An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.

Effects of a water-soluble formulation of tylvalosin on disease caused by porcine reproductive and respiratory syndrome virus alone in sows or in combination with Mycoplasma hyopneumoniae in piglets.

BACKGROUND: The effect of a water-soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) on disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (Mhyop) was investigated in two animal studies. In a PRRSV challenge model in pregnant sows (n = 18), six sows received water medicated at target dose of 5 mg tylvalosin/kg body weight/day from 3 days prior to challenge until the end of gestation. Six sows were left untreated, with a third group remaining untreated and unchallenged. Sows were challenged with PRRSV-2 at approximately 85 days of gestation. Cytokines, viremia, viral shedding, sow reproductive parameters and piglet performance to weaning were evaluated. In a dual infection study (n = 16), piglets were challenged with Mhyop on days 0, 1 and 2, and with PRRSV-1 on day 14 and euthanized on day 24. From day 10 to 20, eight piglets received water medicated at target dose of 20 mg tylvalosin/kg body weight/day and eight piglets were left untreated. Cytokines, viremia, bacteriology and lung lesions were evaluated. RESULTS: In the PRRSV challenge study in pregnant sows, tylvalosin significantly reduced the levels of serum IL-8 (P < 0.001), IL-12 (P = 0.032), TNFα (P < 0.001) and GM-CSF (P = 0.001). IL-8 (P = 0.100) tended to be lower in uterus of tylvalosin sows. All piglets from tylvalosin sows surviving to weaning were PRRSV negative in faecal swabs at weaning compared to 33.3% PRRSV positive piglets from untreated sows (P = 0.08). In the dual challenge study in piglet, tylvalosin reduced serum IL1ß, IL-4, IL-6, IL-8, IL-10, IL-12, IL-1α, IL-13, IL-17A, IL-18, GM-CSF, TGFß1, TNFα, CCL3L1, MIG, PEPCAM-1 (P < 0.001) and increased serum IFNα, IL-1ra and MIP-1b (P < 0.001). In the lungs, tylvalosin reduced IL-8, IL-10 and IL-12 compared to untreated pigs (P < 0.001) and tended to reduce TNFα (P = 0.082). Lung lavage samples from all tylvalosin treated piglets were negative for Mhyop (0 cfu/mL) compared to the untreated piglets which had mean Mhyop counts of 2.68 × 104 cfu/mL (P = 0.023). CONCLUSION: Overall, tylvalosin reduced both local and systemic proinflammatory cytokines after challenge with respiratory pathogens in sows and in piglets. Tylvalosin was effective in reducing Mhyop recovery from the lungs and may reduce virus shedding in piglets following transplacental PRRSV infection in sows.

Evaluation of the clinical efficacy of a water soluble formulation of tylvalosin in the control of enzootic pneumonia associated with Mycoplasma hyopneumoniae and Pasteurella multocida in pigs.

BACKGROUND: The efficacy of a water soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) was evaluated in the treatment and metaphylaxis of Enzootic Pneumonia (EP) in pigs. In all four trials, pigs in the tylvalosin group were administered 10 mg tylvalosin/kg bodyweight in drinking water daily for 5 consecutive days (TVN). In a single-challenge study, pigs were inoculated with lung homogenate containing Mycoplasma hyopneumoniae. In a dual challenge study, pigs were sequentially inoculated with pure culture of M. hyopneumoniae and Pasteurella multocida. Efficacy was evaluated based on reduction of lung lesions compared to unmedicated control pigs (CTL). In two field studies at European commercial farms with confirmed outbreaks of EP, treatment efficacy in clinically affected fatteners was evaluated based on improved clinical conditions compared to pigs treated with tylosin at 10 mg/kg by injection for 3 consecutive days (TYL). In these field trials, healthy in contact pigs were enrolled for metaphylaxis efficacy evaluation based on reduction in incidence of new clinical cases of respiratory disease compared to unmedicated pigs (CTL). RESULTS: In the M. hyopneumoniae-only challenge study, pigs in TVN group had lower lung lesion scores than CTL (6.52 vs. 14.97; p <  0.001). In the dual challenge study with M. hyopneumoniae and P. multocida, pigs in TVN group had lower lung lesion scores than CTL (3.32 vs. 8.37; p <  0.01) and the recovery of both challenge bacteria from the lungs was lower in TVN compared with CTL group (p <  0.01). In field outbreaks of EP, multicentre analysis showed that 13 days after the start of medication, treatment success for TVN pigs was significantly better than for TYL pigs (80.0% vs 48.7% p = 0.03) and metaphylactic administration of TVN significantly reduced the incidence of new clinical cases (2.1% vs. 7.8%; p <  0.01) compared with unmedicated controls. CONCLUSIONS: Tylvalosin at 10 mg/kg daily for 5 days in drinking water was safe and effective in the treatment and metaphylaxis of EP in pigs associated with infections of M. hyopneumoniae either alone or in combination with P. multocida under both experimental challenge and field natural infection conditions.

Population medicine and control of epidemics.

Population medicine is an important component of veterinary care in livestock (farm animals) and companion animals (pets). This chapter covers some of the chemotherapeutic approaches undertaken at population level to control infectious diseases in domestic animals. Optimisation of health, productivity and welfare in livestock commonly entails implementation of whole-herd or whole-flock strategies to effectively counter the negative impact of infectious diseases. Gastro-intestinal and liver parasites of grazing cattle and sheep are endemic in most parts of the world and can result in significant production losses. Strategically timed anthelmintic treatments are instituted with the double objective of reducing worm burdens in infected animals and ensuring reduction of pasture contamination with infective larvae. Mastitis is another major endemic problem, particularly in cattle, which causes significant economic losses to dairy farmers globally. As a painful inflammatory condition of the cow's udder, clinical mastitis also raises animal welfare concerns. Prevention of clinical mastitis requires rigorous post-milking hygiene, identification and culling of chronically infected cows, attention to the cow's environment and therapeutic management of udder health during the dry period. A third condition that can cause high levels of morbidity and mortality is bacterial respiratory disease. Pneumonia in young livestock is often exacerbated by stressful transportation and co-mingling of animals from different herds. The welfare consequences and production losses can be significant. Antimicrobial treatment of pneumonic animals and, when appropriate, of in-contact animals living in the same air-space is an integral part of whole-herd respiratory disease management. The role of the veterinary profession is to also ensure that principles of population medicine are understood and adhered to by pet owners. The increase in pet ownership and the importance of the human-animal bond in modern developed societies give rise to zoonotic risks, which require vigilance and intervention. Regular internal parasite control in dogs and cats, particularly in endemic areas, contributes to animal welfare and minimises public health hazards.

Minimum inhibitory concentrations of tulathromycin against respiratory bacterial pathogens isolated from clinical cases in European cattle and swine and variability arising from changes in in vitro methodology.

The in vitro activity of tulathromycin was evaluated against common bovine and porcine respiratory pathogens collected from outbreaks of clinical disease across eight European countries from 1998 to 2001. Minimum inhibitory concentrations (MICs) for one isolate of each bacterial species from each outbreak were determined using a broth microdilution technique. The lowest concentrations inhibiting the growth of 90% of isolates (MIC90) for tulathromycin were 2 microg/ml for Mannheimia (Pasteurella) haemolytica, 1 microg/ml for Pasteurella multocida (bovine), and 2 microg/ml for Pasteurella multocida (porcine) and ranged from 0.5 to 4 microg/ml for Histophilus somni (Haemophilus somnus) and from 4 to 16 microg/ml for Actinobacillus pleuropneumoniae. Isolates were retested in the presence of serum. The activity of tulathromycin against fastidious organisms was affected by culture conditions, and MICs were reduced in the presence of serum.

Evaluation of the therapeutic activity of tulathromycin against swine respiratory disease on farms in Europe.

The clinical efficacy of tulathromycin in the treatment of natural outbreaks of swine respiratory disease (SRD) was evaluated at five European sites. Pigs (1 to 6 months of age) exhibiting clinical signs of SRD were treated intramuscularly with tulathromycin (n = 247) at 2.5 mg/kg on day 0 versus either tiamulin (n = 102) at 15 mg/kg on days 0, 1, and 2 (Germany, the Netherlands, and the United Kingdom) or florfenicol (n = 20) at 15 mg/kg on days 0 and 2 (France). Actinobacillus pleuropneumoniae, Pasteurella multocida, and Mycoplasma hyopneumoniae infections were the most frequently diagnosed pathogens. For both tulathromycin-treated animals and those treated with tiamulin or florfenicol, there were significant (P = .0001) reductions in mean rectal temperature and the severity of abnormal clinical signs on days 2 and 10 compared with day 0. There were no significant differences (P > .05) between treatments in average daily weight gain. Tulathromycin was found to be safe and highly effective in the treatment of natural outbreaks of SRD.

Pharmacokinetics and lung tissue concentrations of tulathromycin, a new triamilide antibiotic, in cattle.

The pharmacokinetics of the new triamilide antibiotic tulathromycin was investigated in two cattle studies. Following a single subcutaneous injection, the drug was rapidly absorbed and bioavailability was excellent. High and persistent levels of the drug in lung tissue were observed as well. These attributes are advantageous for an antimicrobial drug indicated for the treatment of bacterial and mycoplasmal respiratory diseases in cattle.

An analytical method for the analysis of tulathromycin, an equilibrating triamilide, in bovine and porcine plasma and lung.

Tulathromycin is a novel member of the triamilide class of antibiotics that was developed as a safe and effective single-dose treatment of bovine and porcine respiratory disease. An accurate and precise analytical method was developed for the extraction and measurement of tulathromycin in livestock plasma and lung homogenates. Analytes were solid-phase extracted onto a weak cation exchanger after aqueous dilution of samples and addition of heptadeutero-tulathromycin as an internal standard. Following HPLC with a narrow bore C8 column, quantitative detection of tulathromycin was accomplished by monitoring the transition of a doubly charged precursor ion to a singly charged product ion by tandem mass spectrometry using a triple quadrupole mass spectrometer. Procedures were validated for a dynamic range of 0.1 to 25 ng on column. Observed accuracies were between 90 and 110% of nominal and precision (RSD) varying 7% or less. Response and stability experiments showed that deuterated tulathromycin did not parallel the chemical behavior of tulathromycin. Applicability of the method to livestock studies was tested with plasma and lung samples from cattle and swine dosed with tulathromycin at multiple doses. The results demonstrated that the analytical method performed well in a range of sample concentrations spanning over 3 orders of magnitude and provided dose-exposure relationships for cattle and swine.