When avian patients present with illness, they usually show non-specific symptoms which makes pinpointing an exact diagnosis difficult without diagnostics. Common examples of non-specific clinical signs of avian illness include fluffed-up feathers, dyspnoea, green droppings or regurgitation. It can be difficult for veterinarians to identify a specific disease from history and physical examination alone, similar to our canine and feline patients. Knowledge of common diagnostic tests and how to best collect samples is therefore important for any veterinarian treating avian patients.
In this article we will discuss common diagnostic tests in avian patients and how best to collect samples for these tests, as well as applying these ideas to a commonly diagnosed infectious disease: psittacosis.
Faecal sampling is a very useful and non-invasive diagnostic modality. Droppings are readily produced by bird species and contain urine, uric acid and faeces (Figure 1).
Faecal egg counts can be useful in birds of prey, poultry or ratites, especially when investigating weight loss, poor appetite or poor performance. A pooled sample from several birds in a flock or several samples from a single bird are more useful than a single sample. Egg count estimates can be performed with a known volume of faeces and flotation fluid. Faecal cytology with Gram staining (Figure 2) can also be useful for evaluating changes in the consistency of droppings, for example diarrhoea.
Care must be taken not to over-interpret samples (Isaza, 2000), especially as faecal flora varies markedly between species, but bacterial ratios can be assessed, as well as looking for evidence of fungal organisms. Faeces can also be used for polymerase chain reaction (PCR) testing for certain infectious diseases, such as Chlamydia psittaci.
Crop swabs can be assessed cytologically in a similar way to faeces, and crop washes can be used to identify parasitic organisms, for example Trichomonas in budgerigars. With crop washes, 0.5 to 5ml of warmed sterile saline is instilled into the crop using a rigid crop tube, then aspirated and assessed as a wet mount (Hochleithner and Hochleithner, 2021) or dried and stained with Gram or Romanowski stains.
Blood sampling is incredibly useful for assessing overall health, and blood samples are relatively easy to gain from avian patients.
Common blood sampling sites include the jugular veins on the lateral aspects of the neck (Figure 3), the ulnar or basilic veins on the ventral surface of the wings or the medial metatarsal vein which runs medially over the intertarsal joint.
As a general rule of thumb, 1 percent of a patient’s body weight can be safely taken via venepuncture in a healthy patient or 0.5 percent of a patient’s body weight in a bird showing signs of disease (Samour, 2016). Most patients will tolerate blood sampling while manually restrained; however, some clinicians prefer sedation or anaesthesia prior to venepuncture.
A blood sample can be a highly useful diagnostic tool, allowing for haematology, biochemistry, blood film analysis to assess for haemoparasites, heavy metal testing, DNA sexing, blood gas analysis, and serology and PCR for certain infectious diseases.
Cytology – skin masses and organs
Fine needle aspirates of skin masses or organs can be useful for cytological analysis, as can sticky tape preparations, skin scrapes or feather plucks. Aspiration of coelomic effusions can also differentiate a transudate and exudate in the same way as companion animal practice. This is particularly useful when assessing backyard chickens with coelomic effusion to differentiate egg yolk coelomitis, reproductive neoplasia or cardiac disease.
As an example of the usefulness of diagnostic sampling in avian patients, infection with the bacteria Chlamydia psittaci often presents as a non-specific illness and requires diagnostic sampling to determine if it is the cause of the clinical signs. This is especially important if the patient has symptoms of increased respiratory effort, presence of yellow or green droppings, regurgitation or upper respiratory signs with oculonasal discharge.
C. psittaci is a Gram-negative bacteria that targets epithelial cells from mucous membranes and mononuclear macrophages, acting as an obligate intracellular organism (Yang et al., 2022). It can be transferred by close contact, faeces or aerosols (West, 2011). Definitive diagnosis is important due to its implications as a zoonotic disease, and the risk factors for human contraction include owning birds or working with birds, in particular pigeons and doves (England, 2017),poultry and parrots. The most commonly affected companion parrots include parakeets, lories, cockatiels and budgerigars (Beeckman and Vanrompay, 2009).
How can psittacosis be diagnosed?
Theoretically, one of the most accurate tests that can be used to reveal the C. psittaci pathogen in a sample is cell culture, which is more sensitive and specific than the tests available in clinical practice but also far less practical.
Other possible diagnostic methods include identification of the bacteria in samples taken from tracheal or cloacal swabs (Figure 4) and/or fresh faeces (Pearson et al., 1989). An alternative is direct immunofluorescence staining from conjunctival and cloacal smears (Vanrompay et al., 1992). However, all these diagnostic techniques are often difficult to perform, as many labs do not offer these diagnostic services, and results can take several weeks to be returned.
Most C. psittaci diagnoses are achieved with PCR and serology, which are readily available tests provided by several different laboratories in the United Kingdom. PCR has been proven to be an effective way to detect pathogens in faecal samples as it is highly sensitive and specific (Çelebi and Ak, 2006). Choanal, conjunctival and cloacal samples can be taken with a plain swab. PCR has even been shown to be more sensitive than cell culture isolation (McElnea and Cross, 1999).
Serology is considered a complementary diagnostic tool. The most common serology method is the direct complement fixation, which measures IgG titres. Thus, it is advisable to pair samples, as patients with symptoms can have low antibody titres that may increase within seven days after taking the first sample. Otherwise, diagnosis with serology can be vague and have a significant number of false-negative patients (Grimes et al., 1996). The recommendation for diagnosis via serology is two blood samples taken at least two weeks apart, run at the same time in the same laboratory (Balsamo et al., 2017).
Although treatment is sometimes started before diagnostic sampling, it is advisable to collect samples prior to starting antibiotics, as antibiotic administration has been shown to affect serology and PCR results
Although treatment is sometimes started before diagnostic sampling, it is advisable to collect samples prior to starting antibiotics, as antibiotic administration has been shown to affect serology and PCR results, increasing the odds of false negatives (Fudge, 1991).
Which samples and laboratory tests are needed for C. psittaci?
PCR is a straightforward diagnostic test that can be obtained consciously, either via a pooled faecal sample or plain conjunctival, oral and/or cloacal swabs. Choanal, cloacal and conjunctival sterile plain swabs can be taken aseptically. Pharyngeal samples are the most reliable in order to avoid false-negative results (Andersen, 1996).
A pooled faecal sample of three to five days or repeated cloacal swabs may decrease the chances of obtaining false-negative PCR results, compared to single samples (Hewinson et al., 1997). This is because faecal shedding can be intermittent for days or months depending on the species (Speer et al., 2016). Unfortunately, the exact time of shedding is unknown. Hence, this presents a diagnostic challenge due to the increasing odds of having false-negative cases when PCR is performed during routine health checks because carriers do not show any symptoms and can be negative on serology.
It has been shown that it is preferable to combine PCR and serology to maximise the diagnostic efficiency of the samples obtained (Piasecki et al., 2012).
Should the size of my patient affect my diagnostic approach?
For PCR testing, the size of the patient will not determine or affect the outcome as no blood sampling or other invasive procedures are needed.
In terms of patient size and serology, the patient’s weight does matter. Some laboratories require large volumes of serum for serology, while others just need a drop of whole blood which makes the approach widely different. Discussion with your lab of choice will help determine if serology is a viable diagnostic test for your patient. Commercial kits, such as the ImmunoComb which measures IgG titres (Biogal Galed Laboratories, 2019) and uses only 10 micrograms of whole blood, allow for serological testing in smaller birds. With larger patients that weigh 100 grams or more, a sufficient blood sample should be able to be obtained using the venepuncture sites discussed previously.
Discussion with your lab of choice will help determine if serology is a viable diagnostic test for your patient
What is considered as a positive result?
Once samples are collected and sent, it is crucial to know how to interpret those results.
Balsamo et al. (2017) determined criteria for what is considered a confirmed case versus a suspicious case of C. psittaci. Confirmed cases fulfil at least one of the following criteria:
- Isolation of C. psittaci from a clinical specimen
- Identification of chlamydial DNA by use of in situ hybridisation to detect chlamydial DNA followed by specific C. psittaci DNA detection using PCR-based testing of in situ hybridisation-positive tissues or secondary C. psittaci-specific DNA probes in combination with characteristic pathology. The commercial antibodies used for immunohistochemistry staining cross-react with non-chlamydial epitopes and are not diagnostic
- A fourfold or greater change in serological titre in two specimens from the bird obtained at least two weeks apart and assayed simultaneously at the same laboratory
- Identification of suggestive intracellular bacteria in diseased cells in smears or tissues (eg liver, conjunctival, spleen, respiratory secretions) stained with Gimenez or Macchiavello stains in combination with detecting C. psittaci DNA in the same tissue sample using a C. psittaci-specific PCR-based detection assay
Suspected cases are defined as a compatible illness and observe at least one of the following criteria:
- Identification of chlamydial nucleic acid by PCR-based testing in conjunctival, choanal or cloacal swabs, blood or faeces
- Chlamydiaceae antigen (fluorescent antibody) in faeces, a cloacal swab specimen or respiratory tract or ocular exudates
- The case is epidemiologically linked to a confirmed case in a human or bird
On the other hand, there are cases that are positive to neither PCR nor serology, but if they respond to specific treatment, manifest symptoms and/or the owner or other birds within the flock are positive, then it would be considered as a suspected case (Crosta et al., 2016).
Diagnostic testing in avian patients is essential for determining the underlying cause of illness to appropriately treat disease conditions. As an example, psittacosis is a zoonotic disease that has to be included as a differential diagnosis in avian species. It often presents with non-specific clinical signs and diagnostic sampling is important to diagnose a clinical case.
Diagnostic testing in avian patients is essential for determining the underlying cause of illness to appropriately treat disease conditions