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Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed. [ABSTRACT FROM AUTHOR]

The Kunjin strain of West Nile virus (WNV KUN ) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNV KUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNV KUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNV KUN and MVEV transmission by FTA TM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNV KUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTA TM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTA TM card system as a sensitive and accurate method to monitor the transmission of WNV KUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a "One Health" issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTA TM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

On the 26th of November 2021, the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69-70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

West Nile virus, Kunjin strain (WNV KUN ) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNV KUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles ( Crocodylus porosus ), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 10 5 ( n = 10) or 10 4 ( n = 11) TCID 50 -doses of WNV KUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNV KUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNV KUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

High-throughput sequencing (HTS) provides the opportunity, once a diagnostic result is obtained, to extract additional information from a virus-containing sample. Hence, it offers advantages over established quantitative amplification technology, such as quantitative PCR, particularly in a public health environment. At this early stage of its clinical application, there have been limited studies comparing HTS performance to that of the more established quantitative PCR technology for direct detection of viruses. In this pilot-scale study, we tested HTS with a range of viruses and sample types routinely encountered in a public health virology laboratory. In comparison with quantitative PCR, our HTS method was able to sensitively (92%) detect all viruses in any sample type with the exception of certain tissues. Moreover, sufficient nucleotide sequence information was obtained to enable genotyping of strains detected, thus providing additional useful epidemiological information. While HTS sensitivity may not yet match that of PCR, the added value through enhanced epidemiological data has considerable potential to enable real-time surveillance of circulating strains so as to facilitate rapid and appropriate response to outbreaks and virus zoonotic spillover events.

A male patient in his 50s who traveled from Papua New Guinea (PNG) to Australia in 2016 was diagnosed with a dengue virus serotype 4 (DENV-4) infection, and the virus was isolated from his acute-phase serum. Here, we describe the first complete genome sequence of a DENV-4 strain from PNG.

To better understand the diversity of bunyaviruses and their circulation in Australia, we sequenced 5 viruses (Gan Gan, Trubanaman, Kowanyama, Yacaaba, and Taggert) isolated and serologically identified 4 decades ago as members of the family Bunyaviridae. Gan Gan and Trubanaman viruses almost perfectly matched 2 recently isolated, purportedly novel viruses, Salt Ash and Murrumbidgee viruses, respectively. Kowanyama and Yacaaba viruses were identified as being related to members of a large clade containing pathogenic viruses. Taggert virus was confirmed as being a nairovirus; several viruses of this genus are pathogenic to humans. The genetic relationships and historical experimental infections in mice reveal the potential for these viruses to lead to disease emergence.

A 5' Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0x10(-2) median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5' Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

A 5' Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5' Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5' Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Surrogate motherhood is a controversial subject, and has not previously been formally modelled by economists. In this paper, a neoclassical model of the market for surrogate motherhood contracts is developed, based on the utility maximizing decisions of potential surrogate mothers and commissioning parties. The presence of both altruistic and self-interested behaviour generates unusual market outcomes.