Toxoplasma gondii is not an important contributor to poor reproductive performance of primiparous ewes from southern Australia: a prospective cohort study.

BACKGROUND: Toxoplasma gondii causes reproductive losses in sheep worldwide, including Australia. The reproductive performance of primiparous ewes is typically lower than for mature, multiparous ewes, and younger ewes are more likely to be immunologically naïve and therefore more susceptible to reproductive disease if T. gondii infection occurs during pregnancy. The aim of this study was to assess the impact of infection with T. gondii on the reproductive performance of primiparous ewes in southern Australia using a prospective cohort study. This will inform the need for targeted control strategies for T. gondii in Australian sheep. RESULTS: Toxoplasma gondii seropositivity using indirect ELISA was detected at 16/28 farms located across southern Australia. Apparent seropositivity to T. gondii was lower in primiparous ewes (1.1, 95% confidence interval (CI) 0.6, 1.8) compared to mature, multiparous ewes (8.1, 95% CI 6.0, 10.5; P < 0.001). Toxoplasma gondii seroconversion during the gestation and lambing period was confirmed for 11/1097 (1.0, 95% CI 0.5, 1.7) of pregnant primiparous ewes that failed to raise a lamb, and 1/161 (0.6, 95% CI 0.1, 2.9) primiparous ewes with confirmed mid-pregnancy abortion. CONCLUSIONS: Low frequency of detection of T. gondii seroconversion during gestation and low frequency of seropositivity to T. gondii suggests that toxoplasmosis was not an important contributor to reproductive losses in primiparous ewes on farms located over a wide geographical area in southern Australia.

Seropositivity to Coxiella burnetii in primiparous and multiparous ewes from southern Australia: A cross-sectional study.

The role of infectious diseases including coxiellosis in causing poorer reproductive performance of primiparous ewes are not well studied. The aims of this study were to determine if natural exposure to Coxiella burnetii is widespread in breeding ewes and whether seropositivity is associated with poor reproductive performance of primiparous ewes. Seropositivity to Coxiella burnetii was 0.08% (CI95% 0.01, 0.36) in primiparous ewes and 0.36% (CI95% 0.07, 1.14) in mature ewes. Coxiella burnetii was not detected in aborted or stillborn lambs using qPCR. These findings suggest C. burnetii infection was unlikely to be an important contributor to abortion and perinatal mortalities observed for primiparous ewe flocks, and exposure to C. burnetii was not widespread in ewes on farms located over wide geographical region of southern Australia. Whilst ewes on these farms were not an important reservoir for C. burnetii, sporadic zoonotic transmission from sheep is reported and has public health implications.

Neospora caninum is not an important contributor to poor reproductive performance of primiparous ewes from southern Australia: evidence from a cross-sectional study.

Neospora caninum has been implicated as a sporadic cause of abortion and perinatal deaths in sheep flocks globally. However, its significance as a reproductive pathogen for sheep in Australia remains unknown. The aims of this study were to (i) determine the seroprevalence of N. caninum in Australian breeding ewes and (ii) examine if natural exposure to N. caninum is associated with poor reproductive performance of primiparous ewes in southern Australia. Thirty flocks of primiparous ewes (aged 1-2 years old at lambing) from 28 farms in three states (Western Australia, South Australia and Victoria) were monitored between mating and lamb marking. Blood samples were also collected from multiparous mature ewes (aged 3 years or older) at each farm. Seroprevalence for anti-N. caninum IgG using indirect ELISA was determined for a subset of primiparous ewes that were predominantly determined to be pregnant and subsequently failed to rear a lamb (n = 1279) and randomly selected mature multiparous ewes with unknown reproductive status (n = 558). Neopsora caninum apparent seroprevalence was 0.16% (95% confidence interval 0.03%, 0.5%) in primiparous ewes, with seropositivity identified in two ewes from farms located in South Australia and Victoria. There was no evidence of seropositivity in mature ewes with apparent seroprevalence 0% (0%, 0.45%). These findings suggest that N. caninum infection was not widespread in primiparous ewes or mature multiparous ewes on these farms, and exposure to N. caninum infection was unlikely to explain abortion and perinatal mortalities observed for primiparous ewes.

Abortion and Lamb Mortality between Pregnancy Scanning and Lamb Marking for Maiden Ewes in Southern Australia.

The contribution of abortions to the overall mortality of lambs born to maiden (primiparous) ewes in Australia remains unclear. This cohort study aimed to quantify abortion and lamb mortality for ewe lambs and maiden Merino two-tooth ewes. Lamb mortality from pregnancy scanning to marking were determined for 19 ewe lamb and 11 Merino two-tooth ewe flocks across southern Australia. Average lamb mortality from scanning to marking was 35.8% (range 14.3-71.1%) for the ewe lambs and 29.4% (range 19.7-52.7%) for the two-tooth ewes. Mid-pregnancy abortion was detected in 5.7% of ewes (range 0-50%) in the ewe lamb flocks and 0.9% of ewes (range 0-4.4%) in the two-tooth ewe flocks. Mid-pregnancy abortion affecting ≥2% of ewes was observed in 6/19 ewe lamb flocks and 2/11 two-tooth ewe flocks. Lamb mortality from birth to marking represented the greatest contributor to foetal and lamb mortality after scanning, but mid-pregnancy abortion was an important contributor to lamb mortality in some ewe lamb flocks. Variability between the flocks indicates scope to improve the overall reproductive performance for maiden ewes by reducing foetal and lamb losses. Addressing mid-pregnancy abortion may improve the reproductive performance in some flocks.

Late therapeutic intervention with a respiratory syncytial virus L-protein polymerase inhibitor, PC786, on respiratory syncytial virus infection in human airway epithelium.

BACKGROUND AND PURPOSE: Effective anti-respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium. EXPERIMENTAL APPROACH: Air liquid interface-cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS-8112, an anti-RSV nucleoside analogue, and GS-5806, a fusion-protein inhibitor, which were treated basolaterally. KEY RESULTS: Late intervention with GS-5806 did not show significant anti-viral effects, but PC786 produced potent, concentration-dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS-8112. PC786 showed inhibitory activities against RSV-induced increases of CCL5, IL-6, double-strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium. CONCLUSION AND IMPLICATIONS: Late therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.

Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC50], <0.09 to 0.71 nM) and RSV-B (IC50, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC50, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus.

The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use.

Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness.

BACKGROUND: Resistance to the neuraminidase inhibitor oseltamivir can be conferred by a well-characterized mutation in the neuraminidase gene, H275Y. In human H1N1 viruses that circulated in the first years of the 21st century, this mutation carried a fitness cost and resistant viruses were rare. During the 2007-08 influenza season, oseltamivir-resistant viruses of H1N1 phenotype emerged and predominated. March 2009 saw the emergence of a novel H1N1 influenza pandemic. We examined whether the H275Y mutation affected neuraminidase enzyme activity or replication of the pandemic influenza virus. METHODS: Using reverse genetics we engineered the H275Y mutation into the neuraminidase of a 2009 pandemic H1N1 virus and assessed the ability of this enzyme to desialylate mono- and multivalent substrates. The growth kinetics of wild-type and mutant viruses were assessed in Madin-Darby canine kidney (MDCK) and fully differentiated human airway epithelial (HAE) cells. RESULTS: The presence of H275Y was associated with a 1.3-fold decrease in the affinity of the neuraminidase for a monovalent substrate and a 4-fold compromise in desialylation of multivalent substrate. This was associated with a fitness cost to viral replication in vitro, which only became apparent during competitive replication in the mucus-rich HAE culture system. CONCLUSIONS: The neuraminidase protein of pandemic influenza isolates tolerates the H275Y mutation and this mutation confers resistance to oseltamivir. However, unlike seasonal H1N1 viruses isolated since 2007, the mutation is not associated with any fitness advantage and thus is unlikely to predominate without further antigenic drift, compensating mutations or intense selection pressure.