Influence of Freezing Temperature, Freezing Duration, and Repeated Freeze/Thaw Cycles on Electrophoretic Profiles in the White Stork (Ciconia ciconia).

Plasma electrophoresis is an ancillary diagnostic tool in avian medicine, with agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) being the most common techniques. Frozen samples can be used for quantitative studies or comparative diagnostic purposes, but stability of avian plasma proteins under freezing is poorly described. To evaluate the influence of plasma freezing on electrophoretograms in white storks (Ciconia ciconia), heparin blood was sampled from 30 individuals during annual health examinations. Plasma samples were obtained after centrifugation of fresh samples and divided into aliquots. Both AGE and CZE were performed on fresh aliquots. The remaining aliquots were frozen at -20°C (-4°F) or -180°C (-292°F) and thawed following different protocols: 1 freeze/thaw cycle after 6 months at -20°C; 1, 2, 4, and 7 cycles over 12 months at -20°C; and 1 cycle after 18 months at -180°C. For both techniques, electrophoretic profiles obtained from these thawed aliquots were compared to fresh electrophoretograms. Quantitatively, significant differences (P < 0.05) in most fractions were seen from 6 months postfreezing at -20°C for both techniques. Fewer statistically significant differences were observed after 18 months under cryogenic preservation (-180°C). Qualitatively, AGE provided more repeatable and stable results than CZE over time on samples stored at -20°C, and electrophoretograms were stable after 18 months of cryogenic storage. An electromigration distortion associated with freezing was seen with CZE only. Plasma samples stored in a conventional freezer (-20°C) should not be compared to fresh plasma. For quantitative studies, cryogenic storage should be privileged.

Unraveling the history of the genus Gallus through whole genome sequencing.

The genus Gallus is distributed across a large part of Southeast Asia and has received special interest because the domestic chicken, Gallus gallus domesticus, has spread all over the world and is a major protein source for humans. There are four species: the red junglefowl (G. gallus), the green junglefowl (G. varius), the Lafayette's junglefowl (G. lafayettii) and the grey junglefowl (G. sonneratii). The aim of this study is to reconstruct the history of these species by a whole genome sequencing approach and resolve inconsistencies between well supported topologies inferred using different data and methods. Using deep sequencing, we identified over 35 million SNPs and reconstructed the phylogeny of the Gallus genus using both distance (BioNJ) and maximum likelihood (ML) methods. We observed discrepancies according to reconstruction methods and genomic components. The two most supported topologies were previously reported and were discriminated by using phylogenetic and gene flow analyses, based on ABBA statistics. Terminology fix requested by the deputy editor led to support a scenario with G. gallus as the earliest branching lineage of the Gallus genus, instead of G. varius. We discuss the probable causes for the discrepancy. A likely one is that G. sonneratii samples from parks or private collections are all recent hybrids, with roughly 10% of their autosomal genome originating from G. gallus. The removal of those regions is needed to provide reliable data, which was not done in previous studies. We took care of this and additionally included two wild G. sonneratii samples from India, showing no trace of introgression. This reinforces the importance of carefully selecting and validating samples and genomic components in phylogenomics.

STRESS INFLUENCE ON PLASMA PROTEIN ELECTROPHORESIS IN THE RED-BREASTED GOOSE (BRANTA RUFICOLLIS) AND HAWAIIAN GOOSE (BRANTA SANDVICENSIS).

The objective of this study was to evaluate the effects of stress (as measured by total heterophil + eosinophil counts [THECs] and plasma corticosterone [PC] levels) on plasma protein electrophoresis (PPE) in two Anseriform species. Ten red-breasted geese ( Branta ruficollis ) and eight Hawaiian geese (Branta sandvicensis) were received into quarantine at Beauval Zoo and housed together with other Anseriformes. Two days later, all were examined as part of routine quarantine procedures, and blood was collected. THECs were performed using a Malassez hemocytometer after a 1 : 200 dilution with an eosinophil dilution liquid containing phloxine B. PPE, as well as total protein and PC assays, was performed on lithium heparin plasma. Twenty days after arrival, all birds were similarly restrained, examined, and sampled. Pododermatitis lesions were identified on four geese that were excluded from statistical analysis. For each goose, THECs and PC values were sorted by value (lower or higher) independently from the day of sampling. A Wilcoxon signed rank test showed no significant differences between lower and higher values of THECs for any of the PPE fractions. Higher values of corticosterone were associated with higher values of prealbumin, but none of the other fractions were significantly different. A Spearman rank correlation coefficient showed that THECs and PC were not correlated; this suggested differences in kinetics between these stress markers. Results did not show significant alterations of electrophoresis patterns associated with stress as measured with THECs and PC. The results also highlight the complexity of precisely assessing acute and chronic stress in avian species.

GREATER KUDU (TRAGELAPHUS STREPSICEROS) MORTALITY IN EUROPEAN ZOOLOGICAL INSTITUTIONS: A RETROSPECTIVE STUDY.

A questionnaire was sent to 39 European institutions holding greater kudus (Tragelaphus strepsiceros), in order to determine the causes of captive greater kudu mortality. All reported macroscopic lesions and histopathologic observations, as well as other information regarding individuals that died, were analyzed to determine the most affected body systems and causes of death. Overall response rate was 31%, and 131 individuals were included in the study. The most frequently affected body systems were the digestive system (47%), respiratory system (38%), musculoskeletal system (37%), and cardiovascular system (32%). Most frequent causes of death were infectious diseases (27%) and trauma/accidents (18%); the cause was undetermined in 28% of cases. Nutrition-related disorders were difficult to assess, but results highlight possible nutritional imbalances. This retrospective study represents the first overview of greater kudu mortality in a captive population.

Plasma protein electrophoresis in birds: comparison of a semiautomated agarose gel system with an automated capillary system.

Plasma agarose gel electrophoresis (AGE) is recognized as a very reliable diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary zone electrophoresis (CZE) have emerged in human laboratory medicine but have never been investigated in birds. To investigate the use of CZE in birds and to compare it with AGE, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 racing pigeons (Columba livia) were analyzed by both AGE and CZE. For the 3 species studied, values determined by AGE and CZE were well correlated for albumin and beta and gamma fractions whereas other values differed significantly. Values for alpha-3 fraction in the rooster, alpha-1 fraction in the black kite, and alpha fractions in the pigeon obtained by AGE were very well correlated with the prealbumin fraction values obtained by CZE. Repeatability and reproducibility appeared higher with CZE than with AGE. Although the interpretation of CZE electrophoresis patterns seems to produce results similar to those obtained with AGE, some proteins present in the alpha fraction measured with AGE migrated to the prealbumin fraction found with CZE. Although CZE requires the use of specific reference intervals and a much higher sample volume, this method has many advantages when compared with AGE, including better repeatability and reproducibility and higher analysis output.

Influence of molt on plasma protein electrophoretic patterns in bar-headed geese (Anser indicus).

Plasma protein electrophoresis is recognized as a reliable diagnostic tool in avian medicine; however, the influence of circannual phenomena such as molt on protein electrophoregrams is poorly documented. The molt is a period of heavy hormonal and metabolic change in birds. The purpose of this study was to investigate the effects of molt on total protein concentration and electrophoresis patterns in birds. Blood samples were taken from 19 Bar-headed Geese (Anser indicus) from mid-May to mid-August, at 15-day intervals. At the same time, molting stage of each bird was recorded. Total protein concentrations were measured and plasma agarose gel electrophoresis was performed on these samples. The Bar-headed Goose was chosen as a model, because they molt over a very short period. The total protein concentration and albumin, alpha-2, beta, and gamma fractions were at their minimum values during molt, whereas the prealbumin and alpha-1 fractions rose to their maximum levels. This study provides baseline information relevant to changes occurring in avian proteinograms throughout the molt. The increase in prealbumin and alpha-1 fractions may be related to an increase in plasma thyroid hormones during molt. The decrease observed in albumin, alpha-2, beta, and gamma fractions may be related to protein and energy shifts toward feather growth, as well as to an expansion of the circulatory system located around the feather follicles with secondary dilutional effects on protein fractions. From a clinical point of view, the observed changes associated with molting were less significant than initially expected, and would not likely results in incorrect diagnoses based on interpretation of the protein electrophoretic patterns.

Identification of apolipoprotein A-I in the alpha-globulin fraction of avian plasma.

BACKGROUND: Plasma protein electrophoresis is frequently used in birds as a tool for the diagnosis and monitoring of disease. Identification of proteins in individual peaks can help improve our understanding of changes in protein concentration in physiologic and pathologic conditions. OBJECTIVE: The aim of this study was to verify the presence and identity the protein(s) in the prominent alpha-globulin peak of orange-winged parrots (Amazona amazonica), black kites (Milvus migrans), and rock pigeons (Columba livia). METHODS: Heparinized plasma samples were obtained from 12 birds of each species. Agarose gel electrophoresis and total protein concentration were determined using standard techniques. One plasma sample from each species was then electrophoresed using high-resolution agarose gels to isolate the alpha-globulin band. Gel strips were digested in trypsin and peptides were extracted and analyzed using liquid chromatography with tandem mass spectrometry. De novo sequencing was used to identify the protein based on homology scoring against a protein database. RESULTS: Electrophoresis verified the presence of a single prominent alpha-globulin peak, usually in the alpha(1)-region, that had a median concentration of 9.4 g/L (range, 2.1-11.7 g/L, 21.6% of total protein) in parrots, 12.2 g/L (10.4-13.2 g/L, 35.9%) in kites, and 10.7 g/L (9.0-11.5 g/L, 40.0%) in pigeons. Mass spectrometry and sequencing analysis unequivocally identified the protein as a mature circulating form of apolipoprotein A-I (apo A-I) in all 3 species. CONCLUSIONS: Apo A-I accounts for the prominent alpha-globulin peak and comprises a major proportion of total protein concentration in diverse avian species. As a high-density lipoprotein and negative acute phase protein with a pivotal role in cholesterol homeostasis, further study is warranted to determine the significance of changes in apo A-I concentration in avian electrophoretograms.

Effect of hemolysis on plasma protein levels and plasma electrophoresis in birds.

Protein electrophoresis is recognized as a reliable diagnostic tool for birds even though results are seldom pathognomonic. Unfortunately, this technique is underused in avian medicine because many factors interfere with electrophoresis patterns; hemolysis is one of these factors and is often associated with improper specimen handling. In human laboratory medicine, hemolysis is a known interference factor that can lead to erroneous results. Published data on the influence of hemolysis on protein electrophoresis in birds is currently restricted to a single study in Psittacidae. The aim of this study was to further investigate this effect and to analyze potential interspecific differences. Blood samples were drawn from 28 Black Kites (Milvus migrans) and 19 Bar-headed Geese (Anser indicus) and separated into two aliquots. One aliquot was dipped into liquid nitrogen for 5 sec in order to cause freeze-thawing hemolysis before centrifugation. Total plasma protein concentration, plasma hemoglobin concentration, and plasma protein electrophoresis patterns were determined for both hemolyzed and nonhemolyzed samples. In both species, hemolysis resulted in falsely elevated total plasma protein concentration. In Bar-headed Geese, hemolysis caused a rise in the gamma fraction. In Black Kites, this rise involved not only the gamma fraction but also the beta fraction, stressing the potential for species-related differences. In both species, the effects of hemolysis mimicked a chronic inflammatory condition with resulting antigenic stimulation.

Novel gamma-2-herpesvirus of the Rhadinovirus 2 lineage in gibbons.

We obtained 475 nucleotides of the DNA polymerase gene of a novel human herpesvirus 8 homolog sequence in a gibbon. The finding of this new gibbon virus, which clusters with a related chimpanzee virus in the rhadinovirus 2 genogroup, suggests the existence of a novel gamma-2-herpesvirus in humans.