Sample Collection

Collection of blood volume equivalent to 1% of a bird's body weight. Many practitioners use tuberculin or insulin syringes that do not have detachable needles. These needles can easily be cut from the syringe using a pair of large veterinary nail clippers.

Avian plasma samples are frequently yellow due to carotenoid pigments, not bilirubin. Pink or red plasma is indicative of hemolysis. Needles must be detached and blood volume must be adequate for the tube. Biochemical samples should always be separated within 30 minutes to prevent artifact that may cause misdiagnosis. Lipemia is rarely observed in avian samples, however, obese Amazons and other birds may produce lipemic plasma. This may be indicative of underlying metabolic disease such as hypothyroidism or diabetes mellitus.

Liver/Muscle

Liver disease is common in birds. Interpretation of standard hepatic analytes used in companion animals must be modified due to physiological differences.

As in large animals, alanine aminotransferase (ALT) is found in hepatic cytosol as well as in muscle and other tissues of birds. Intramuscular injection of doxycycline in pigeons can cause plasma ALT levels to increase above the reference range for greater than 200 hours.⁶ This results in a test with poor specificity and does not add any clinically relevant information to an AST value.

Aspartate aminotransferase is also not specific for hepatocellular damage but returns to normal reference ranges within 100 hours after muscle trauma in pigeons.⁶ It is highly sensitive in detecting liver damage caused by ethylene glycol in pigeons.⁶ It is currently considered to be a very sensitive indicator of hepatocellular disease in avian species and is frequently used with the muscle specific creatine kinase to differentiate liver and muscle damage. Increases in AST may be caused by hepatocellular damage, muscle damage, Vit E/Selenium deficiency, toxins/pesticides, and other.

Most birds produce very little biliverdin reductase and therefore do not produce bilirubin in health. There are numerous reports, especially in chicken, of jaundiced birds with increased bilirubin. Though generally at trace levels, bilirubin may be useful in diagnosis of hepatic pathology. Biliverdin (the tetrapyrrole-dehydrobilirubin) can be measured but may not prove to be clinically more useful than bile acids (Ritchie, personal communication).

Glutamate dehydrogenase (GDH) is found in hepatocyte mitochondria and is considered the most specific indicator of hepatocellular damage. There are also high concentrations in renal tissue; however, most of this is excreted directly into urine and never reaches the blood stream.¹ This enzyme is generally elevated only when there is hepatic necrosis and therefore has low sensitivity.⁶

Gamma glutamyl transferase (GGT) is probably specific to biliary and renal epithelium similar to companion animals. Although considered "insensitive and inconsistent in the diagnosis of liver disease" by many practicing avian veterinarians, Lumeij found that GGT was increased in the majority of pigeons with experimentally induced liver disease.⁶ Marked elevations of GGT in clinical cases of bile duct carcinoma have been reported.⁷ Though no reference ranges have been officially established, 0–15 U/L is considered normal at the Schubot Exotic Bird Health Center. This appears to be slightly different in aging Amazon parrots that may have up to 18 U/L without other evidence of liver disease (Phalen, personal communication). GGT is most likely elevated in cholestatic conditions and biliary epithelial disorders. Therefore, it will not be sensitive in situations of hepatocellular damage alone. It may be very useful to diagnose the most common hepatic neoplasia in birds, biliary carcinoma.

Cholesterol metabolism is similar to that of mammals, but there are some specific differences in clinical presentation. In oviparous species, such as iguanas and birds, a marked elevation of cholesterol and triglyceride concentration can be seen during vitellogenesis and egg formation. Increases may be seen before the egg(s) can be visualized on radiographs.

Though reference ranges have been established for bile acids using the enzymatic method, neither the enzymatic method nor any of the radioimmunoassay (RIA) kits have been validated for use in avian or reptilian species. RIA generally measures nonsulfated conjugated bile acids. There is variability in human test kits caused by the different antibodies binding different amino acid conjugates.

Additionally, different bird species have different predominant bile acids than humans. Consequently, different kits will yield different values that may or may not be representative of total bile acids in that species. The enzymatic method, validated in dogs, cats, and humans, measures the 3 alpha hydroxyl group present in most predominant bile acids. This test will likely best approximate total bile acids present in most avian species. Using the enzymatic method, bile acid values > 100 umol/L are considered abnormal and > 75 umol/L are suspect.^5,6 Amazon parrots normally have slightly higher bile acid concentrations. Of course, reference ranges for individual species should be generated in each laboratory and validation is necessary.

Electrolytes

There has been extensive study on intestinal absorption of electrolytes and calcium transport in chickens. One may access the literature through Whittow. The predominant anions and cations are similar to mammals. Reference values in the literature should be critically evaluated since many are based on studies performed prior to the existence of ISE electrodes.

Serum Electrophoresis and Protein Evaluation

Reference ranges for birds are significantly lower than those for mammalian species. The total protein value generated using a refractometer is frequently inaccurate due to high concentrations of other refractile compounds in plasma, such as chromogens, lipids, and glucose. (Recall that a hummingbird normally has a blood glucose of 800 mg/dl.) The biuret method is the most accurate method to quantify total protein.⁶

Plasma gel electrophoresis is used to accurately quantitate albumin and evaluate globulin distribution. It is also a useful aid for monitoring therapeutic response in animals that frequently show few overt clinical signs. Plasma proteins identified in the classic banding pattern of avian species include transthyretin in the prealbumin fraction; albumin; alpha1-anti trypsin in the alpha1 zone; alpha2-macroglobulin in the alpha2 zone; fibrinogen, beta-lipoprotein, transferrin, complement, and vitellogenin in the beta zone; immunoglobulin and complement degradation products in the gamma zone.^2,3

Transthyretin has replaced "prealbumin" in human medical vocabulary. This protein binds thyroid hormones and retinol with varying affinities for the different hormones across mammalian, avian, and reptilian species. It has greater than 98% homology and has been shown to have a very similar banding pattern across species.^2,4

The bromocresol green (BCG) methodology is not validated in birds. Significant discrepancies have been shown between BCG and gel electrophoresis. This disparity is caused, in part, by the use of a human albumin standard and control which has a different binding affinity for the dye than does avian albumin.⁸ Gel electrophoresis is the recommended method of albumin determination in avian species at this time.^3,6 Reference ranges for species of birds should be established in each laboratory.

Fibrinogen is an acute phase protein which may be used to assess inflammatory illness in birds.

Albumin:globulin (A:G) ratio can be decreased in disease states such as inflammation, protein losing nephropathy, and liver failure. However, females of oviparous species can also have a physiological decrease in A:G ratio. The majority of the yolk proteins and chalazae band in the globulin region. Albumin is only very mildly increased during egg formation. This results in a decreased A:G ratio that does not indicate a diseased state.

Recently Beckman Coulter, the main manufacturer of electrophoresis equipment used in North American veterinary laboratories for 20–30 years announced discontinuation of the Paragon electrophoresis system. There are two remaining options, the Helena SPIFE 300 system using Split Beta gels (Helena Laboratories) or the Sebia Hydrasys system using Hydragel 7 protein gels (Sebia, Inc.), neither of which is directly correlated to the Beckman system due to differences in voltage, buffer composition, staining procedure, and gel concentration. There are different thoughts on how to proceed with change to new equipment. While both the systems offer benefits, differing philosophies guide the adoption of the two units. One thing is certain. The dissimilarities inherent in the systems will cause proteins to migrate differently and the different fractions therefore have different quantitative values on the same sample. The Helena system is better correlated with the Beckman system though there are quantitative differences which require reference interval generation. The benefit of the Sebia system is that it separates the albumin fraction better with minimal overlap of the globulin proteins allowing for more precise and accurate albumin quantitation. This is the primary reason that I recommend gel electrophoresis and so this is the unit that we have chosen. Mammals are more similar in quantitation to the Helena as mammals do not have the same issues with transthyretin and the previous "prealbumin" fraction. Transthyretin migrates to the alpha 1 position using the Sebia instrument as it does in mammals so there will be minimal to no measurement of the prealbumin fraction in birds in the future. As these instruments are used over time, further evaluation will be possible.

Chlamydophila

The following section is based on the 2010 NASPHV Compendium of Measures to Control Chlamydophila psittaci Infection Among Humans and Pet Birds.⁸

Chlamydophila psittaci is a member of the family Chlamydiaceae. There are at least eight serovars and nine genotypes described which in the future may prove to be of importance in the epidemiology of the disease in animals and humans. Chlamydial organisms have been isolated from over 460 bird species from 30 orders but are more commonly found in psittacines, especially cockatiels and budgies.

This disease is occasionally zoonotic causing influenza-like symptoms that can lead to pneumonia and nonrespiratory health problems. With appropriate antibiotics, the disease is rarely fatal and is cleared from the body. Before antimicrobial agents were available, 15–20% of humans with C. psittaci infection died. From 2005–2009, 66 human cases of psittacosis were reported to the CDC though this is likely an underrepresentation. Most infections are typically acquired from psittacine birds, although transmission has also been documented from free ranging birds including doves, pigeons, shore birds, and birds of prey. Human infection usually occurs when a person inhales organisms that have been aerosolized from dried feces or respiratory tract secretions, though mouth to beak contact has been documented. In humans differential diagnosis of the typical signs includes Coxiella burnetti, influenza, Histoplasma capsulatum, Mycoplasma pneumonia, Legionella sp, C. pneumoneia, and other respiratory viruses.

C. psittaci is excreted in the feces and nasal discharges of birds and while it does degrade, it can remain stable for over a month. Subclinical carriers exist but stress increases shedding and so factors such as reproduction, rearing of young, relocation, shipping, crowding, and chilling may all increase shedding. The usual incubation period in birds is 3 days to several weeks. Clinical signs include lethargy ruffled feathers, anorexia, with respiratory discharge, diarrhea, and other GI and respiratory signs.

A confirmed case of avian chlamydial infection is defined on the basis of one of the following: isolation of C. psittaci from a clinical specimen using culture, IFA identification of the organism in tissues, fourfold or greater rise in titer in two specimens from the bird obtained at least 2 weeks apart and assayed simultaneously at the same lab, identification of Chlamydiaceae within macrophages in smears or tissues (liver, conjunctival, spleen, respiratory) stained with Gimenez or Macchiavello stain. A probable case is defined as a single high titer with clinical signs, or identification of Chlamydiaceae antigen (identified by ELISA, PCR or IFA) in feces, a cloacal swab specimen, or respiratory tract or ocular exudates. A suspected case is defined as a compatible illness that is not laboratory confirmed but is epidemiologically linked to a confirmed case in a human or bird, a bird with no clinical signs and a single high serological titer or detection of chlamydial antigen, compatible illness with positive results from a new investigational test or response to therapy.

Culture is difficult. Chlamydophila species are obligate intracellular bacteria that must be isolated in tissue culture or embryonating chicken eggs. Specialized lab facilities and training are necessary for reliable identification of chlamydial isolates and adequate protection of microbiologists. The proper handling of specimens is critical for maintaining the viability of the organism and a special transport medium is required. Specimens should be refrigerated and sent to the lab on ice but not frozen.

Positive serologic tests only indicate exposure though very high titers or rising titers indicate active disease. False negatives occur in birds that have acute infection when specimens are collected prior to seroconversion or in budgies and the smaller birds that produce less cross-reactive humoral compounds. Titers should be compared to WBC (marked leukocytosis), and plasma liver enzymes (typically increased but not always). Fourfold increase between paired titers is considered a confirmed case. There are several different serologic assays which should be interpreted and used differently. Elementary body agglutination (EBA) detects IgM antibodies in early infection. The elementary body is the infectious form of C. psittaci. Titers greater than 10 in budgies, cockatiels, and lovebirds and > 20 in larger birds indicate recent infection. Indirect fluorescent antibody (IFA) uses polyclonal secondary antibody and is used to detect host antibodies (primarily IgG). Sensitivity and specificity varies with the immunoreactivity of the polyclonal antibody to various avian species. Low titers occur because of nonspecific reactivity. IFA is my least favorite test due to assay controls and subjectivity based on technician opinion. Complement fixation is more sensitive than agglutination methods. False negative results are possible in specimens from parakeets, young African gray parrots, and lovebirds due to lack of production of antibody in the animals. High titers can persist after treatment which complicates interpretation of positives. Modified direct CF is more sensitive than direct CF.

Tests for antigen detect the organism but do not require live whole organisms to be positive. ELISAs were originally developed for identification of Chlamydia trachomatis in humans. The exact sensitivity and specificity of these tests (immune tests with multiple manufacturers) are not known. These are screening tests that should be followed with further testing such as titers or PCR. IFA uses either monoclonal or polyclonal antibodies with fluorescein staining techniques to identify the organism in impression smears or other specimens. This test is utilized by some state diagnostic laboratories.

Test for DNA include polymerase chain reaction, and it should be noted that the organism does not need to be alive for detection. PCR can be performed on conjunctival, choanal and cloacal swab specimens, and blood. Fecal samples can be pooled to increase the likelihood of detection. There are no standardized PCR primers and lab techniques and sample handling may vary. Because of the sensitivity of the assay, samples for PCR must be collected using techniques to avoid contamination from the environment or other birds. This test should be interpreted with clinical signs and hematologic and biochemical data.

References

1.  Battison AL, Buchzkowski S. Plasma bile acid concentration in the cockatiel. Can Vet J. 1996;37:233–234.

2.  Chang L, Munro SLA, Richardson SJ, Schreiber G. Evolution of thyroid binding by transthyretins in birds and mammals. Eur J Biochem. 1999;259:534–542.

3.  Cray C, Tatum L. Applications of protein electrophoresis in avian diagnostics. J Avian Med Surg. 1998;12(1):4–10.

4.  Farer LS, Robbins J, Blumberg GS, Rail JE. Thyroxine serum protein complexes in various animals. Endocrinology. 1962;70:686–96.

5.  Hoeffer H. Bile acid testing in psittacine birds. Semin Avian Exot Pet Med. 1994;3(1): 33–37.

6.  Lumeij JT. Avian clinical biochemistry. In: Clinical Biochemistry of Domestic Animals. Kaneko, Harvey, Bruss, ed. Academic Press, Harcourt. San Diego. 1997:857–877.

7.  Phalen DN, Homco L, Jaeger L. Investigations into the etiologic agent of internal papillomatosis of parrots and untrasonographic and serum chemical changes in Amazon parrots with bile duct carcinomas. Proceedings Assoc of Avian Vet. 1997:53–56.

8.  Smith KA, Campbell CT, Murphy J, Stobierski MG, Tengelsen LA. Compendium of measures to control Chlamydophila psittaci infection among humans (psittacosis) and pet birds (avian chlamydiosis), 2010 National Association of State Public Heath Veterinarians (NASPHV). J Exot Pet Med. 2011;20(1):32–45.

9.  Spano JS, Whitesides JF, Pedersoli WM, Krista LM, Ravis WM. Comparative albumin determinations in ducks, chickens, and turkeys by electrophoretic and dye-binding methods. Am J Vet Res. 1988;49(3):325–6.