Allen E. Page

Immunogenicity and clinical protection against equine influenza by DNA vaccination of ponies

Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza.

Serial use of serologic assays and fecal PCR assays to aid in identification of subclinical Lawsonia intracellularis infection for targeted treatment of Thoroughbred foals and weanlings

OBJECTIVE To assess the serial use of serum immunoperoxidase monolayer assays (IPMAs) and fecal PCR assays, combined with other diagnostic methods, to identify subclinical Lawsonia intracellularis infections for targeted treatment of Thoroughbred foals and weanlings at farms in which the pathogen was endemic or nonendemic. DESIGN Evaluation study. ANIMALS 100 foals and weanlings (53 and 47 at farms in which L intracellularis was endemic and nonendemic, respectively). PROCEDURES Serum was collected every 4 weeks and tested via IPMA, for antibodies against L intracellularis. Fecal samples were collected every 2 weeks and tested by use of an L intracellularis-specific PCR assay. When results for IPMAs or PCR assays were positive or clinical signs compatible with equine proliferative enteropathy (EPE) were detected, clinicopathologic testing was performed to determine treatment. RESULTS No foals had positive results for the L intracellularis-specific IPMA until after weaning; 32 of 53 (60.4%) weanlings at the farm in which L intracellularis was endemic and 8 of 47 (170%) at the farm in which L intracellularis was nonendemic had positive IPMA results, whereas the number of weanlings that tested positive via fecal PCR assays at those farms was 6 and 0, respectively. Nineteen of 32 weanlings with positive IPMA results at the farm in which L intracellularis was endemic were treated for EPE; 5 of these had clinical signs of EPE. No weanlings at the nonendemic farm had clinical signs of or were treated for EPE. CONCLUSIONS AND CLINICAL RELEVANCE IPMA appeared to be a useful means of identifying weanlings exposed to L intracellularis.

Acute Deterioration and Death with Necrotizing Enteritis Associated with Lawsonia intracellularis in 4 Weanling Horses

An 8-month-old Thoroughbred colt was evaluated in October 2010 with a less than 1-day history of inappetence. Physical examination in the field revealed throat latch edema, lethargy, and fever (103.8°F) (ref 99–101.5°F). A complete blood count revealed leukocytosis (18.0 × 10/lL; ref 5.0–12.6 × 10/lL) with a relative neutropenia (49%; ref 55–65%) and lymphopenia (26%; ref 35–45%), as well as a toxic left shift (25% bands; ref 0–5%). Serum biochemistry abnormalities included hypoproteinemia (3.3 g/dL; ref 6–7.9 g/dL), hypoalbuminemia (1.2 g/dL; ref 3.4–4.1 g/dL), and an increased BUN (45 mg/dL; ref 11–26 mg/dL), along with other abnormalities (Table S1). Lawsonia intracellularis-induced equine proliferative enteropathy (EPE) was suspected because of the combination of hypoproteinemia, hypoalbuminemia, inappetence, and the autumn presentation. Treatment consisted of intravenous oxytetracycline (6.6 mg/kg IV q24hr), flunixin meglumine (1 mg/kg IV q12h), oral omeprazole (1 mg/kg PO q24hr), dexamethasone (0.1 mg/kg IV q24hr), intravenous crystalloid fluids (10 mL/kg IV bolus once), and intravenous colloids (10 mL/kg IV bolus once). Despite treatment, the weanling was euthanized within 48 hours of presentation because of continued deterioration and signs of pulmonary disease characterized by nasal discharge, epistaxis, and tachypnea. Blood work submitted the morning of euthanasia revealed a worsening leukocytosis (35.4 9 10/lL) with an unchanged differential, as well as continued hypoproteinemia (3.5 g/dL), hypoalbuminemia (1.1 g/ dL), and an increased BUN (59 mg/dL). Case 2

Adaptation and validation of a bacteria-specific enzyme-linked immunosorbent assay for determination of farm-specific Lawsonia intracellularis seroprevalence in central Kentucky Thoroughbreds

REASONS FOR PERFORMING STUDY Lawsonia intracellularis is the causative agent of equine proliferative enteropathy (EPE), a disease for which no large-scale seroprevalence studies have been conducted. OBJECTIVES To validate and use an equine-specific enzyme-linked immunosorbent assay (ELISA) for L. intracellularis to determine the seroprevalence of L. intracellularis on numerous farms. METHODS An ELISA, in which purified antigen was used, was adapted from previous work in swine. A total of 337 Thoroughbreds from 25 central Kentucky farms were enrolled and monthly serum samples collected from August 2010 to January/February 2011. Samples were screened for L. intracellularis-specific antibodies using a modified ELISA. Farms were classified into one of 3 groups based on 3 year prior history with EPE. RESULTS The ELISA intra-assay coefficient of variation (CV) was 6.73 and inter-assay CV was 9.60. An overall seroprevalence of 68% was obtained, with farm-specific seroprevalances ranging from 14 to 100%. A significant difference was found in the average seroprevalence (P<0.05) on farms with a confirmed recent history of EPE cases. Additionally, both lower average ELISA unit (EU) values (P = 0.079) and maximum EU values (P = 0.056) were detected on farms with no recent EPE history when compared to the other groups. A bimodal exposure distribution to L. intracellularis was detected in the fall and winter months. CONCLUSIONS Recent history of EPE was associated with higher average seroprevalence indicating increased exposure on farms with prior cases of EPE. Seasonally bimodal exposure was also observed. POTENTIAL RELEVANCE The adapted ELISA appears to be useful for determination of L. intracellularis-specific antibody levels. The high farm-specific seroprevalences and bimodal distribution of exposure to L. intracellularis were unexpected and suggest that farms with a previous history of EPE remain at risk due to heightened exposure levels beyond early winter.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

Comparison of Feces versus Rectal Swabs for the Molecular Detection of Lawsonia Intracellularis in Foals with Equine Proliferative Enteropathy

The purpose of the current study was to compare the molecular detection rate of Lawsonia intracellularis between feces and rectal swabs collected from 42 foals with suspected equine proliferative enteropathy (EPE). Fecal samples and rectal swabs were processed for DNA purification by using an automated extraction system. The purified DNA was then analyzed by real-time polymerase chain reaction (PCR) for the presence of the aspartate ammonia lyase (aspA) gene of L. intracellularis. Absolute quantitation was calculated by using a standard curve for L. intracellularis and expressed as copy numbers of the aspA gene of L. intracellularis per microliter of purified DNA. The combined PCR detection rate for L. intracellularis was 90%, with 38 foals testing PCR positive in feces (33 samples), rectal swabs (32), or both (27). Six foals tested PCR positive only in feces, whereas 5 tested positive only in rectal swabs. Feces yielded a significantly higher aspA gene copy number of L. intracellularis than rectal swabs. Feces and rectal swabs tested PCR negative from 4 foals. In conclusion, the results showed that feces yielded similar numbers of PCR-positive results, with a higher L. intracellularis aspA gene load than rectal swabs. By analyzing dual samples, the PCR detection rate for L. intracellularis increased from 76% and 79% for rectal swabs and feces, respectively, to 90%. Rectal swabs should be considered as an alternative sample type for EPE-suspected patients with decreased or no fecal output.

The use of streptolysin O (SLO) as an adjunct therapy for Rhodococcus equi pneumonia in foals

Rhodococcus equi is a soil borne bacterium that causes severe morbidity and death in young foals. The economic costs of the disease include loss of life, treatment expenses, veterinary monitoring expenses and, perhaps most importantly, potential reduction in future athletic performance in horses that suffer severe lung abscessations caused by R. equi. Current standard of care for pneumonia caused by R. equi is treatment with a macrolide antimicrobial and rifampicin. However, the hallmark of pneumonia caused by R. equi is severe formation of pyogranulomas and a walling off effect that can prevent systemic antibiotics from reaching antimicrobial concentrations in lung tissues. It is hypothesized that streptolysin O (SLO) used as an adjunct therapy with antibiotics will reduce the duration and severity of disease caused by R. equi pneumonia compared to antibiotic therapy alone. Addition of SLO to the antibiotic enhanced clinical responses compared to the other groups, including the antibiotic alone group. Of particular significance were lower bacterial counts in the lungs and longer survival time in those foals treated with SLO and antibiotics.

Lawsonia intracellularis and Equine Proliferative Enteropathy

Lawsonia intracellularis is the etiologic agent for equine proliferative enteropathy (EPE), which typically affects weanling and yearling horses. In North America, EPE cases often occur between August and January, although cases outside of this time frame have been reported. Clinical signs of EPE are usually nonspecific and include lethargy, pyrexia, anorexia, peripheral edema, weight loss, colic, and diarrhea. Diagnosis is based on the presence of hypoproteinemia and hypoalbuminemia along with clinical signs and positive commercial serologic and/or molecular testing. Treatment requires the use of antimicrobials with good intracellular penetration and supportive care to prevent or decrease secondary complications.

The effect of passively acquired antibodies on Lawsonia intracellularis infection and immunity in the horse

REASONS FOR PERFORMING STUDY Multiple hypotheses into the age-based susceptibility of animals to Lawsonia intracellularis exist, including the decline of passively acquired antibodies. OBJECTIVES To determine whether the decline in passively acquired antibodies in horses is responsible for the age predilection of equine proliferative enteropathy (EPE). Additional objectives included examination of various risk factors for the development of EPE as well as the determination of naturally occurring attack rates for clinical and subclinical EPE. STUDY DESIGN Prospective, multifarm field study. METHODS A total of 369 mare and foal pairs from 15 central Kentucky Thoroughbred farms were used in this study, which took place from January 2012 to February 2013. Serum samples were collected from mares and foals within 48 h of parturition, and then monthly from foals to February of their yearling year. Lawsonia intracellularis-specific antibodies were measured using an enzyme-linked immunosorbent assay. RESULTS No effect of passively acquired antibodies on the occurrence of presumptive clinical or subclinical EPE was noted. In total, 5.3% and 6.3% of seropositive horses developed presumptive clinical or subclinical EPE, respectively. In multiple logistic regression models, colts were at a significantly greater risk than fillies of developing presumptive clinical EPE (odds ratio [OR] 5.468, 95% confidence interval [CI] 1.134-26.362, P = 0.034) or a combination of either presumptive clinical or subclinical EPE (OR 3.861, 95% CI 1.461-10.206, P = 0.006) while foals that were weaned in September or beyond were at a lower risk of developing presumptive EPE (OR = 0.281, 95% CI 0.0807-0.981, P = 0.05). CONCLUSIONS This is the first study to show that passively acquired antibodies to L. intracellularis do not have an effect on the occurrence of clinical or subclinical EPE. A number of novel findings, including identification of the disease rate among naturally exposed horses, warrant additional work as they may help to identify potential risk factors for L. intracellularis exposure and/or the reservoir host(s) of the bacterium.

The effect of environment on interferon-gamma production in neonatal foals

While interferon-gamma (IFNγ) plays an important role in protection against viral and intracellular bacterial infections, its production in neonates is deficient. Exposure to environmental antigens can promote the maturation of the immune system of neonatal humans and mice. We hypothesize that exposure to high level of microbial components would increase the production of IFNγ in neonatal foals. To test this hypothesis, one group of foals was placed into stalls three times a week for 8 weeks. A second group of foals remained on pasture. Air samples were collected from the barn and pasture for microbial culture. There were more bacteria and fungi in the air samples collected from the barn compared with those from the pasture. Bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) were collected from both groups of foals at various times to assess IFNγ production. The frequency of IFNγ(+) lymphocytes in BAL cells and PBMC was higher for foals kept in the stalls.