 2018, primarily among small-scale beekeepers and hobbyists. In 2019 the number of cases increased to 8, but were mostly seen in commercial operations, 4 of which were investigated here. Many factors associated with the pathogen, host, and environment are thought to play a role in determining the development of clinical disease, including strain virulence, the hygienic behavior of worker bees within a colony (social immunity), and basic management practices, respectively (7,25–27). How these factors interplay in the presence of chronically treated colonies remains unknown. Investigations of potential risk factors for disease have helped to shed light on management practices critical to American foulbrood management and control in countries such as New Zealand and Belgium, but because antibiotic use in these regions is prohibited, the applicability of the results of these investigations to Saskatchewan and Canadian beekeeping is uncertain (10,27).

With the recent regulatory changes on December 1, 2018, requiring all medically important antimicrobials for veterinary use to be sold by prescription only, the current use of antibiotics in the honey-bee industry must be re-evaluated, and Canadian veterinarians have an opportunity to establish themselves as leaders in the judicious use of antimicrobials in this industry (28). Common to all operations investigated here was the variability of approaches taken with regard to antibiotic use. Operation A had discontinued antibiotic metaphylaxis in their management program, and although operations B, C, and D were using regular antibiotic metaphylaxis, all were doing so with different, off-label protocols. It is possible that the cessation of antibiotics in operation A, or under-dosing of antibiotic in the other operations, may have contributed to the clinical appearance of AFB. Until there is a better understanding of the risk factors associated with clinical outbreaks of AFB in antibiotic-managed systems, Canadian veterinarians are uniquely poised to build effective, working veterinary-client-patient relationships with hobbyist and commercial beekeepers around communicating the importance of on-label use of antibiotic therapy to minimize the likelihood of clinical disease and slow the development of antimicrobial resistance within the apiculture industry.

Conversely, if beekeepers wish to cease the use of antibiotics for metaphylactic control of AFB, veterinarians can be relied upon to consider this decision in an evidence-based manner through risk assessment, the testing of hive products, and the implementation of frequent brood frame examination to minimize the likelihood of occurrence of clinical disease. Here, Operation A had discontinued the use of oxytetracycline due to concern about the development of antimicrobial resistance and saw an emergence of clinical disease less than 3 y after cessation of treatment in an operation that had had no clinical disease for the previous 25 y. It is suspected that the long-term viability of bacterial spores, combined with a silent accumulation of spores through clinical masking with chronic antimicrobial use, can create a false sense of contamination-free colony status (24). Removing the suppressive effects of antimicrobials in these hives without an understanding of underlying contamination of P. larvae spores and relevant risk factors could potentially be disastrous. If antibiotic use is discontinued, then a rigorous health management plan must be in place that emphasizes frequent and thorough examination of brood frames to improve the early detection of disease and prompt destruction of affected colonies to reduce the spread of AFB. This approach for control of AFB has been implemented in New Zealand and many European countries and it is being adopted successfully by a few commercial beekeepers in Saskatchewan and Alberta (10,23). Veterinarians again have an opportunity to educate and aid in the implementation of such plans, and can advocate the testing of hive products such as honey and bees to detect the presence of spores of P. larvae and the presence of antimicrobial resistance that may influence the decision to use antibiotic metaphylaxis or not (23,29).

Practitioners are advised to follow the procedures listed in suspected AFB cases:

1. Contact the Provincial Specialist in Apiculture to establish a working relationship with their team. Provincial apiculture teams are a valuable resource in both knowledge and skills concerning beekeeping practices and the detection/diagnosis and management of honey-bee diseases. Strong collaboration is necessary for efficient prevention, early detection, and optimal management of AFB. Provincial apiculturists also perform a regulatory role to implement and enact provincial acts related to beekeeping.

2. Inspect the colony frames for presence of pathognomonic signs: ropiness and/or scales.

3. Submit suspect frame(s) to an appropriate microbiology laboratory to confirm the presence of P. larvae.

4. If a diagnosis of AFB is confirmed and is a notifiable disease within your province, report to the Chief Veterinary Officer within 24 h of laboratory diagnosis.

5. Burn affected colonies and equipment or irradiate exposed equipment if this service is available. Treat the remaining colonies with antibiotics or implement frequent and rigorous inspections to identify and destroy bacteria in new cases at early stages to prevent spread of the disease.