The Veterinarian Magazine August 2023

TheVeterinarian

The evolutionary fight against megafires

The northern hemisphere summer this year has seen out-of-control wildfires raging across parts of Europe, while scientists warn Australia faces an El Niño event in coming months, increasing the risk of severe and destructive bushfires such as those experienced in 2019/2020 that resulted in the loss of an estimated three billion animals, birds and reptiles.

A multidisciplinary team of ecology experts reviewed nearly 100 papers to identify examples of animal evolution in response to fire, and to analyse and categorise the different ways in which this evolution can take place. This growing body of evidence for fire as a ‘force in animal evolution’ provides an insight into the implications and impacts fire has for wildlife and conservation due to climate change. The results of the study were published recently in the journal Trends in Ecology & Evolution

Researchers found fires were burning more frequently, more severely, across larger areas, and with different seasonality, and they identified over 4400 species across the world that were vulnerable to extinction because of changing fire regimes. These included 1000 species of birds, amphibians, reptiles and mammals.

Lead author Gavin Jones, a Research Ecologist for the US Department of Agriculture’s Forest Services, said there was a wide range of animal adaptations to fire and that evolution was happening – and will continue to happen –‘right in front of our eyes’.

“The Australian frilled lizard, for example, hid in trees to escape grassfires, which is behavioural adaptation, but this behaviour could turn deadly when larger, modern fires burn their once-safe hiding places. Other species such as the Temminck’s courser, a wader bird from subSaharan Africa that lays its eggs the colour of

recently burnt ground, has evolved by changing its basic biological characteristics,” he said.

Jones said a better understanding of the variations in how animal species are evolving was important as it would enable more effective conservation efforts.

“We must acknowledge that fire is not just a process that influenced evolution long ago. We need to incorporate evolutionary thinking into approaches for conserving populations

under changing fire regimes, including detecting symptoms of fire naivety, and identifying potential fire savvy-traits that could be leveraged for conservation. Doing so will allow for improvements to conservation efficacy,” he said.

Further reading: doi.org/10.1016/j.tree.2023.06.003

■ ANNELAYTON-BENNETT

WSAVA award winners named

WSAVA was established in 1959 and has grown to include 115 veterinary association members globally, representing more than 200,000 small companion animal veterinarians.

The 2023 WSAVA Award for Global Meritorious Service has been awarded to Laurel Kaddatz, a veterinarian from Florida. Kaddatz spent 25 years of his career as a senior partner in a large hospital group in Rochester, New York, before serving in the tole of Hospital Director in a practice he bought.

Kaddatz has also been committed to promoting the wider work of veterinarians, publishing articles in the media, making presentations to community groups and organising initiatives such as shadowing programs for high school students.

Leader Award in recognition of his strong contribution to the veterinary community in Canada, in particular his work to highlight issues around mental health in the profession and to promote technological innovation in the sector.

In his role on the board of the Ontario Veterinary Medical Association (OVMA), Laing has been an advocate for technological advancements and updates to regulations which allowed the industry to adopt telemedicine to treat animals during the COVID pandemic.

The World Small Animal Veterinary Association (WSAVA) have announced the recipients of several of their annual awards ahead of this year’s WSAVA World Congress in Lisbon, Portugal in late September.

“Community-building is at the heart of everything the WSAVA does and, in bestowing this prestigious Award on Mr Kaddatz, we are recognising an individual who has personally exemplified community-building throughout his career,” WSAVA President Ellen van Nierop said.

Meanwhile, Brendon Laing, a Canadian veterinarian and co-owner of several clinics in Ontario, is to receive the 2023 WSAVA Future

Commenting on the Award, Mike Topper, Chair of the WSAVA Membership and Nomination Committee, said: "Mr Laing stood out because of his engaged leadership at the provincial and national levels of organised veterinary medicine; his continuous efforts to improved wellness and wellbeing in his small animal hospitals, and his on-going promotion of telemedicine to improve the accessibility of veterinary care."

The WSAVA World Congress is hosted by a member association and rotates yearly through three regions of the world – the Americas, Europe/Middle East and Africa and Oceania.

■ JULIA GARDINER

Laurel Kaddatz

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Stingless bee research highlighted

Australia is home to native stingless bees, which are the subject of scientific studies being carried out to determine what they like to eat, and how diet impacts overall hive health and their ability to pollinate crops effectively.

The research comes on the back of the discovery of Varroa mite in NSW in 2022. The mites wreak havoc not only to bees themselves, feeding on the bees internally and externally, as well as carrying viruses which can infect the bees, but also to crops, which are impacted by lower numbers of bees which are required to pollinate them.

TheVarroa mite outbreak and the controls put in place to protect the agricultural industry have impacted horticulture growers’ access to access to Australia’s most common crop pollinator, the European honey bee, leading researchers to study commercial management and capabilities of alternative pollinators.

The research is being delivered through Hort Innovation and led by Western Sydney University and Griffith University in partnership with key commercial stingless beekeepers and industry partners.

“The incursion of Varroa mite has placed us in a position where we have had to reassess how we think about pollination,” Hort Innovation chief executive officer Brett Fifield said. “Unlocking

the potential of alternative pollinators, like the stingless bee, is going to be instrumental in providing the horticulture sector with new crop pollinating options.”

Research into maintaining honey bee health and boosting alternative pollination methods is a priority for Hort Innovation, with $61 million of investments currently underway across various activities, including the development of a Varroa mite pesticide that is safe for bees.

Western Sydney University Professor James Cook said the research project will identify the nutritional choices of stingless bees by detecting the pollen species and essential nutrients collected during their foraging activities.

“We are exploring the relationship between stingless bees’ dietarychoices and their colonies’ wellbeing,” Cook said. “By understanding this relationship, we can identify new opportunities to optimise hive health, such as introducing nutritional supplements into stingless bees’ colonies, and thereby improve propagation of the bees and pollination services.”

Inaddition to working on stingless bee nutrition, crop pollination trials will be conducted at a state-of-the-art research glasshouse at the National Vegetable Protected Cropping Centre on the Western Sydney University Hawkesbury campus.

Sugarbag Bees’ business owner and beekeeper Tim Heard said the facility’s experimental hives dedicated to bee rearing will equip beekeepers with knowledge to expand on stingless bee management and provide new options for the industry.

“By equipping beekeepers with valuable knowledge about rearing and managing these incredible pollinators, we will pave a sustainable future for beekeeping and as a result, the horticulture industry,” Heard said.

■ JULIA GARDINER

Marine heatwaves a threat for seabirds

Although the focus in recent months has been the unseasonal and extreme weather events like floods, storms and wildfires so many countries around the world have experienced, a study published in the international journal Marine Ecology Progress Series shows the world’soceans are also not immune to our changing climate and the risks this poses for marine species.

Marine ecologist Eric Woehler OAM from the Australasian Seabird Group, and lead author of the study, said marine heatwaves are

a global phenomenon that are increasing in frequency, intensity and spatial extent, and are impacting on fish stocks, marine invertebrates, water temperatures and seabirds.

“Marine heatwaves have probably been happening longer than we appreciated but it was only in the last ten or 15 years that we started to take note and document them. Seabirds are acting as the climate canaries in the ocean in terms of providing critical insights into how they will deal with future scenarios when these MHW conditions become the norm,” Woehler said.

Co-author Alistair Hobday, Research Director and Senior Principal Research Scientist at CSIRO, is a world expert on MHWs. He was part of the scientific team that defined, characterised and classified these phenomena, that showed they could no longer be considered one-off events or an anomaly,but were an environmental stressor that posed a serious threat to all marine life, including seabirds.

“Migratory and long-distance species such as albatrosses and

shearwaters appear to be more likely to survive a marine heatwave than species that are confined to nearshore areas around their colonies, such as penguins,” Hobday said.

The study found MHWs fundamentally change the marine environment on which seabirds depend, by measuring how the birds responded to them and how their responses could be projected into their life historystrategies such as foraging and breeding.

“It’sclear some species are in deep trouble because they haven’t got the options to bypass or avoid MHWs. It’s partly due to the traditional breeding sites for near-shore foragers, and the availability of their food sources which are being pushed either into deeper water or further offshore. Or there’s an overlap, or mismatch, in the breeding season of the birds with the availability of their prey species. So although all marine species are impacted, seabirds are giving us the most obvious signal in terms of what’s happening in the marine ecosystem,” Woehler explained.

The study concluded that although, ‘Marine Protected Areas

provide opportunities for seabird conservation, given the trajectoryof environmental stressors that impact seabirds, investigation and testing of interventions is urgent. Marine heatwaves provide contemporary insights into potential future scenarios for seabirds. These scenarios are also an opportunity to predict the impacts of MHWs and, finally,to investigate potential management responses that will further the conservation of seabirds.’

‘Impacts of marine heatwaves may be mediated by seabird life history strategies’ is available at www.int_res.com/ articles/meps_oa/m14333_advview.pdf

■ ANNE LAYTON-BENNETT

Pictures Eric Woehler

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UQ’s experimental canine cancer vaccine test

University of Queensland veterinary researchers are recruiting pet dogs diagnosed with cancer totest an experimental vaccine.

The clinical research trial is for dogs recently diagnosed with canine appendicular osteosarcoma, acommon bone tumour that affects 10,000 dogs ayear globally.

Abhilasha Dadhichfrom UQ’sSchool of Veterinary Sciencesaid researchers will assess two vaccine formulations.

“These treatments aim to stimulate a dog’s immune system to fight the spread of cancerous cells to other tissues of the body,” Dadhich said.

“We’re essentially ‘waking up’ the dog’s immune system to fight the cancer.

“This has real potential to enhance the life expectancy of the pets in the trial.”

To be eligible, dogs need to have been diagnosed with appendicular osteosarcoma of long bones of the limbs with no evidence of metastasis, orcancer spread, based on a physical exam and radiographs.

What’s that ribbit? FrogID it!

Weare all familiar with the different accents and speech rhythms among humans, that can offer a hint about which part of the country or the world people come from, but research led by scientists from University of New South Wales and The Australian Museum, and with support from data collected by citizen scientists who participated in the Museum’s FrogID Project, has

found individual frog calls can also have distinct accents.

FrogID is an app developed by the Museum that invites citizen scientists to record frog calls from around the country, and submit them both for verification, and for assessing species’ distribution.

Researchers analysed the calls of nearly 700 banjo frogs, a species that is common throughout Australia across arange of habitats, and found that counter to common theory,variation in calls was not strongly linked to habitat structure but was more likely to be influenced by the interplay between amultitude of factors, such as noise from other animals, or anthropogenic noise, including wind and water.

Lead author of the study Grace Gillard, who completed the project as part of her honours at UNSW, said the results were surprising because the link between habitat and variation inanimal calls had quite Topage 30

Dogs can be any breed but must be proceeding to surgery to treat their cancer.

“If the dog meets these parameters, their owner will simply need to sign an informed consent document after the trial has been explained,” Dadhich said.

“The veterinary clinic will then collect blood and cancerous bone tissue from the dog at the time of surgery.

“Our prepared treatment is provided for free, administered to the dog in two doses.

“The first vaccine dose will be given within two weeks of their surgery and the second dose as abooster three weeks later.

“Further blood samples from the dog will be collected to assess its immune response.”

Professor Rachel Allavena, who is leading the team conducting the trial, said it was hoped immune treatments would one day be a straightforward process for veterinarians around the world.

Topage 30

Reef still vulnerable, AIMS warns

Despite increases in coral coverage across the Great Barrier Reef in 2022, the Australian Institute of Marine Science (AIMS) warns that this year’s survey of the reef shows a pause in recovery, with the reef still vulnerable to degradation.

The AIMS monitoring team spent 120 days at sea during the survey period this year, traveling over 1000 kilometres around the perimeter of the 111 reefs they surveyed. Their findings showed that hard coral cover across the Great Barrier Reef remains at similar levels to those recorded in 2022, when the northern and central regions of the reefs recorded their highest amount of coral cover since AIMS began monitoring 37 years ago.

AIMS Research Program Director David Wachenfeld said that while continued recovery on some reefs was good news, the pause in recovery showed that even relatively milder mass bleaching events had consequences for the Reef.

“The 2022 coral bleaching event was not as severe as the 2016 or 2017 events but caused enough mortality to pause recent regional gains in hard coral cover. The heat stress during the bleaching event also likely had sub-lethal effects, including reductions in coral growth and reproduction,” Wachenfeld said.

AIMS research has shown much of the recent recovery has been driven by fast-growing branching and plate corals, or Acropora; however, the species remain susceptible to threats including cyclones, crown-of-thorns starfish To page 30

Barney, owned by Professor Allavena, was given an early version of the vaccine when diagnosed with an aggressive bone cancer and 10 months later is alive and well

Insurer makes submission to parliamentary inquiry

Standalone pet insurer PetSure has made a submission to a NSW Parliamentary Inquiry into the shortage of veterinarians across the state. The insurer has proposed several initiatives to support the growth and sustainability of the veterinary workforce in NSW. In addition to apublic education campaign, greater support for digital telehealth services, and legislative changes to allow better pet access to public transport, the insurer has also put forward a new model to improve equitable access to veterinary services for disadvantaged pet owners.

Convened by the Upper House of the NSW Parliament and chaired by the Hon Mark Banasiak MLC, the Parliamentary Committee has been tasked with investigating workforce shortages across all areas of veterinary practice in NSW, including small and large animal practice as well as government, academic, research, industry and pathology roles. The inquiry was prompted by media coverage of burnout in the veterinary profession, reported workforce shortages and increased demands for veterinary care in the face of burgeoning pet ownership. The inquiry’s terms of reference are reasonably broad, encompassing not only the shortage of veterinarians across the profession, but also taking into account such things as the mental health and wellbeing of veterinary workers, impacts of current legislative and regulatory frameworks, geographic and socioeconomic barriers to accessing veterinary care in NSW, and the particular challenges posed by the shortage of vets in regional, rural and remote areas of the state.

PetSure is well positioned to understand the challenges facing both veterinarians and pet owners, not least because it underwrites more than 80 per cent of the Australian pet insurance market and helps to cover the treatment costs of more than 4,000 dogs and cats everyday. Moreover, PetSure and its associated entities also employ around 80 veterinaryprofessionals across the country, including Mara Neale, General Manager of GapOnly, the insurer’s on-the-spot claiming solution. “Given our position in the market, including our deep knowledge, industry relationships and collaboration with veterinary professionals, we saw an opportunity to put forward our ideas and recommendations to improve sustainability within the veterinaryindustry,” Neale said in relation to PetSure’s parliamentary submission. “The Australian veterinar y workforce faces several challenges, and we believe the government, businesses and stakeholders within the broader pet health ecosystem can work more closely together to drive real change in this space.”

One of the main initiatives included in the submission to the NSW parliamentaryinquiry is apublic education campaign aimed at raising awareness of the benefits and costs of veterinary

care and of the importance of being financially prepared to meet those costs. “Global studies show difficult financial conversations are asource of stress for vets, often driven by the financial constraints of the owner.A public education campaign, aimed at driving greater awareness of costs for common pet health ailments, could help alleviate some of this pressure for vets,” Neale said. “We believe a public education campaign focused on early health programs for preventative care, could have many benefits for the veterinary profession. It could support better pet health outcomes, improved veterinarian wellbeing and mental health, and reduced treatment costs. The long-term economic benefits of an early health approach are demonstrated in the human health system.”

The insurer has already developed its GapOnly product as a means of reducing financialstresses affecting pet owners and veterinarians. It has been rolled out in vet clinics nationwide, enabling pet insurance claim benefits to be paid directly to clinics at the point of care, reducing upfront out-of-pocket costs for customers and making veterinary services more accessible. In its submission to parliament, PetSure suggested its proposed public education campaign regarding financial awareness could also be complemented by early intervention programs highlighting safety and preventative care for animals. Such programs would likely result in improved outcomes not only in terms of animal health, but also in relation to veterinarians’ mental health and wellbeing.

Another proposal made is that the State Government provides greater support for digital telehealth services across the veterinary industry, particularly after lockdowns during the COVID-19 pandemic normalised such consultations in both human and animal health systems. “Developing effective digital innovation in telehealth offerings, while protecting the

Native birds lost and stranded

More than 90 aquatic birds have been found mysteriously stranded across Victoria during the past two months. The rescued grebes, which are duck-like native waterbirds, have often been debilitated or injured, and in need of veterinary care.

The strandings have been occurring throughout this year’s autumn and winter seasons, when grebes traditionally migrate from inland freshwater locations to coastal areas; however, Werribee Open Range Zoo associate veterinarian Paul Eden said the grebes were being rescued well away from their usual freshwater habitats.

“People are finding these waterbirds inside their backyards, around industrial estates, and we’ve even had birds turning up at the casino,” Eden said.

Many of the injured birds have been rescued

by teams of veterinarians and wildlife experts at Melbourne Zoo, Werribee Open Range Zoo and Healesville Sanctuary and are being released back into the wild after the rehabilitation provided by Zoos Victoria.

One of the theories behind the sudden rash of strandings could be climate change, according to Eden.

“A breeding and population boom of the species, caused by the wetter weather conditions of La Niña throughout the past few years is resulting in grebes dispersing to find new wetland habitats,” Eden said. “At this time of year,grebes like to come together to congregate insocial flocks, however they are sadly losing their way on that journey.”

■ JULIA GARDINER

veterinarian-client-patient relationship, requires regulatory change in this space,”Neale said. “When used in addition to in-person consultations, vet telehealth provides several benefits to vets including reduced after-hours calls and unnecessary visits, improved work life balance through effective triaging of emergencies and in managing non-complex cases where diagnostics are not required, and greater flexibility for vets unable to work full-time in clinics.” Expanded telehealth services also have the potential to deliver greater patient reach and accessibility, particularly in regional, rural, and remote areas of NSW and for patients who require access to veterinary services outside normal business hours. Legislative change to improve pet access to public transport was another area highlighted by the submission. “Currently in NSW, the ability of animals to travel on buses, light rail, ferries, and taxis, is up to the discretion of the driver and crew members while animals are prohibited from travelling on trains and coaches,” Neale said. “There is increasing public discourse about the impact of these restrictions on the human rights of so many Australians who rely on public transport, and we advocate for change in this space.” Similarly, PetSure has called for greater support and more inclusive access to veterinary services for vulnerable and disadvantaged Australians, and used its parliamentary submission to put forward a new model to promote equal access to veterinary services for Australians who need them most. “Within a new model, pet owners could access a dedicated tele-health service, be verified as eligible (for example as a Centrelink or NDIS client) and receive immediate access to high quality virtual pet healthcare,” Neale said. “Where necessary, the client would be referred, based on their postcode, to the nearest participating veterinarian in their town to receive essential or emergency pet healthcare under an arrangement like bulk-billing, on a pre-agreed fee schedule.” In this regard, Neale readily acknowledged veterinarians are critical partners in providing equitable access, and that anecdotal evidence suggests many vet clinics already provide services to vulnerable community members at no charge. “By participating in this model, veterinarians would be empowered to provide services at a pre-agreed rate to eligible clients, in some cases avoiding the trauma of economic euthanasia of the animal,” she said. Submissions to the NSW Parliamentar y Inquiry closed in July 2023, with Committee hearings scheduled for late August 2023. A release date for the Committee’sfindings and recommendations has not yet been set, but it can only be hoped that the inquirywill help create a more sustainable veterinary workforce and a better-informed pet owning public in NSW.

■ JAI HUMEL

Mark Banasiak

Picture JJ Harrison

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Kiwi Post

In Aotearoa, things are beginning to hot up ahead of the election. Literally. While Southern Europe burns, and Maui is tragically consumed by wildfires, a recent report noted that the Southern Ocean has been up to 4o warmer this past summer than normal. If for some strange reason you weren’t quite convinced by the science of climate change, this reality may have started to help.

The Southern Ocean is notoriously cold and hostile, but over summer this year the waters around Rakiura/Stewart Island were often 4o higher than their 25-year average. If you wanted toget into your togs and brave the Great Whites around Oban on Rakiura, you’d normally be looking at a sea temperature of just over 14o This summer, on two, prolonged occasions, the temperature reached over 18o.Toput it in context, for those of us who surf or swim around the Southern tip of New Zealand, this change in temperature is physically noticeable as the sea reaches your more sensitive bits.

The North Island has seen a series of unprecendented rainfall events. ‘Events’ is not really the best way to describe them- it really started raining before Christmas and hasn’t stopped since. The only variation has been the intensity. At a meeting with a group of farmers in Rotorua earlier this month, a number of them mentioned they’d recorded 4m of rain over last season on their properties. This is more than double their normal and is approaching the legendarybiblical rainfall of the South Island West Coast.

Added to that was the impact of Cyclone Gabrielle in Februarywhich hit the upper North Island, just as they were mopping up from an extreme rainfall event at the end of January. Sadly,these events didn’tjust bring devastation, but also death, including a veterinarian who was working as a firefighter and helping evacuate houses at the time.

For a few years now we’ve not allowed overseas investors to invest in developing farms, but we

have allowed them to invest in wrecking them- by planting pine trees all over good quality farmland. No, I dont pretend to understand this either. This has been good for overseas investors, and for those selling farms (the average value of sheep and beef farms sold for trees is approximately 3-4 times those sold for real farming), but disastrous for ecology, rural communities, ecosystems, infrastructure and anyone living downstream. North West Scotland tried this 50 years ago, and ended up with a vast acidic, boggy monoculture where nothing grew or lived- except pine trees. Weavoided the acidic bog and ended up with $10bn worth of infrastructure damage instead as the externalised costs of pine trees destroyed roads, bridges and communities downstream after the last cyclone.

Now, with the election looming, politicians are suddenly turning green. Except for the Greens, who are suddenly turning to tax restructure. But that’s ok, a key feature of this election seems to be that every party is out of ideas and is stealing the others’ policies. What this means of course, is there is the (admittedly vague) possibility that we may get some good policies that support environmental sustainability.

Of course in general, planting trees is generally agood idea. So, rather than stop it, it could easily be amended. Planting native species would have only upside encouraging the redevelopment of a struggling ecosystem, as well as providing carbon sink. Of course it’snot as profitable as pine trees, but only if you only count financial capital. If you dared consider social, community,ecosystem and human capital you may find this a far more ‘profitable’ approach. Finally, with election time coming, some politicians are at least considering this.

They’re also considering our approach to Predator Free 50. This is our aspirational goal to remove a bunch of small predators from Aotearoa by 2050. Sadly not all predators but possums, stoats and rats. These 3 introduced

species decimate our native bird species kiwi, kakapo, kokako, kaka, kereru, toutouwai (Robin), tieke (Saddleback) are all threatened; and they also impact species of native bats, reptiles, snails and insects.

Sadly, we have other predators, which aren’t (currently) covered by PF2050- deer destroy the understory of the native bush, and cause immense damage. They’re not the only introduced ungulate doing this- pigs, thar, goats and chamois are also contributors but deer are having the most impact. Currently, the understory on Rakiura is virtually eliminated, and on the point of ecosystem collapse. Critically, it’s been estimated that the understory of native bush in New Zealand contributes more to carbon sequestration than the rest of the bush. So losing this is not just an ecosystem disaster, but a climatic disaster too.

Wild wallabies, once localised around Waimate in the mid-South Island, have now spread way beyond that immediate area and multiplied, causing similar damage. And cats are probably the most contentious of all introduced species. They are of course consummate hunters. But cats are also some of our best friends, and there is little difference in impact between your best tabby and the average feral moggy. Trapping and killing the latter often leads to inadvertent killing of some of the former, and the recent feral- cat hunting section of a North Canterbury school fundraiser had to be cancelled when it faced a viral global backlash from cat lovers.

The complexity of the intertwining of ecosystems, native and introduced populations, and climate change means that these large issues are often overlooked in our rush for ‘growth’. But, if we are to live sustainably in this amazing corner of the world, these are issues that are critically important and need to be addressed. I’m not daft enough to think that any politician will keep their green promise beyond October, but at least they’re discussing it. And hopefully this isn’t just more warm air.

DO YOU HAVE SOMETHING TO SAY?

Is there a topic

you’ve wanted to tell everyone about?

Have you ever thought you would like to write aclinical review but don’tknow whereto start?

Do you have a hobby or history that you think vets everywhere would like to learn more about?

Then don’t delay – email The Veterinarian ateditor@vetmag.com.au and let us know –because your words could be in the pages of next month’sissue!

Mark Bryan qualified in Glasgow in 1988, and moved to New Zealand in 1995.

The long and winding

g

FINDING PERSPECTIVE AND BALANCE WITH WILDLIFE VET AND ARTIST

AMBER GILLETT

ver the past decade, the vast majority of people I have the privilege to interview for The Veterinarian have known from an early age that they want to be veterinarians. All of these individuals have an abiding love of animals, most have access to educational opportunities that – with hard work and dedication –allow them to progress to their chosen veterinary science program at university, many then transition smoothly into working as veterinarians in practices around the country or overseas, and some go on to undertake specialist qualifications inareas of veterinary science that particularly interest them.

Every now and then, however, I come across aperson whose road to veterinary school and practice has not been nearly so straight forward. Irealise I am generalising (though hopefully not too wildly), but these people tend to fall into three broad categories. The first of these categories is made up of people who pursue a career path entirely different from vet science and subsequently ‘change lanes’: one of the veterinarians I interviewed started out as a naval lieutenant; another had accepted a cadetship to work in a major bank before going to vet school. The second category is occupied by those whose love of veterinary science competes with another of their passions. Their road to becoming a veterinarian comes to a fork and forces them to choose one path over the other – like the vet who opted to put his career ahead of the possibility of representing Australia in kayaking at the Olympics. And the third category, which is perhaps the rarest of all, is comprised of people who are passionate about becoming veterinarians, but their road to achieving that goal is long and winding, sometimes difficult to see, and – more often than not – strewn with unexpected obstacles. It is to this third category that wildlife veterinarian and artist Amber Gillett belongs. Like many vets I interview, Gillett knew she wanted to be a vet since she was a small child. Born in Cairns, her family moved to a rural area of the Atherton Tablelands in Far North Queensland when she was three years old. As time went on, she became what she described as the “bossy big sister” to two younger brothers, onethree years and one eight years younger than her. “We grew up as real country kids, surrounded by farming families, riding motor bikes and horses,” she said. “We didn’t have a farm, but we had a little bit of acreage and a menagerie of animals throughout my childhood. My mother was a major animal lover as well –she was always out there, milking the cows and feeding the ducks and the chickens and the geese.” One of the first pets Gillett can recall was a rooster named Jack, who would sit on the couch with her and watch Play School when she was a toddler. Later on, she had her own horse, as well as peacocks, guinea pigs, dogs, cats and apiglet.

The township Gillett grew up in was relatively remote and isolated, and the family did not travel out of the area often – she recalls playing flute on an orchestra trip to the NorthernTerritory when she was in primaryschool, and visiting the Gold Coast once with her mother in her early teens. That said, since both Gillett’s parents were English, the family did fly to the UK to visit relatives, and these trips brought her into contact with another animal lover. “My mum’s sister was akeen collector of all sorts of exotic and unusual animals,” she said. “I’m not surehow she came to have so many, but some of the most memorable species included an African bull frogthat was as big as the palms of both your hands, and a huge boa constrictor that use to roam the lounge room and would curl up in the bay window and look out at passers-by.” Her aunt had a variety of reptiles, including ball pythons, iguanas and other frog species, and also had lots of rabbits and rats. “She treated them all as treasured

animals in her life and taught me respect for animals of all shapes and sizes,” Gillett said.

Unsurprisingly, the only career path Gillett can ever remember wanting to pursue was to become a vet. “It was probably a culmination of a few things: my love for animals, and my appreciation for all these exotic weird species my aunt had to expose me to, and the amount of times we had to take animals to the vet and be involved with the vet – it just seemed like a very natural thing for me to want to do.” At school, agricultural and science subjects were among Gillett’s favourites, but as her high schooling progressed, she became aware that it was unlikely she would achieve the grades she needed to get into veterinary science.

Gillett’s predicament was exacerbated by the fact that her parents had split up around the time she started high school. Sadly, her upbringing was marred by domestic violence, protracted Family Court disputes and “all sorts of stuff” that happened to her, her mother and her brothers. Totop things off, money was tight. “I wasn’t from a wealthy background, so there was no way of paying to get a position in courses like vet science,” she said. “If I wanted to get in, I knew it was going to be purely on academic grades. Thinking back now, I didn’t really understand the university system and the opportunities that were available once you were in the system, and neither did my family as my mum had not gone to university either.” Despite putting loads of effort into her classes, particularly in maths and physics, Gillett had to contend with the knowledge that she was not as gifted in these areas as she was in others. “I had agreat physics teacher who was very supportive of me, but no matter how

young, and she was also a florist. She had a really good eye for colour and context and all that sort of stuff, so I guess all that kind of rubbed off on me,” she said. “But when you come from a rural background, making a living from art is not exactly something you see or think of.”

Gillett can still recall the specific task her teacher set her that sparked her interest in art at the beginning of high school: using a grid system to draw an enlarged image of a cartoon character. While she can’t recollect exactly what she drew (though she thinks it was Mickey Mouse), Gillett does remember the boost of confidence she received when her teacher saw the finished product. “The exercise was a bit technical for that age group, but I remember my teacher was very impressed with how I was able to enlarge the image,” she said. “It must have been a huge source of encouragement for me, because it stuck with me – and that’skind of whereit started.”

much I tried I was lucky to get a B minus in those subjects, even though I was used to getting A’s in art and agriculture,” she said. “It was disappointing, because I tried so hard, and it crushed my enthusiasm for being able to get into vet school, because I couldn’t do really well in those subjects and the OP 1 needed to get straight into vet school was out of my reach.”

Art, however,was a class Gillett did excel at, even though she does not remember doing anything particularly artistic or creative before the end of her junior schooling. “My mum was acreative person; she did a little bit of drawing and sketching and photography when she was

From fairly early on in her high school years, Gillett gained confidence in her artistic abilities and in the artworks she was drawing and painting, guided by a supportive art teacher. “He was a gentle, softly spoken kind of person, but he really did foster my passion and enthusiasm for art,” she said. “Certain teachers can obviously see some kids have potential, and they spend a lot of time working with them, and that happened for me.” At around age 15, Gillett plucked up the courage to enter some of her artworks in the local Show, taking out First Prize and Best in Show. With her confidence in her artistic abilities boosted by her success, she began to explore other options for tertiary study. “I thought if Ididn’tget the marks, I wouldn’tbe able to be a vet,” she said. “So, I looked at my other strengths – and that was my art.”

Gillett’s artistic endeavours did open the door to university for her,though that door did not lead directly to vet school. She was successful in obtaining a bursarywhich stipulated that it must be used for an arts program, and even though it

‘Ithought if I didn’t get the marks, I wouldn’t be able to be a vet,” she said. “So, I looked at my other strengths –and that was my art. ’

was not a large amount of money it was sufficient to cover the first couple of semesters of an undergraduate degree. “I was really interested in media and documentaries, and I wanted to find a way that I could be part of documentary filmmaking or TV, combining my artistic and creative side with animals. By that stage I’d done a little bit of commercial stuff, as I was doing some modelling and minor TV commercial appearances towards the end of high school, and I was trying to figure out how to blend everything I knew and liked together,” she said. “So, with the vision of wanting to do some sort of documentary that was conservation related or animal related, I chose a media studies degree.” Family finances were still tight, meaning a plane fare to Brisbane to start university was out of the question. Aged only 17, therefore, Gillett boarded a train and travelled for two days from Far North Queensland to Brisbane. “It was massive shock having to say goodbye to my mum, then hoping on a train and going to aplace in the country I had never been to before,” she said. “It was very daunting, scary even, when the train arrived at Roma Street Station in the middle of the night.” Gillett knew only one person in Brisbane – a student who had been a couple of years ahead of her at high school. “He had started studying architecture atuniversity in Brisbane and he had a room I could rent,” she explained. “I think he might have actually picked me up from the station, which was very helpful.”

The first year Gillett spent in Brisbane was tough going. She commenced her media studies degree at the University of Technology, but was battling depression and struggling to deal with the various issues affecting her family. Gillett quickly recognised the degree pathway she had embarked upon was not one she wanted to continue with, and the coursework was definitely not helping. “They put you to the test, examining and analysing all these really dark movies” she said. “None of that was really helping my mental health.” After a few months, Gillett found herself crying hysterically on the phone to her mother,desperately wanting to come home. With no money for a train fare, she used afriend’sconnections with a trucking company to make her way north: “I got in the cab with atruck driver I had never met and drove all the way back to Far North Queensland because Iwas so homesick.”

Despite the odds, Gillett made it through the first year of her media studies degree but began having conversations with people about how much she disliked the course, particularly because it did not allow her to express her artistic creativity the way she had hoped to. She was surprised to discover it was possible to swap to adifferent degree and university, one which might offer a pathway to the veterinary program she had always wanted to attend. After making some inquiries, Gillett spent her second year in Brisbane enrolled in a Bachelor of Science in Zoology degree at the University of Queensland. “It was at the St Lucia campus, which is just a breath of fresh air compared with the city – it’s an amazingly beautiful campus,” she said. “I started to settle: I found a better living arrangement, I got a job in a hospitality role that I really enjoyed, and I was able to excel in my course.” With a high grade-point average to her name, Gillett was allowed to transfer into the veterinary science degree the following year

“Although it took me two years of faffing around at university,Ifinally got into the degree I wanted, and I loved it,” Gillett said. “The vet school at St Lucia was a quaint little school, it had its own area within the bigger campus. You werestill connected to other

groups within the campus, but everyone within the course was really close.” Now that she was on the path she had always wished to be on, Gillett found life much easier and her mental health improved noticeably. Her cohort was comprised of around eighty students, with a good mix of males and females, and many of them – like her – came from different parts of Queensland, not just from Brisbane. Going out to the University’s country veterinary facility at Pinjarra Hills also provided her with a welcome surprise: “It reminded me of home a lot, because there were rolling green hills, and cattle and sheep, and all sorts of open areas – and there was a little koala population as well.”

Although Gillett loved the first few years of her veterinary course and was convinced she was on the road she was always meant to take, she was also plagued by a nagging feeling that neither small animal practice nor large animal practice was a good fit for her. “I didn’t really like learning about dogs and cats and cows, I didn’t want to learn about abattoirs, and I started wondering

the program. “We were based at the University of Pretoria in South Africa, and we would go out on different game capture experiences,” she said. “This was my first big adventure on my own, and it was amazing!”

Gillott was hooked. After returning to Brisbane, she was homesick for Africa – a feeling that was as weirdly unexpected as it was galvanising. “Irealised if I was going to make something different of my career other than becoming a dog/cat/horse/cow kind of vet, I needed to make something happen,” she said. “And I knew it would have to be something drastic.” She wrote to the University of Pretoria Veterinary School and asked whether she would be able to enrol in their school as an international student, and subsequently asked the University of Queensland whether she was able to defer her studies. Despite risking not being able to rejoin her vet science cohort at the University of Queensland when she returned, Gillett took a leap of faith and headed back to South Africa for a year, living on campus at the University of Pretoria and using as many of her contacts from the game capture course as possible to further her learning and career.

Within a few weeks of arriving in South Africa, Gillett had an opportunity to start working weekends at the DeWildt Cheetah and Wildlife Centre as a cheetah handler and tour guide, an experience that became a highlight of her year studying overseas. “They had a lot of cheetahs and wild dogs there, and I couldn’t believe how lucky I was to have a chance to work with these animals,” she said. “I was driving this massive, rusty bus full of tourists around the Centre, spruiking all the information I had learned about the animals they had in the facility, and feeling like a bit of an imposter.” The added benefit to working at the DeWildt Centre was that Gillett was able to observe and participate in any veterinary procedures and animal treatments that were taking place. “I learned so much, particularly about cheetah medicine and disease and African wild dogs,” she said, “and since they had another couple of facilities in South Africa I also got to travel around and visit these also.”

At the end of twelve months, Gillett’smoney had run out and she was forced to come back to Brisbane. "I was devastated,” she said, but she also felt fortunate to be able to rejoin her veterinary science degree with her original classmates at the University of Queensland, and subsequently graduated with first class honours in 2005. Over the course of the following decade, she returned to Africa five or six times to volunteer with vets and game capture crews she knew, cementing a strong sense of connection to the continent that endures to this day.

whether this would be what my vet career would be like,” she said. Recalling all the exotic animals she had been exposed to by her aunt on visits to the UK, Gillett began wondering how she could learnmoreabout such species, and identified that she wanted to learn more about birds, and wildlife, and animals more likely to be found in zoos than regular veterinary practices.

An opportunity to attend a game capture course in South Africa open only to vet students provided a major turning point in Gillett’s second year of veterinaryschool. She scraped together her savings and worked extra hours at her job and, with the supportof her grandmother from whom she borrowed some additional funds, successfully secured a place in

Gillett also spent a considerable amount of time in South East Asia during the final years of her university degree because she was in a long distance relationship with a veterinary student she had met at the University of Pretoria. He had also left South Africa, and ended up establishing veterinary practices in Macau, near Hong Kong. “His practice was verybusy,and I would assist regularly with consultations and surgical procedures” she explained. “Things work a little differently in Macau, and I was basically able to function as extra vet therewhich gave me abit of a window into exactly what I didn’t want to be involved in – which was small animal practice.” Not surprisingly,with Gillett and her partner clearly moving in different directions and living in different countries, the relationship did not last.

As Gillett continued to grapple with her aversion to so-called “regular” veterinary practice in the final year of her degree, she turned to

what she now knew was one of her greatest resources as she searched for the road that would suit her best – other people. One of Gillett’s university lecturers proved instrumental in inspiring her to follow her passion for African wildlife, which resulted in her completing her final year Honours project on tuberculosis in lions. Shortly before she graduated in 2005, the same lecturer suggested Gillett speak to the senior veterinarian working at the recently opened wildlife hospital at Australia Zoo to get some advice on how to get into the wildlife/zoo industry. That vet was Dr Jonathan Hanger, who had founded the conservation organisation Wildcare and also helped establish the wildlife hospital at Australia Zoo. After an unexpected and very informal interview, Gillett was invited to spend a few days working at the wildlife hospital in December 2005, not long after she had finished her degree. By January 2006, she had been offered a full-time position.

Looking back, Gillett is still astounded that the wildlife hospital took her on as a new graduate. “There were so many lucky stars that fell into place for me around that time,” she reflected. “They knew I was a new grad, but they also recognised I was passionate about working in the zoo/wildlife industry.” By this stage, Gillett also had her time in Africa, experiences in Macau and an externship she had completed at Taronga Zoo under her belt, which was probably more veterinary experience than other new graduates had. “Whatever it was, from my first day at the wildlife hospital I felt I had found my place and still feel that way today,” Gillett said. Even with the experience Gillett had gained prior to starting her working life as a vet, there was still much for her to learn, from basics tasks like learning drug dosages and prescribing, to applying her veterinary knowledge and skills to the multitude of Australian native and exotic species that were part of the zoo collection or had brought in from the wild for treatment.

“I remember being excited and feeling very comfortable when a cheetah from the zoo came over for treatment, but I had no idea what to do with kangaroos or koalas!” she said. “I had so much to learn about our native wildlife – not just from a medical perspective, but knowing all the different anatomy, biology and normal versus abnormal behaviours in such a vast variety of native species.” Gillett appreciated Hangar’s mentorship when she started out in the industry, and recognises how lucky she was to have an opportunity to learnfrom someone with his skill level in a world class facility treating nothing but non-domestic animals. “He could be a pretty up-front teacher, and I still remember the hard lessons I learned and the direct advice I received –which sometimes brought me to tears – but he constantly reinforced going back to first principles with my cases, and helped me immensely with improving my diagnostic, surgical and pathological skills,” she said. “He shaped me as a vet and without his advice and guidance Iwouldn’tbe the vet that I am today,and for that I am extremely grateful.”

Since 2006, Gillett’s familiarity with and interest in Australian native species has flourished, to the point where she is now more comfortable treating Australian wildlife than exotic species. Her main area of expertise is now koala medicine. “Koala medicine, surgeryand conservation is my passion and is probably what I have published or presented most on in my career,” she said. That said, Gillett has continued to build on her wildlife knowledge through her role as research coordinator the Australia Zoo wildlife hospital, and had the opportunity to pursue a PhD on sea snake disease and pathology. “Right from the first day I started at the hospital we would have

sick sea snakes coming in, and there was no literature or reference material on them and they had all sorts of pathologies nobody understood,” she said. “It was so fascinating to me and my interest in sea snakes eventually rolled into a PhD. I started with the basics like anatomy reviews and reference ranges, conducted a field trip where I spent five nights out on a boat with two colleagues fishing sea snakes out of a river mouth with crocodiles all around us, and had a heap of data to analyse.” For five years, Gillett worked full time and was doing her PhD part time, before she dropped down to working part time at the wildlife hospital to finish the degree. “It was a pretty hard slog,” she said, “but a rewarding process to come out with adoctorate at the end.”

An unexpected upside to Gillett’s PhD was that she rediscovered her love of art. “Towards the end of my PhD I was getting so overwhelmed with data analysis, running scenarios and staring at computer screens, along with the grind of working. It was then I realised I needed some sort of outlet,” she said. “I wanted to start making art again, but I was terrified to try to even put pen to paper to be honest. I thought Ididn’t know how to draw any more as it had been more than 15 years since I had done any

well as cards, calendars and other products, and also takes commissions for original works. “The most rewarding thing about being an artist is hearing and seeing the impact that a piece of my art can have on a person,” Gillett said. “I will hear people comment on things like, ‘I never knew the animal’s eyes looked like that’ or, ‘I felt like I could reach out and touch its fur’ or‘I wonder what that animal was thinking’–that’s when I know I have been able to reach them with my drawings and that they will forever have a connection with that drawing and those animals. That is the best feeling in the world to me, and my absolute aim with my art.”

Rediscovering her art has brought Gillett to a place of perspective and balance. At this stage of her journey, she is still working a couple of days a week at the Australia Zoo Wildlife Hospital, and is the veterinarian for the Koala Ecology Group at the University of Queensland. She also works casually with Endeavour Veterinary Ecology and also works on private consultancy field projects (often with koalas) with her partner Sean. Then, when time permits –or simply when she feels the need to – Gillett retreats to her art studio and works on her art projects. “I’m an ambitious person and have worked hard to get to a point in my career where I am content, but I also know I don’t want the pressure of art being a job for me,” she said. “I have finally found a really good balance, splitting my time between different areas of interest and expertise and mixing up the elements of creating art and working as a vet. Also, I am now a mother to a little girl, which presents its own challenges, especially with respect to time, but my daughter gets to come along on many of our field trips and I absolutely love having her in my life: she is the best part.”

drawing.” Gillett’s partner Sean, an ecologist who also works with wildlife and focuses particularly on koalas, provided her with the encouragement she needed to give herself a chance to create, drawing on the animals she treated at the wildlife hospital for inspiration. Talent, as it turns out, is something that lies dormant until it is reawakened. “It slowly came back to me, over the course of a few months,” Gillett said. “I realised that art is a really good outlet for me, and it’s something I have such a personal connection with: I am not interested in drawing apictureof an animal from a magazine, it’s about creating a drawing of an animal that I have a connection with and bringing its personality or personal traits to life. I can do this best when I know the background behind the animal or been personally involved in its care.”

Gillett’s images of Australian native animals, which she does primarily in charcoals and pastels, are not only beautiful but are also incredibly lifelike. A few years ago, she successfully obtained membership to the Queensland Wildlife Artist Society.The Society usually hosts four exhibitions a year, and Gillett described having her artdisplayed amongst others as a “very proud moment”. She launched her website, www.ambergillettart.com.au, and uses that platform along with social media to sell her original drawings, prints of her drawings, as

Gillett may have taken a long and winding road from her childhood on the Atherton Tablelands to the fulfilling space she occupies today,but she had never lost sight of what is most important to her along the way. She loves that her artwork enables people to see the animals she cares for from a different perspective. “The animals I draw arethe shining stars in our wildlife world, the ones that really need our attention,” she said. “Bringing beauty through art has the potential to be a powerful way to show people how special these species are and make lifelong connections with them, because people will protect what they care about, and Iwant more than anything for them to care about the wildlife I bring to life in drawings.” What makes Gillett’sartso poignant, of course, is that the animals who inspireher artworks are often animals she is studying in the wild or her patients at the hospital. “The most rewarding part about being a vet is bringing a patient back from the brink and knowing your intervention has given them a second chance at life,” she said. “Seeing a critically ill patient you’ve been working to recover starttobreathe normally, or hearing their heart clearly again gives any vet a feeling of accomplishment, satisfaction and relief, because healing animals is what we do this job for. The cherry on top for me is getting to release those animals back into the wild and, in some cases, to follow their journey through life.”

Amber Gillett’s artworks can be viewed and purchased at www.ambergillettart.com.au. She can also be followed on Facebook and Instagram. If aspects of this article have raised concerns for you, please contact Lifeline (13 11 44), Beyond Blue (1300 224 636) or the Domestic Violence Helpline (1800 737 732).

■ JAI HUMEL

How to understand them? Areview of emotional indicators in horses

Stabled horses often experience negative emotions due to the inappropriate living conditions imposed by humans. However, identifying what emotions horses experience and what can trigger positive and negative emotions in stabled horses can be challenging. In this article we present a brief history of the study of emotions and models that explain emotions from a scientific point of view and the physiological bases and functions of emotions. Wethen review and discuss physiological and behavioral indicators and cognitive bias tests developed to assess emotions in horses. Hormone concentrations, body temperature, the position of the ears, facial expressions and behaviors, such as approach and avoidance behaviors, can provide valuable information about emotional states in horses. The cognitive bias paradigm is a recent and robust tool to assess emotions in horses. Knowing how to evaluate the intensity and frequency of an individual's emotions can guide horse owners and caretakers to identify practices and activities that should be stimulated, avoided or even banned from the individual's life, in favor of a life worth living. The development and validation of novel indicators of emotions considering positive and negative contexts can help in these actions.

Letícia Santos Maurício1,Denise Pereira Leme1,Maria José Hötzel2

JEquine Vet Sci. 2023 Jul; 126:104249. doi: 10.1016/j.jevs.2023.104249.

1Laboratory of Applied Ethology and Animal Welfare, Department of Animal Science and Rural Development, Federal University of Santa Catarina, Florianópolis, SC, Brazil.

2Laboratory of Applied Ethology and Animal Welfare, Department of Animal Science and Rural Development, Federal University of Santa Catarina, Florianópolis, SC, Brazil. Electronic address: maria.j.hotzel@ufsc.br.

Investigating the relationship between inbreeding and life expectancy in dogs: mongrels live longer than pure breeds

This study aimed to investigate the establishment of relationship between inbreeding and life expectancy in dogs. A dataset of N = 30,563 dogs sourced from the VetCompass™ Program, UK was made available by the Royal Veterinary College, University of London, containing information about breed and longevity and was subject to survival analysis. A Cox regression proportional hazards model was used to differentiate survivability in three groups of dogs (mongrel, cross-bred and pure breed). The model was found highly significant (p <0.001) and we found that mongrel dog had the highest life expectancy, followed by cross-bred dogs with only one purebred ancestor and purebred dogs had the lowest life expectancy.A second Cox regression was also found highly significant (p <0.001) differentiating the lifespan of different dog breed and correlating positively the hazardratio and the Genetic Illness Severity Index for Dogs (GISID). The results show that survivability is higher in mongrel dogs followed by cross-bred with one of the ancestor only as a pure breed, and purebreed dog have the highest morbidity level. Higher morbidity is associated with higher GISID scores, and therefore, higher inbreeding coefficients. These findings have important implications for dog breeders, owners, and animal welfareorganizations.

Fernando Mata1,Andreia Mata2

PeerJ. 2023 Jul 19;11: e15718.doi: 10.7717/peerj.15718.

1Center for Research and Development in Agrifood Systems and Sustainability,Instituto Politécnico de Viana do Castelo, Viana do Castelo, Portugal.

2Department of Biosciences, Durham University, Durham, United Kingdom.

Ear health and quality of life in pet rabbits of differing ear conformations: A UK survey of owner-reported signalment risk factors and effects on rabbit welfare and behaviour

The impacts of ear disease on animal welfare and behaviour are little documented. Ear disease may be common in rabbits, but difficult to recognise, and lop-ears have previously been indicated as a risk factor for ear disease. We aimed to better understand the range of ear conditions in pet rabbits, signalment risk factors, and impacts on welfareand behaviour. Through an online questionnaire, we investigated owner-reported signalment, veterinary diagnosis of ear conditions, impaired hearing, and ear pain for UK pet rabbits. Relationships between ear condition measures and ear conformation, quality of life, and behaviour were analysed using logistic regression. Of 551 valid responses, 28.5 per cent of rabbits reportedly had experienced ear conditions; 21.2 per cent diagnosed or mentioned by vets, with otitis and excess cerumen most common. Approximately 25 per cent of lop-eared rabbits had ear conditions indicated by a vet versus 10 per cent of erect-eared rabbits. Lop-eared, half-lop, and older rabbits weremost at risk (P<0.050). Rabbits reported as showing ear pain responses had reduced owner-reported quality of life compared with other rabbits (P<0.050). Rabbits with ear problems were less likely to be responsive to relevant sounds, and performed binky behaviour (joy jumps) less frequently, than rabbits without such issues. Understanding prevalence and risk factors for ear conditions is critical to improving welfare standards across this widely owned pet species. The findings suggest that improved recognition and treatment of ear conditions, and avoiding breeding from rabbits with early signs, or a family history, of ear disease are necessary to help combat this animal welfare issue.

Benedict D Chivers1,Melissa R D Keeler1,Charlotte C Burn1 PLoS One. 2023 Jul 19;18(7): e0285372.doi: 10.1371/journal.pone.0285372.

1Animal Welfare Science and Ethics, Royal Veterinary College, Hertfordshire, United Kingdom. Free PMC article

Rescued cats prefer to scratch fabrics commonly used to cover upholstered furniture

Preference responses of cats for scratching fabrics commonly used on furniture were evaluated during four consecutive days in three Non-Governmental Organizations (NGOs) that rescue companion animals. Cats were grouped and their choices were registered at a group level (no individual identification). Daily choices for chenille, suede, synthetic leather, or waterproof grosgrain fabrics were evaluated for the cats' groups. A preference for chenille and non-preference for synthetic leather and waterproof grosgrain was found, independent of the NGO. In conclusion, although not using chenille does not assure that cats stop scratching furnitureespecially if no other option to scratch is available - synthetic leather and waterproof grosgrain seem to be less attractive fabrics for these animals. Further studies are needed to investigate whether these findings apply to cats in a home scenario, when just one or a few individuals are usually present and only one type of fabric covering furniture is commonly available. Although we did not investigate the effect of providing scratching posts for these animals, we recommend such posts are available in the environment as scratching behavior is important to cats. To page 30

When ‘It’s not that bad!’ makes it worse

All too often, veterinary practice owners only make the decision to put their practice for sale after several years of declining revenue and profit.

■ Why is it that so many vet practice owners wait so long?

■ They know that the declining revenue and profit will be depreciating their asset.

■ Why is it that when they see this happening, they wait?

■ It isn’t because they have a master plan to turn it around.

■ It isn’t because they see market forces turning in their favour.

■ It isn’t because they are blissfully happy in ownership….

■ It isn’t because they love the practice more than they love their financial wellbeing….

All too often it is because of a phenomenon known as the Region-Beta Paradox

The term Region-Beta Paradox was first illustrated by Daniel Gilbert, a Professor of Psychology atHarvard University, in a paper titled The Peculiar Longevity of Things Not So Bad (www.scn.ucla.edu/ pdf/PeculiarLongevity.pdf). The Region-Beta Paradox is a cognitive bias that explains how:

People will get stuck in a bad or declining set of circumstances because it isn’t deemed bad enough to do anything about.

AND

This often means that people will get better outcomes from a worse set of circumstances, simply because a worse set of circumstances will be acted upon faster.

Once you hear about this paradox, examples of it are everywhere:

■ If you would see a doctor if your discomfort was greater than five out of 10 but would put up with the discomfort if it was two out of 10. (You could be recovering more quickly from a 5/10 injury than a 2/10 injury.)

■ People working at jobs that are OK (but not good) are unlikely to quit to seek out better employment, but people with bad jobs are. Thus, people with bad jobs are much more likely to get to good jobs faster.

■ People in OK (but unfulfilling) relationships are less likely to break up and seek better relationships than people with bad relationships. And, when a business owner holds on to a business that is in decline and not making them happy, they would often get a better result (financially and emotionally) the faster they act.

How can we overcome the Region-Beta Paradox in business ownership?

The first step is to recognise if you are in it.

■ We need to be honest with ourselves when the business is in decline and will be worth less in a few years. An easy indicator of this is when revenue is stable or declining over time. (If your revenue is stable over time when expenses are going up, this is an indicator of a business in decline.)

■ Weneed to be honest when we don’t have an effective strategy (or the energy) to do anything about it.

■ We need to be honest about whether running the practice is making us happy, or if we are simply used to it and have become comfortably numb.

If, upon analysis, we discover that the business is in decline and owning it isn’t making us happy,

but things haven’t gotten bad enough yet for us to do anything about it… then we are in the Region-Beta Paradox.

The next step is to question why we are stuck in this paradox

We need to ask ourselves: “Why, if I admit that things are currently not good, and if I don’t think that things are getting better, do I wait for things to get worse before I take action?”

Surely we should all be striving to get to good as fast as we can, rather than staying where we are mildly deteriorating and complacent.

The final step is to take action

This is not always easy. It often involves stepping out of the ambivalent, mediocre but declining business condition that you are in, before it reaches the threshold of bad:

■ Reinvesting time, effort and energy into your business to turn it around or

■ Selling the business before it depreciates further, and reallocating your time: - towards other more rewarding business interests -towards interests that make you happier -working as a veterinarian without being a business owner -phasing into retirement.

The lesson to learn from the Region-Beta Paradox is that the thing that is usually standing in the way of getting to good,in business and in life, isn’t bad We will usually spring into action and overcome bad.

The thing standing in the way of getting to a good result is usually our amazing ability to put up with a deteriorating set of circumstances for far too long.

Simon Palmer is the Managing Director of Practice Sale Search, Australia’s largest vet practice brokerage. If you’d like more information on practice sales or want tohave a confidential discussion about your practice’s circumstances, email Simon Palmer at info@practicesalesearch. com.au or call 1300 282 042.

Management

ADAYINTHELIFEOF AMANDAMcLUNE:

MOBILEACUPUNCTURE &CHIROPRACTICVET

This month ADay in the Life heads to Brisbane, where Amanda McLune works as a mobile veterinarian specialising in acupuncture and chiropractic treatments for small animals. McLune is also a Senior Veterinary Officer for the Australian Defence Force, and in that capacity oversees the care of military working dogs as well as providing veterinary assistance to remote Aboriginal communities and emergency care for animals affected by natural disasters, such as the 2020 bushfires on Kangaroo Island. Most of the time, however, a typical day in McLune’s not so typical veterinary practice sees her travelling all over Brisbane treating four legged patients with a combination of conventional and integrative veterinarymedicine.

From the age of 12, McLune knew she wanted to be a veterinarian. She began her career on the outskirts of Brisbane in 2004 after graduating from the University of Queensland and started working in mixed practice. Her initial interest was in horse surgery, and she completed an internship inequine medicine and surgery before turning her attention to biomechanical and Chinese medicine. McLune subsequently completed post-graduation studies in animal chiropractic, veterinary acupuncture and clinical rehabilitation, and is currently finishing a Graduate Diploma in Veterinary Chinese Medicine. After fifteen years of working in veterinary clinics, McLune started her mobile practice, VETMACS, and now incorporates Western medicine, Eastern and allied health modalities in her veterinary work, and focuses solely on small animal care.

McLune’s workday usually begins at around 7:00am, when she logs onto her computer to prepare for the day’s consultations. Her first appointment is usually scheduled for 8:00am, and she sees anywhere between five and twelve patients in a workday – depending on where they arelocated. As a mobile veterinarian, McLune spends a significant portion of each day driving from one appointment to the next. “My travel area is approximately 40 kilometres to the north, 30 kilometres to the south, 30 kilometres to the west and 25 kilometres to the east,” she said. “I try to stick to heading in one direction per day, but it doesn’t always work out that easily.On an average day I will accumulate two to three hours of driving time.”

Appointments with McLune run longer than a typical face to face consultation in a veterinary clinic, because acupuncture and chiropractic treatments take between 45 minutes and an hour to complete. These treatments are also different from regular clinical appointments because they take place in the pet owner’s home. “Pets are often different in their home environment than theyare in a clinic: mostly they are more relaxed,” McLune observed. “It is also helpful to see how they live so I can offer specifically tailored advice regarding things like injury management.” That said, performing consultations in pet owner’s homes – without all the equipment and assistance that come with a clinical setting – can also bechallenging. “I mostly work alone without an assistant, so it can be difficult performing procedures such as blood tests when an uncooperative pet needs to be restrained,” she said.

McLune finds mobile practice rewarding –particularly since she has combined conventional Western veterinary knowledge with integrative techniques and Chinese medicine. Examining patients using both systems of medicine has provided McLune with a broader, more multifaceted point of view when diagnosing and treating animals. “Since incorporating integrative medicine into my treatments I have been amazed at how much better I have been able to care for my patients,” McLune said. “It has changed my perspective on what an acceptable quality of life can be.” In particular, McLune has noticed massive

benefits for patients suffering from arthritis, which has been rewarding for her as well as for the pets she treats and their owners.

“Acupuncture has made a huge improvement in quality of life over what I had historically been able to achieve with conventional medicine,” she said. “Owners are hugely appreciative of being offered more treatment options.”

McLune’s day usually finishes sometime between 5:00pm and 7:00pm, depending on how far afield she has had to travel to treat her clients. “I usually finish up with some paperwork or inventory management tasks,” she said. “As a small business owner it can be challenging managing all the ordering, accounting and client communication on

topof providing the professional services.” Even so, McLune is more than happy to do the work –particularly because she plans to expand her business in the future. “Next year I will be opening a bricks and mortar practice with an emphasis on rehabilitation, but I still intend to keep the mobile practice going. I really enjoy what I do!”

And now, as always, we conclude our Day in the Life of Amanda McLune with our Creature Questions:

Do you currently have any pets?

Ilive vicariously through my clients’ pets! This will change once our house renovations are complete. Once the renovations are done, Iwill get a mini schnauzer for myself and a cat for hubby.

What is your favourite animal?

Horses are my favourite animals. They are so trusting and amazingly gentle for such large animals.

If you could transform into any animal, what would you be and why?

I’d probably be a dog. They get to go on so many adventures with us as their human companions without the stress of taxes or the threat of unemployment. Their joyful spirit reminds us what is most important in life.

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Evaluation of plasma nucleosome concentrations in dogs with a variety of common cancers and in healthy dogs

M. Wilson-Robles1,2,T.

Abstract

Background: Cell free DNA, in the form of nucleosomes, is released into circulation during apoptosis and necrosis in a variety of diseases. They are small fragments of chromosomes that are composed of DNA wrapped around a histone core made of four duplicate histone proteins forming an octamer. The nucleosome compartment is a relatively uninvestigated area of circulating tumor biomarkers in dogs. The objectives of this study were to quantify and better characterize nucleosome concentrations in 528 dogs with various common malignancies and compare them to 134 healthy dogs.

Results: The sensitivity of increased circulating nucleosome concentrations for the detection of cancer in all dogs

Background

Biomarkers for the early detection of cancer have revolutionized cancer screening in healthy and high-risk human populations. These cancer screening tests enable enhanced opportunities for earlier treatment and higher cure rates in a variety of cancers [1] . Tumour specific markers such as prostate-specific antigen [2], carcinoembryonic antigen [3], CA-15.3, and CA-27.29 have allowed for quick, non-invasive and inexpensive screening in large at risk populations in human medicine. Unfortunately, liquid biopsy techniques are rare in veterinary medicine.

Many platforms have been utilized to assess blood-based biomarkers for cancer Traditionally, enzyme-linked immunosorbent assays (ELISAs) or polymerase chain reactions (PCR) have been utilized to detect specific biomarkers. However, cancers are multifactorial and veryfew cancers share biomarker expression between them or even within the same histology. Whole genome sequencing can cast a wider net to detect a variety of biomarkers in the blood, but this approach is costly and time consuming. ELISAs directed at acancer surrogate, such as nucleosomes, can provide a rapid, cost effective and widely applicable approach for biomarker detection in the blood of both humans and veterinarypatients.

Nucleosomes are small fragments of chromosomes released into the blood during cell death or white blood cell activation. These fragments consist of a histone octamer core with a short segment of DNA wrapped around it. Nucleosomes have demonstrated utility as epigenetic biomarkers for the detection and monitoring of a variety of human cancers including pancreatic, lung, and colorectal cancer [4–7]. Furthermore, plasma nucleosome concentrations have been shown to be predictive of outcome in patients with breast cancer. A multivariate analysis of 92 patients with breast cancer showed preoperative plasma nucleosome concentrations were as consistent as hormone receptor (HER2) status, lymph node status and tumour grade in predicting disease free survival [8]. Plasma nucleosome levels can also predict response to therapy in patients with non-small cell lung cancer (NSCLC). In a study of 134 patients with advanced NSCLC, treated with chemotherapy,those with significant decreases in nucleosome concentrations after their first cycle of chemotherapy had significant improvement on imaging after their 3rd cycle. Those with insufficient decreases in plasma nucleosome concentrations were more likely to have stable or progressive disease and a significantly shorter progression free survival [9]. Circulating plasma nucleosomes have also been used to identify genome- and exome-wide cancer specific mutations as well as longitudinal

was 49.8% with a specifcity of 97% with an area under the curve of 68.74%. The top 4 malignancies detected by the test included lymphoma, hemangiosarcoma, histiocytic sarcoma and malignant melanoma. The malignancies least likely to be detected were soft tissue sarcomas, osteosarcoma and mast cell tumors.

Conclusions: Avariety of tumor types may cause increased nucleosome concentrations in dogs. Tumors of hematopoietic origin aremost likely to cause elevations and local tumors such as soft tissue sarcomas are least likely to cause elevations in plasma nucleosome concentrations.

Keywords: Nucleosome, Histone, H3.1, Canine, Cancer, Osteosarcoma, Mast cell tumor, Histiocytic sarcoma, Melanoma, Soft tissue sarcoma.

changes throughout the course of treatment that can be used to capture clonal evolution and identify mechanisms of resistance [10, 11].

Plasma nucleosome concentrations are increased in a variety of diseases, including cancer. Letendre et al. published some of the earliest work describing the use of nucleosomes in dogs with trauma and sepsis in 2018 demonstrating that plasma nucleosome concentrations were positively correlated with a worse outcome in dogs with sepsis and trauma whereas cell free DNA did not demonstration a correlation [12, 13]. Additionally, similar to humans, increased plasma nucleosomes have been detected in cases of autoimmune disease and severe inflammation [12, 14–18]. Another study evaluating plasma nucleosome concentrations in dogs with acute and chronic gastrointestinal diseases found no statistical differences in nucleosome concentrations between the groups. However, dogs with acute and chronic GI disease tended to have slightly higher concentrations than the healthy control dogs. The one dog in this study with gastrointestinal lymphoma had a 10–20 fold increase in plasma nucleosome concentrations compared to the other groups [18].

Few studies exist defining the plasma nucleosome compartment in canine cancer. Recently, our group published data demonstrating increased plasma nucleosome concentrations in dogs with lymphoma and hemangiosarcoma [19–21]. In these studies, increased plasma nucleosome concentrations were found in the majority of cases with cancer, even in early stages of disease. The assay correctly identified 82 per cent of hemangiosarcoma cases and 74 per cent of lymphoma cases with 97 per cent specificity. For dogs with hemangiosarcoma the assay was able to detect 67 per cent of dogs with stage I, 76 per cent of dogs with stage II and 90 per cent of dogs with stage III disease. The assay was also able to distinguish LSA patients from healthy patients in 63 per cent of stage I patients, 14.3 per cent of stage II patients, 75.7 per cent of stage III patients, 81.6 per cent of stage IV patients, and 81.8 per cent of stage V patients. Performance was also evaluated by immunophenotype and the threshold could distinguish LSA patients from healthy patients in 95.3 per cent of B-cell LSA and 55.6 per cent of T-cell LSA.

The goal of the current study was to expand upon that initial study to evaluate circulating nucleosome concentrations in dogs with a variety of common cancers. Understanding which cancer types are associated with increased nucleosome levels will not only expand the use of nucleosome screening but will also increase our understanding of nucleosomes and their role in cancer development.

Results

Atotal of 662 dogs were included in this study (528 dogs with cancer and 134 healthy dogs). The most common cancers evaluated included lymphoma (LSA; n =126), Hemangiosarcoma (HSA; n =77), osteosarcoma (OSA; n =49), soft tissue sarcoma (STS; 51), malignant melanoma (n =49), mast cell tumours (MCT; n =126) and histiocytic sarcoma (n =26) (Fig. 1). A variety of carcinomas (mammary (n =1), pulmonary (n = 3), hepatocellular (n =1), squamous cell (n =3), urothelial cell (n =3) and anal sac carcinomas (n =3)) as well as a few miscellaneous tumours (multiple myeloma (n =1), acute leukemias (n =2), insulinoma (n =1), nasal chondrosarcoma (n =1), sertoli cell tumour (n =1) and others) were also evaluated. These cases are described here however, due to the low case numbers of any specific histology,they are not included in the statistical analyses reported here. Of the 528 dogs with cancer, 244 were spayed females, 15 were intact females, 237 were neutered males and 30 were intact males. Gender was unknown for 2 of the dogs. These dogs ranged in age from 1 – 19 years (median 9years, mean 9.06 years) and ranged in weight from 5 – 74.5 kg (median 30.9 kg, mean 29.63 kg). The most commonly represented breeds included mixed breed dogs (n =126), Labrador retriever (n =58), golden retriever (n =53) and boxers (n =19).

Atotal of 134 healthy dogs were recruited for this study ranging in age from 10 months to 14 years (median 6 years). There were 61 spayed females, 4 intact females, 66 neutered males and 3intact males. The most common breeds represented were mixed breed dogs (n = 28), Labrador retrievers (n =15) and Australian cattle dogs (n =10). Dogs were determined to be healthy based on results from a client questionnaire as well as a physical exam by the attending veterinarian. The median nucleosome concentration for all healthy dogs was 31.1 ng/mL (mean 32.07 ng/mL, SEM 1.118). For a specificity of 100 per cent the cut off for the healthy range was set at 67.5 ng/mL (nucleosome range for all healthy dogs was 6.33 – 67.42 ng/mL). Neither age, neuter status, gender or size had an effect on nucleosome concentrations in either cohort of dogs[21] .

When all cancer cases were considered together in comparison to healthy dogs the sensitivity of this assay was 49.8 per cent with a specificity of 97 per cent (Fig. 1). A receiver operator characteristic (ROC) curve was generated and the AUC was determined to be 68.74 per cent (Fig. 2). The top 4 malignancies detected by the test included lymphoma, hemangiosarcoma, histiocytic sarcoma and malignant melanoma. The malignancies least likely to be detected

H.

Bygott3,T. K. Kelly2,T. M. Miller1,P. Miller1,M. Matsushita1,J. Terrell2,M. Bougoussa4 and T. Butera2

using this assay were soft tissue sarcomas, osteosarcomas and mast cell tumours.

For the purposes of group comparisons, a single specificity of 97 per cent was chosen and the sensitivity was then calculated using this parameter. The cancers with the highest sensitivities were lymphoma (76.98 per cent), hemangiosarcoma (81.82 per cent), visceral histiocytic sarcoma (61.9 per cent) and malignant oral melanoma (50 per cent). The cancers with the lowest sensitivity were cutaneous melanoma (0per cent), Mast cell tumours (19.05 per cent all grades, grade 1: 33.33 per cent, grade 2: 11.49 percent and grade 3: 34.62 per cent) and primary bone histiocytic sarcomas (20 per cent) (Table 1). When considering the plasma nucleosome concentrations of dogs with a variety of carcinomas, eight of the sixteen cases had increased levels at the time of diagnosis. Two out of three of the anal sac apocrine gland adenocarcinomas, two of the three urothelial cell carcinoma cases and two of the 5 had sinonasal/oral carcinomas had increased plasma nucleosome concentrations at the time of diagnosis. The two dogs with increased levels were both diagnosed with oral squamous cell carcinomas. There were 3 dogs in this group with primary lung carcinomas, all of which had increased nucleosome concentrations and one each with mammarycarcinoma and hepatocellular carcinoma that did not have increased nucleosome concentrations (Fig. 3).

Several other tumours were included in this study as well. Both dogs with acute leukemia had increased plasma nucleosome concentrations (lymphoblastic leukemia – 262.8 ng/mL, myeloblastic leukemia 423.5 ng/ mL). We also saw increased plasma nucleosome concentrations in a dog with a sertoli cell tumour (118 ng/ mL), a dog with a pharyngeal sarcoma (135.4 ng/mL), a dog with nasal chondrosarcoma (109.25 ng/mL) and a dog with multiple myeloma (71.7 ng/mL). There were three cases that did not have increased plasma nucleosome concentrations including a dog with an undifferentiated splenic sarcoma (16.4 ng/mL), a dog with insulinoma (8.5 ng/mL) and a dog with multilobular osteochondrosarcoma (20.3 ng/mL).

Detailed evaluations of plasma nucleosome concentrations in dogs with lymphoma and hemangiosarcoma by stage, location or phenotype have been described elsewhere [20, 21]. For the cases with melanoma there were two main groups. The first represents cutaneous melanoma of haired skin which are often considered benign (n =7) and those in the oral cavity (n =42) which are often malignant. The median plasma nucleosome concentration for the dogs with cutaneous melanoma was 24.8 ng/mL (mean 27.6 ng/mL, range 7.3–43.9) and the median size of these tumours was 3 cm (mean 4.7 cm, range 1.8–12 cm) (Fig. 3). The median plasma nucleosome concentration for

Fig. 1. Box and whisker plot representing the various common cancers relative to the healthy cases. The y axis represents the plasma concentration of H3.1 nucleosomes in ng/mL and the x axis represents the different malignancies evaluated. (LSA- lymphoma, HSA- hemangiosarcoma, OSA- osteosarcoma, STS- soft tissue sarcoma, MCT- mast cell tumor, Hist Sarc- histiocytic sarcoma)

dogs with oral melanoma was 60.0 ng/mL (mean 130.422, range 14.0–1234.5 ng/mL) and the median size of these tumours was 4 cm (mean 4.5 ng/mL, range 1.5–15 cm) (Fig. 3). While the highest nucleosome concentration was seen in the largest tumour (1234.5 ng/mL, 15 cm), size of the lesion was not associated with an increased nucleosome concentration for those cases for which size was available. Mitotic index was not available for evaluation.

Mast cell tumours were divided by recorded grade. All tumours in the DCTD biobank were categorised according to the Patnaik 3 tier grading scheme. Grade 3 tumours had the highest median and mean plasma nucleosome concentrations (38.2 and 149.7 ng/mL respectively) followed by grade 1 tumours. Grade 2 tumours had the lowest nucleosome concentrations of the group (Fig. 4, Table 2). When considering low grade mast cell tumours along with those of unknown grade (n =4), the grade 3 mast cell tumours had a significantly higher nucleosome concentration than both the low grade tumour cases and the healthy dogs (p = 0.005) (Table 3, Fig. 5). Tumour size and stage of disease was not available for comparison. Follow up information regarding overall survival was not available for the dogs with increased plasma nucleosome concentrations.

Overall, visceral histiocytic sarcomas as a group had increased plasma nucleosome concentrations similar to lymphoma and hemangiosarcoma. Those cases involving the spleen had the highest median plasma nucleosome concentrations. Histiocytic sarcomas involving the bone had the lowest median plasma nucleosome concentrations (Fig. 6) [21]. Stage and size of

Fig. 2. ROC curve demonstrating the AUC of 68.74% for all cancers when compared to healthy animals

Fig. 3. Graphic representation of plasma nucleosome concentrations in a variety of canine carcinoma cases. The green dotted line represents the normal cut off value of 67.4 ng/mL. Eight of the 16 carcinoma cases had increased plasma nucleosome concentrations. AGASACA- Apocrine gland anal sac adenocarcinoma, UCC- Urothelial Cell Carcinoma, Ca- Carcinoma, HCC- hepatocellular carcinoma

the tumour was not available for comparison. Of the 50 soft tissue sarcomas evaluated in the study, 15 had increased plasma nucleosome concentrations. Six of these 15 cases were located in the skin/subcutis. Five were primary splenic sarcomas (non-hemangiosarcoma). Two were malignant peripheral nerve sheath tumours. One was ocular, one was primary renal and two had no primary site listed. Tumour size, grade and stage were not available for comparison.

Of the 50 STS cases, 18 were primary skin/ subcutis tumours with 33 per cent (6/18) having increased plasma nucleosome concentrations. Ten of the cases were primary muscle STS and none of them had increased plasma nucleosome concentrations. Seven of the cases were primar y splenic non-hemangiosarcoma sarcomas with 71.4 per cent (5/7) having

increased plasma nucleosome concentrations. Five were primary bone non-osteosarcoma sarcomas and none of these cases had increased plasma nucleosome concentrations. There was one case each of primaryrenal, auricular and ocular sarcomas. Seven of the STS cases had no reported location.

Just under 35 per cent (17/49) of the osteosarcoma cases demonstrated increased plasma nucleosome concentrations at diagnosis. Interestingly,the dog with the highest plasma nucleosome concentration in the osteosarcoma group (446 ng/mL) was the one dog that presented with metastatic disease at diagnosis (Tables 1 and 2). When evaluated based on location, there were 9 cases with primary femur OS, 33 per cent (3/9) had increased plasma nucleosome concentrations. One of these dogs was the one with metastatic disease. Sixteen were primary humeral OS cases and 37.5 per cent (6/16) had increased plasma nucleosome concentrations. Eleven cases had primary radius OS and only one (9 per cent) had increased plasma nucleosome concentrations. Eleven cases had primary tibial OS and 4 (36 per cent) had increased plasma nucleosome concentrations.

Discussion

Nucleosome levels were evaluated in plasma samples from patients with seven of the most common canine cancers. The dogs with the most frequent elevations in plasma nucleosome concentrations were dogs with hemangiosarcoma and lymphoma, consistent with previous publications [20, 21]. Other cases with frequent elevations in plasma nucleosome concentrations include those with histiocytic sarcoma and oral malignant melanoma.

In this cohort, the H3.1 ELISA was able to correctly identify 174 or 229 cases of systemic cancer (76 per cent) including lymphoma, hemangiosarcoma and histiocytic sarcoma. Overall, the test was able to correctly identify 49.8 per cent of all of the cancers evaluated in this study.This compares favorably with other similar tests in the veterinary and human space.

In the PATHFINDER study published by GRAIL, the Galleri test had a positive predictive value of 49 per cent of the 50 cancers studied in humans [23] . In the veterinary space, the CANDID study was able to detect 54.7 per cent of all the cancers they studied in that cohort and 85.4 per cent of lymphoma, hemanogiosarcoma and osteosarcoma cases [24] .

Plasma nucleosome concentrations are not specific for cancer.In humans, a variety of other diseases have been shown to cause increases in plasma nucleosome concentrations such as diabetes mellitus type 2, pediatric acute respiratory

distress syndrome, chronic kidney disease, pancreatitis, trauma, and COVID19 infections among other diseases [25–27] [26, 28–30]. Indogs, increased plasma nucleosome concentrations have been noted in dogs with sepsis, after trauma and in cases of hemolytic anemia [12–15, 31] . Because these diseases are often associated with inflammation and frequent cell death, it is not surprising that plasma nucleosome concentrations are increased in these cases. Very little has been published in the literature regarding elevated plasma levels of nucleosomes in diseases other than trauma or inflammation in canines. Our group is currently working to evaluate many other common chronic and acute diseases in canines.

Not surprisingly, the majority of dogs with visceral disease had increased plasma nucleosome concentrations. However, only one of the cases with primary osseous histiocytic sarcoma had increased plasma nucleosome concentrations. This is in contrast to the fact that 100 per cent of primaryosseous hemangiosarcomas had increased plasma nucleosome concentrations in one study [21]. In this study, only 34.7 per cent of dogs with osteosarcoma had increased plasma nucleosome concentrations at diagnosis as well. Though osteosarcoma in dogs is often an aggressive tumour with a rapid growth rate, the local nature of this disease at the time of diagnosis is most likely part of the reason why so few of these cases have increased nucleosome concentrations. Interestingly the dog diagnosed with osteosarcoma that had the highest elevations in nucleosome concentrations (446 ng/mL) was the only dog in the osteosarcoma cohort that had evidence of metastatic disease, suggesting that as this disease becomes more systemic it may be more likely correlated with elevations in plasma nucleosome concentrations. Additional cases of metastatic disease are needed to determine the value of plasma nucleosome concentrations as apredictor of metastatic disease in dogs with osteosarcoma as well as other cancers.

Overall, the assay was only able to correctly predict 19 per cent of the cases with mast cell tumours. Dogs with grade 1 tumours were detected 33 per cent of the time and dogs with high grade tumours were detected 34 per cent of the time, whereas grade 2 tumours were only detected 11.5 per cent of the time. It is unclear from the patient information available why there is a difference between grade 2 tumours and the other grades. Interestingly,the high grade tumours had a significantly higher mean nucleosome concentration compared to low grade tumour cases and healthy dogs. The samples used for this particular study were graded using the older Patnaik grading scheme.

Fig. 4. Dot plot expressing mean with SEM of plasma nucleosome concentrations (ng/mL) for melanoma cases by location

Fig. 5. Dot plot expressing mean with SEM of plasma nucleosome concentrations from dogs with mast cell tumors according to grade

Fig. 6. Dot plot of mean with SEM for plasma nucleosome concentrations of histiocytic sarcoma based on location

Additional work is needed to see if this same difference holds true when the newer two-tier grading system is applied. Unfortunately, we do not have the outcome data associated with this group and cannot determine whether those cases with increased plasma nucleosome concentrations have a better or worse prognosis.

Interestingly, in this cohort of samples none of the benign cutaneous melanomas had increased plasma nucleosome concentrations, while just over half of the malignant oral melanomas demonstrated increased plasma nucleosome concentrations. This finding highlights the possibility of using nucleosome levels to discriminate between benign and malignant processes indogs with melanoma. All of the malignant melanomas in this study were oral in origin.

Oral tumours, in particular melanomas and squamous cell carcinomas, often have significant inflammation associated with them. This may be adding to the plasma nucleosome concentrations in the 21/42 cases of oral melanoma and 2 oral squamous cell carcinomas described in this study. Additional cases from other commonly malignant locations such as the ungual process or the perianal region need to be evaluated to determine if this trend holds true for all cases of malignant melanoma.

Similarly to what has been noted in humans, the three cases with primary lung carcinomas had elevations in plasma nucleosome concentrations [32–35]. Two of the three dogs with anal sac tumours also had significant elevations in plasma nucleosome concentrations. The one dog with a plasma nucleosome concentration over 800 ng/mL had stage I disease that was treated with surgery demonstrating that this may also be a useful tool for dogs with early stage anal sac neoplasia. Two of the 5 cases of oral/ sinonasal carcinomas had increased nucleosome concentrations. Given the local nature of these diseases, we did not expect to see elevations in any of these cases. However, both of these cases were squamous cell carcinomas. These tumours tend to have a large inflammatory component that may be responsible for the elevations of plasma nucleosome concentrations seen here. It was also surprising to see increased nucleosome concentrations in 2/3 of the cases with urothelial carcinoma. Again, these cases had local disease only. It was not surprising to see increased nucleosome concentrations in the one case of multiple myeloma given that it is a systemic tumour and a tumour of plasma cells. However, the fact that the stage III nasal chondrosarcoma, which tends to be slow growing, displayed elevations in plasma nucleosomes was unexpected. Local inflammation at the site of the tumour may play a role here and additional cases of sinonasal tumours as well as non-melanoma oral malignancies are being enrolled. Several cases did not have increased plasma nucleosome concentrations. These include the cases of insulinoma, mammary carcinoma, MLO, undifferentiated splenic sarcoma and hepatocellular carcinoma did not demonstrate elevations in plasma nucleosome concentrations. This may be due to the low disease burden or the slow cellular turnover rate associated with most of these tumours. Additional cases of non-hemangiosarcoma splenic masses are being actively recruited to better understand these diseases.

Conclusions and futuredirections

In summary, although the majority of information published regarding plasma nucleosome concentrations in dogs with cancer have been centered around lymphoma and hemangiosarcoma, this test is likely useful for detecting other forms of cancer as well. The test performs best for tumours that are systemic (higher metastatic rate) and for those that have a high cellular turnover rate. This is consistent with the

observation that nucleosomes are released into the plasma at a higher rate when there is a rapid cellular turn over and high cellular death rates. This is also true for non-cancerous diseases including severe infections or inflammation. Additional studies are ongoing to determine how noncancerous concomitant diseases affect the plasma nucleosome compartment in dogs. Evaluation of H3.1 plasma nucleosome concentrations using a low-cost simple ELISA test requiring low blood sample volume and may also provide additional information regarding the overall health and well-being of companion dogs. While this test was only able to detect approximately half of the cancers it tested for, it iswell positioned as a companion test to other wellness tests and has the potential to provide valuable additional information that can inform the clinical decision-making process. Additional assays are needed to improve the sensitivity of all liquid biopsy techniques in both the human and veterinary spaces.

Methods

Sample collection and processing

All animal studies were approved by the Texas A&M University Institutional Animal Care and UseCommittee (36 2019–0211 and AUP# 2017–0350). A detailed characterisation of the participants has been included in the results section. The lymphoma and hemangiosarcoma cases were previously described in two publications from 2021 [20, 21]. These cases were included here in order to generate a broad AUC and sensitivity and specificity of this assay for the most common canine cancers. Canine plasma samples from the National Cancer Institute Division of Cancer Treatment and Diagnosis (NCI-DCTD) biorepository and from active patients or healthy volunteers at the Texas A&M University Small Animal Teaching Hospital were purchased or collected with owner consent, respectively, for this study. A minimum of 0.5 mL of plasma was collected from each patient. Dogs were fasted for a minimum of 4 h before blood collection. Samples were drawn from a peripheral or jugular vein into a K2 EDTAtube (BD Vacutainer, Franklin Lakes, NJ) and centrifuged at 3000 g for 10 min within 1 h of collection (Clinical 100 centrifuge, VWR, Radnor,PA). The plasma samples were labeled and frozen at -80°C until samples were run in batches.

All samples were tested using the Nu.Q™ H3.1 assay (Belgian Volition, SRL, Isnes, Belgium). This enzyme-linked immunosorbent assay (ELISA) contains a capture antibody directed at histone 3.1 and a nucleosome specific detection antibody [36]. Frozen samples were thawed and allowed to come to room temperature for at least 30 min prior to analysis. Assays were performed according to the manufacturer’s protocol. Briefly,astandard curve was generated using the positive control stock (recombinant H3.1 nucleosomes) provided. Samples were vortexed and centrifuged at 11,000 x g for 3 min at 4°C. HSA samples were diluted threefold to ensure that they could be measured within the detection limits of the assay.The nucleosomes were bound to the capture antibody and the plates were washed 3 times using 1 x wash buffer. Twenty microliters of each diluted sample were pipetted in duplicate into wells on the 96 well plates. Next, 90µLof the assay buffer was added to each well. The plate was covered with sealing film and incubated on an orbital shaker for 2.5 h at 700 rpm. Plates were then emptied and washed 3times using 1 x washing buffer.Next, 100 µLof the detection antibody was added to each well, the plate was resealed and incubated for 1.5 h on the orbital shaker. The plates were then washed as described above. Streptavidin HRP conjugate was incubated for 30 min in each well and washed before applying the colorimetric substrate solution and incubating the plates in

the dark for 20 min. A stop solution was added to the wells and the plates were read on a plate reader at 450 nm (BioTek Synergy H1 plate reader, BioTek Instruments, Winooski, VT). The standard curve was linearised and fitted to a 4-parameter logistic curve using statistical software (Graphpad Software, version 8, San Diego, CA). Two kit controls (KC) are included for each plate. KC1 has a value near the lower level of detection and KC2 has a value near 200 ng/mL. The kit control values must fall within a predetermined range for the plate to beused to limit interplate variability.

Data processing of H3.1 plasma measurements

Duplicate raw values recorded by the lab were expressed in optical density (OD) values, that is the color intensity related to the quantity ofthe biomarker present in the sample. The transformation of OD values was done using the standards with assigned concentrations, allowing the preparation of quantification curves for a calculation of relative concentration. The final record for each subject is a mean of duplicate concentration measurements for each sample tested on each biomarker. Any concentration values recorded with a co-efficient of variation [37] percentage above [20 per cent] were rejected and the analysis repeated. The standard curve was linearised and fitted to a 4-parameter logistic curve using statistical software (Graphpad Prism Software, version 9, San Diego, CA, USA, www.graphpad.com). Concentration values calculated in this way are expressed in ng/ml.

The dynamic range is defined as the usable range of an assay between the upper and lower limits of quantification (LLOQ = lower, ULOQ = upper). Where a reading is above the ULOQ, there is a procedure for the serum sample to be diluted and measured again in relation to the standard curve to provide a good quantity of biomarker in the tested serum. Where a reading is below the LLOQ, the value for that sample is set to a concentration of zero.

Outliers and missing values

No samples were removed from the analysis if they were calculated to be outliers for any variable. Subjects with missing values for a variable used for a calculation were removed from that particular calculation only, where the result is undefined for missing values. Subjects with missing values for variables other than those used in the calculation being performed were not removed.

Data analysis

Descriptive statistics for the patient populations were performed using Microsoft Excel for Mac (v. 16.16.27, 2016). A Shapiro–Wilk test was performed, and the data was determined to not be normally distributed (no group had a test value above 0.1) For this reason, nonparametric tests were utilised for data comparisons. For data sets containing only two cohorts, such as the healthy controls versus all mast cell tumour cases, a Wilcoxon rank sum test was used to determine whether there is a statistical difference between the orderings of the two sets (with a null hypothesis that the probability that arandomly drawn observation from one group is larger than a randomly drawn observation from the other group is equal to 0.5 against an alternative that this probability is not 0.5). For data sets where multiple conditions were compared, such as when all cancers were considered, a Kruskal–Wallis test for repeat measures with a Dunn’s multiple comparisons test was performed using GraphPad Prism Software version 9, San Diego, California USA, www.graphpad. com.

To assess the diagnostic performance of the Nu.Q H3.1 assay, Receiver Operator

Charatceristic (ROC) curves were calculated, along with the Area Under the ROC Curve (AUC). The sensitivity was calculated for each level of specificity. To make it easy to compare different types of cancer, a standard sensitivity of 97per cent was used, and it was noted that this was not far from the cut-off for whcih Youden’s Index (J = sensitivity + sepcificity -1) was maximised (53.57 per cent sensitivity at 94.78 per centspecificity).

Wilcoxon rank sum tests, ROC curves and specificity/ sensitivity calculations were only performed on cancer groups for which there were at least 20 cases. These calculations were performed using R version 3.4.3 and the pROC package [38, 39].

Abbreviations

DNA: Deoxyribose Nucleic Acid; PSA: Prostate Specific Antigen; CEA: Carcinoembryonic Antigen; ELISA: Enzyme Linked Immunosorbent Assay; PCR: Polymerase Chain Reaction; NSCLC: Non Small Cell Lung Cancer; LSA: Lymphoma/Lymphosarcoma; OSA: Osteosarcoma; STS: Soft tissue Sarcoma; MCT: Mast Cell Tumour; SEM: Standard Error of the Mean; ROC: Receiver Operator Characteristic Curve; NCI?DCTD: National Cancer Institute Division of Cancer Treatment and Diagnosis biorepository; EDTA: Ethylenediaminetetraacetic acid; g: Relative Centrifugal Force; C: Celsius; ANOVA: Analysis of Variance.

Acknowledgements

The authors would like to acknowledge the Fred and Vola Palmer Chair in Comparative Oncology for funding support of this work.

All methods were carried out in accordance with relevant guidelines and regulations

■ No humans were involved in this study.

■ No agents were administered to dogs in any way during this study.

Author details

1College of Veterinary Medicine, Small Animal Clinical, Sciences Department, Texas A&M University, College Station, TX 77843, USA.

2Volition America & Volition Veterinary Diagnostic Development, 13215 Bee Cave, Parkway,Galleria Oaks B, Suite 125, Austin, Texas 78738, USA. 3Volition Diagnostics UK Ltd, 93-95 Gloucester Place, London W1U 6JQ, UK. 4Belgian Volition SRL, ParcScientifiqueCrealys, 22 Rue Phocas Lejeune, 5032 Isnes, Belgium. This article previously appeared in BMC and was provided to The Veterinarian by Heska.

References

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2.Bremer HD, Lattwein E, Renneker S, Lilliehook I, Ronnelid J, Hansson-Hamlin

H. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme linked immunosorbent assay Vet Immunol Immunopathol. 2015;168(3–4): 233–41.

3.Barker DA, Foale RD, Holmes MA, Demetriou JL. Survey of UK based veterinary surgeons’ opinions on the use of surgery and chemotherapy in the treatment of canine high grade mast cell tumour, splenic haemangiosarcoma and appendicular osteosarcoma. Vet Rec. 2016;179 2016;179(22):572.

4.Bauden M, Pamart D, Ansari D, Herzog M, Eccleston M, Micallef J, et al. Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer.Clin Epigenetics. 2015;7:106.

5.Rahier JF, Druez A, Faugeras L, Martinet JP,Géhénot M, Josseaux E, et al. Circulating

nucleosomes as new blood based biomarkers for detection of colorectal cancer. Clin Epigenetics. 2017;9:53.

6.Rasmussen L, Christensen IJ, Herzog M, Micallef J, Nielsen HJ, Danish Collaborative Group on Early Detection of Colorectal C. Circulating cell free nucleosomes as biomarkers for early detection of colorectal cancer. Oncotarget. 2018;9(12):10247–58.

7.Cristiano S, Leal A, Phallen J, Fiksel J, Adleff V, Bruhm DC, et al. Genome wide cell free DNA fragmentation in patients with cancer. Nature. 2019;570(7761):385–9.

8.Mego M, Kalavska K, Karaba M, Minarik G, Benca J, Sedlackova T, Gronesova P, Cholujova D, Pindak D, Mardiak J, Celec P. Plasma Nucleosomes in Primary Breast Cancer. Cancers (Basel). 2020;12(9):2587. https://doi.org/ 10.3390/cancers12092587.

9.Kumar S, Guleria R, Singh V, Bharti AC, Mohan A, Das BC. Plasma nucleosome levels might predict response to therapy in patients with advanced non small cell lung cancer. Clin Lung Cancer. 2010;11(1):36–44.

10. Murtaza M, Caldas C. Nucleosome mapping in plasma DNA predicts cancer gene expression. Nat Genet. 2016;48(10):1105–6.

11. Ulz P, Thallinger GG, Auer M, Graf R, Kashofer K, Jahn SW, et al. Inferring expressed genes by whole genome sequencing of plasma DNA. Nat Genet. 2016;48(10):1273–8.

12. Letendre JA, Goggs R. Determining prognosis in canine sepsis by bedside measurement of cell free DNA and nucleosomes. J Vet Emerg Crit Care (San Antonio). 2018;28(6):503–11.

13. Letendre JA, Goggs R. Concentrations of plasma nucleosomes but Not Cell?Free DNA Are prognostic in dogs following trauma. Front Vet Sci. 2018;5:180.

14. Letendre JA, Goggs R. Measurement of plasma cell free DNA concentrations in dogs with sepsis, trauma, and neoplasia. J Vet Emerg Crit Care (San Antonio). 2017;27(3):307–14.

15. Martiny P, Goggs R. Biomarker guided diagnosis of septic peritonitis in dogs. Front Vet Sci. 2019;6:208.

16. Monestier M, Novick KE, Karam ET, Chabanne L, Monier JC, Rigal D. Autoantibodies to histone, DNA and nucleosome antigens in canine systemic lupus erythematosus. Clin Exp Immunol. 1995;99(1):37–41.

17. Jeffery U, Kimura K, Gray R, Lueth P, Bellaire B, LeVine D. Dogs cast NETstoo: canine neutrophil extracellular traps in health and immune mediated hemolytic anemia. Vet Immunol Immunopathol. 2015;168(3–4):262–8.

18. Krogh AKH, Lyngby JG, Bjørnvad CR, Nielsen LN. Presence of nucleosomes in plasma and increased thrombin generation in dogs with acute and chronic gastroenteropathies. Res Vet Sci. 2021;135:504–10.

19. Wilson Robles H, Miller T, Jarvis J, Terrell J, DewsburyN, Kelly T,et al. Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer. PLoS ONE. 2020;15(8):e0236228.

20. Dolan C, Miller T, Jill J, Terrell J, Kelly TK, Bygott T, et al. Characterizing circulating nucleosomes in the plasma of dogs with lymphoma. BMC Vet Res. 2021;17(1):276.

21.Wilson Robles H, Miller T, Jarvis J, Terrell J, Kelly TK, Bygott T, et al. Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma. BMC Vet Res. 2021;17(1):231.

22. Morales Molina A, Gambera S, Leo A, García Castro J. Combination immunotherapy using GCSF and oncolytic virotherapy reduces tumour growth in osteosarcoma. J Immunother Cancer. 2021;9(3):e001703. https://doi. org/10.1136/ jitc 2020?001703.

23. Nadauld LD, McDonnell CH 3rd, Beer TM, Liu MC, Klein EA, Hudnut A, Whittington RA, Taylor B, Oxnard GR, Lipson J, Lopatin M,

Shaknovich R, Chung KC, Fung ET, Schrag D, Marinac CR. The PATHFINDER Study: Assessment of the Implementation of an Investigational Multi Cancer Early Detection Test into Clinical Practice. Cancers (Basel). 2021;13(14):3501. https://doi.org/10.3390/ cancers13143501.

24. Flory A, Kruglyak KM, Tynan JA, McLennan LM, Rafalko JM, Fiaux PC, et al. Clinical validation of a next generation sequencing based multi cancer early detection “liquid biopsy” blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study. PLoS ONE. 2022;17 (4):e0266623.

25. Phan T, McMillan R, Skiadopoulos L, Walborn A, Hoppensteadt D, Fareed J, et al. Elevated extracellular nucleosomes and their relevance to inflammation in stage 5 chronic kidney disease. Int Angiol. 2018;37(5):419–26.

26. Cavalier E, Guiot J, Lechner K, Dutsch A, Eccleston M, Herzog M, et al. Circulating nucleosomes as potential markers to Monitor COVID?19 Disease Progression. Front Mol Biosci. 2021;8:600881.

27. Goswami J, MacArthur T, Bailey K, Spears G,Kozar RA, Auton M, et al. Neutrophil extracellular trap formation and Syndecan 1 shedding are increased after trauma. Shock. 2021;56(3):433–9.

28. Carestia A, Frechtel G, Cerrone G, Linari MA, Gonzalez CD, Casais P, et al. NETosis before and after hyperglycemic control in Type 2 Diabetes Mellitus Patients. PLoS ONE. 2016;11(12):e0168647.

29. Yehya N, Fazelinia H, Lawrence GG, Spruce LA, Mai MV, Worthen GS, et al. Plasma nucleosomes are associated with mortality in pediatric acute respiratory distress syndrome. Crit Care Med. 2021;49(7):1149–58.

30. Penttilä AK, Rouhiainen A, Kylänpää L, Mustonen H, Puolakkainen P,Rauvala H, et al. Circulating nucleosomes as predictive markers of severe acute pancreatitis. J Intensive Care. 2016;4:14.

31. Goggs R. Effect of sample type on plasma concentrations of cell free DNA and nucleosomes in dogs. Vet Rec Open. 2019;6(1): e000357.

32. Bowman RV, Yang IA, Semmler AB, Fong KM. Epigenetics of lung cancer.Respirology. 2006;11(4):355–65.

33. Holdenrieder S, Nagel D, Schalhorn A, Heinemann V,Wilkowski R, von Pawel J, et al. Clinical relevance of circulating nucleosomes in cancer.Ann N Y Acad Sci. 2008;1137:180–9.

34. Tufman A, Huber RM. Biological markers in lung cancer: a clinician’s perspective. Cancer Biomark. 2010;6(3–4):123–35.

35. Wang K, Shan S, Wang S, Gu X, Zhou X, Ren T. HMGB1 containing nucleosome mediates chemotherapy?induced metastasis of human lung cancer.Biochem Biophys Res Commun. 2018;500(3):758–64.

36. Arimura Y, Ikura M, Fujita R, Noda M, Kobayashi W, Horikoshi N, et al. Cancer associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome. Nucleic Acids Res. 2018;46(19):10007–18.

37. Wells JW, Evans CH, Scott MC, Rutgen BC, O’Brien TD, Modiano JF,et al. Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation. PLoS ONE. 2013;8(1):e54464.

38. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2017. https:// www.R-project.org/.

39.Xavier Robin NT,Alexandre Hainard, et al. pROC: an open source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;7(77).

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worked for five years in a combination of mixed and small animal practices, before joining the team at PAW by Blackmores as aTechnical Services Veterinarian. She now works as Head Vet for PAW, and works on product development, education, and the technical advisory services. Howard has two young children, a horse called Flora and averyhuman-like Whippet called Winnie. She is passionate about natural health and studies Naturopathy in her (very sparse!) sparetime. Howard lives in the Southern Highlands of NSW.

BUSINESS

Nutrients and stress

Introduction

In this month’s edition of The Veterinarian, the fourth article in our mental health series explores specific nutritional supplements that can support mental wellbeing. We build on our previous article where we discussed foods that can positively impact mood, and dive into the importance of specific nutrients that can nurture brain health throughout life. Essential micronutrients can add a further layer of support for the brain, especially in times of stress.

Micronutrients

Micronutrients play major roles in cellular function and physiological processes within the body. Deficiencies can contribute to pathology within the body,including the brain, and have been implicated in several psychological conditions including anxiety and depression1

Micronutrients are typically obtained from a balanced diet, however more than 90 per cent of Australian adults do not meet the recommended vegetable intake2 and in many areas soils are depleted of nutrients, leaving food that has been grown in these soils deficient1,-this can necessitate the need for supplementation.

Additionally, stress exposure increases the requirements for many micronutrients; therefore, chronic stress can contribute to nutrient deficiency3.Specifically, B group vitamins, vitamin C, magnesium, zinc, and iron are required at increased levels during times of stress3

Supplementing the diet with these micronutrients can improve the stress response and restore balance to the nervous system3

The role of gut health

During times of acute or chronic stress, the hypothalamic-pituitary axis, or stress regulation system of the body, becomes hyperactivated. This state of nervous system activation has direct negative implications on gut function and can lead to intestinal dysbiosis. Chronic stress disrupts the integrity of the intestinal mucosal barrier.

Wellness

These changes can lead to inadequate digestion and absorption of dietary nutrients, including fatty acids. In addition to altered digestive function, when the gut is in a state of dysbiosis, neurotransmitter production becomes affected, and this can directly impact neuronal health and emotional regulation4

To page 30

Table 1 lists some easily accessible nutrients which can be added to a diet to further support the brain and body to tackle issues such as stress, poor sleep, and anxiety.

Table 1. Nutritional supplementation for supporting mental health

Nutrient Need state

Vitamin B complex

Magnesium

Probiotics (specific strains selected for need state)

Omega-3 fatty acids

GABA (gamma aminobutyric acid)

L-theanine

L-tyrosine

Stress

Stress and sleep support

Stress and anxiety

Dose/adult/day

Choose a high dose complex3

300-350 mg (elemental magnesium) for minimum of four weeks3

1x109- 3 x 109 CFU (colony-forming units)3

Depression

Stress

Stress, sleep support

Stress

Maximising sterilisation success

From smaller veterinary clinics with benchtop autoclaves to larger sites with packing, prep and sterilisation rooms, achieving efficiencies and proper sterilisation is vital.

According to sterilisation specialist Andrew Elford from Mediquip/MQ Vet – which has provided veterinary sterilisation set ups for decades, some initial considerations include the type of instruments the clinic needs to sterilise, and their length and volume.

“While S-class sterilisers can do the trick for basic solid instruments like scissors and forceps, clinics utilising more complex hollow bodied instruments should look at B class,” Elford said.

Effective steam sterilisation relies on the right amount of steam, pressure, temperature, and time. And it’s not just steam in the chamber – it’s penetration of steam into the sterilisation load, inside packs and hollow chambers.

Elford said this is why correct use of chemical indicators is important: outside the pack to indicate if it has been subject to the sterilisation process (but not whether successful) and inside the pack to verify penetration of sterilisation agent.

While steam sterilisation is most common, Mediquip/MQ Vet is seeing an increase in

use of plasma or low temp sterilisers for materials and devices that cannot tolerate high temperatures and humidity such as some plastics,electrical devices, and corrosionsusceptible metal alloys.

Elford said washers are also growing in popularity, particularly in clinics with large surgeryloads.

“Having a washer can save vet nurses significant time from manualhandling, with devices that wash, disinfect and dry.”

The overall volume and size of the clinic’s instruments and packs will help determine the appropriate size of steriliser,Elford said, adding chamber sizes can vary from 18 litres to over 600 litres.

Clinics shouldn’tunderestimate the importance of water quality. While some vet centres use tap water with their steam sterilisers, this practice can lead to costly downtime and expensive repairs over time.

“When hard water is boiled into pressurised steam, it leaves behind salt and mineral deposits within the chamber,steam generator, pipes and valves and as this builds up, it decreases the efficiency and functionality of the steriliser,” Elford said.

There are two mains type of water suitable for sterilisation – reverse osmosis and dem-

1000 mg EPA

300-500 mg DHA4

00-200 mg for 4-12 weeks3

200-400 mg for up to 8 weeks3

100-150 mg per kg bodyweight3

ineralised – each using filters with different physical reactions to clean water Another reverse osmosis solution is the optional integrated Reverse Osmosis Steam Injection ROSI feature in VetAssure sterilisers, allowing the steriliser to be connected to tap water supply. Elford said general maintenance of sterilisers, including regular cleaning, filter changing, and annual servicing helps keep them performing well for many years. With Australia-wide field service engineers, Mediquip offers servicing and repairs for vet clinics across Australia.

To learn more, visit mqvet.com.au/collections/ sterilisation-set-ups.

Sarah Howard graduated as a veterinarian from the University of Sydney in 2012. She

What’s that ribbit? FrogID it!

From page 5

a strong theoretical background.

“We had thousands of recordings of banjo frog calls at our fingertips so were able to use the data from around 700 of them, covering an area from Western Australia to Tasmania to far North Queensland and everywhere in between. Importantly, we’ve also shown that citizen science data provides a novel opportunity to examine important ecological theories across a huge spatial scale. And that the venue – be it dense forest or open plain – doesn’t matter for a banjo frog gig!” Gillard said.

Researchers paired FrogID data with remote sensing imagery to get a measure of canopy cover, to establish if there was any correlation between the level of canopy cover and the acoustic parameters of each frog call. Using a sound visualisation system, they were able to analyse dominant frequency, the duration of each call and the rate at which a frog calls.

“Our findings have suggested that other factors may have a greater influence over the variation ofbanjo frog calls. It’s likely to be a combination of all different factors like more fine-scale features of the environment, acoustic competition from other frogs, and noise interference from wind, water, and other animals,” she said.

Animals rely on communication

Wellness

From page 29

Probiotics and omega-3 fatty acids

In recent years, there has been plenty of research published deepening our understanding of the bidirectional relationship between the gut and the brain. This has paved the way for specific recommendations of how probiotics behave as “psychobiotic agents” and can modulate the gut-brain axis offering possible therapeutic applications for mental ill health. Correcting the gut-brain axis compromise that causes bacterial imbalance (dysbiosis) specific to acertain health condition, such as depression or anxiety, is the primary goal of probiotic therapy5

Omega-3 fatty acid deficiency has also been linked to depression and supplementation is frequently recommended as adjunctive therapy4

Conclusion We hope our series is helping you understand the many ways you can support your mental wellbeing. Taking an integrated approach, including maximising nutrition through food and nutritional supplementation, is a great way to provide reliable, long-term support for mental health.

for survival and Gillard said frogs depended almost entirely on their acoustic communication, and they all have unique calls.

“It’s like an accent, but while we’re aware of the variation, we don’t really know why they vary so much, although one theory is the acoustic adaptation hypothesis. This suggests animals that communicate acoustically adapt their vocalisations to the local conditions to optimise transmission through their habitat,” she explained.

The research showed that habitat plays an important role in the transmission of acoustic signals, either bydistorting and reflecting sound waves, echoing, or decreasing the distance from where the call can be heard. Surrounding habitat can influence calls so much they might not reach their intended audience, whether it be to attract a mate or defend territory.

Gillard said the physical environment is likely to be at least partially responsible for the evolution of variation in frog advertisement calls, but further research involving different frog species, and frogs with more complex calls, might reveal more of a signal for habitat.

‘Assessment of the acoustic adaptation hypothesis in frogs using large-scale citizen science data’ is available at doi.org/10.1111/jzo.13088

■ ANNE LAYTON-BENNETT

In the next article in our mental health series, we will add another layer to your mental health toolkit and explore some lifestyle recommendations such as sleep practices and exercise.

References

1. Shayganfard M. Are Essential Trace Elements Effective in Modulation of Mental Disorders? Update and Perspectives. Biological Trace Element Research [Internet]. 2002 [cited 2023 Jul 19];200(3): 1032–1059. Available from: https://doiorg.torrens.idm.oclc.org/ 10.1007/s12011-021-02733-y

2. Australian Bureau of Statistics. Dietar y behaviour [Internet]. Canberra: ABS; 2020-21 [cited 2023 August 1]. Available from: https:// www.abs.gov.au/statistics/health/h ealth-conditions-and-risks/dietarybehaviour/latest-release.

3. Braun L. Mental Wellbeing – The essential guide to using herbs and nutritional supplements. Blackmores Institute, a division of Blackmores Limited; 2021.

4. Low Dog T. The role of nutrition in mental health. Alternative Therapies in Health and Medicine [Internet]. 2010. [cited 2023 Jul 19];16(2):42–46.

5. Johnson D, Letchumanan V, Thum CC, Thurairajasingam S, Lee LH. (2023). A Microbial-Based Approach to Mental Health: The Potential of Probiotics in the Treatment of Depression. Nutrients [Internet]. 2023 [cited 2023 Jul 19];15(6). Available from: https://doi-org.torrens.idm.oclc. org/10.3390/nu15061382

Reef still vulnerable, AIMS warns

From page 5 and coral bleaching, the latter increasing in frequency since 2016. “The 2022 bleaching event was the first ever recorded during a La Niña year, which are usually characterised by cooler temperatures,” AIMS Long-Term Monitoring Program leader Mike Emslie said.

“The best hope for the future of the Great Barrier Reef and all coral reefs globally requires reduction in greenhouse gas emissions to stabilise temperatures, best practice management of local pressures, and the development of interventions to help boost climate tolerance and resilience for coral reefs,” Wachenfeld said.

■ JULIA GARDINER

UQ’s experimental canine cancer vaccine test

From page 5

“This technique has incredible potential to reduce suffering in our pets and eventually a variation may even be able to help humans,” Allavena said.

“We’re very eager to kick off this trial, as we have promising results from prior trials for dogs with cancer.”

Pet owners able to take their dog to theUQ VETS Small Animal Hospitalat Gatton for surgery and treatment will be preferentially selected, however the team would also like to recruit patients from elsewhere in Australia, except NSW (due to an ethics requirement).

The project team includes experienced veterinarians, board-certified pathologists and clinical trial experts who will provide regular monitoring and follow ups.

Applications for the trial can be made at sur vey.app.uq.edu.au/79E20EFEDE3A-4652-8937-F992FA11EAE7 or questions can be directed to Dadhich at a.dadhich@uq.edu.au.

Abstracts - rescued cats

From page 16

Alexandre P Rossi1,Cassia R C Dos Santos1,Caroline M Maia12, Claudia C B Terzian1,Deisy F Predebon1,Juliana S C de Queiroz1, Laraue P M Pereira1,Marina Z N Bastos1,Maurício Choinski1, Patricia E Tsapatsis1,Samantha R Mde Assis1,Tatiana M Corrêa1

J Appl Anim Welf Sci 2023 JulSep;26(3): 313-324.doi: 10.1080/ 10888705.2021.1949595.

1Group of Scientific Studies (GEC), Cão Cidadão Company, São Paulo, Brazil.

2Consciência Animal Initiative –Advisor y, Consultancy and Solutions on Animal Behavior and Welfare, Botucatu, SP, Brazil.

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CREATURE Feature Caribbeanreefsquid ( Sepioteuthis sepioidea )

Withalengthofapproximately 20cm,theCaribbeanreefsquid possessestheabilitytopropel themselvesupoutofthewater by2mandflyforroughly 10mbeforereentry. Thesesquidtypicallycongre- gateinshallowturtlegrass throughouttheCaribbeansea toavoidbirdpredation.

PictureBettyWills