Karrie Rose

1 APPROACH TO WILDLIFE HEALTH INVESTIGATION

1.1 Why study wildlife health?

In a global environment where approximately 75% of recently emerging and re-emerging zoonotic diseases emanate from wildlife reservoirs, there has never been more interest in wildlife health (Woolhouse 2002). Additional compelling factors driving an interest in wildlife health include public concern regarding animal welfare, zoonotic diseases and food safety issues associated with endemic and exotic disease, biodiversity protection, bioterrorism detection, climate change and its effects on animal and human health and disease, and the development of a nationally integrated biosecurity strategy (Bengis et al. 2004).

The significance of disease in wildlife populations and its impact on the conservation of biodiversity in Australia is poorly understood. Disease-causing organisms are a component of most natural ecosystems. A balance among the relationships of host, pathogen and environment most often meters the occurrence of disease. As wildlife habitats are modified through human activity and an increasing number of species are intensively managed, the possibility of disturbing these relationships increases. Wildlife health investigations are thus evolving through our increased involvement with wildlife management. The investigation of wildlife health and disease provides the opportunity to view individual species as indicators of environmental contamination, habitat degradation or climate change.

Wild animals may be subclinically infected with a variety of potential pathogens and can function as reservoirs for some disease-causing agents. Undiagnosed disease in wildlife may pose a significant risk to human health, to the agricultural industry, international trade, and to in situ and ex situ species recovery programs.

Some examples of the potential effects of wildlife disease include:

• potentially fatal respiratory illness in humans and horses in 1994, 1999 and 2004 caused by Hendra virus, a virus resident in flying fox populations (Murray et al. 1995; Breed et al. 2006);
  
• a 1998 outbreak of infertility and deformity in pigs caused by Menangle virus, a virus carried by bats (Philbey et al. 1998);
  
• the fifth Australian outbreak of highly pathogenic avian influenza in poultry occurred in Tamworth in 1997, and probably originated from free-living waterfowl (Sellek et al. 2003);
  
• Australian bat lyssavirus, found in a variety of bat species throughout Australia, has caused the death of two people in Queensland (Fraser et al. 1996; Allworth et al. 1996);
  
• a global decline in amphibian populations, including the extinction of at least one Australian species, has been associated with the presence of Chytrid fungus (Daszak et al. 1999; Lips 1999; Bonaccorso et al. 2003; Bosh et al. 2000).

Wildlife management involving capture and animal translocation in Australia includes animal research and targeted disease surveillance, the rehabilitation and release of injured or illegally held animals, the reintroduction of species that are depleted or extinct in their natural range, the creation of safe populations of wildlife where they will not be threatened by disease, harvesting or habitat degradation, the dispersal or relocation of animals deemed pests, and the movement of feral or native animals in attempts to reduce population density to control disease or protect habitat quality.

Regardless of the circumstances that initiate your involvement in a health investigation, examining a wild animal should be viewed as an opportunity to collect as much information as possible (Woodford 2001). Collecting blood and faecal samples during restraint of an animal to allow a radio-collar to be fitted provides the opportunity to screen for blood-borne and intestinal parasites, and natural food sources. Serum samples can be tested for antibodies that may reflect exposure to potential pathogens. Some nutrient concentrations can be assessed in serum, and stored serum samples can prove invaluable in defining emerging disease syndromes.

Ultimately, the collaboration of biologists, veterinarians, researchers and diagnosticians can provide cost-effective health assessment, even if this was not the primary goal of the project.

Through our involvement with wildlife health initiatives we can attempt to maximise the success of wildlife conservation activities, contribute to successful reproductive programs and participate in the banking of rare genomic materials. Each wildlife management activity can provide animal health information that is useful as a surveillance tool to help us understand the occurrence of disease and provide information to plan disease control and eradication programs (Zepeda & Salman 2003).

Information on wildlife health from all zoological parks, universities, agriculture agencies, conservation departments and wildlife rehabilitation facilities is being systematically collated by the Australian Wildlife Health Network (AWHN 2007) to contribute to animal health information systems and national disease surveillance programs.

1.2 Health screening or surveillance

A baseline health assessment can be conducted on wild animals captured for management purposes, or it can be targeted to answer specific questions regarding the health of a population.

Common goals of a baseline health assessment in live animals could include determining:

• physiological data (temperature, heart rate, blood pressure, respiratory rate, reproductive details etc.);
  
• morphological assessment;
  
• haematological and serum biochemical data and establishment of reference ranges, and identification of haemoparasites;
  
• identification of ectoparasites;
  
• the best method of external examination to detect evidence of disease;
  
• repeated faecal examination for evidence of internal parasites;
  
• bacterial fauna and potential pathogens in the upper respiratory tract, eyes, ears, lower gastrointestinal tract or lower urogenital tract;
  
• analysis of genetic variability using blood, skin or oral swabs;
  
• radiographic examination or skeletal analysis;
  
• stomach content or scat examination for diet analysis;
  
• fur or blood examination for the presence of nutrients or toxins;
  
• serology to determine what organisms the animal has been exposed to in the past;
  
• polymerase chain reaction (PCR), bacterial, fungal or viral culture to determine whether specific organisms are present within the animal (AAZV 2001).

An experienced wildlife veterinarian and a veterinary pathologist should be consulted during the planning phases of a health screening or surveillance study to ensure that the diagnostic testing is conducted in a humane fashion and that the laboratory findings will be rewarding.

• land owners;
  
• local interest groups and non-profit organisations;
  
• researchers involved in similar projects;
  
• species recovery programs (it is always best if the project is actually generated by the recovery group and designed to answer specific operational questions);
  
• an animal ethics committee (if live animals will be handled or observed);
  
• state conservation and agriculture agencies (if animal samples will be transported interstate);
  
• Department of Environment and Water Resources (DEWR) administers the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2007) and should be contacted if wildlife samples will be transported overseas;
  
• the laboratories that will be receiving the samples (to determine optimum samples to collect, transport medium, transport packaging and shipping details).

The state conservation agency should be consulted before any wild animals, particularly marine mammals or migratory birds, are handled.

Projects designed to assess population health are becoming more common. Collecting samples from different populations of wildlife provides a fantastic opportunity to accumulate baseline normal data for haematology, biochemistry, parasite burdens and parameters of a general physical examination.

Baseline physiologic data determination and specific disease testing can be expensive. Collaborating with interested veterinarians and researchers can decrease the costs of investigation. However, it is important to encourage conservation agencies and recovery groups to include funding for health assessment and monitoring in their wildlife management budgets.

It can be very difficult to decide which specific disease-causing agents, toxins or nutrient concentrations to include in a health assessment. Test selection will rely on careful consideration of the species’ susceptibility to a range of agents. A thorough literature and resource search of existing health information is the basis of test selection.

The sensitivity and specificity of each potential test should be evaluated. When tests have poor sensitivity, the use of several diagnostic tests to assess exposure to and infection with a single infectious agent may be required. Often, combining several tests such as culture or PCR with serology will help to differentiate:

• animals that may have been exposed to an infectious agent (and now have antibodies towards that agent) in the past, but are not necessarily still carrying the organism;
  
• animals that are actively infected but remain healthy;
  
• animals that are actively infected but have reduced function or disease;
  
• animals that have not been exposed to the infectious agent and could be considered susceptible due to a lack of circulating antibodies.

Always ensure that the tests you are using have been validated for the species involved in your study. If at all possible, diagnostic tests should be thoroughly evaluated in captive wildlife prior to being used in field studies. If you are not confident that the test has been validated, speak with a pathologist or microbiologists regarding means to validate new tests through your proposed study.

Carefully consider how you will interpret your findings, both positive and negative. Identifying the presence of a potential pathogen does not necessarily indicate a clinically significant infection in the animal sampled. Negative findings do not always indicate that an individual animal is free of infection. It is also wise to contact the state chief veterinary officer in advance when you plan to test for diseases listed as notifiable.

After test selection has taken place, it can take time to find laboratories that can assist with your investigation. Canvassing laboratories for the tests that they conduct, the costs involved and the optimum method of sample collection can take several weeks for a large-scale project. Some researchers can be encouraged to test samples on a collaborative basis to reduce costs. Some large commercial laboratories offer a discount for large numbers of samples shipped in bulk, if negotiated in advance.

A field trip to gather data and animal samples requires a great deal of preparation. Personnel participating in a trip must be identified and trained well in advance, as this is often required in the animal ethics committee application. Generally, at least three people are required for any venture in animal handling: 1) animal handler or anaesthetic monitor; 2) examiner and sample collector; 3) recorder. The recorder can complete the examination checklist, properly label the samples, take photographs and pass any additional equipment required during the procedure (Fig. 4.1).

Planning and preparing the equipment for fieldwork can be a mind-racking experience. Obviously, it is very disappointing to be caught without a vital piece of equipment out in the field. It is important to begin the planning early so that any unusual items can be ordered and received. An equipment list is given in Table 4.1, but if this is your first trip talk to more experienced researchers and have them check your list. All equipment should be tested prior to being packed.

When samples can’t be taken to a laboratory, it may be necessary to set up a remote lab. A variety of manual and electrical instruments can be taken into the field to assist with sample collection and analysis. Small generators can power most equipment. Centrifuges, microscopes and even serum biochemistry analysers can be transported quite readily. Gas anaesthetic machines can be small and portable.

Packing equipment into sealed plastic bins will protect the materials from water and pests. You may not be allowed onto some off-shore islands if your equipment is not in pest-proof cases.

Figure 4.1 a) Sample collection from a northern hairy-nosed wombat (Lasiorhinus krefftii) in the field. b) Cetacean necropsy set-up on a beach.

When planning any project, from a single necropsy in the field to a long journey, it is important to remember your own safety and comfort. Simply including a few extra tarps to erect a makeshift tent can keep you dry or shaded. Research what resources will be available on-site, and what you will have to bring.

Table 4.1 Field trip equipment checklist

Usually it is very difficult to sample the entire wildlife population, and only a small proportion of animals is examined as a study population. Prior to designing a health investigation it is important to define:

If you can only collect samples from a proportion or subset of the animals at risk or of interest, then you are sampling the population. Your study population will provide only an estimate of the prevalence of specific diseases or traits within the population. Sampling from a study population is not a precise measurement throughout the population at risk, and selection bias can be introduced (Wobeser 1994; Cameron et al. 2003).

When using a study population, it is very important to either limit your comments or inference to the population sampled, or attempt to collect a study population that accurately reflects the population at risk. Study populations can be affected by selection bias when sampling methods are not random. Ensuring that the age and sex distribution in the study group is similar to that in the entire population is important. Other factors such as ecosystem, geographic distribution and genetic variability should also be considered when identifying a study population.

Wildlife health sampling may be undertaken on a convenience basis rather than trying to select a study population that reflects the entire population. Animals are often sampled due to accessibility (they are already being handled or are the only animals found or trapped) or in a targeted program based on presenting signs that make them more likely to have the disease of interest. These sampling techniques, however, make it very difficult to make inferences regarding disease prevalence in the population as a whole. We can only comment with certainty on the prevalence of disease within the target group, rather than the entire population (i.e. the prevalence of lyssavirus infection in bats found with behavioural abnormalities).

Random sampling techniques are difficult if not impossible to apply to wildlife. The best option is to find a method that reduces bias and represents the population as closely as possible.

Many wildlife health surveillance projects are designed to detect specific disease. Determining the number of animals that need to be sampled to detect or rule out the presence of an infectious agent depends upon:

• an estimate of the prevalence of the disease within the population;
  
• the population size;
  
• the accuracy of the test—sensitivity and specificity;
  
• the level of certainty (confidence) in results, usually set by convention to 0.95 so that there is a 95% chance of detecting the disease (Wobeser 1994).

To determine the number of animals to test to detect a specific disease, use the following formula:

where n = sample size, P = probability of detecting at least one case of the disease if it is present in the population (usually set to 95%) and d = number of detectable cases in the population. Thus, d = population size × prevalence × sensitivity of the test. N = population size.

Example

We want to determine if an army of 500 frogs is infected with chytrid fungus, which we suspect will have a 10% prevalence (approximately 10% of the frogs would be infected if the population had been exposed to the fungus). The diagnostic test is a real-time PCR with a sensitivity of 98%. We want to be 95% certain that we will detect the disease if it is present. Thus N = 500, P = 95%, d = 500 × .1 × 0.98 = 49

At least 28 frogs would have to be tested to determine if the population was infected with the fungus, with 95% confidence, where the prevalence is 10%. The sample size required increases with level of confidence required. It also increases as the prevalence of disease decreases and estimating the sample size for very low-prevalence diseases (<0.1%) with a high level of confidence can be difficult and very expensive. For wildlife, there may also be animal and population welfare concerns, the capacity of the diagnostic laboratory may be limiting and appropriate levels of funding and resources may simply not be available. In these instances there is often a shift from sampling to detect disease at a known level of confidence, towards determining the probability of disease freedom. These studies require targeted investigation into disease events or surveillance of high-risk individuals or populations (Salmon 2003).

The motto of fieldwork is ‘Above all, do no harm’. It is important not to spread infectious agents to wildlife populations, domestic animals or humans during health investigations. Some infectious agents and parasites can be very hardy in the environment or adhere to clothes and equipment. Each site where wildlife is handled should be considered a quarantine site (CCWHC 1998; CCWHC 1999).

Protocols for the prevention of disease spread should be incorporated into written operating procedures and animal ethics and research application forms.

Outer clothing and equipment worn in the field should be removed, and placed into plastic bags in the field. Hands and boots should be washed and disinfected in the field. Animal-handling bags or boxes should be cleaned and disinfected between uses with different animals. All equipment and clothing should be thoroughly washed and sanitised prior to being used in another location. Once organic material has been removed from equipment, a solution of household bleach (1 part bleach to 9 parts water) is an effective disinfectant. This solution can also be used to scrub boats, vehicle tyres and work surfaces. The external surfaces of sample vials should be cleaned and disinfected in the field. Sample vials should be leak-proof, and should be double-bagged prior to submission to the laboratory. Carcasses should be individually bagged in leak-proof heavy-duty plastic bags to be submitted to the laboratory. The carcasses should be placed into plastic tubs, in case leakage does occur en route.

Carcass disposal should be undertaken with great care. Animal carcasses are difficult to burn, and can contaminate groundwater if buried improperly. Often the easiest way of managing excess carcasses is to double-bag them at the site and organise incineration by a commercial contaminated waste disposal company. If carcasses must be burned or buried, seek prior approval from the local council, state agriculture department and rural fire service.

2 PATHOLOGICAL APPROACH TO DISEASE INVESTIGATION

Although the procedures and tests used to investigate disease in wildlife are similar to those used in domestic animals, the approach is dramatically different.

Veterinarians are often trained that diagnosis requires them to obtain a biopsy or collect a lump of the diseased tissue into formalin and send it to a pathologist. The pathologist will examine the tissue, make observations and interpret the lesions in comparison with a large dataset of the known diseases of that species. A sample of intestine from a dog with parvovirus usually has lesions that are characteristic of that disease, so there is generally no need to pursue viral culture. The difficulty with this approach is that we know relatively little about wildlife health and there is no definitive dataset for most non-domestic species.

Specialised diagnostic testing is often required for a definitive diagnosis of illness in wildlife. It is vital to collect appropriate samples that allow conclusions in the event of a disease outbreak in wildlife. It is also important to collect samples from healthy animals to produce baseline normal health indicators that will complete the dataset. Table 4.2 gives a list of the samples that should be collected during a routine necropsy.

Table 4.2 Routine necropsy checklist—in most cases, the following samples are sufficient

The collection of appropriate information is just as important as the collection of diagnostic samples. Having as much information as possible about the animal and how samples were collected will greatly improve the pathologists’ ability to interpret their findings.

• mass or unexpected mortalities/morbidities of unknown causes;
  
• significant clusters of deaths;
  
• suspect livestock-associated notifiable diseases;
  
• undiagnosed syndromes;
  
• suspected human/zoonotic connection;
  
• diseases likely to spread and be difficult to eradicate if they become established;
  
• suspected exotic and OIE list diseases;
  
• diseases with overseas events or international drivers;
  
• diseases listed as key threatening processes by DEWR (AWHN 2007).

Mass mortality, behavioural abnormality, granulomas, vesicles and ulcers should trigger an investigation. If investigation of a disease syndrome in wildlife produces negative test results for relevant endemic diseases, exotic or emerging diseases should be considered.

In the face of an unusual disease event, or disease in a highly threatened species, a very detailed and thorough post mortem procedure is required. Multiple sample sets must be collected from each animal so that a wide variety of diagnostic testing can be undertaken, potentially using several different laboratories. You may be investigating a new disease. In these situations it is equally important to rule out all other possible explanations for the event as to identify the agent of concern. A complete set of samples from as many animals as possible will facilitate the investigation. Table 4.3 provides a comprehensive post mortem checklist for these situations.

2.2 Diagnostic flow

Reaching a diagnosis in wildlife involves the following:

• observers to monitor wildlife, report any abnormalities and submit carcasses to an appropriate laboratory;
  
• a necropsy conducted by a veterinarian, veterinary pathologist or trained layperson. Sometimes a good understanding of what killed the animal is evident at the time of examination, but this technique is not very sensitive. You might determine that an animal died due to severe trauma when it was hit by a car, but not determine that the animal was hit because it was affected by an infectious disease or exposure to toxins;
  
• samples collected during the necropsy are usually submitted first for histopathology Sometimes these tissues can be examined with special stains that help to define the type of reaction in the tissues, or the type of organism. It is, however, often difficult to determine the exact organism inside a lesion using this technique;
  
• a pathologist most often forms a tentative diagnosis at this stage. Histopathology can take a few days to several weeks to complete;
  
• some diseases can be confirmed with histopathology, but additional diagnostic testing is often required for wildlife. The tentative diagnosis can be confirmed using tissues collected during the PM and kept frozen. These tissues can be submitted to specialised laboratories for bacterial, fungal or viral culture, parasite identification, electron microscopy or PCR to detect a variety of infectious agents, or toxicology testing;
  
• additional diagnostic tests can be as much value in ruling out some possibilities of the disease process (e.g. underlying exposure to toxins) as they are in ruling in or finding the cause of the event. These additional tests add time and expense to the diagnostic process. While most bacterial and fungal culture can be completed in 48–72 hr, it can take many weeks to isolate viruses or mycobacteria in culture;
  
• the pathologist will review the case history and the results of all examinations and tests. Their interpretation of complex findings is very important. If a sea eagle is shot, but dies of a fungal infection and other complications of being injured and in care, the cause of death is still the primary event—gunshot wound;
  
• after interpreting the findings, the pathologist will prepare a final report that may contain a definitive or confirmed diagnosis, but for various reasons it may not be possible to confirm the suspected diagnosis. If samples were inadequate, tests were not available, or the disease-causing agent was no longer present in the tissues etc., a definitive diagnosis may not be determined;
  
• when submitting samples to a veterinarian or a laboratory, ask how long they expect the tests to take and who will provide and discuss the results.

Table 4.3 Comprehensive necropsy checklist