Transplacental transmission of Theileria orientalis occurs at a low rate in field-affected cattle: infection in utero does not appear to be a major cause of abortion.

BACKGROUND: Bovine theileriosis, caused by the haemoprotozoan Theileria orientalis, is an emerging disease in East Asia and Australasia. Previous studies have demonstrated transplacental transmission of various Theileria spp. but molecular confirmation of transplacental transmission of T. orientalis has never been confirmed in the field. In this study, cow-calf (< 48 h old) pairs were sampled across 3 herds; opportunistic samples from aborted foetuses or stillborn calves were also examined. Molecular (multiplex qPCR) and serological (ELISA) methods were used to determine infection prevalence and the presence of anti-Theileria antibodies in each herd. In addition, pregnant heifers and foetal calves were sampled at abattoir and tested for the presence of T. orientalis by qPCR. RESULTS: The qPCR results indicated that, even though there was a high prevalence of T. orientalis infection in cows, the rate of transplacental transmission to their calves was low, with only one newborn calf from one herd and one foetus from the abattoir testing positive for T. orientalis DNA. Five aborted foetuses and stillborn calves, 3 of which were derived from a herd experiencing a high number of clinical theileriosis cases at the time of sampling, all tested negative for T. orientalis by qPCR. This suggests that in utero infection of calves with T. orientalis may not be a major driver of abortions during theileriosis outbreaks. Temporal monitoring of 20 calves born to T. orientalis-positive mothers indicated that T. orientalis was detectable in most calves between 10 and 27 days post-partum, consistent with prior field studies on adult cattle introduced to Theileria-affected herds. There was a positive correlation between the ELISA ratio of newborn calves and their mothers within 48 h of calving; however, maternal antibodies were only detectable in some calves and only for 4-4.5 weeks post-partum. All calves displayed high parasite loads peaking at 4-8 weeks post-partum, with only some calves subsequently mounting a detectable adaptive antibody response. CONCLUSIONS: These findings indicate transplacental transmission of T. orientalis appears to play only a minor role in persistence of T. orientalis infection in the field; however calves are highly susceptible to developing high level T. orientalis infections at 4-8 weeks of age regardless of whether maternal antibodies are present post-partum.

Longitudinal study of winter mortality disease in Sydney rock oysters Saccostrea glomerata.

Winter mortality (WM) is a poorly studied disease affecting Sydney rock oysters Saccostrea glomerata in estuaries in New South Wales, Australia, where it can cause significant losses. WM is more severe in oysters cultured deeper in the water column and appears linked to higher salinities. Current dogma is that WM is caused by the microcell parasite Bonamia roughleyi, but evidence linking clinical signs and histopathology to molecular data identifying bonamiasis is lacking. We conducted a longitudinal study between February and November 2010 in 2 estuaries where WM has occurred (Georges and Shoalhaven Rivers). Results from molecular testing of experimental oysters for Bonamia spp. were compared to clinical disease signs and histopathology. Available environmental data from the study sites were also collated and compared. Oyster condition declined over the study period, coinciding with decreasing water temperatures, and was inversely correlated with the presence of histological lesions. While mortalities occurred in both estuaries, only oysters from the Georges River study site showed gross clinical signs and histological changes characteristic of WM (lesions were prevalent and intralesional microcell-like structures were sometimes noted). PCR testing for Bonamia spp. revealed the presence of an organism belonging to the B. exitiosa-B. roughleyi clade in some samples; however, the very low prevalence of this organism relative to histological changes and the lack of reactivity of affected oysters in subsequent in situ hybridisation experiments led us to conclude that this Bonamia sp. is not responsible for WM. Another aetiological agent and a confluence of environmental factors are a more likely explanation for the disease.

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs.

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel(®) or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n=10 per group) or Suvaxyn(®) M. hyo (n=12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n=12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn(®) M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3P216, but not against the remaining vaccine subunit antigens. Alhydrogel(®) and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn(®) M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn(®) M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1ß, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn(®) M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia.

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.

Plasmin activity in the porcine airways is enhanced during experimental infection with Mycoplasma hyopneumoniae, is positively correlated with proinflammatory cytokine levels and is ameliorated by vaccination.

In Mycoplasma hyopneumoniae (Mhp) infection of swine, the host immune response is considered a major driver of lung pathology; however the underlying inflammatory mechanisms are not well understood. The serine protease plasmin is being increasingly recognised as a significant player in inflammatory processes. Here we compare plasmin activity in tracheobronchial lavage fluid (TBLF) from pigs experimentally challenged with Mhp that were either unvaccinated (n=10), or vaccinated with the commercial vaccine Suvaxyn(®) M.hyo (n=10). TBLF collected immediately prior to challenge and at 21 d and 35 d post-challenge was also assayed for levels of proinflammatory cytokines (TNF-α, IL-1ß and IL-6), and for bacterial load (by qPCR). Clinical signs, pathology, cytokine analyses and qPCR all indicated that vaccinated pigs had significantly reduced disease relative to unvaccinated animals. Plasmin activity increased significantly in TBLF collected at 21 d post-challenge compared to pre-challenge TBLF in unvaccinated (P<0.01), but not vaccinated animals (P>0.05). A significant correlation was observed between bacterial load and plasmin activity in the 21 d (r=0.66; P<0.01) and the 35 d post-challenge samples, (r=0.62; P<0.01). Plasmin activity was also significantly correlated with levels of TNF-α, IL-1ß and IL-6 at 21 d (r=0.78, P<0.0001; r=0.77, P<0.0001; r=0.64, P<0.005) and with TNF-α and IL-1ß at 35 d post-challenge (r=0.77, P<0.0001; r=0.74, P<0.0005). Our results indicate that plasminogen is activated to plasmin in the respiratory tract of pigs as part of the host inflammatory response to Mhp infection and that this effect is ameliorated by vaccination.

Evaluation of clinical, histological and immunological changes and qPCR detection of Mycoplasma hyopneumoniae in tissues during the early stages of mycoplasmal pneumonia in pigs after experimental challenge with two field isolates.

Differences in Mycoplasma hyopneumoniae strain virulence and infection patterns will affect experimental challenge systems used to evaluate vaccine efficacy. Two strains (Hillcrest and Beaufort) were assessed by experimental pig challenge for their ability to induce clinical and pathological lesions and cytokine responses. Tracheobronchial lavage fluid (TBLF) was collected before and 17-18 days after challenge with Hillcrest (n=8), Beaufort (n=8) or no organisms (n=3). Coughing was assessed twice daily, and at slaughter 21 (n=9) or 28 (n=10) days post-challenge, gross and histopathology of lungs were quantified and a quantitative PCR (mhp183 qPCR) was applied to detect M. hyopneumoniae DNA in tissues and TBLF. Hillcrest was clearly superior to Beaufort in its ability to induce coughing and pneumonic lesions. At 17-18 days, interleukin (IL)-1ß and IL-6 concentrations in TBLF were only significantly higher (8.7 and 5.1 fold respectively) than controls (P<0.001) in Hillcrest-challenged pigs. Lungs of all Hillcrest-challenged pigs were qPCR positive at either slaughter date, but only at day 28 in Beaufort-challenged pigs. M. hyopneumoniae DNA was highest in concentration in lungs 21 days after Hillcrest challenge, and was detected in the spleen, kidney and/or liver of Hillcrest-challenged pigs, but not in Beaufort pigs. While M. hyopneumoniae DNA concentration in TBLF was elevated following Hillcrest and Beaufort challenge, there was no significant difference in mean mycoplasmal DNA concentration detected in TBLF from pigs challenged with either isolate (P>0.05). Thus a suitable challenge strain, coupled with lung pathology and cytokine assays, are valuable in assessing post-challenge responses. Assessment of M. hyopneumoniae DNA in lung and abdominal tissues by mhp183 qPCR, in conjunction with histopathology, were valuable in confirming M. hyopneumoniae infection.

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle.

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.