No Evidence of Hendra Virus Infection in the Australian Flying‐fox Ectoparasite Genus Cyclopodia

Hendra virus (HeV) causes potentially fatal respiratory and/or neurological disease in both horses and humans. Although Australian flying‐foxes of the genus Pteropus have been identified as reservoir hosts, the precise mechanism of HeV transmission has yet to be elucidated. To date, there has been limited investigation into the role of haematophagous insects as vectors of HeV. This mode of transmission is particularly relevant because Australian flying‐foxes host the bat‐specific blood‐feeding ectoparasites of the genus Cyclopodia (Diptera: Nycteribiidae), also known as bat flies. Using molecular detection methods, we screened for HeV RNA in 183 bat flies collected from flying‐foxes inhabiting a roost in Boonah, Queensland, Australia. It was subsequently demonstrated that during the study period, Pteropus alecto in this roost had a HeV RNA prevalence between 2 and 15% (95% CI [1, 6] to [8, 26], respectively). We found no evidence of HeV in any bat flies tested, including 10 bat flies collected from P. alecto in which we detected HeV RNA. Our negative findings are consistent with previous findings and provide additional evidence that bat flies do not play a primary role in HeV transmission.     

Radiographic evaluation of cardiac size in flying fox species (Pteropus rodricensis, P. hypomelanus, and P. vampyrus).

Dilated cardiomyopathy is a relatively common pathology in captive flying foxes (Pteropus spp.). The goal of this study was to establish quantitative reference range measurements that could be used to support a diagnosis of cardiac disease in these animals. Lateral and ventrodorsal thoracic radiographs from apparently healthy flying foxes (n = 66) of three species (Rodriguez island flying fox, P. rodricensis, n = 18; small island flying fox, P. hypomelanus, n = 16; and Malaysian flying fox, P. vampyrus, n = 32) were evaluated objectively to describe the cardiac appearance. Absolute and relative cardiac dimensions also were measured. The same methods were used to evaluate radiographs from flying foxes (n = 9) with known dilated or acute cardiomyopathy. The following ratios were most appropriate for categorizing normal cardiac silhouette size. In the ventrodorsal projection, heart width to thoracic width and heart width to clavicle length were the preferred measurements. In the lateral projection, heart width compared with thoracic height was the preferred measurement. From radiographs of the bats with known dilated and acute cardiomyopathy, the apicobasilar heart length compared with thoracic height and heart width compared with thoracic height on lateral films were the most sensitive ratios for diagnosing cardiomegaly.     

Molecular detection of bat coronaviruses in three bat species in Indonesia

Bats are an important reservoir of several zoonotic diseases. However, the circulation of bat coronaviruses (BatCoV) in live animal markets in Indonesia has not been reported. Genetic characterization of BatCoV was performed by sequencing partial RdRp genes. Real-time polymerase chain reaction based on nucleocapsid protein (N) gene and Enzyme-linked immunosorbent assay against the N protein were conducted to detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and antibody, respectively. We identified the presence of BatCoV on Cynopterus brachyotis, Macroglossus minimus, and Rousettus amplexicaudatus. The results showed that the BatCoV included in this study are from an unclassified coronavirus group. Notably, SARS-CoV-2 viral RNA and antibodies were not detected in the sampled bats.     

Clinical and pathologic findings in an outbreak in rabbits of natural infection by rabbit hemorrhagic disease virus 2 in the northwestern United States

Rabbit hemorrhagic disease virus 2 (RHDV2) causes an often-fatal disease of rabbits that has resulted in outbreaks in rabbitries in Europe, Africa, Australia, and Asia. RHD has historically been characterized as a foreign animal disease in the United States. In July 2019, RHDV2 was detected in rabbits on Orcas Island along the northwestern coast of Washington (WA) State following reports of deaths in multiple feral and domestic rabbits. We document and highlight here the unique clinical presentation and gross and histologic lesions observed in this recent WA outbreak. Affected rabbits died without premonitory signs or displayed hyporexia and/or lethargy for ≤1 d prior to death. The most consistent pathologic finding was random, multifocal hepatocellular necrosis, often with concurrent multifocal-to-diffuse splenic necrosis. The lack of significant clinical signs in conjunction with the random distribution of hepatic necrosis in the WA outbreak contrasts with previous reports of RHDV2 disease progression.     

Dynamics of invasion and dissemination of H5N6 highly pathogenic avian influenza viruses in 2016–2017 winter in Japan

Large highly pathogenic avian influenza (HPAI) outbreaks caused by clade 2.3.4.4e H5N6 viruses occurred in Japan during the 2016–2017 winter. To date, several reports regarding these outbreaks have been published, however a comprehensive study including geographical and time course validations has not been performed. Herein, 58 Japanese HPAI virus (HPAIV) isolates from the 2016–2017 season were added for phylogenetic analyses and the antigenic relationships among the causal viruses were elucidated. The locations where HPAIVs were found in the early phase of the outbreaks were clustered into three regions. Genotypes C1, C5, and C6–8 HPAIVs were found in specific areas. Two strains had phylogenetically distinct hemagglutinin (HA) and non-structural (NS) genes from other previously identified strains, respectively. The estimated latest divergence date between the viral genotypes suggests that genetic reassortment occurred in bird populations before their winter migration to Japan. Antigenic differences in 2016–2017 HPAIVs were not observed, suggesting that antibody pressure in the birds did not contribute to the selection of HPAIV genotypes. In the late phase, the majority of HPAI cases in wild birds occurred south of the lake freezing line. At the end of the outbreak, HPAI re-occurred in East coast region, which may be due to the spring migration route of Anas bird species. These trends were similar to those observed in the 2010–2011 outbreaks, suggesting there is a typical pattern of seeding and dissemination of HPAIV in Japan.     

Spatial and temporal variation in the use of supplementary food in an obligate termite specialist,the bat-eared fox

The bat-eared fox (Otocyon megalotis) is considered a termite specialist. However, studies of its diet have been limited to indirect methods, such as scat and stomach content analyses, resulting in intraspecific dietary variations due in part to methodological differences. Because diet plays a central role in the social dynamics of these canids, we hereby contribute further to our knowledge about their dietary habits. We present 2-year data of direct observations of foraging bouts of 19 habituated bat-eared foxes in the kalahari desert of South Africa, as well as data on seasonal variation in invertebrate prey communities obtained through pitfall and sweep net trapping. Despite showing a diet breadth reflective of a specialised forager across all seasons, foxes exhibited substantial seasonal variation in diet breadth with a broader range of food categories utilised in summer compared to the other seasons. Supplementary food categories appear to not have been utilised opportunistically, but it is unclear what drove the preference for some food categories over others. A literature review indicated strong effects of local conditions on the utilisation of supplementary food across southern Africa. Our data support bat-eared foxes as obligate termite specialists but highlight that they appear to have the ability to show dietary flexibility based on both temporal and spatial variations in food abundance.     

Work-related increases in titer of Campylobacter jejuni antibody among workers at a chicken processing plant in Miyazaki prefecture,Japan, independent of individual ingestion of edible raw chicken meat

Workers in poultry abattoirs may be frequently exposed to Campylobacter jejuni, which is a leading cause of bacterial food poisoning in Japan. The present study was conducted to measure the titers of IgG and IgA antibodies against C. jejuni among 104 female workers in a chicken processing plant in Miyazaki prefecture, Japan. Information regarding habitual ingestion of raw chicken meat and potential occupational risk factors was collected using a questionnaire. Acid extracts of four C. jejuni strains representing the genotypes most dominant in Miyazaki were used as antigens. The levels of both immunoglobulins measured by ELISA were not correlated with ingestion of edible raw chicken meat, the amount consumed in one sitting, or its frequency. Although age was correlated with antibody levels, the length of employment was not. Furthermore, the IgG and IgA levels in workers at the evisceration step were significantly higher than those at other locations in the plant. To identify the bacterial proteins recognized by the workers’ IgG and IgA antibodies, Western blotting followed by LC/MS was conducted. Flagellin was identified as the common protein recognized in the sera of workers for whom ELISA demonstrated both the highest and lowest antibody levels. We concluded that the titers of IgG and IgA against C. jejuni in workers at the processing plant had been increased by occupational exposure to Campylobacter, regardless of raw chicken meat ingestion.     

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.     

Antimicrobial resistance in Escherichia coli isolated from brown rats and house shrews in markets,Bogor, Indonesia

The prevalence of antimicrobial resistance (AMR) in small mammals dwelling in the city was used as an indicator of AMR bacteria in the environment. We captured 87 small mammals (79 brown rats and 8 house shrews) in four markets, Bogor, Indonesia in October 2019, and we obtained 20 AMR Escherichia coli (E. coil) from 18 brown rats and two house shrews. Of these, eight isolates were determined to be multi-drug resistant (MDR) E. coli, suggesting the potential contamination of AMR E. coli in the markets in Bogor, Indonesia, and that mammals, including humans, are at risk of infection with AMR E. coli from environment.     

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.     

Handheld mechanical nociceptive threshold testing in dairy cows – intra‐individual variation,inter‐observer agreement and variation over time

ObjectiveTo examine the use of handheld methodology to assess mechanical nociceptive threshold (MNT) on cows kept loose-housed.Study designProspective randomized partial cross-over experimental study. A one-factor (test day) design was used to evaluate MNT over time.AnimalsOne hundred and fifteen healthy, loose-housed Danish Holstein cattle.MethodsWe evaluated intra-individual variation, inter-observer agreement and variation over time of MNT using two handheld devices and two stimulation sites. Mechanical, ramped stimulations were performed with an algometer (6.5 mm diameter steel probe, 0–10.0 kgf) or an electronic von Frey device (plastic tip with diameter 0.8 mm, 0–1000 gf). Each cow received 5–6 consecutive stimulations within a 2 × 5 cm skin area on the dorsal or lateral aspect of the left third metatarsus until an avoidance reaction occurred. We investigated the difference in precision [expressed as coefficient of variation (CV)] between the combinations of devices and stimulation sites. The inter-observer agreement and the difference in MNT between test day 1, 3, 7, 10 and 24 were investigated for selected combinations. Data were analysed in mixed models and Bland-Altman as relevant.ResultsThe CVs did not differ [range 0.34–0.52 (p = 0.1)]. Difference between observers (95% limits) was 0.2 kgf (2.8) and 4 gf (369) for the algometer and von Frey device, respectively. Mechanical nociceptive threshold increased from 361 on test day one to 495 gf on test day 24 (p < 0.01).Conclusion and clinical relevanceAll methods showed a high degree of intra-individual variation, and no combination of device and stimulation site showed superior precision. Mean difference between observers was low, and MNT was not consistent over time. Further development of the methods is required before they can be used in research to investigate possible relations between claw lesions and hyperalgesia.     

Epidemiology and control of Menangle virus in pigs

OBJECTIVE: To describe the epidemiology and eradication of Menangle virus infection in pigs. DESIGN: Field observations and interventions, structured and unstructured serological surveys, prospective and cross-sectional serological studies and laboratory investigations. PROCEDURE: Serum samples were collected from pigs at a 2600-sow intensive piggery in New South Wales that experienced an outbreak of reproductive disease in 1997. Serum samples were also collected from piggeries that received pigs from or supplied pigs to the affected piggery and from other piggeries in Australia. Serum and tissue samples were collected from pigs at piggeries experiencing reproductive disease in New South Wales. Sera and faeces were collected from grey-headed flying foxes (Pteropus poliocephalus) in the region of the affected piggery. Serum samples were tested for neutralising antibodies against Menangle virus. Virus isolation was attempted from faeces. RESULTS: Following the outbreak of reproductive disease, sera from 96% of adult pigs at the affected piggery, including sows that produced affected litters, contained neutralising antibodies against Menangle virus. Neutralising antibodies were also detected in sera from 88% of finisher pigs at two piggeries receiving weaned pigs from the affected piggery. No evidence of Menangle virus infection was found in other piggeries in Australia. In cross-sectional studies at the affected piggery, colostral antibodies were undetectable in most pigs by 14 to 15 weeks of age. By slaughter age or entry to the breeding herd, 95% of pigs developed high antibody titres (> or = 128) against Menangle virus in the virus neutralisation test. Menangle virus was eradicated from the affected piggery following a program of serological testing and segregation. Neutralising antibodies against Menangle virus were also detected in P poliocephalus from two colonies in the vicinity of the affected piggery. Two piggery workers were infected with Menangle virus. There was no evidence of infection in cattle, sheep, birds, rodents, feral cats and a dog at the affected piggery. CONCLUSIONS: Serological evidence of infection with Menangle virus was detected in pigs at a piggery that had experienced reproductive disease, in pigs at two associated piggeries and in fruit bats in the region of the piggery. Two humans were infected. The mode of transmission between pigs is unknown, but spread by faecal or urinary excretion is postulated. This virus can be eradicated by the segregation of pigs into discrete age groups.     

Egg trait variation in a large hawk‐cuckoo (Hierococcyx sparverioides) host population of Chinese babax (Babax lanceolatus)

Mutual interaction between brood parasites and their hosts is a well‐known model system for studying host–parasite coevolution. Both parties have acted reciprocally, resembling an evolutionary arms race, in which adaptations and counter‐adaptations have evolved as a result of host–parasite dynamics, such as the classical cuckoo–host system. Discrimination among parasite and cuckoo eggs and rejection of foreign eggs is regarded as an important anti‐parasitism strategy. The Chinese babax (Babax lanceolatus) is a large hawk‐cuckoo (Hierococcyx sparverioides) host distributed in southwest China. A previous study shows that the babax is an intermediate egg rejector, and most cuckoo eggs are accepted by the Chinese babax, although a small proportion of hosts reject cuckoo eggs. Interestingly, the large hawk‐cuckoo lays non‐mimetic eggs in contrast to the uniform blue eggs of babaxes. Because egg coloration is a critical cue used by host species in favor of the recognition of parasitic eggs by hosts, we used a spectrometer to quantify egg color variation to understand the differentiation in discrimination ability between the egg rejectors and acceptors. We found that the chroma of intra‐clutch variation of babax eggs was more consistent in egg rejectors than in acceptors. However, no statistical significance was found in inter‐clutch variation between these two types of hosts. Our results suggest that hosts lay eggs with a low level of intra‐clutch variation without the necessity of a high level of inter‐clutch variation simultaneously as predicted by the egg signature hypothesis. This study may further indicate that selection pressures from evolutionarily recent parasites can drive individual‐based differences in an anti‐parasitism strategy.     

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/tat region represents an important target for the detection of non-clinical horses.     

Seroprevalence of antibody to NcSAG1 antigen of Neospora caninum in cattle from Western Java,Indonesia

Neospora caninum can cause fetal abortion and neonatal mortality incattle, and is a cause of economic concern worldwide. This study aimed to determine theprevalence of Neospora caninum-specific antibodies in cattle from WesternJava, Indonesia. Serum samples from 991 cattle from 21 locations were tested forantibodies to N. caninum by using an enzyme-linked immunosorbent assay(ELISA) on the basis of recombinant NcSAG1. The overall seroprevalence was 16.6%, rangingfrom 0 to 87.5% in the sampled locations. The results of this study indicate latentinfection rates of sampled animals were different in each location. Further studies arenecessary to elucidate the relationship between N. caninum infection andabortion in cattle, and to identify risk factors for infection in high-prevalenceenvironments.     

Viral RNA extraction using an automatic nucleic acid extractor with magnetic particles and genetic characterization of bovine viral diarrhea virus in Tokachi Province,Japan, in 2016–2017

In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016–2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.     

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.