Use of the rSpaA415 antigen for detection of IgG anti-Erysipelothrix rhusiopathiae in farmed cattle indicates a low prevalence in dairy and feedlot cattle in the United States of America and Great Britain

Background Clinical cases of Erysipelothrix rhusiopathiae, a zoonotic gram-positive bacterium, have been reported in many ruminant species, including in cattle, deer, moose and muskoxen. Fatal cases have been repeatedly reported in cattle over the years but to date there is only one Japanese study investigating the seroprevalence of this bacterium in bovine using the growth agglutination test (GAT). This technique is subjective, time-consuming, expensive and hazardous compared to modern serological tests such as enzyme-linked immunosorbent assays (ELISA) or the newly developed fluorescent microbead-based immunoassays (FMIA). Results A FMIA based on the surface protein SpaA (rSpaA415) antigen of E. rhusiopathiae developed in this study had an almost perfect agreement with the GAT (k=0.83) and showed a sensitivity of 89.7% and a specificity of 92.9% when compared to the GAT. Overall, detection rates of E. rhusiopathiae antibody positive samples were 13.8% (51/370) in British herds and 6% (12/200) in US herds. Positive cattle were present in 34.3% (24/70) of the investigated British farms and in 34.7% (8/23) of the US farms with an on-farm prevalence of 7.1% to 100% for the British and 8.3-30% for the US. Conclusions FMIA is a fast, safe and economic alternative to the GAT for the diagnosis of E. rhusiopathiae in cattle. This work is the first seroprevalence study of E. rhusiopathiae in healthy farmed cattle in the Great Britain and the US and revealed that infection occurs at a low level. Further investigations to evaluate risks of zoonotic transmission when handling cattle are needed.

, Erysipelothrix sp. strain 1serotype13) and Erysipelothrix sp. strain 2 (serotype18) [1]. Erysipelothrix rhusiopathiae is considered to be the pathogenic species of the genus and known as the etiologic agent of swine erysipelas which can be associated with sporadic cases or larger outbreaks of major economic importance [2]. Besides pigs, E. rhusiopathiae can cause a wide range of diseases in other species such as sheep, fish, poultry, cattle and humans [3][4][5][6]. Infections in humans are primarily a result of contact with infected animals and are presented either as a localised cutaneous lesion called erysipeloid, as a generalised cutaneous lesion, or as a septicaemic form which is often associated with endocarditis [7].
Recently, E. rhusiopathiae has been isolated in increasing frequency from ruminants, especially from farmed cattle (Bos taurus) [4,5]. Most of the clinical cases in cattle are observed in young calves presenting septicaemia [8] with abscesses in the liver and lungs [9], encephalomeningitis [10], or polyserositis and arthritis [11].
Erysipelothrix rhusiopathiae has been associated with multiple unusual mortality events in muskoxen (Ovibos moschatus wardi) in theCanadian Arctic Archipelago [12]. During the years 2009-2011, a total of 22 muskoxenwere founddead during multiple expeditions in theCanadian Arctic Archipelagoand in 2012 approximately 150 muskoxen were found dead; E. rhusiopathiae serotype 5 was confirmed by serotyping, PCR and/or histopathology on tissues from these animals [12]. Interestingly, E. rhusiopathiae serotype 5 was also isolated from afatal case of metritis in a Norwegian heifer [13] and from a fatal case of acute multifocal necrotic hepatitis in a white tailored reindeer in Iowa, USA [14]. In Canada, the death of three elks (Alces Alces) was linked to a septicaemia caused by Erysipelothrix of serotype 17 [15]. The bacterium was isolated from liver, kidney, lungs and lymph nodes from the three animals [15].
During studies in Japanese abattoirs, Erysipelothrix was isolated from 6.4% of 1236 4 healthy, slaughtered cattle [16] which demonstrates that cattle may be subclinically infected with the bacterium. An epidemiological follow-up study using the growth agglutination test (GAT) to detect antibodies anti-Erysipelothrix in Japanese cattle found that 76% of 854 healthy cattle had detectable antibodies [3]. The same study also found a higher rate of seropositive cattle in areas with concurrent swine industry [3]. This data could indicate that Erysipelothrix is mainly transmitted by pigs although cattle may also act as a vehicle for its distribution [5,16]. In support of this, Erysipelothrix was isolated from cattle slurry [3] which could enhance the bacterium's ability to spread as Erysipelothrix can survive in soil contaminated with faecal material [4]. Current methods of antibody detection in cattle have been carried out using solely GAT. GAT has been extensively used in pigs and chickens and it has shown a good correlation between the antibody titres and immune status in vaccinated pigs [17] and challenged chickens [18] but this correlation has not yet been studied in cattle. The use of GAT in pigs and chickens was replaced by recently developed enzyme-linked immunosorbent assays (ELISAs) and fluorescent microbead-based immunoassays (FMIAs) [6,[19][20][21][22] due to their ability to permit the testing of large numbers of samples in a short time, while giving objective results.
Although Erysipelothrix and antibodies against it have been detected in healthy cattle in Japan [3][4][5], data is lacking for the distribution of Erysipelothrix in cattle across Europe and North America where its epidemiological importance is not known.  [23].
A highly sensitive (96.5%) and specific (100%) ELISA was recently developed for the detection of Erysipelothrix in swine using a recombinant SpaA (rSpaA415) [6]. This was then enhanced by adapting it into an FMIA [21]. This study aimed to investigate the antibody distribution against Erysipelothrix in cattle in Great Britain and the USA to increase the knowledge of its epidemiological significance and to develop an ELISA and FMIA test using rSpaA415 antigen for the detection of antibodies against Erysipelothrix in cattle to overcome the disadvantages of GAT.

Results
Development and optimisation of rSpaA415 FMIA and ELISA and cut-off evaluation using the GAT as the reference assay.
A first subset of 300 samples were tested with the ELISA and FMIA to evaluate the performance of both tests. The FMIA had superior performance compared to the ELISA. Specifically, the FMIA had an area under the curve (AUC) of 94.8% (95%CI 91%-98%) indicating a high accuracy of the test. The optimal cut-off was determined to be 1073.5 MFI giving a diagnostic sensitivity and specificity of 89.7% and 92.9% respectively. The ELISA had an AUC of 64.7% (95%CI 55%-74%) and a sensitivity and specificity of 61% and 53% for the optimal cut-off which had an S/P value of 22%.
ELISA and FMIA had an agreement of 79% and a kappa value of 0.31 showing a fair agreement. FMIA was preferred because it was more accurate, sensitive, specific, and easy to operate than the ELISA and was selected to test the remaining field samples. The percentage of agreement of the FMIA and GAT was 93% (95%CI, 88%-96%) which was almost perfect (κ = 0.83). McNemar's test showed that the paired discordances occurred randomly and that therefore, both tests were comparable (McNemar's p value = 1). The 6 CVs obtained for the FMIA test for the positive (13.1%) and negative (14%) reference sera were considered good suggesting a high level of assay reproducibility.

Antibody titres on field and experimental samples
Experimentally vaccinated cattle developed antibodies against E. rhusiopathiae as demonstrated by the FMIA, ELISA and GAT (Fig. 1, Table 1). Both cows had detectable antibodies on 14 dpv and antibody levels remained detectable until the 42 dpv. MFI and GAT titres were higher in experimentally vaccinated cattle compared to non-vaccinated field cattle with natural exposure. The distribution of GAT titres against E. rhusiopathiae for the studied bovine samples is summarized in Table 1. The majority of positive field samples had GAT titres of 32 (22%, 36/162), followed by 64 (13%, 21/162) and 128 (13%, 5/16). The majority of the samples tested showed agglutination below the cut-off (50%, 81/162). High antibody titres were detected more frequently in British cattle compared to US cattle (Table 1).
MFI and GAT titres were plotted and showed a good correlation (Fig. 2) which was supported by Pearson coefficient (r = 0.93).

Prevalence of Erysipelothrix antibodies in US and British cattle herds by FMIA
Antibodies against E. rhusiopathiae were present in both populations of farmed cattle at a low level. Overall, the prevalence in the studied British cattle population was 13.8%

Discussion
The growth agglutination test was used as the reference test as it is the only methodology used so far to evaluate the Erysipelothix antibody responses in cattle. The FMIA developed in this study is an improved tool for the detection of Erysipelothix antibodies in cattle and had almost perfect agreement with the GAT, with a high sensitivity and high specificity.
There are three main disadvantages of the GAT. First, the assay is time consuming as only 8 samples can be included in a plate with two overnight incubations, one to prepare the inoculum and a second one to read the results. Second, the GAT is hazardous since live pathogenic bacteria have to be used. Finally, and third, the results are read by naked eye which may be subjective and variable between operators. In contrast, the FMIA permits the testing of large numbers of samples in less than 4 hours and gives precise and objective results. For these reasons, we propose in here the use of MFI as an alternative to GAT.
The specificity of the GAT in cross-reaction with antibodies against organisms other than E. rhusiopathiae has not been studied in detail. The cross-reactivity of rSpaA415 has been validated with pig pathogens but it remains unknown for pathogens infecting bovine.
Higher specificity is predicted by using FMIA than GAT as the GAT involves an extent of non-specific agglutination since a titre of less of 32 indicates a negative reaction.
Differences in sensitivity and specificity between GAT and MFI could be due to the  (11) and E. sp. strain 8 2containingSpa Cserotype 18). In contrast, low antibody responses were observed in sera from rabbits inoculated with E. tonsillarum serotype 20 (no Spa type) and no antibody response was observed in sera from rabbits inoculated with the remaining serotypes without Spa type (E. tonsillarum and E. sp. strain 1) [6]. The knowledge of the Erysipelothrix serotypes infecting cattle is limited; one study in Japan recovered 79 Erysipelothrix isolates from the tonsils of healthy slaughtered cattle of which only 43 out of these isolates were typeable and were classified into the serotypes 1b, 2, 3, 5, 9, 12, 13, 19 and 21. Responses against rSpaA415 were detected in rabbits infected with serotypes 1b, 2, 5, 9, 12, 19 and 21 but not with serotypes 3 and 13 [6]. There is no data of the serotypes of the Erysipelothix serotypes circulating among the US and British herds but in Norway, a serotype 5 was isolated from an heifer with fatal metritis [13].
This study showed that FMIA was more sensitive and specific than the ELISA based on the same antigen. Higher sensitivity of FMIA assays than ELISA have been reported in other studies [21,[24][25]. Higher sensitivity of the FMIA in comparison to the ELISA using the rSpaA415 protein was observed in experimentally infected pigs, where the FMIA could detect more pigs with an Erysipelothrix IgG antibody response than the ELISA at day 7 post challenge [21]. The higher specificity of FMIA assays has been suggested due to proteins covalently coupled to microspheres, and antigen purity eliminates nonspecific reactions that often cause high background problems in ELISA [26]. The lower background may add increase the detection limit on samples with a low antibody concentration [21].
Because there are no studies outside Japan that have investigated the seroprevalence of E. rhusiopathiae in bovine samples, we considered this knowledge gap important. This work represents the first study investigating E. rhusiopathiae in British and US cattle. The results suggest that E. rhusiopathiae infections in cattle occurs at low likely subclinical levels. Although the percentage of farms with seropositive cows was nearly the same in both countries; the overall prevalence in the British herd (13.8%) was significantly higher than in the US (6%) and positive British cattle had higher antibody titres when compared to the US herd. The reason for this is not clear; in Japan, higher cattle antibody rates were obtained where there were also areas of swine industry [3]. The seroprevalence in Japanese cattle (76%) was much higher than the obtained present prevalence [3].
Challenge studies in pigs have shown that a titre above 32 in the GAT indicates protective immunity against infection but it is unknown whether this applies also to cattle. The cattle vaccinated with the pig vaccine showed much higher antibody titres (GAT 256-1024, MFI 10,000-19,000) than the E. rhusiopathiae positive unvaccinated field cows (GAT 32-128,

MFI 1074-6800).
Seroprevalence studies in other ruminant species besides cattle are virtually absent; only one Japanese study found a seroprevalence of Erysipelothix of 13.5% among 52 wild deer (Cervus nippon yesoensis and Cervus nippon centralis),, also using the GAT [27].
The obtained results suggest that 13.8% and 6% of the farmed British and US cattle have had exposure to Erysipelothrix spp. and could be carriers of the bacteria and be a potential source of infection for other animals and humans. Furthermore, 34.4% and 34.7% of the sampled farms in Great Britain and the US had at least one seropositive animal. This data is of epidemiological importance as the bacterium has been isolated from cow slurry [3] and it is very resistant to environmental challenges [4], being able to survive in contaminated soil for several months [8]. Preventive measures should be implemented to avoid the transmission of the pathogen, especially among, abattoir workers, butchers, farmers and veterinarians that are at higher risk of exposure [28].

Conclusions
This work is the first one investigating the seroprevalence of E. rhusiopathiae among North American and British cattle and results showed that, although at low frequency, cattle are exposed to the bacterium and therefore could act as a reservoir of transmission of the disease. The newly developed FMIA test represents an improvement on the serodiagnosis of this bacterium in cattle as it is fast, proving objective results, sensitive (89.7%), specific (92.9%) and has a good agreement with the reference assay GAT.

Serum samples
Cattle with known Erysipelothrix exposure

Serological assays ELISA and FMIA development
The recombinant protein rSpaA415, based on the major surface protective antigen A (SpaA) [6,21] was used as antigen in both ELISA and FMIA. The optimal dilution of the serum sample and regents was determined by a checkerboard titration in both assays.

Growth agglutination test
The growth agglutination test was conducted to determine the agglutinating antibody titres of the sera as described elsewhere [3,[17][18]

Consent for publication
Not applicable.

Availability of data and material
All data generated during this study is presented in an analysed format is this manuscript.
Raw datasets generated during the current study are available from the first author