Alan T. Loynachan

Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine

A single-cycle, propagation-defective replicon particle (RP) vaccine expressing a swine influenza virus hemagglutinin (HA) gene was constructed and evaluated in several different animal studies. Studies done in both the intended host (pigs) and non-host (mice) species demonstrated that the RP vaccine is not shed or spread by vaccinated animals to comingled cohorts, nor does it revert to virulence following vaccination. In addition, vaccinated pigs develop both specific humoral and IFN-γ immune responses, and young pigs are protected against homologous influenza virus challenge.

Replicon particle vaccine protects swine against influenza.

An alphavirus derived replicon particle (RP) vaccine expressing the cluster IV H3N2 swine influenza virus (SIV) hemagglutinin (HA) gene induced protective immunity against homologous influenza virus challenge. However, pigs with maternal antibody had no protective immunity against challenge after vaccination with RP vaccines expressing HA gene alone or in combination with nucleoprotein gene.

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

The effect of bacterial dose and foal age at challenge on Rhodococcus equi infection.

While Rhodococcus equi remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study R. equi infection has proven difficult. The objective of this study was to identify a challenge dose of R. equi that resulted in slow progressive disease, spontaneous regression of lung lesions and age-dependent susceptibility. Foals less than one-week of age were challenged intratracheally using either 10(6), 10(5), 10(4), 10(3) or 10(2) cfu of R. equi. Two doses (10(3) cfu and 10(5) cfu) were used to challenge 2 and 3-week-old, and 3 and 6-week-old foals, respectively. Physical examination, thoracic ultrasound and blood work were performed. Foals were euthanized at the end of the study or when clinical signs of pneumonia developed. All foals were necropsied and their lung lesions scored. Foals challenged with low concentrations of R. equi developed slow progressive pneumonia and approximately 50% of the foals recovered spontaneously. Likewise, macroscopic (>1cm diameter) pyogranulomatous lesions were only observed when low doses of R. equi were used. Clinical pneumonia was not seen after low dose challenge in the 3-week-old foals or in the 6-week-old foals. This study demonstrates that the use of low doses of R. equi to challenge neonatal foals provides an improved model for studying this disease. Furthermore, susceptibility to R. equi infection was shown to diminish early in the foals life, as has been reported in the field.

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.

Three Equine Cases of Mixed Hepatoblastoma with Teratoid Features

Hepatoblastoma was diagnosed in 3 Thoroughbreds at the University of Kentucky Livestock Disease Diagnostic Center (LDDC) since 1997. Case #1 involved a fetus with a well-demarcated, multilobulated, solitary mass that extended from the left liver lobe. Case #2 was observed in a neonate with a primary hepatic mass and multiple metastases in the skin, brain, meninges, and stylohyoid bone. Case #3 was a solitary hepatic mass incidentally discovered in a neonate at necropsy. Microscopically, the masses were similarly composed of sheets and cords of fetal and embryonal epithelial cells that frequently formed sinusoid-like structures. Intermixed with the neoplastic epithelial cells were variable amounts of hemorrhage, necrosis, osteoid, and bone. Immunohistochemically, the epithelial cells stained variably positive for alpha- fetoprotein, frequently positive for vimentin, and occasionally positive for cytokeratin. All 3 cases were diagnosed as mixed hepatoblastoma with teratoid features.

Haemagglutinin and nucleoprotein replicon particle vaccination of swine protects against the pandemic H1N1 2009 virus

The recent emergence of the pandemic H1N1 (pH1N1) and H3N2 variant influenza A viruses (IAV) in 2009 and 2011–2012, respectively, highlight the zoonotic potential of influenza viruses and the need for vaccines capable of eliciting heterosubtypic protection. In these studies, single-cycle, propagation-defective replicon particle (RP) vaccines expressing IAV haemagglutinin (HA) and nucleoprotein (NP) genes were constructed and efficacy was evaluated in homologous and heterologous pig challenge studies with the pH1N1 2009 influenza virus (A/California/04/2009). Homologous HA RP vaccination eliminated virus shedding and decreased pulmonary pathology in pigs following pH1N1 2009 challenge. An RP vaccine expressing an H3N2-derived NP gene was able to decrease nasal shedding and viral load following heterosubtypic pH1N1 2009 challenge in pigs. These studies indicate that although homologous vaccination of swine remains the most effective means of preventing IAV infection, other vaccine alternatives do offer a level of heterosubtypic protection, and should continue to be evaluated for their ability to provide broader protection.

Evaluation of gadodiamide versus gadobutrol for contrast-enhanced MR imaging in a rat brain glioma model at 1.5 and 3 T.

Purpose:To compare equivalently-dosed (0.1 mmol/kg) gadobutrol (Gadovist) and gadodiamide (Omniscan) in a rat brain glioma model with respect to lesion signal-to-noise (SNR), contrast-to-noise (CNR), and contrast enhancement (CE) at 1.5 and 3 T. Lesion enhancement with standard-dose gadobutrol in scans performed at 1.5 T was also compared with that of half-dose gadobutrol in scans performed at 3 T. Materials and Methods:Fifty-four rats were injected with glioma cells via a plastic brain cannula and divided into 3 groups. In the first group, each animal was studied using gadodiamide and gadobutrol, with 24 hours separating injections. The 2 agents were administered in random order at a dose of 0.1 mmol/kg. Each animal was scanned using a 3 T MR system. The procedure for the second group was similar, but scanning was performed at 1.5 T. For the third group, rats were given standard or half-dose gadobutrol and scanned at 1.5 and 3 T, respectively. For all MR examinations, T1-weighted images were obtained precontrast and at 1, 3, 5, 7, and 9 minutes postcontrast administration. Results:At 3 T improvements in SNR, CNR, and CE with gadobutrol ranged from 11.8% to 16.0%, 30.5% to 35.4%, and 27.1% to 31.5%, respectively, and at 1.5 T from 7.0% to 11.1%, 27.1% to 35.8%, and 23.8% to 29.5%, respectively. Differences between these parameters with gadobutrol and gadodiamide were statistically significant (P < 0.0001–0.05) at all time points following contrast administration. In group 3, no significant differences in CNR or CE were found between full dose gadobutrol at 1.5 T and half-dose at 3 T, although SNR was significantly greater (28.5%–35.1%; P < 0.0008) at 3 T. Conclusion:Gadobutrol (Gadovist) demonstrates superior lesion enhancement to equivalently-dosed gadodiamide (Omniscan) in the rat brain glioma model. These results are complemented by the improved observed and theoretical safety profile of the first agent, in particular with regard to nephrogenic systemic fibrosis. The ability to image with half-dose gadobutrol at 3 T without a statistically significant decrease in lesion enhancement, compared with 1.5 T, offers an additional theoretical safety margin and potential cost-savings.

Complete Diphallia, Imperforate Ani (Type 2 Atresia Ani), and an Accessory Scrotum in a 5-Day-Old Calf

A 5-day-old Angus calf was submitted to the necropsy service at the University of Kentucky Livestock Disease Diagnostic Center. At birth, the calf was noted to have 2 scrota. Five days post-calving the calf developed severe tenesmus and an umbilical infection. Clinical examination revealed atresia ani. At necropsy, complete diphallus, imperforate ani (type 2 atresia ani), and remnants of 2 scrota were identified. This report describes the gross pathologic appearance of a rare case of complete diphallia, imperforate ani (type 2 atresia ani), and an accessory scrotum in a 5-day-old calf.

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.