Selected complete blood cell count and plasma protein electrophoresis parameters in pet psittacine birds evaluated for illness.
|Subject:||Blood protein electrophoresis (Methods)|
Briscoe, Jeleen A.
Rosenthal, Karen L.
Shofer, Frances S.
|Publication:||Name: Journal of Avian Medicine and Surgery Publisher: Association of Avian Veterinarians Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Association of Avian Veterinarians ISSN: 1082-6742|
|Issue:||Date: June, 2010 Source Volume: 24 Source Issue: 2|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
Abstract: Veterinarians rely on results of both the complete blood
cell count (CBC) and plasma protein electrophoresis (EPH) in conjunction
with the results of the plasma biochemical analysis to evaluate the
health status of avian patients. Because the CBC and protein EPH measure
different aspects of the immune response to disease, both tests are
recommended in avian patients to rule out infectious or inflammatory
disease. To evaluate results of the CBC and protein EPH in pet
psittacine birds, the records of 144 pet psittacine birds, comprising 11
genera, that were presented for suspected illness were reviewed. Results
of the CBC (total white blood cell count and packed cell volume) and
protein EPH (alpha, beta, and gamma globulin concentrations) from
submitted blood samples from each bird were evaluated. Of the 144 birds,
63 (43.8%) had abnormal CBC results, and 25 (17.4%) had abnormal EPH
measurements. Results of the CBC and protein EPH were within reference
ranges in 73 birds (50.7%). Abnormal results of the CBC in conjunction
with normal EPH results were present in 46 birds (31.9%), compared with
8 birds (5.6%) with normal results of the CBC and abnormal EPH results.
The findings of this study could aid practitioners in evaluating
psittacine patients and prioritizing the value of individual diagnostic
Key words: complete blood cell count, plasma protein electrophoresis, immune response, diagnostic, avian, psittacine birds
Diagnosis of disease in pet psittacine birds can be challenging. Psittacine birds are well known to mask signs of illness so as to appear to potential predators to be healthy members of the flock. (1) This characteristic of wild birds continues to be expressed in psittacine birds kept as pets. Consequently, the veterinarian may miss signs of illness if the health assessment solely depends on the results of the physical examination. The potential to erroneously conclude that a sick bird is healthy imparts value to diagnostic testing in fully evaluating the health status of an individual bird)
Two tests commonly used by veterinarians to measure the immune response of birds are the complete blood cell count (CBC) and plasma protein electrophoresis (EPH). The white blood cell component of the CBC helps to measure the cellular response to certain diseases. The packed cell volume (PCV) can decrease in cases of blood loss or hemolysis, but a low PCV can also develop with chronic disease and thus serves as an indicator of the duration and severity of disease. (3,4) The globulin portion of the protein EPH measures the acute-phase proteins and humoral, or adaptive, response to disease. (3,5,6) The alpha-1, alpha-2, and beta globulin fractions of the EPH contain most of the acute-phase proteins, including fibrinogen and serum amyloid A, whereas the gamma fraction contains antibodies to viral, bacterial, or fungal pathogens. (7-9) Because the CBC and protein EPH measure different aspects of the immune response to disease, both tests are recommended in avian patients to rule out infectious or inflammatory disease. (3,5,8-11)
The decision as to which diagnostic tests to perform is based on both the clinical signs described by the owner and the physical examination findings. The volume of blood that can be safely obtained from many psittacine patients can be limited on the basis of their size, and the number of diagnostic tests run is also often limited by the willingness of the owner to pay for them. This requires that veterinarians prioritize the importance of various tests.
The goal of this retrospective study was to evaluate the number of parrots that were examined with a presenting complaint of illness that displayed abnormal values on the CBC, the protein EPH, or both, as measured from a single blood sample. Our purpose was to provide a clinical review of how often the results of these tests were abnormal, individually and in combination, in a group of psittacine birds that were presented for illness.
Materials and Methods
Records of pet psittacine birds examined during 2000-2005 at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania (MJR-VHUP) were reviewed. Only records from birds that were presented for illness were included in the study; however, if a bird was brought in for a wellness check but subsequently was found to have signs of illness on physical examination, it was also included. The owner's description of clinical signs was the basis for inclusion in the study. Only cases in which the CBC and EPH were performed on the same blood sample were included. Blood test results for repeat visits were not included; thus, every bird in the study was evaluated only once. For each bird, the clinical signs provided by the owner were recorded along with species and sex, when known. Most birds evaluated were adult psittacine birds (ie, > 1 year old); however, the accurate age was often not known by the owner and was not evaluated in the study. Although egg-laying proteins can cause a marked increase in the globulin fraction of the EPH, (12) birds with a history of egg laying were included in the study.
In most birds, blood samples were obtained by jugular venipuncture, but in some birds, samples were collected from the basilic or medial metatarsal vein. Each sample was immediately placed into a microtainer containing ethylenediaminetetraacetic acid (EDTA) for the CBC and lithium heparin for the protein EPH (Microtainer, Becton Dickinson and Co, Franklin Lakes, NJ, USA). The CBC was analyzed in house (Clinical Pathology Service, MJR-VHUP, Philadelphia, PA, USA) with a manual hemocytometer after red blood cells were lysed with Natt and Herrick solution. The heparinized sample was centrifuged at 2000g at 21[degrees]C, and the plasma was decanted, transferred to a sterile Eppendorf tube, refrigerated at 4[degrees]C, and transported to a reference laboratory overnight for evaluation of the protein EPH (Antech Diagnostics, Lake Success, NY, USA). The reference laboratory used biuret methodology for measuring plasma total protein concentration. Agarose gel electrophoresis was used to measure prealbumin, albumin, and alpha, beta, and gamma globulins. This method has been described in detail elsewhere. (13)
Data are presented as frequencies and percentages with Clopper Pearson 95% confidence intervals (CIs) around the percentages where appropriate.
A total of 144 birds met the inclusion criteria for the study. More than 40 different species were included, representing 11 genera, most of which were medium to large psittacine birds (Table 1). The sex of 90 birds was known; of those, 53 of 90 (59%) were intact females, 36 of 90 (40%) were intact males, and 1 of 90 (1%) was a salpingectomized female. Five birds (3%) were less than 1 year of age. Table 2 lists the clinical signs provided by the owner as the reason the bird was brought to the hospital. Of the 144 birds, 37 (26%) were presented for nonspecific signs, including anorexia or lethargy. Fourteen birds (9.7%) had a history of chronic egg laying, but the owners of each of these birds reported clinical signs in addition to egg laying, which included cloacal prolapse, polydypsia, blindness, and lethargy.
Of the birds evaluated for illness, 63 of 144 (43.8%) had abnormal values on the CBC, whereas 25 of 144 (17.4%) of the birds had abnormal values on the EPH (Table 3). Results of both the CBC and EPH were considered normal in 73 (50.7%) of the birds (Figure 1). Forty-six (31.9%) of the birds had abnormal values on the CBC and normal values on the EPH compared with 8 (5.6%) of the birds that had abnormal values on the EPH with normal values on the CBC.
In the group of 144 psittacine birds evaluated for illness, abnormal results for a single test were more common on the CBC than the EPH (31.9% and 5.6%, respectively). The CBC is performed to evaluate for inflammation or infection, which is ultimately indicated by the WBC count and the differential. Leukocytosis can occur with inflammation, stress, and steroid administration. (4) In a study comparing normal African grey parrots (Psittacus erithacus) with those presumed to have infectious disease, all of the sick birds exhibited leukocytosis. (15) At least several species of parrots can exhibit a left shift in reaction to severe inflammation. (14) Stress from handling and transport has been associated with an increase in the heterophil-lymphocyte (H/L) ratio in psittacine birds but a decrease in the WBC count in pigeons (Columba livia). (16) In chickens, there is evidence of a potentially biphasic leukocyte response to stress; mild to moderate stress produces a heterophilia and increased H/L ratio (17) and severe stress produces heteropenia, lymphocytosis, and basophilia. (18) To our knowledge, although stress-induced increases in the WBC count and relative heterophilia have been reported in psittacine birdsy, (4-19-23) a peer-reviewed study of the correlation between high corticosteroid levels and changes in the CBC has not been performed in psittacine birds. Whether or not stress influenced the WBC count of the birds in this study is unknown, but the inclusion of only birds with illness as reported by their owners makes this possibility less relevant to the purpose of the study.
The CBC will also reveal changes in the PCV. In addition to blood loss or hemolysis, a nonregenerative anemia occurs commonly in birds with chronic inflammation or other disease. (3,4,24) In this study, 25.7% of the birds had an abnormal PCV, which is similar to the percentage of birds with an abnormal WBC count (27.8%) but almost twice as high as the percentage of birds with abnormal EPH values (17.4%). Because this study did not evaluate the diagnoses of each case, individuals with a low PCV from hemolysis or blood loss (as opposed to chronic infection) were not eliminated from analysis. This could have affected the final results. Regardless, the PCV is a rapid and technically simple test to perform on a small amount of blood; thus, its value as a potential indicator of illness of any type, as suggested by the findings of this study, is notable.
For more than 10 years, the EPH has been recommended as part of the minimum database in the health evaluation of psittacine birds presented to the avian veterinarian. (3,5,9,10,25) Evaluation of the serum EPH is used in domestic mammalian medicine to characterize dysproteinemias associated with diseases such as hepatic cirrhosis, (26) tumors of the reticuloendothelial system, canine ehrlichiosis, (27) parvovirus, (28) and fungal disease. (7,29) The EPH is used to evaluate the albumin and globulin levels in pet birds because most biochemistry analyzers are unable to accurately measure the low levels of albumin in avian plasma. (10.30-32) The release of acute-phase proteins in response to external or internal challenges to the animal (eg, infection or inflammation) represents a nonspecific component of innate immunity. Measure of the concentration of acute-phase proteins in response to disease provides diagnostic and prognostic information on the health status of an animal. (33,34) Acute-phase proteins, such as alpha-lipoprotein and haptoglobin, are said to migrate within the alpha-1 and alpha-2 globulin fraction, whereas the acute-phase proteins fibronectin and transferrin migrate through the beta globulin fractions of the EPH. (5) Thus, the EPH is recommended as a measure of the acute-phase protein response. (7) The EPH is said to be useful in pet bird medicine in the evaluation of infection, neoplasia, toxicosis, nutritional deficiencies, and even behavioral problems. (25)
It has been stated that prealbumin should be included in the calculation of the albumin:globulin ratio (10,31,32) and that it can act as a negative acute-phase reactant; (13) however, because the role of prealbumin in the psittacine immune response could be species-specific and is as yet unclear, (13) prealbumin was not evaluated in this study. Evaluation of the albumin :globulin ratio was also not done in this study because its value can be negatively influenced by the presence of egg-laying proteins and thus might not necessarily reflect infection or inflammation.'2 As stated above, birds with a history of egg laying were not excluded from this study.
The premise for the use of EPH in the diagnostic workup of pet birds is that a WBC elevation might not occur with infection or inflammation. In a study evaluating changes in heterophil number (cellular response) and avian fibrinogen concentrations (humoral response) during bacterial infection, 21% of birds had
elevations in fibrinogen levels without heterophilia. Thus, the authors of that study recommended the evaluation of cellular and humoral immune responses to diagnose illness in birds. (35) It has been estimated that 30% of apparently healthy birds will have elevated EPH values without changes on the hematologic and biochemical analyses. (5) Thus, the veterinarian could miss illness in approximately one-third of avian patients if an EPH is not performed in conjunction with a CBC and biochemical analysis.
The overall low percentage of birds presenting for illness with abnormal EPH values in this study does not seem to support this theory. A possible explanation is that the EPH might not be a reliable diagnostic test for a variety of reasons, including human error and the difficulty in measuring avian plasma proteins, which are lower than those of mammals. (30) A more recent evaluation of psittacine EPH values with a different statistical method to evaluate reliability found variable protein concentrations and migration patterns; thus, the use of species-specific reference ranges was suggested. (13) One limitation of our study could have been that the reference ranges used for the EPH were incorrect and did not take into account possible genus- or species-specific variations. (32) On the basis of difference in response of exotic birds to intradermal tuberculin testing compared with the response seen in poultry, it has been proposed that inflammatory responses among even avian orders are actually quite heterogeneous. (36) The avian tuberculin testing study also reported that hemolysis and lipemia might cause artifactual decreases in the albumin:globulin ratio and an increase in the beta globulin fraction. (13) Hemolysis and lipemia were not evaluated in this study, but eliminating birds with lipemia evident on their blood test results might have negatively affected the results by decreasing the number of birds with abnormal beta globulin levels (approximately 14% reported in this study).
Although we found results of the CBC to be abnormal more frequently than the EPH values in psittacine birds evaluated for illness, less than 50% of the birds had abnormal CBCs on the basis of our criteria. Analysis of the WBC differential possibly would have revealed abnormalities in more of these birds. A study of wild black cockatoos (Calyptorhynchus magnificus and Calyptorhynchus funereus) found the most common hematologic changes in response to trauma and inflammation were heterophilia and monocytosis, in addition to a mild anemia. (37) In a study of gentoo penguins (Pygoscelis papua) comparing results of the CBC and fibrinogen values in birds with bumblefoot to those without, several birds with severe bumblefoot exhibited a relative heterophilia not reflected in an elevated WBC. One of these birds also had a relative basophilia. (38) Evaluation of the WBC differential was not performed in our study, because consensus and evidence is lacking in the avian literature regarding species-specific relative or absolute reference ranges for heterophils, lymphocytes, monocytes, eosinophils, and basophils in psittacine birds. Lymphocytes rather than heterophils might be the predominant leukocyte in certain psittacine species, (39) making it difficult to interpret changes in the WBC differential in response to illness. In a study of Cuban Amazon parrots (Amazona leucocephala leucocephala), a lack of monocytes, basophils, and eosinophils were found in the differential counts, leading the authors to suggest that cell lines might differ among species. (40) Furthermore, even with an equal distribution of WBC types across a blood smear, differential counts are not reliable among technicians performing an avian WBC count. (41) In a study providing species-specific reference ranges for hematologic and plasma biochemical values for psittacine birds, the authors cautioned against drawing conclusions from the small number of species reported in their study and stated that there was a wide variety in the ranges of certain parameters, including WBC and percentages of heterophils and lymphocytes. (39) The authors further stated that the staining technique and the specific laboratory that performs the WBC differential might be factors that influence the interspecific variation rather than actual taxonomic differences. Because of this, our study relied on the reference ranges provided by the laboratories used in the study. Once precision and reliability studies identifying species-specific absolute and relative WBC differential reference ranges are performed for psittacine species, this study should be repeated with those values.
Overall, the results of this study showed that in 50.7% of the birds that presented for illness, results of both the CBC and EPH were within reference ranges. In addition to the points discussed above, several possibilities exist as to why half of the birds in this study did not demonstrate abnormal values. Although birds were presented for examination because of presumed illness by the owner, the prevalence of infectious or inflammatory disease might have been low in this patient population; thus, changes in CBC and EPH values would not have been expected. A limitation of this study was that illness as defined in this study was based on clinical signs described by the owner. This was done to reflect what happens in a clinical situation, in which decisions on what diagnostic tests to perform are not based on final diagnoses but rather on the history as described by the owner, as well as abnormal results of the physical examination. Evaluation of the CBC and EPH in conjunction with a plasma biochemical analysis, infectious disease testing, radiographs, histopathologic examination, or necropsy might have helped determine which birds in this study group had a disease that stimulated an immune response and thus would have elicited changes in the CBC or protein EPH. Because the patients in this study were from a referral patient population, some of the birds might have been medicated previously by the referring veterinarian, which could have biased our findings. Repeating this study using other large collections of psittacine birds presented for illness, while controlling for administration of medications or other therapies known to influence the immune system, would be useful. The patient population at a referral hospital also might consist of more chronic cases relative to other populations; therefore, the immune systems of these birds possibly were already impaired at the time of evaluation, limiting the ability of the bird to produce a cellular or humoral immune response.
Within research on the immune system, trends exist that suggest the diagnostic utility of such methodologies as the CBC and EPH is limited and might not be applicable to the vast number of avian species comprising different genera and even orders. Over time, it has been shown that reference ranges and reagents for these tests are species specific, making their application to a broad range of avian patients impractical.32,42 Evaluation of diagnostic modalities that do not depend on species-specific reagents (cg, assays measuring phagocytic or bactericidal capacity) is underway and shows promise. The further benefit of such assays is the small amount of plasma that is required for testing, making them applicable to smaller psittacine species.42 However, until such assays become more widely available, the CBC and EPH will continue to be used as measures of health status in psittacine birds.
This study reflects the decision-making process in the clinical setting in which diagnostic tests are often done before the practitioner knows the cause of the bird's clinical signs. Knowing early in the course of the diagnostic workup which tests will be most valuable in providing information on the health status is imperative. Results of this study could help practitioners prioritize which diagnostic tests are more likely to reveal illness in the psittacine patient and thus guide appropriate treatment.
In this study, we examined the relative likelihood of documenting abnormal results with the use of 2 diagnostic tests in psittacine birds. Our results show that in this population of birds, abnormal results were more common in the CBC than in the plasma protein EPH. On the basis of these results, further studies should focus on the species-specific immune response, involve a larger sample size, and include only birds in which inflammatory or infectious disease is confirmed by additional blood tests, histopathologic analysis, or postmortem examination results.
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From the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104, USA. Present Address: 1400 Irving Street, NW, Washington, DC 20010 (Briscoe).
Jeleen A. Briscoe, VMD, Dipl ABVP (Avian), Karen L. Rosenthal, MS, DVM, and Frances S. Shofer, PhD
Table 1. Distribution of genera for 144 pet psittacine birds evaluated for illness during 2000-2005 at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania. Common name Genera included of genus in category Frequency, % Macaw Ara, Anodorhyncus 32 (22.2) Cockatoo Cacatua, Eupholus 30 (20.8) African grey Psittacus 30 (20.8) Amazon Amazona 20 (13.9) Cockatiel Nymphicus 10 (6.9) Conure Aratinga, Nandayus 8 (5.6) Eclectus Eclectus 6 (4.2) Caique Pionites 4 (2.8) Poicephalus Poicephalus 2 (1.4) Asian parakeet Psittacula 1 (0.7) Pionus Pionus 1 (0.7) Table 2. Clinical signs provided by owners of 144 pet psittacine birds evaluated for illness during 2000-2005 at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania. Clinical signs provided by owner n (%) (a) Nonspecific signs (eg, anorexia, lethargy, or "fluffed") 37 (25.7) Gastrointestinal signs (eg, regurgitation) 37 (25.7) Respiratory 24 (16.7) Feather destructive behavior (FDB) 24 (16.7) Integumentary (other than FDB) 17 (11.8) Neurologic (eg, nystagmus or ataxia) 14 (9.7) Reproductive (eg, chronic egg laying) 14 (9.7) Trauma (eg, bite wound) 10 (6.9) Musculoskeletal (eg, bony mass or crooked toes) 8 (5.6) Behavioral (other than FDB) 3 (2.1) Some birds had more than 1 clinical sign on presentation. Table 3. Abnormal CBC and EPH parameters for 144 pet psittacine birds evaluated for illness during 2000-2005 at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania. Abnormal parameters on the Abnormal values CBC and EPH n % 95% CI WBC, cells/[micro]L <2000 or >14 000 or presence of bands) 40 27.8 20.6%-35.8% PCV (<42%) 37 25.7 18.8%-33.6% Alpha-1 globulins (>0.8 g/dL) 0 0 0%-2.5% Alpha-2 globulins (>0.8 g/dL) 1 0.7 0.02%-3.8% Beta globulins (>0.57 g/dL) 20 13.9 8.7%-20.6% Gamma globulins (>0.57 g/dL) 10 6.9 3.4%-12.4% Total number of birds with abnormal parameters on the CBC (a) 63 43.8 35.5%-52.3% Total number of birds with abnormal parameters on the EPH (b) 25 17.4 11.6%-24.6% Abnormal parameters on the CBC and EPH Mean [+ or -] SD Range WBC, cells/[micro]L <2000 or >14 000 or presence of bands) 19275 [+ or -] 7674 1800-36500 PCV (<42%) 34.2 [+ or -] 8.3 6-41 Alpha-1 globulins (>0.8 g/dL) NA Alpha-2 globulins (>0.8 g/dL) NA Beta globulins (>0.57 g/dL) 0.88 [+ or -] 0.27 0.59-1.6 Gamma globulins (>0.57 g/dL) 1.09 [+ or -] 0.74 0.58-2.93 Total number of birds with abnormal parameters on the CBC (a) NA Total number of birds with abnormal parameters on the EPH (b) NA Abbreviations: CBC indicates complete blood cell count; CI, confidence interval; EPH, plasma protein electrophoresis; PCV, packed cell volume; SD, standard deviation; WBC, white blood cell; NA, not applicable. (a) WBC, PCV, or both abnormal. (b) Alpha, beta, gamma, or a combination of these fractions abnormal. Figure 1. Pie chart of selected complete blood cell count and plasma protein electrophoresis parameters for 144 pet psittacine birds evaluated for illness during 2000-2005. Reference values for parameters evaluated are white blood cell count, 2000-14 000 cels/[micro]L; packed cell volume, 42%; alpha-1 or alpha-2 globulin fractions, [less than or equal to] 0.8 g/dL; and beta or gamma globulin fractions, [less than or equal to] 0.57 g/dL. Normal 1WBC and PCV Normal alpha, beta, gamma globuline 73 (50.7%) Abnormal WBC and PCV Abnormal alpha, beta, gamma globuline 17 (11.8%) Abnormal WBC and PCV Normal alpha, beta, gamma globuline 48 (31.9%) Normal 1WBC and PCV Abnormal alpha, beta, gamma globuline 8 (5.8%) Note: Table made from pie chart.
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