Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.).

Herpesviruses have been detected in bat species from several countries, with a limited number of studies examining herpesviruses in Pteropus spp. (flying foxes) and no investigation of herpesviruses in Australian flying foxes. We examined the presence and prevalence of herpesviruses in the four mainland Australian flying fox species. A nested PCR targeting highly conserved amino acid motifs in the DNA polymerase (DPOL) gene of herpesviruses was used to analyze 564 samples collected from 514 individual Pteropus scapulatus, Pteropus poliocephalus, Pteropus alecto, and Pteropus conspicillatus. The prevalence of herpesvirus DNA in blood, urine, oral, and fecal swabs from the four species was 17% in P. scapulatus, 11% in P. poliocephalus, 10% in P. alecto, and 9% in P. conspicillatus (31% in P. conspicillatus spleen tissue). Five putative novel herpesviruses were detected. Following PCR amplicon sequence analysis, four of the herpesviruses grouped phylogenetically with the gammaherpesviruses, with nucleotide identities between 79% and 90% to gammaherpesviruses from Asian megabats. A betaherpesvirus was detected in P. scapulatus with 99% nucleotide identity to the partial DPOL gene sequence of an Indonesian fruit bat betaherpesvirus. This study lays the foundation for future epidemiology research of herpesviruses in Australian Pteropus spp. and adds to the discussion of hypotheses surrounding the evolutionary epidemiology of bat-borne viruses on a global scale.

Cleft Palate Syndrome in the Endangered Spectacled Flying Fox (Pteropus conspicillatus): Implications for Conservation and Comparative Research.

Cleft palate syndrome, first observed in the spectacled flying fox population in 1998, has produced sporadic neonatal mortality events over the past two decades, with an estimated incidence of up to 1/1000 births per year. This study presents a rudimentary characterisation of the syndrome, presenting gross pathology of syndromic signs upon visual inspection, a histological examination of palate malformations, and syndrome incidence data representing the past two decades. The syndrome presents with a range of signs, primarily congenital palate malformations ranging from a pinhole cleft to a complete hard and soft palate deficit, resulting in the death or abandonment of neonates shortly after birth. The congenital palate malformations are often associated with claw deformities, wiry facial hair, and in some instances, muscle weakness and neurological signs. The natural occurrence of the lethal congenital orofacial birth defects in the spectacled flying fox presents a unique opportunity for the investigation of putative aetiologies, drawing parallels between bat and other mammalian cleft palate risk factors. Further syndrome investigation has the potential to deliver both biodiversity conservation and comparative veterinary and biomedical outcomes.

Genome Reference Assembly for Bottlenecked Southern Australian Koalas.

Koala populations show marked differences in inbreeding levels and in the presence or absence of the endogenous Koala retrovirus (KoRV). These genetic differences among populations may lead to severe disease impacts threatening koala population viability. In addition, the recent colonization of the koala genome by KoRV provides a unique opportunity to study the process of retroviral adaptation to vertebrate genomes and the impact this has on speciation, genome structure, and function. The genome build described here is from an animal from the bottlenecked Southern population free of endogenous and exogenous KoRV. It provides a more contiguous genome build than the previous koala reference derived from an animal from a more outbred Northern population and is the first koala genome from a KoRV polymerase-free animal.

Canine parvovirus is shed infrequently by cats without diarrhoea in multi-cat environments.

Whether subclinical shedding of canine parvovirus (CPV) by cats might contribute to the epidemiology of canine CPV infections, particularly in facilities housing both cats and dogs, requires clarification. Conflicting results are reported to date. Using conventional PCR (cPCR) to amplify the VP2 gene, shedding of the CPV variants (CPV-2a, 2b, 2c) by healthy cats in multi-cat environments was reportedly common in Europe but rare in Australia. The aim of this study was to determine whether low-level faecal CPV shedding occurs in multi-cat environments in Australia and Italy using a TaqMan real-time PCR to detect Carnivore protoparvovirus 1 (CPV and feline parvovirus, FPV) DNA, and minor-groove binder probe real-time PCR assay to differentiate FPV and CPV types and to characterize CPV variants. In total, 741 non-diarrhoeic faecal samples from shelters in Australia (n = 263) and from shelters or cat colonies in Italy (n = 478) were tested. Overall, Carnivore protoparvovirus 1 DNA was detected in 49 of 741 (6.61 %) samples. Differentiation was possible for 31 positive samples. FPV was most common among positive samples (28/31, 90.3 %). CPV was detected in 4/31 samples (12.9 %) including CPV-2a in one sample, CPV-2b in another and co-infections of FPV/CPV-2b and CPV-2a/CPV-2b in the remaining two samples. A high rate of subclinical FPV infection was detected in one shelter during an outbreak of feline panleukopenia, during which 21 of 22 asymptomatic cats (95.5 %) sampled were shedding FPV. Faecal shedding of CPV by cats in multi-cat environments is uncommon suggesting that domestic cats are not significant reservoirs of CPV.

Transport of Moving Duck Flocks in Indonesia and Vietnam: Management Practices That Potentially Impact Avian Pathogen Dissemination.

Highly pathogenic avian influenza (HPAI) virus is endemic in Indonesia and Vietnam, where "moving" duck production is commonly practiced. Questionnaire surveys were conducted with transporters of "moving" duck flocks in Indonesia (N = 55) and Vietnam (N = 43). The main purpose of transportation was to transport duck flocks between rice paddies used for scavenging. Trucks were commonly utilized for transport in both countries (Indonesia: 98.2%, 54/55; Vietnam: 37.2%, 16/43), while boats were only used in Vietnam (62.8%, 27/43). Transporters in Vietnam moved larger flocks and traveled over longer distances. Deaths of ducks due to diseases were reported in both countries (Indonesia: 16.4%, 9/55; Vietnam: 4.7%, 2/43; p = 0.11). Throwing away of carcasses was the primary method of disposal of dead birds in Indonesia (60.0%, 33/55), but was not practiced in Vietnam (p < 0.001), while more transporters in Vietnam (34.9%, 15/43) buried carcasses compared to Indonesia (6.8%, 4/55; p = 0.001). Consumption of carcasses (20.9%, 9/43), sale of dead ducks (14.0%, 6/43) and processing of ducks for fish feed (9.3%, 4/43) was conducted in Vietnam, but not in Indonesia. Vehicles were predominantly cleaned in rivers and stored outside in Vietnam, while cleaning and storage was usually conducted in houses/garages in Indonesia. In conclusion, we identified management practices that potentially impact transmission of avian pathogens, such as HPAI virus. In Indonesia, unsafe management practices were related to multipurpose usage of transport vehicles and disposal of birds in the environment, while in Vietnam, they were related to the mixing of birds during transport, the processing of dead carcasses and the storage and cleaning of transport vehicles.

Detection of porcine circovirus type 2 (PCV2) in the Philippines and the complexity of PCV2-associated disease diagnosis.

Porcine circovirus type 2 (PCV2), an important pig viral pathogen, can cause porcine circovirus-associated disease (PCVAD), resulting in economic losses associated with decreased growth and mortalities. The diagnosis of PCVAD is complex requiring clinical, pathological and virological approaches. This study assessed PCV2 infection using histopathology and immunohistochemistry (IHC) on tissue samples and quantitative polymerase chain reaction (qPCR) on serum samples from 47 grower-finisher pigs allocated in three clinical groups in the Philippines. Typical PCV2 histopathological lesions were observed in mediastinal lymph nodes (MLN) of eight of 47 pigs. Lymphoid depletion was seen in all eight pigs and granulomatous inflammation in one of these pigs. Four of these eight pigs were PCV2 positive by both IHC and qPCR. IHC revealed PCV2 antigen in 8 pigs in at least one of the following tissues: MLN (5/8), spleen (3/8), tonsils (4/8) and lungs (5/8). PCV2 antigen was observed in 3/8 MLN with lymphoid depletion and in one MLN with depletion and granulomatous inflammation. The qPCR test showed that 33 sera had a non-detectable level, twelve had < 106 and two had > 106 PCV2 DNA copies/ml serum. One pig with lymphoid depletion had > 106 PCV2 DNA copies/ml serum, and another pig without MLN lesions also had > 106 PCV2 DNA copies/ml serum. These findings suggest that PCVAD is present in the Philippines and confirm the challenges of PCVAD diagnosis as different patterns of results were obtained from the different tests.

Development of a Luminex microbead-based serotyping assay for Glaesserella parasuis.

Glaesserella parasuis consists of 15 serovars with some of them highly virulent and some of them avirulent. As killed vaccines do not provide crossprotection across serovars, serotyping is of importance. Serotyping, previously done by gel diffusion, is now done by multiplex PCR followed by electrophoresis. Accurately differentiating 15 serovars by electrophoresis is problematic. To overcome this problem, a Luminex microbead-based multiplex assay was used to differentiate the serovars. The assay consisted of a multiplex PCR assay followed by hybridisation to microbeads which were then analysed on a Luminex machine. The newly developed assay was compared to the multiplex serotyping PCR and the gel diffusion/indirect haemagglutination assay (GD/IHA). The microbead-based assay worked very well for the 15 reference strains but when used on the 74 Australian field strains displayed some problems. The main problems were with the eight out of nine serovar 4 field isolates and the five serovar 7 and three serovar 14 field isolates. While the microbead-based assay could differentiate between the serovar 5 and 12 reference strains, which the serovar multiplex PCR could not, all four field isolates identified by GD/IHA as serovar 12 were identified as serovar 5 by the microbead-based assay. Serovar 4 has been noted to have a high diversity especially among strains from different countries. Our work clearly shows that the diversity of strains at both the national and the international level has to be taken into account when developing diagnostic assays.

Analysis of canine parvoviruses circulating in Australia reveals predominance of variant 2b and identifies feline parvovirus-like mutations in the capsid proteins.

Canine parvovirus (CPV) is a major enteric pathogen of dogs worldwide that emerged in the late 1970s from a feline parvovirus (FPV)-like ancestral virus. Shortly after its emergence, variant CPVs acquired amino acid (aa) mutations in key capsid residues, associated with biological and/or antigenic changes. This study aimed to identify and analyse CPV variants and their capsid mutations amongst Australian dogs, to gain insights into the evolution of CPV in Australia and to investigate relationships between the disease and vaccination status of dogs from which viruses were detected. CPV VP2 sequences were amplified from 79 faecal samples collected from dogs with parvoviral enteritis at 20 veterinary practices in five Australian states. The median age at diagnosis was 4 months (range 1-96 months). Only 3.7% of dogs with vaccination histories had completed recommended vaccination schedules, while 49% were incompletely vaccinated and 47.2% were unvaccinated. For the first time, CPV-2b has emerged as the dominant antigenic CPV variant circulating in dogs with parvoviral enteritis in Australia, comprising 54.4% of viruses, while CPV-2a and CPV-2 comprised 43.1% and 2.5%, respectively. The antigenic variant CPV-2c was not identified. Analysis of translated VP2 sequences revealed a vast repertoire of amino acid (aa) mutations. Several Australian CPV strains displayed signatures in the VP2 protein typical of Asian CPVs, suggesting possible introduction of CPV strains from Asia, and/or CPV circulation between Asia and Australia. Canine parvoviruses were identified containing aa residues typical of FPV at key capsid (VP2) positions, representing reverse mutations or residual mutations retained from CPV-2 during adaptation from an FPV-like ancestor, suggesting that evolutionary intermediates between CPV-2 and FPV are circulating in the field. Similarly, intermediates between CPV-2a-like viruses and CPV-2 were also identified. These findings help inform a better understanding of the evolution of CPV in dogs.

Latent class analysis identifies multimorbidity patterns in pigs with respiratory disease.

Respiratory disease is one of the major causes of losses to the pig industry worldwide. The pig subsector is the largest component of the livestock sector in the Philippines. Using lung scoring, this study aimed to estimate the prevalence of thoracic lesions in slaughter-age pigs in two provinces in the Philippines (Batangas and Albay) and define classes for respiratory health of pigs characterised by different patterns of thoracic lesions. A total of 260 pigs from Batangas and 300 pigs from Albay from either commercial or backyard farm types were included in this cross-sectional study. Lungs were scored for cranio-ventral pneumonia (0-55) and pleurisy (0-3). Presence or absence of pericarditis as well as focal dorso-caudal pneumonia were recorded. Latent class analyses considering four indicator variables, and province and farm type as covariates were used to explore different patterns of thoracic lesions across the study populations. Using a threshold of ≥7, the prevalence of a high lung score was 51.9% (95% confidence interval [CI]: 42.3-61.4%) and 13.7% (95% CI: 8.1-22.2%) in Batangas and Albay, respectively. Similarly, the prevalence of a pleurisy score of ≥1 was 56.9% (95% CI: 37.5-74.4%) and 5.0% (95% CI: 2.9-8.4%), pericarditis 24.6% (95%CI: 10.1-48.6%) and 1.7% (95%CI: 0.3-6.7%) and focal dorso-caudal pneumonia lesions 7.7% (95% CI: 3.7-15.5%) and 0% (97.5% one-sided CI: 0-1.2%), respectively. Latent class analyses identified four classes based on lung score, pleurisy score and the presence/absence of pericarditis: "healthy", "mild respiratory disease", "moderate pneumonia", and "multi-lesion". The relative frequency of these classes differed with province and farm type. Most pigs from Albay were "healthy", whereas in Batangas most pigs from commercial farms were "multi-lesion" and those from backyard farms were in the "mild respiratory disease" class. This study has provided baseline data on thoracic lesions in slaughter-age pigs for the provinces of Batangas and Albay in the Philippines. Targeting farms and areas where "multi-lesion pigs" are most common and further research to identify risk factors for particular classes should maximize impact of future control measures. The latent class analysis approach used could be applied more widely and could add value to analysis of multi-morbidity data collected routinely as part of ongoing monitoring schemes.

An unprecedented cluster of Australian bat lyssavirus in Pteropus conspicillatus indicates pre-flight flying fox pups are at risk of mass infection.

In November 2017, two groups of P. conspicillatus pups from separate locations in Far North Queensland presented with neurological signs consistent with Australian bat lyssavirus (ABLV) infection. These pups (n = 11) died over an 11-day period and were submitted to a government laboratory for testing where ABLV infection was confirmed. Over the next several weeks, additional ABLV cases in flying foxes in Queensland were also detected. Brain tissue from ABLV-infected flying foxes during this period, as well as archived brain tissue, was selected for next-generation sequencing. Phylogenetic analysis suggests that the two groups of pups were each infected from single sources. They were likely exposed while in crèche at night as their dams foraged. This study identifies crèche-age pups at a potentially heightened risk for mass ABLV infection.

Combining conventional and participatory approaches to identify and prioritise management and health-related constraints to smallholder pig production in San Simon, Pampanga, Philippines.

Pork is the main meat produced and consumed in the Philippines. The majority of pigs are raised by smallholders who experience a range of constraints to their pig production. This study presents the findings of the first part of an overarching project that used an Ecohealth approach and aimed to improve the production and competitiveness of the smallholder pig system in an area of the Philippines. A participatory approach was embraced, combining conventional and participatory epidemiology methods followed by a stakeholder discussion. The first aim was to identify management and health-related constraints to pig production among smallholder famers in San Simon, Pampanga, Philippines. The second aim was for the project team and stakeholders to jointly prioritise activities for the immediate future to address these constraints. Key management and health-related constraints identified included inadequate water supply to pigs, particularly lactating and gestating sows, and a range of feeding-related issues. Diarrhoea was recognised as the disease syndrome of highest priority and limited record keeping meant that farmers were unable to assess the productivity and profitability of their pig farming enterprises. Actions jointly prioritised by stakeholders and the project team were: the appointment of a project coordinator within each barangay; conduct two sets of seminars, the first covering water and nutrition and the second piglet management and diarrhoea, to be delivered by technical experts but with farmer "trusted sources" also sharing their experiences; development of easily understandable leaflets and posters covering key technical information; promotion of nipple drinkers attached to five-gallon water containers and creep boxes for piglets, and conduct of a record keeping workshop with a small group of innovative farmers to develop a useful and usable tool for record keeping. The use of multiple approaches to data-gathering enabled triangulation of study findings. Without any one of these components the understanding of the pig production system would have been less complete and it is possible that the proposed actions would not have been as well-tailored to the needs of the farmers. The participatory approach, in particular the stakeholder discussion, provided the opportunity to embrace the "deciding together" and "acting together" stances of participation rather than the lower "information giving" stance, thereby giving stakeholders greater ownership of the future activities of the overarching project and beyond.

Novel insights into viral infection and oncogenesis from koala retrovirus (KoRV) infection of HEK293T cells.

Koala retrovirus is thought to be an underlying cause of high levels of neoplasia and immunosuppression in koalas. While epidemiology studies suggest a strong link between KoRV and disease it has been difficult to prove causality because of the complex nature of the virus, which exists in both endogenous and exogenous forms. It has been difficult to identify koalas completely free of KoRV, and infection studies in koalas or koala cells are fraught with ethical and technical difficulties, respectively. This study uses KoRV infection of the susceptible human cell line HEK293T and RNAseq to demonstrate gene networks differentially regulated upon KoRV infection. Many of the pathways identified are those associated with viral infection, such as cytokine receptor interactions and interferon signalling pathways, as well as viral oncogenesis pathways. This study provides strong evidence that KoRV does indeed behave similarly to infectious retroviruses in stimulating antiviral and oncogenic cellular responses. In addition, it provides novel insights into KoRV oncogenesis with the identification of a group of histone family genes that are part of several oncogenic pathways as upregulated in KoRV infection.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations.

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.

Distinct Lineages of Feline Parvovirus Associated with Epizootic Outbreaks in Australia, New Zealand and the United Arab Emirates.

Feline panleukopenia (FPL), a frequently fatal disease of cats, is caused by feline parvovirus (FPV) or canine parvovirus (CPV). We investigated simultaneous outbreaks of FPL between 2014 and 2018 in Australia, New Zealand and the United Arab Emirates (UAE) where FPL outbreaks had not been reported for several decades. Case data from 989 cats and clinical samples from additional 113 cats were obtained to determine the cause of the outbreaks and epidemiological factors involved. Most cats with FPL were shelter-housed, 9 to 10 weeks old at diagnosis, unvaccinated, had not completed a primary vaccination series or had received vaccinations noncompliant with current guidelines. Analysis of parvoviral VP2 sequence data confirmed that all FPL cases were caused by FPV and not CPV. Phylogenetic analysis revealed that each of these outbreaks was caused by a distinct FPV, with two virus lineages present in eastern Australia and virus movement between different geographical locations. Viruses from the UAE outbreak formed a lineage of unknown origin. FPV vaccine virus was detected in the New Zealand cases, highlighting the difficulty of distinguishing the co-incidental shedding of vaccine virus in vaccinated cats. Inadequate vaccination coverage in shelter-housed cats was a common factor in all outbreaks, likely precipitating the multiple re-emergence of infection events.

Genetic diversity of Koala retrovirus env gene subtypes: insights into northern and southern koala populations.

Koala retrovirus (KoRV) is a recently endogenized retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next-generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the 'northern' and 'southern' koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV's ongoing entry into the koala genome.

Hematology and Plasma Biochemistry of Wild Spectacled Flying Foxes ( Pteropus conspicillatus) in Australia.

The spectacled flying fox ( Pteropus conspicillatus) is listed as vulnerable to extinction in Australia. The species' restricted population is in decline, putatively attributed to decreasing habitat, climatic extremes, anthropogenic activities, and more recently, mass mortality events associated with tick paralysis and neonatal cleft palate syndrome. Knowledge of fundamental physiologic parameters of the species is limited. To address this knowledge gap, we sampled 50 wild-caught adult spectacled flying foxes in June (winter) in Far North Queensland, Australia. Hematologic and plasma biochemistry reference ranges were established, and a suite of urine biochemistry analytes were measured. Analyte values were compared within spectacled flying fox sex cohorts and between the spectacled flying fox and the paraphyletic black flying fox ( Pteropus alecto). Significant differences in multiple analytes (including erythrocyte, leucocyte, plasma, and urine biochemistry) were found between spectacled flying fox sex cohorts. The majority of spectacled flying fox analyte values did not differ significantly from black flying fox values. Of those analytes that differed between species (erythrocyte, platelet, eosinophil, liver enzyme, and triglyceride levels), the majority were plausibly explained by intraerythrocyte parasite burden and food resource type. Our findings provide baseline data essential to measure and meaningfully interpret flying fox population health in ecologic, conservation, and epidemiologic contexts.

Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.

Prevalence and Risk Factors Associated with Gross Pulmonary Lesions in Slaughtered Pigs in Smallholder and Commercial Farms in Two Provinces in the Philippines.

A cross-sectional study of lungs from 1,887 randomly selected pigs from 471 farms from two provinces in the Philippines was carried out to estimate the prevalence of gross pathological lesions, identify potential risk factors and spatial clustering associated with high lung or pleurisy score farms. Lungs from pigs were scored at slaughter. Interviews with the farm managers were conducted to collect information about farm management and biosecurity practices. Of lungs examined, 48% had a lung score above 6 (maximum was 55) and 22% showed pleurisy. When data were aggregated at the farm level, commercial farms were at higher risk of being high lung score farms and high pleurisy farms compared to smallholder farms (P < 0.01). Variables that were associated with an increased risk of a farm being a high lung score farm included the presence of a market pen on the farm, not vaccinating against hog cholera and the presence of another piggery within 500 m. Practicing "feedback" (feeding pig manure, viscera or aborted material to pigs), presence of another piggery within 500 m, and allowing commercial livestock vehicles on farm were all associated with an increased risk of being a high pleurisy farm. Spatial analyses revealed a primary 9.6 km-radius cluster of 39 farms with high lung and pleurisy scores in the southeast of Bulacan province. High lung and pleurisy score farms could be targeted to improve on-farm disease control programs to reduce the risk of respiratory diseases. Clusters of high scoring farms could be prioritized for further investigations or for coordinating intervention efforts.

Emergence of canine parvovirus subtype 2b (CPV-2b) infections in Australian dogs.

Tracing the temporal dynamics of pathogens is crucial for developing strategies to detect and limit disease emergence. Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs around the globe. Previous work in Australia reported that the majority of cases were associated with the CPV-2a subtype, an unexpected finding since CPV-2a was rapidly replaced by another subtype (CPV-2b) in many countries. Using a nine-year dataset of CPV-2 infections from 396 dogs sampled across Australia, we assessed the population dynamics and molecular epidemiology of circulating CPV-2 subtypes. Bayesian phylogenetic Skygrid models and logistic regressions were used to trace the temporal dynamics of CPV-2 infections in dogs sampled from 2007 to 2016. Phylogenetic models indicated that CPV-2a likely emerged in Australia between 1973 and 1988, while CPV-2b likely emerged between 1985 and 1998. Sequences from both subtypes were found in dogs across continental Australia and Tasmania, with no apparent effect of climate variability on subtype occurrence. Both variant subtypes exhibited a classical disease emergence pattern of relatively high rates of evolution during early emergence followed by subsequent decreases in evolutionary rates over time. However, the CPV-2b subtype maintained higher mutation rates than CPV-2a and continued to expand, resulting in an increase in the probability that dogs will carry this subtype over time. Ongoing monitoring programs that provide molecular epidemiology surveillance will be necessary to detect emergence of new variants and make informed recommendations to develop reliable detection and vaccine methods.

Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia.

Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.