In vitro Evaluation of Candidate Bacillus Strains Against Erysipelothrix rhusiopathiae from Erysipelas Outbreaks in Layer Flocks Open Access

Erysipelas is an emerging zoonotic disease causing acute and fulminating infections in swine, poultry, and a wide range of animals, including humans (1). Erysipelothrix rhusiopathiae is the causative agent of erysipelas; it is ubiquitous in nature, with soil, water, litter, and wild animals being the main sources of infection (2). The pathogen uses compromised skin or other mucosal membranes to enter the body and it can pass through the stomach without having its viability affected; thus carrier animals can reinfect soil and water continuously (3). Behaviors such as cannibalism and feather pecking, in flocks, have been suggested as causes of increased transmission (1); biting insects can also be vectors and reservoirs of the pathogen, increasing its persistence in the environment and the potential of transmission among animals (4).

In layers, erysipelas affects birds 43–73 wk old, resulting in up to 50% overall mortality of affected flocks (5). Its incidence is higher in systems going from conventional to alternative production, with the latter having reportedly higher numbers of bacterial infections (6). Typical erysipelas clinical signs include hepatomegaly and splenomegaly, septicemia, and acute death. Reduced egg production, pale combs, and diarrhea have also been described in outbreaks of laying hen flocks (1). Antibiotics such as small-spectrum penicillin are highly effective to control the disease; however, antibiotic treatment has been found ineffective in chronic cases (3). In addition, the increased use of broad-spectrum antibiotics in the poultry industry has led to the development of antimicrobial resistance, reducing their efficacy to control erysipelas infection (7).

Direct-fed microbials (DFMs) are a current alternative to the use of antibiotics in poultry production (8). DFMs are defined as “live microorganisms that in adequate amounts confer benefits to the host by improving its intestinal microbial balance” (9). Lactic-acid-producing bacteria and Bifidobacterium spp. are the most commonly used bacterial DFMs. However, there has been a growing interest in the use of Bacillus DFMs because of their resistance to harsh environmental conditions by their spore-forming ability (10,11). Additionally, Bacillus spp. produce 1) antimicrobial compounds with broad spectrum against bacterial pathogens, and 2) enzymes that enhance feed digestibility and nutrients absorption (11,12). This study aimed to evaluate the inhibitory effect of 20 novel Bacillus DFMs against E. rhusiopathiae isolated from laying hens with erysipelas.

Outbreaks of Erysipelothrix rhusiopathiae were investigated in two flocks with outdoor access. Flocks were not vaccinated; they started showing signs of disease around 40–50 wk of age. Initial performance indicators for disease included: slight reductions in water intake (5.3 gallons to 5) and feed consumption (from 26 to 27 lb/100 to 25 lb), decreased egg production, and increased mortality. Both flocks were diagnosed shortly before flock depopulation at Week 75, mortality ranged from 0.5 to 1% per week for Flock A, and from 1 to 1.29% for Flock B (14.56% accumulated mortality), production was 3–10% below breed standards.

Depressed birds or recent mortality were necropsied from each flock (5–10 birds). During necropsy, generalized septicemia including pericarditis, hepatitis, and, most commonly, mottled splenomegaly, were observed (as shown in Fig. 1).

Tissue samples including femur, spleen, and liver were aseptically collected in sterile bags at necropsy and shipped to the Veterinary Diagnostic Laboratory at Iowa State University for bacterial culture and isolation. Upon receiving, organs were swabbed, and swabs were incubated aerobically at 35 C for 48 hr in a selective brain heart infusion broth (BHI, Becton Dickinson) supplemented with 5% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 400 mg/ml kanamycin (Sigma-Aldrich), and 50 mg/ml neomycin (Sigma-Aldrich) as previously described (13,14). After incubation, 100 μl of each incubated culture was plated onto Columbia agar supplemented with 5% defibrinated sheep blood, incubated aerobically at 35 C for 7 d, and checked daily for colony formation (13,14). Presumptive E. rhusiopathiae colonies were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) spectrophotometry.

Erysipelothrix rhusiopathiae confirmed isolates, recovered from femur, spleen, and liver were shipped overnight in tryptic soy agar (TSA, Sigma-Aldrich) slants + 5% defibrinated sheep blood to the Emerging Technology Center at Purina Animal Nutrition Center (ETC: PANC) for further screening. Upon arrival, an isolated colony from each agar slant was restreaked onto TSA + 5% defibrinated sheep blood agar plates. Plates were incubated aerobically at 37 C for 24–48 h. A single well-isolated colony from each plate was selected, grown in 9 ml of tryptic soy broth (TSB, Sigma-Aldrich) + 5% defibrinated sheep blood, and incubated aerobically at 37 C for 24–48 hr. A 500-µl aliquot of the overnight culture was added to 50% glycerol and stored at −80 C for further testing.

A total of 20 proprietary Bacillus strains (15 individual strains and 5 combinations) were selected from an internal library of novel DFMs, based on their broad antimicrobial effect against a set of Gram-positive and Gram-negative bacterial pathogens from our internal pathogen repository (data not published). Bacillus isolates were streaked onto Luria-Bertani (LB, ThermoFisher Scientific, Waltham, MA) agar and incubated aerobically at 37 C for 24 hr. A single isolated colony was transferred to 9 ml of LB broth and incubated aerobically at 37 C for 24 hr. Overnight cultures at 10⁹ colony-forming units (CFU)/ml, were spread plated onto LB agar, and diluted once on buffered peptone water (BPW, Sigma-Aldrich) to 10⁸ CFU/ml. Diluted cultures were used for AWD assay, as described below.

AWD screening as described by Vinderola and others (15) with slight modifications was used in this study. Erysipelothrix rhusiopathiae isolates were streaked on TSA + 5% defibrinated sheep blood and incubated aerobically overnight at 37 C. A single isolated colony was incubated overnight in TSB broth + 5% defibrinated sheep blood at 37 C for 24 hr; overnight cultures were then diluted to 10⁶ CFU/ml. Erysipelothrix rhusiopathiae isolates were spread plated onto BHI agar to a final concentration of 10⁵ CFU/ml. Plates were dried for 5 min in a biosafety cabinet, then five 6-mm-wide wells were made in the agar and filled with 100-μl aliquots of 1) 10⁸ CFU/ml of each Bacillus DFMs in duplicate wells (two Bacillus strains screened per plate) and 2) TSB broth + 5% defibrinated sheep blood (control) in a single well. Plates were incubated at 4 C for 2 hr to allow suspensions to diffuse in the agar followed by a 24-hr incubation at 37 C. Antimicrobial effect was determined by the development of a clear zone of inhibition around each well. DFMs were ranked for their overall antimicrobial activity by calculating the sum of the individual inhibition (mm) scores across all E. rhusiopathiae isolates. The effectiveness score was determined by calculating the percentage of isolates that were susceptible to each DFM tested in the study.

Birds sampled exhibited the typical erysipelas lesions; at necropsy hepatomegaly and splenomegaly were observed. No petechial hemorrhages were noticed on the heart, abdominal fat, skeletal muscles, and viscera. Follicles on affected birds were broken in some cases, but did not appear regressed. Samples were confirmed by bacteriology and MALDI-TOF for the presence of E. rhusiopathiae. Mortality in these affected flocks can be as high as 2% per week with egg production drops to 15% below breed standards (authors’ observations).

Erysipelothrix rhusiopathiae was isolated from femur, spleen, and liver from layers from both flocks analyzed in this study. Presumptive E. rhusiopathiae isolates on selective plates were confirmed positives by MALDI-TOF. Two E. rhusiopathiae representative isolates from each farm (Farm A: femur and liver; Farm B: femur and spleen) were used for the inhibitory tests.

The antimicrobial results of the Bacillus DFM screening against E. rhusiopathiae isolates from femur, spleen, and liver from layer hens with erysipelas are shown in Table 1. A total of 9 out of the 20 (45%) Bacillus tested (7 individual strains and 2 combinations) were inhibitory against all 4 E. rhusiopathiae isolates, 5 Bacillus (4 individual strains and 1 combination) were inhibitory against 3 isolates, 2 Bacillus (1 individual strains and 1 combination) were inhibitory against 2 of the 4 E. rhusiopathiae isolates, and 3 Bacillus (2 individual strains and 1 combination) were inhibitory against only 1 of E. rhusiopathiae isolates. The overall score was obtained by summing across the E. rhusiopathiae isolates and it ranged from 11 to 83.50 mm with B. amyloliquefaciens 4183A and B. amyloliquefaciens 4589D, respectively. Two out of the five Bacillus combinations were inhibitory against all E. rhusiopathiae isolates used in this study (Table 1). Bacillus amyloliquefaciens 4589D was the most inhibitory with clear zones of inhibition ranging from 20 to 22.5 mm. Bacillus amyloliquefaciens 4577C was not inhibitory against the E. rhusiopathiae isolates screened in this study.

Erysipelas has long been considered a disease of economic importance for turkey production; however, layer flocks are experiencing increased economic losses from this disease (2). Erysipelothrix rhusiopathiae, the causative agent of erysipelas, is a soil-borne micro-organism that can survive long periods of time in soil and manure; therefore, flocks with outdoor access, such as organic and pasture-raised layer flocks, are at greater risk for developing this infection (2,5,16,17). In birds, E. rhusiopathiae is shed in the feces of infected birds for up to 41 days and can survive in soil for up to 35 days. This provides ample opportunity for other birds within these flocks to come into contact with the contaminated ground (18,19).

Erysipelas outbreaks in two flocks from nonconventional (pasture-raised) systems were investigated in this study. In this study, E. rhusiopathiae isolates were recovered from spleens of sampled birds at both flocks; our findings agree with those observed by Erickson and others (2), who, in a study to determine association between housing systems and outbreaks of erysipelas in laying hens, isolated E. rhusiopathiae from the spleen of all sampled hens from farms with ongoing erysipelas outbreaks.

A live oral registered vaccine is the suggested preventive treatment for turkeys. This vaccine may also be effective to control erysipelas in other poultry species (19). Autogenous vaccine could also be pursued, or an inactivated swine vaccine could be beneficial. Unfortunately, vaccination is not a standard practice for layer flocks, and only confers protection to few erysipelas-causative strains (2). Antibiotics can be used successfully for treating erysipelas in conventionally raised flocks. Penicillin, cephalosporins, and clindamycin have been found effective to control E. rhusiopathiae (1,3); however, antibiotic therapy may be ineffective in chronic cases (3). Broad-spectrum antibiotics, such as danofloxacin, ceftiofur, and enrofloxacin have been reported as most active against E. rhusiopathiae (3). However, it is worth noting that as of the time of this writing there is no penicillin product approved for use in the United States for egg production; therefore, other effective control strategies are necessary.

The use of Bacillus DFMs in layer hen diets has shown benefits at regulating gut microbiota, enhancing egg production, and improving performance and overall health (12,20,21). Pathogen control is of great importance for the poultry industry from an economic and public health aspect. DFMs reduce pathogens by competitive exclusion mechanisms and the production of antimicrobial peptides, such as bacteriocins, and other compounds that suppress pathogens in the environment (11,20). In this study, 20 proprietary Bacillus DFMs (15 individual strains and 5 Bacillus combinations) were screened against E. rhusiopathiae isolates from birds with erysipelas; 9 DFMs were found effective at inhibiting/reducing all 4 E. rhusiopathiae isolates. As observed in other characterization studies (20,22), the antimicrobial effect of the panel of DFMs was found strain dependent, with B. amyloliquefaciens 4589D being the most inhibitory with zones of inhibition ranging between 20 and 22.50 mm, and B. amyloliquefaciens 4577C being the least effective, with no reductions of any the E. rhusiopathiae isolates screened in this study. Further research to classify E. rhusiopathiae at strain level and the potential effect of E. rhusiopathiae strain variability on response to DFMs should also be conducted. In a study to characterize the causative agents of focal ulcerative dermatitis (FUDS) in commercial laying hens, the use of a targeted Bacillus combination from our library of novel DMFs showed in vitro inhibition of the causative agents of FUDS. Results have been found replicable in the field, as the customized product is used at multiple farms with history of FUDS with effective reduction of the pathogens and decrease of FUDS mortality (20). The in vitro inhibition results presented in this note highlight multiple Bacillus strains and combinations as alternatives to be used in vivo to evaluate their efficacy as biocontrol technologies to reduce the growth E. rhusiopathiae in the field.

AWD =

agar-well diffusion;

BHI =

brain heart infusion;

BPW =

buffered peptone water;

CFU =

colony-forming units;

DFM =

direct-fed microbial;

ETC:PANC =

Emerging Technology Center at Purina Animal Nutrition Center;

FUDS =

focal ulcerative dermatitis;

LB =

Luria-Bertani;

MALDI-TOF =

matrix-assisted laser desorption ionization time-of-flight mass spectrometry;

TSA =

tryptic soy agar;

TSB =

tryptic soy broth