Veterinary Immunology and Immunopathology 162 (2014) 162–167

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Short communication

Sub-isotypic differences in the immunoglobulin G response to Lawsonia intracellularis in vaccinated, seropositive, and equine proliferative enteropathy-affected horses Allen E. Page a , Harold F. Stills Jr b , David W. Horohov a,∗ a b

University of Kentucky, Maxwell H. Gluck Equine Research Center, Lexington, KY 40546, United States University of Kentucky, Department of Microbiology, Immunology, and Molecular Genetics, Lexington, KY 40536, United States

a r t i c l e

i n f o

Article history: Received 1 August 2014 Received in revised form 19 September 2014 Accepted 23 September 2014 Keywords: Lawsonia Equine ELISA IgG Sub-isotype

a b s t r a c t In the horse, Lawsonia intracellularis infection results in equine proliferative enteropathy (EPE). While upwards of 100% of weanlings on an endemic farm may seroconvert, only a small percentage (approximately 5%) will develop clinical disease. Cell-mediated immune mechanisms likely play a role in resistance to L. intracellularis and the absence of a L. intracellularis-specific IFN-␥ response has been associated with the development of EPE. The goal of this study was to determine whether protection from clinical EPE is associated with the induction of a systemic IgG sub-isotypic response consistent with a Th1-type cytokine response. To describe their L. intracellularis/EPE status, horses enrolled in this study were placed into one of three categories: seropositive-only, vaccinated, and presumptive clinical EPE. An existing ELISA method was modified to detect L. intracellularis-specific IgG(a), IgG(b), and IgG(t) antibodies using the mouse anti-equine hybridomas CVS-48, CVS-39, and CVS-40, respectively. Additionally, the existing ELISA method was used to quantify total IgG antibodies specific for L. intracellularis for comparison between the groups. Total L. intracellularis-specific IgG was found to be significantly higher (p < 0.05) in presumptive clinical EPE cases (n = 21) when compared with seropositive (exposed but unaffected) (n = 36) and vaccinated horses (n = 27). Further, a similar pattern for IgG(a) was seen in that the presumptive clinical EPE horses had significantly more L. intracellularis-specific IgG(a) (p < 0.05) than the seropositive or vaccinated horses. With IgG(b), however, the vaccinated horses had significantly more IgG(b) (p < 0.05) than the presumptive clinical or seropositive horses. No L. intracellularis-specific IgG(t) was detected in samples from any of the groups. While the results presented here with respect to IgG(a) response in the presumptive clinical EPE group were expected, a higher concentration of IgG(a) was anticipated in the seropositive horses that failed to develop clinical disease as well as in the vaccinated horses. Future work utilizing newer reagents against a broader range of equine IgG subisotypes may provide additional information once these become commercially available. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Tel.: +1 859 257 4757; fax: +1 859 257 8542. E-mail addresses: [email protected], [email protected] (D.W. Horohov). http://dx.doi.org/10.1016/j.vetimm.2014.09.004 0165-2427/© 2014 Elsevier B.V. All rights reserved.

Lawsonia intracellularis is an obligate, intracellular, gram-negative rod that invades intestinal crypt cells and causes proliferation of the intestinal epithelium

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(McOrist et al., 1995). While initially described as a pathogen of pigs (McOrist et al., 1995), L. intracellularis is now viewed as a cause of proliferative enteropathy in a variety of mammalian species (Drolet et al., 1996; Hotchkiss et al., 1996), including horses (Brees et al., 1999; Cooper et al., 1997; Frank et al., 1998; Williams et al., 1996). In the horse, L. intracellularis infection results in equine proliferative enteropathy (EPE), which has been reported worldwide (Feary et al., 2007; Steinman et al., 2014; Wuersch et al., 2006). Clinical signs in infected horses can be non-specific and include anorexia, fever, lethargy, depression, and peripheral edema, with colic and diarrhea infrequently seen (Lavoie et al., 2000). Affected horses suffer weight loss while some ultimately die despite aggressive treatment (Page et al., 2011b, 2012). Equine proliferative enteropathy typically affects weanlings and is rarely seen in older horses (Dauvillier et al., 2006; McGurrin et al., 2007). Preventative measures for EPE are poorly defined, however a porcine vaccine for L. intracellularis has been reported to prevent clinical EPE when used in horses (Pusterla et al., 2009, 2012b). Interestingly, while upwards of 100% of weanlings on an endemic farm may seroconvert (Page et al., 2011c), only approximately 5% will develop clinical disease (Page et al., 2014). The reasons for this differential susceptibility to disease remain unknown, though it is likely due to differences in the individual’s immune response to the bacterium. Unfortunately, the immunological aspects of L. intracellularis infections also remain largely undefined. Cell-mediated immune mechanisms likely play a role in resistance to L. intracellularis (Go et al., 2005; Kroll et al., 2004). The absence of a L. intracellularis-specific IFN␥ response has been associated with the development of clinical disease in both horses (Page et al., 2011a) and mice (Go et al., 2005). Oral vaccination of pigs and horses with an avirulent live strain of L. intracellularis results in bacteriaspecific IFN-␥ expression by PBMCs (Guedes and Gebhart, 2010; Pusterla et al., 2012a). Stimulation of PBMCs from infected horses likewise leads to L. intracellularis-specific IFN-␥ expression (Page et al., 2011a; Pusterla et al., 2012a). While the specific role IFN-␥ might be playing in this infection remains unclear, it is one of three cytokines associated with antibody class switching, the others being IL-4 and TGF-␤. In mice, IL-4 induces B cells to switch to IgG1 and IgE, IFN-␥ induces IgG2a, and TGF-␤ induces IgG2b and IgA expression (Coffman et al., 1989). While isotype regulation in the horse is less well defined (Wagner, 2006), a similar process is likely occurring, as has been found for cattle (Estes, 2010). Thus, characterization of cytokine expression patterns could be predictive of isotypic antibody expression or vice versa. The goal of this study was to determine whether protection from clinical EPE (provided by vaccination or as evidenced by exposure to L. intracellularis without clinical disease) is associated with the induction of a systemic IgG sub-isotypic response consistent with a Th1-type cytokine response. To do this, we adapted our existing ELISA method (Page et al., 2011c) to detect L. intracellularisspecific IgG(a), IgG(b), and IgG(t) antibodies. We then determined IgG sub-isotype responses in horses that seroconvert due to natural exposure in the absence of clinical

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disease, those that seroconvert as a result of vaccination with a modified-live L. intracellularis vaccine, and those that seroconvert due to the occurrence of clinical EPE. 2. Materials and methods 2.1. Study samples Samples for this study were collected from Thoroughbred farms in central Kentucky known to have endemic EPE. Many of these farms were involved in another project (Page et al., 2014) involving the screening of horse serum for antibodies to L. intracellularis. Other samples for this project were collected and submitted by private veterinarians for L. intracellularis antibody testing in our laboratory. Approval to conduct this study was obtained from the University of Kentucky’s Institutional Animal Care and Use Committee and forms documenting informed consent for inclusion of privately owned horses were obtained from the farm managers or farm veterinarians, whom were authorized to act on the behalf of private owners. Blood samples were obtained via jugular venipuncture and stored at 4 ◦ C until they could be centrifuged at 800 × g for 10 min. After centrifugation, serum was removed and stored in duplicate at −20 ◦ C until assayed for L. intracellularis-specific antibodies (see below). The collection of presumptive clinical EPE cases took place over 1½ years, from August 2012 through February 2014, while the collection of seropositive samples took place from August 2013 through February 2014. Clinical samples were obtained from nine farms while a total of six farms provided the seropositive samples. Given the endemic EPE status of these farms, several farms provided both the clinical and seropositive samples. Samples from L. intracellularisvaccinated horses on two EPE-endemic farms were also collected from August 2013 through February 2014. These horses were intra-rectally vaccinated prior to weaning with a modified-live L. intracellularis porcine vaccine1 using a two-dose protocol, one month apart, as previously described (Nogradi et al., 2012); only the sample collected at one-month after the second vaccine administration was used in this study. Samples from presumptive clinical EPE cases were collected immediately following detection of concurrent clinical signs compatible with EPE while seropositive samples were the first sample from a horse that tested positive for L. intracellularis-specific antibodies but had normal total protein and albumin concentrations at that time. All horses in this study were less than 1½ years of age. 2.2. Study horse classification To describe their L. intracellularis/EPE status, horses enrolled in this study were placed into one of three categories: seropositive-only, vaccinated, and presumptive clinical EPE. Presumptive clinical EPE cases were considered as such based on our previous work (Page et al., 2014).

1 Enterisol Ileitis, Boehringer Ingelheim Vetmedica Inc, St. Joseph, MO, USA.

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Since post-mortem examination is required for a definitive diagnosis of EPE, all clinical cases of EPE were considered presumptive. 2.3. Detection of total IgG L. intracellularis-specific antibodies Detection of total IgG (heavy and light chain) antibodies specific for L. intracellularis were carried out using an ELISA, as previously described and modified (Page et al., 2011c, 2014). OD values were converted to an ELISA unit (EU) scale based on a logarithmic regression line obtained from a standard curve run on every plate (Page et al., 2011c). 2.4. Detection of IgG sub-isotypes specific for L. intracellularis While newer nomenclature exists for equine IgG subisotypes, identified as IgG1 through IgG7 (Wagner et al., 2004), the reagents needed to identify these newer subisotypes are not commercially available. Therefore, we opted to use the accepted CVS hybridoma lines in this project (Lunn et al., 1991). Once the materials become available, additional work using the newer sub-isotypes could be used to further examine any differences in the groups presented here. The detection of IgG(a), IgG(b), and IgG(t) sub-isotypes was carried out using a modified version of our previously described ELISA for L. intracellularis-specific antibodies (Page et al., 2011c). Briefly, hybridomas producing monoclonal antibodies directed against equine IgG(a), IgG(b), and IgG(t) (CVS-48, CVS-39, and CVS-40, respectively) (Lunn et al., 1991) were grown prior to this study using standard cell-culture techniques. A checkerboard titration scheme (Kroll et al., 2005; Wattanaphansak et al., 2008) was used to determine the optimal antigen, serum, antiequine antibody, and anti-mouse antibody concentrations. Using this method, it was determined that an antigen concentration of 2.5 ␮g/mL, a serum dilution of 1:100, an anti-equine antibody concentration of 1:2, and an antimouse antibody of 1:2000 produced the most consistent results. A total of six samples from different horses were screened as possible candidates for use as standard curves with each of the three sub-isotypes using serial dilutions from 1:30 through 1:15,360. Ultimately, one horse was identified for use with the standard curves. With respect to the overall procedure, ELISA plates were coated with antigen in carbonate buffer, covered, and allowed to sit overnight at 4 ◦ C. The following day, plates were washed three times with PBS + 0.05% Tween-20 (ELISA wash) using an automatic plate washer. To each well was added 200uL of 1% (w/v) blocking buffer (polyvinyl alcohol2 ) for 1 h at room temperature. After blocking, the plates were washed 3 times, as above. Serum samples were diluted 1:100 in blocking buffer and then added to their respective wells on the ELISA plate for 1 h at room temperature. At the same time, the standard curve

2

Mowiol 6-98, Thermo Scientific, Rochester, NY, USA.

sample, with serially diluted samples ranging from 1:30 to 1:7680, was added to each plate, as was a positive control sample, two negative controls, and one fetal equine serum sample. All samples, including those of the standard curve and controls, were added to the plates in duplicate at a volume of 100 ␮L per well. After 1 h, the plates were again washed, as above. Afterwards, 100 ␮L of a 1:2 dilution of the respective anti-equine sub-isotype specific antibody was added to each well for 1 h at room temperature. The plates were again washed, as above, and then 100 ␮L of a 1:2000 goat anti-mouse conjugated with HRP3 added to each well. Following a 1 h incubation at room temperature in the dark, the plates were washed for the final time. After this wash, 100 ␮L of 3,3 ,5,5 -tetramethylbenzidine (TMB)4 was added to each well and the color reaction allowed to develop at room temperature over 5 min; 100 ␮L of stop solution5 was then added and the plate read at 450 nm within 15 min. Results were converted from OD values to ELISA units based on a logarithmic regression line from each plate-specific standard curve. An r2 ≥ 0.90 was required for the plate’s results to be considered valid. 2.5. Data analysis As the ELISA data was not normally distributed, a oneway analysis of variance6 (ANOVA) on ranks was used to detect significant differences between the classification groups for total IgG as well as the three IgG subisotypes. P values ≤ 0.05 were considered to be statistically significant. 3. Results and discussion Given the difficulty with definitive antemortem diagnosis of EPE, an alternative method for identifying clinically affected horses is needed. As such, the possibility of using L. intracellularis-specific antibody concentrations to diagnose or rule-out disease was examined. Based on data obtained for this study, total L. intracellularis-specific IgG is significantly higher (p < 0.05) in presumptive clinical EPE cases (n = 21) when compared with seropositive (exposed but unaffected) (n = 36) and vaccinated horses (n = 27) (Fig. 1). Since horses may be vaccinated against L. intracellularis, of particular interest was the difference between presumptive clinical EPE cases and seropositive horses. While there was some overlap between the confidence intervals of the two groups and some outliers (Fig. 1), a conservative cutoff of 375–400 EU’s would provide a reasonable value to use in discriminating between clinical EPE cases and exposed but unaffected horses. Even though a value such as this may help to increase the specificity of antibody testing for L. intracellularis, it would decrease the sensitivity given that a number of presumptive clinical cases would fall below

3

Sigma–Aldrich, St. Louis, MO, USA. SureBlue TMB Microwell Peroxidase substrate, KPL, Gaithersburg, MD, USA. 5 TMB Stop solution, KPL, Gaithersburg, MD, USA. 6 SigmaStat, SPSS Inc., Chicago, IL, USA. 4

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Fig. 1. Total IgG box and whisker plots of presumptive clinical EPE, seropositive, and L. intracellularis-vaccinated horses. Data expressed as ELISA units. Letters indicate significant (p < 0.05) differences between groups.

this value. As such, this type of test should be used to confirm clinical EPE rather than rule it out or be used with paired serum samples. It is important to note that the samples reported here were the initial samples taken after the appearance of clinical signs compatible with EPE. As such, the antibody concentrations may not accurately represent the maximum concentration each horse obtained, but they do likely represent the sample that would be submitted by a practitioner for initial diagnosis. Thus, it does appear that veterinarians can rely on the magnitude of the total IgG concentration to differentiate clinically affected horses from those only exposed to L. intracellularis. A similar pattern for IgG(a) (Fig. 2) was seen as for total IgG in that the presumptive clinical EPE horses had significantly more L. intracellularis-specific IgG(a) (p < 0.05) than the seropositive or vaccinated horses. With respect to IgG(b), however, the vaccinated horses had significantly more (p < 0.05) than the presumptive clinical or seropositive horses (Fig. 3). No detectable IgG(t) antibodies to L. intracellularis were identified in any of the three groups of horses (data not shown). It should be noted that the vaccinate outlier in each of the figures (220 EU in Fig. 3) was the same horse. These antibody concentrations were measured in an early October sample and corresponded with concurrent seroconversions from natural exposure to L. intracellularis on a nearby, EPE-endemic farm. Thus, these high concentrations likely represent an anamnestic response from natural exposure to the bacterium following vaccination in this horse. While evidence of a Th1 (IgG(a)) response in the presumptive clinical EPE group was expected based on previous reports of post-infection IFN-␥ production in vitro in horses (Page et al., 2011a; Pusterla et al., 2012a), an even greater IgG(a) response was expected in the seropositive horses that failed to develop clinical disease following exposure or those protected by vaccination from clinical

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Fig. 2. IgG(a) box and whisker plots of presumptive clinical EPE, seropositive, and L. intracellularis-vaccinated horses. Data expressed as ELISA units. Letters indicate significant (p < 0.05) differences between groups.

disease. Indeed, we have previously shown that increased IFN-␥ mRNA production in vitro in response to stimulation with L. intracellularis is associated with protection from clinical disease (Page et al., 2011a). An approach similar to ours has been used to associate IgG(a) with a protective Th1 (IFN-␥) response and IgG(b)/IgG(t) with a non-protective Th2 (IL-4, IL5) cytokine response in foals exposed to Rhodococcus equi (Hooper-McGrevy et al., 2003). Similarly, protective immunity to Strongylus vulgaris has shown a similar relationship between IgG sub-isotypes and a Th2 cytokine response while pathologic responses were associated with

Fig. 3. IgG(b) box and whisker plots of presumptive clinical EPE, seropositive, and L. intracellularis-vaccinated horses. Data expressed as ELISA units. Letters indicate significant (p < 0.05) differences between groups.

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a Th1 response (Swiderski et al., 1998). The increased IgG(b) in horses following vaccination against L. intracellularis would suggest that these horses have more of a Th2 response. As such, these findings appear inconsistent with previous work showing vaccination of horses against L. intracellularis elicits an IFN-␥ recall response in vitro (Pusterla et al., 2012a). Based on our results, it appears that IgG(b) production was not necessary for protection. Given that the presumptive clinical EPE and seropositive horses were likely exposed to an equine-strain of L. intracellularis, whereas the vaccine contains a porcine strain, this strain difference could account for the disparity in the IgG(b) response. Indeed, it has recently been reported that host-adaptation of L. intracellularis is important, especially with the occurrence of clinical disease (Sampieri et al., 2013). It is possible that the IgG sub-isotype profile could have changed over time. However, the goal of the study was to determine whether the initial sub-isotype profile could be used as a predictor or indicator as to the state of each horse. As such, we did not seek to evaluate samples other than the first seropositive sample (both the presumptive EPE and seropositive groups) or the one-month post-second vaccine sample. It remains unlikely that systemic antibodies to an enterically localized L. intracellularis infection would play a direct role in immunity to the bacterium. Here we have shown that clinically affected EPE horses have significantly higher total IgG and IgG(a) antibodies to L. intracellularis compared with exposed but unaffected or vaccinated horses. However, using IgG sub-isotypes, we were unable to definitively demonstrate the induction of a Th1 in immune horses compared to EPE-affected horses. While we were unable to use this method to further explore the reasons behind the variable disease rate amongst horses exposed to L. intracellularis, there remains ample evidence for the importance of a CMI response in immunity to the bacterium (Page et al., 2011a; Pusterla et al., 2012a). Additional work, utilizing the more diverse IgG sub-isotypes IgG 1–7, may prove useful once the reagents become widely available. Conflict of interest The authors have no conflicts of interest to declare. Funding Boehringer Ingelheim Vetmedica Inc. funded this project in its entirety through a competitive grant process. Other than funding, Boehringer Ingelheim Vetmedica Inc. had no role in any portion of this project, including, but not limited to, study design, data analysis, and study presentation. Dr. Page’s post-doctoral fellowship stipend is paid for by the Morris Animal Foundation (Grant D13EQ-401). Acknowledgements The authors would like to thank the farm managers and farm veterinarians who volunteered their time and effort to collect the samples used in this study.

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Sub-isotypic differences in the immunoglobulin G response to Lawsonia intracellularis in vaccinated, seropositive, and equine proliferative enteropathy-affected horses.

In the horse, Lawsonia intracellularis infection results in equine proliferative enteropathy (EPE). While upwards of 100% of weanlings on an endemic f...
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