PLASMA PROTEIN ELECTROPHORESIS IN THE WHITE STORK (CICONIA CICONIA): AGREEMENT BETWEEN AGAROSE GEL VERSUS CAPILLARY ZONE METHODS AND DEVELOPMENT OF REFERENCE INTERVALS.

The white stork (Ciconia ciconia) is a ciconiiform species widely represented in zoological institutions. Plasma protein electrophoresis is widely used in avian patients for assessment of inflammatory conditions, but reference intervals for this testing modality are lacking for the white stork. The two main electrophoretic methods are agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE). This study assessed fresh plasma samples of healthy adult white storks (n = 30). Statistical analyses were performed to evaluate agreement between AGE and CZE. Typical electrophoretic fractions were obtained from both methods (prealbumin, albumin, α1, α2, ß, γ1, and γ2). The AGE and CZE methods were not equivalent for determining major electrophoretic fractions (except ß-globulins) and albumin:globulin ratio on plasma samples. An additional prealbumin fraction was seen with CZE. Reference intervals were established for each method as the smallest n group was 27 individuals for a given value; most values had normal distribution, and robust or parametric methods were used on the data.

Capillary Zone Electrophoresis in Humboldt Penguins (Spheniscus humboldti).

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, ß-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of ß-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

ASSESSMENT OF CAPILLARY ZONE ELECTROPHORESIS AND SERUM AMYLOID A QUANTITATION IN CLINICALLY NORMAL AND ABNORMAL SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM) AND SOUTHERN BLACK RHINOCEROS (DICEROS BICORNIS MINOR).

Capillary zone electrophoresis (CZE) and an immunoassay for serum amyloid A (SAA) were used to examine serum samples from clinically normal and abnormal southern white rhinoceros (Ceratotherium simum simum) and southern black rhinoceros (Diceros bicornis minor) under managed care. CZE resolved seven fractions as well as subfractions for α1 globulins. Reference intervals were calculated for white rhinoceros (n = 33) and found to have some differences over previously reported intervals generated using agarose gel electrophoresis (AGE) methods in sera from free-ranging animals. In addition, the coefficient of variation related to fraction quantitation was found to be overlapping or superior to that reported for AGE. No significant differences were observed in CZE measurands and total protein between clinically normal and abnormal rhinoceros. In contrast to CZE, significant differences in SAA levels (P < 0.001) were observed in samples from the white rhinoceros between clinically normal and abnormal animals. In addition, in limited sample sets with repeated measures, SAA provided prognostic value. Future studies should generate more robust reference intervals and delineate the application of both SAA quantitation and CZE in routine health assessments and in prognostication.

PLASMA CAPILLARY ZONE ELECTROPHORESIS AND PLASMA CHEMISTRY ANALYTES IN TORTOISES (TESTUDO HERMANNI, TESTUDO GRAECA) AND TURTLES (TRACHEMYS SCRIPTA ELEGANS, GRAPTEMYS SPP.) IN FALL.

Clinical health checks including blood testing before brumation in fall are an important tool in reptile medicine and help to reduce the risk of complications during brumation. Electrophoresis may be useful for the evaluation of liver cell function and the detection of antigenic stimulation. The goal of this study was to compare species-specific reference intervals for plasma chemistry analytes and capillary zone electrophoresis (CZE) for a variety of pet chelonian species in fall. Lithium heparinized samples were collected from 68 clinically healthy spur-thighed tortoises (Testudo graeca), 79 red-eared sliders (Trachemys scripta elegans), and 30 map turtles (Graptemys spp.) from September to November 2016 and 2017. During the same period, 128 equivalent samples were collected from Hermann's tortoises (Testudo hermanni) and the obtained data was used for comparison. Chemistry analytes were measured from plasma using an autoanalyzer and electrophoresis was carried out by CZE. Significant variations (P < 0.0001) between the species were found for several analytes including total protein, percent albumin, and albumin: globulin ratio, α-, ß-, and γ-globulin, alkaline phosphatase, glutamate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bile acids, creatine kinase, urea, uric acid, inorganic phosphorus, total calcium, and potassium. The variations in albumin (g/l) measured by CZE were also significant (P = 0.0064). No significant variations were detected for sodium levels. The results demonstrate the importance of species-specific reference intervals and provide reference intervals for the plasma chemistry and capillary zone electrophoresis in three chelonian species.

PARTIAL HEMATOLOGY, BIOCHEMISTRY, AND PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR CAPTIVE SPOTTED TURTLES (CLEMMYS GUTTATA).

Spotted turtles (Clemmys guttata) are an endangered species and are commonly encountered in the pet trade and in many zoological collections across the United States, yet peer-reviewed published reference intervals (RI) for common clinicopathologic tests are unavailable for this species. The objectives of this study were to calculate partial RI for routine hematology, biochemistry, and electrophoretic analyses, as well as to perform an initial comparison of capillary zone electrophoresis (CZE) and agarose gel electrophoresis (AGE) in this species. A single blood sample was obtained from a single collection of 32 apparently healthy captive spotted turtles weighing at least 100 g and was submitted for standard hematologic and biochemistry analyses, as well as electrophoresis via CZE and AGE methods. Partial RI were calculated for corresponding analytes for each type of testing. While CZE and AGE protein fractions were found to have good correlation, some significant differences were observed, reinforcing that RI should be reported with the specific method used for their determination. The spotted turtle electrophoretograms were distinctly different from those previously reported from turtles in the same taxonomic family, including differences in the number and relative prominence of protein fractions.

Comparison of Capillary Zone Electrophoresis in Greater Flamingos (Phoenicopterus roseus) and American Flamingos (Phoenicopterus ruber).

Electrophoresis can be used to aid in the diagnosis of infectious diseases (eg, aspergillosis) in avian species. Reference intervals for blood plasma proteins of 2 different flamingo species (Phoenicopterus roseus and Phoenicopterus ruber) and their hybrids were calculated by capillary zone electrophoresis (CZE) and differences between these species, sexes, and age groups were evaluated. Lithium-heparinized plasma samples from 111 animals from a zoological collection were analyzed by CZE and statistically evaluated. Differences were only found between greater and American flamingos (P = .003) and between greater flamingos and hybrids (P = .001) in the γ-globulin fraction. Male greater flamingos showed significantly higher α-globulins (P = .022) and females higher total albumin by CZE (P = .037). In American flamingos, the percent total albumin (P = .017), total albumin (P = .025), prealbumin (P = .005), and albumin/ globulin (A/G) ratio (P = .008) were higher in females, and α- (P = .023) and ß-globulins (P = .021) were higher in males of the same species. The following parameters differed significantly between the age groups: γ-globulins (P = .048) in greater and α- (P = .021) and ß-globulins (P = .001) in American flamingos increased with increased age and percent total albumin (P = .002), total albumin (P = .024), and A/G ratio (P = .002) decreased with age in American flamingos. The results showed only small differences between the species, but greater differences between the sexes and ages, especially in American flamingos, which must be considered when interpreting laboratory results.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and ß-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and ß-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the ß-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

Technical note: Capillary electrophoresis as a rapid test for the quantification of immunoglobulin G in serum of newborn lambs.

Finding a rapid and simple method of serum IgG determination in lambs is essential for monitoring failure of passive transfer of immunity. The aim of this study was to assess the ability of capillary electrophoresis (CE), an instrument mainly used in blood serum protein analysis, to estimate IgG content in serum of newborn lambs through determination of only total Ig percentage by comparing the results with those obtained with radial immunodiffusion (RID), the reference method for serum IgG quantification. Serum samples were collected at 24 h after birth from 40 Sarda lambs. The IgG concentration measured by RID and serum total Ig concentration measured by CE were (mean ± standard deviation) 29.8 ± 16.1 g/L and 37.8 ± 15.0%, respectively. Data provided by RID and CE analysis showed a polynomial trend (RID = 0.02CE2 - 0.04CE + 4.13; coefficient of determination, R2 = 0.96), displaying a strong relationship between these 2 methods. Applying the polynomial equation, the IgG values were predicted. Predicted IgG values were highly correlated (r = 0.98) and related (R2 = 0.96) to IgG values obtained by RID assay. These data were subjected to Bland-Altman analysis, revealing a high level of agreement between CE and RID methods with a bias that was not different from 0 (-0.04 g/L) and agreement limits of -6.38 g/L (low) and +6.30 g/L (high). In addition, the linear regression analysis between differences (dependent variable) and average of IgG concentration by CE and RID (independent variable) did not show proportional bias (R2 = 0.01). In conclusion, CE is a reliable instrument for a lamb health monitoring program, where Bland-Altman analysis also confirmed that the CE method can be a suitable alternative to RID.

Short communication: Caseins and α-lactalbumin content of camel milk (Camelus dromedarius) determined by capillary electrophoresis.

Camel milk has unique physical, nutritional, and technological properties when compared with other milks, especially bovine. Because proteins confer many of the properties of milk and its products, this study aimed to determine the proteins of camel milk, their correlations, and relative distribution. Raw milk samples were collected from 103 dromedary camels in the morning and evening. Capillary electrophoresis results showed wide variation in the concentrations (g/L) of proteins between samples as follows: α-lactalbumin, 0.3 to 2.9; αS1-casein, 2.4 to 10.3; αS2-casein, 0.3 to 3.9; ß-casein, 5.5 to 29.0; κ-casein, 0.1 to 2.4; unknown casein protein 1, 0.0 to 3.4; and unknown casein protein 2, 0.0 to 4.6. The range in percent composition of the 4 caseins were as follows: αS1, 12.7 to 35.3; αS2, 1.8 to 20.8; ß, 42.3 to 77.4; and κ, 0.6 to 17.4. The relative proportion of αS1-, αS2-, ß-, and κ-caseins in camel milk (26:4:67:3, wt/wt) differed from that of bovine milk (38:10:36:12, wt/wt). This difference might explain the dissimilarity between the 2 milks with respect to technical and nutritional properties.

Evaluation of different analytical methods to assess failure of passive transfer in neonatal calves.

The objective of this study was to evaluate different analytical methods of assessing failure of passive transfer (FPT) in neonatal calves. We hypothesized that 3 different media (i.e., centrifuged serum, centrifuged plasma, filtered plasma) and different analytical methods [i.e., ELISA, capillary electrophoresis (CE), Brix refractometer, and handheld optical refractometer] would be highly correlated with the gold standard radial immunodiffusion (RID) and would generate comparable results. Serum and plasma blood samples were collected from Holstein Friesian calves (n = 216) aged 1 to 7 d, from 2 commercial dairy herds in northeast Germany. The RID analysis showed that 59 of 216 calves (27%) had serum IgG concentrations of <10 mg/mL and 157 calves (73%) had serum concentrations of ≥10 mg/mL. The mean IgG concentration (± standard deviation) was 17.1 ± 9.8 mg/mL, and the range was 0.8 to 47.8 mg/mL. In serum, the correlation between RID and CE was r = 0.97, and between RID and ELISA was r = 0.90; CE and ELISA were also highly correlated (r = 0.89). Both refractometry methods were highly correlated with RID using centrifuged serum, centrifuged plasma, or filtered plasma (Brix refractometer: r = 0.84, 0.80, and 0.78, respectively; handheld optical refractometer: r = 0.83, 0.81, and 0.80, respectively). We determined test characteristics (optimum thresholds, sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve) for CE, ELISA, and the handheld optical and digital refractometers using receiver operating characteristic curve analyses with RID as the reference value. Optimal thresholds for assessing FPT using plasma were higher than for serum, regardless of the method of plasma harvesting. The 4 different devices had comparable areas under the curve, irrespective of the medium used. All analytical methods can be used to assess FPT.

PROTEIN ELECTROPHORESIS OF PLASMA SAMPLES FROM BOA CONSTRICTORS WITH AND WITHOUT REPTARENAVIRUS INFECTION.

Reptarenaviruses infect a variety of boid and pythonid snake species worldwide and have been shown to be the cause of inclusion body disease (IBD). Little is known about the correlations between virus infection and clinical disease, as well as the effects of viral infection on the immune system and the blood protein fractions. The goal of this study was to examine the differences in the plasma protein fractions in reptarenavirus reverse transcription polymerase chain reaction (RT-PCR)-negative and -positive tested snakes with and without clinical signs of disease. Blood from a total of 111 boa constrictors (Boa constrictor) was evaluated. Reverse transcription PCRs and H&E staining for inclusion bodies were carried out on each sample for the detection of reptarenavirus, and the plasma protein fractions were evaluated by capillary zone electrophoresis (CZE). Thirty four of the 111 evaluated snakes were positive by RT-PCR and 19 of the 34 showed clinical signs of disease. In comparison with IBD-negative healthy boa constrictors, the positive snakes with clinical signs had significantly lower albumin levels (P = 0.0052), lower A: G ratios (P = 0.0037), and lower α-globulin levels (P = 0.0073), while their γ-globulin levels were significantly higher (P = 0.0004). In the same comparison, clinically healthy arenavirus-positive boas showed only significantly lower α-globulin (P = 0.0124) and higher γ-globulin levels (P = 0.0394). The results of the present study indicate that reptarenavirus infection may influence plasma protein fractions in boa constrictors.

COMPARISON OF AGAROSE GEL AND CAPILLARY ZONE ELECTROPHORESIS METHODS USING PLASMA FROM GREEN TURTLES (CHELONIA MYDAS).

Agarose gel electrophoresis (AGE) has been widely implemented throughout veterinary medicine and for analysis of plasma proteins of avian and reptile species. Capillary zone electrophoresis (CZE) is becoming a standard method in human clinical pathology laboratories but has not widely been used for the analysis of animal samples. The objective of the present study was to compare protein fractions derived from AGE and CZE methods using plasma from the green turtle (Chelonia mydas). Plasma samples were analyzed by AGE and CZE per manufacturer guidelines. The methods were assessed by CV analysis, Spearman's correlation, Passing-Bablok regression, and Bland Altman plots. CZE consistently resolved more fractions than AGE with three fractions observed in the prealbumin migrating region versus one for AGE and two fractions in the γ globulin region versus one for AGE. Compared with AGE, CZE showed a lower CV in intra-assay tests (1.0-4.9% vs 2.0-28.3%) and a lower or overlapping CV in interassay tests (1.0-10.6 vs 2.3-22.0). The prealbumin, α2 globulin, and ß globulin fractions correlated the least between the methods (for all three fractions: rs ≤ 0.28, P > 0.21). Moderate, significant correlations between AGE and CZE methods were observed for albumin (rs = 0.78, P < 0.0001) and γ globulins (rs = 0.78, P < 0.0001). CZE has a higher precision and ease of use over AGE and offers the opportunity to resolve additional protein fractions. This will necessitate the development of new conventions in placement of fraction delimits, definition of species-specific reference intervals, and evaluation of clinical utility in abnormal turtles.

REFERENCE INTERVALS FOR PLASMA CAPILLARY ZONE ELECTROPHORESIS IN HERMANN'S TORTOISES (TESTUDO HERMANNI) DEPENDING ON SEASON AND SEX.

Electrophoresis is a useful diagnostic tool for the detection of shifts in plasma protein fractions, which can be associated with a variety of diseases. This method is not often used in reptile medicine because there are only a few studies available and no reference intervals have been published for most species. The goal of this study was to establish reference intervals for plasma capillary zone electrophoresis in Hermann's tortoises (Testudo hermanni) under consideration of variations by season and sex. 251 samples from healthy adult Hermann's tortoises were collected from May 2016 to October 2017. Total protein was measured by the biuret method and albumin was measured twice by bromocresol green dye-binding method (BCG) and capillary zone electrophoresis (CZE) in order to compare the detection methods. The globulin fractions were analyzed by CZE. Seasonal variations were significant for total protein (P = 0.0055), albumin (P = 0.0322) and α-globulins (P = 0.0007). The variations between male and female Hermann's tortoises were highly significant for albumin (P < 0.0001), α-globulins (P < 0.0001) and ß-globulins (P < 0.0001). The differences between the measurements of albumin by CZE and BCG also differed depending on the sex and season. This study demonstrates that it is important to include the variations by sex and season when establishing reference intervals and interpreting results. Furthermore, it confirms the importance of using electrophoresis for the correct determination of albumin in tortoises.

Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum.

Capillary electrophoresis (CE) is a technique routinely used in clinical laboratories that allows the separation and quantification of blood serum proteins in a rapid, precise, accurate, and inexpensive manner. Recently, CE has been proposed to separate and measure colostral proteins, but an evaluation of the agreement between CE and radial immunodiffusion (RID) method, currently used to quantify IgG in colostrum, is still lacking. The purpose of this study was to test the ability of a CE instrument, normally used in blood serum protein analysis, to realize the correct quantification of total Ig concentration in ewe colostrum, using RID assay as reference. Colostrum samples (n = 68) were collected from 35 multiparous Sarda ewes at first milking (n = 33) and at 24 h postpartum (n = 35). The mean ± standard deviation of IgG concentration measured by RID and whey colostrum total Ig concentration measured by CE were 54.76 ± 41.82 g/L and 54.70 ± 41.43 g/L, respectively. Lin's concordance correlation coefficient (r = 0.993; 95% confidence interval = 0.989 to 0.996) and linear regression analysis results (RID = 1.0022CE - 0.063; R2 = 0.986) showed an excellent agreement between these 2 methods. Bland-Altman analysis confirmed that CE method can be a suitable alternative to RID: the mean of the differences between CE and RID was -0.055 ± 4.95 g/L (95% confidence interval = -1.25 to 1.14 g/L) and the agreement limits were -9.75 to 9.60 g/L (low limit 95% confidence interval = -11.82 to -7.68 g/L; high limit 95% confidence interval = 7.57 to 11.72 g/L). In conclusion, the current study indicates that CE method may be a reliable tool for the quantification of the total Ig concentration in ewe colostrum.

Detection of hereditary bisalbuminemia in bottlenose dolphins (Tursiops truncatus, Montagu 1821): comparison between capillary zone and agarose gel electrophoresis.

BACKGROUND: Hereditary bisalbuminemia is a relatively rare anomaly characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis. In human medicine, it is usually revealed by chance, is not been clearly associated with a specific disease and the causative genetic alteration is a point mutation of human serum albumin gene inherited in an autosomal codominant pattern. This type of alteration is well recognizable by capillary zone electrophoresis (CZE), whilst agarose gel electrophoresis (AGE) not always produces a clear separation of albumin fractions. The aims of this study is to report the presence of this abnormality in two separate groups of related bottlenose dolphins and to compare the results obtained with capillary zone and agarose gel electrophoresis. RESULTS: Serum samples from 40 bottlenose dolphins kept under human care were analyzed. In 9 samples a double albumin peak was evident in CZE electrophoresis while no double peak was noted in AGE profile. Since only an apparently wider albumin peaks were noted in some AGE electrophoretic profiles, the ratio between base and height (b/h) of the albumin peak was calculated and each point-value recorded in the whole set of data was used to calculate a receiver operating characteristic curve: when the b/h ratio of albumin peak was equal or higher than 0.25, the sensitivity and specificity of AGE to detect bisalbuminemic samples were 87 and 63 %, respectively. The bisalbuminemic dolphins belong to two distinct families: in the first family, all the siblings derived from the same normal sire were bisalbuminemic, whereas in the second family bisalbuminemia was present in a sire and in two out of three siblings. CONCLUSIONS: We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration.

Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha2-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

Serum protein electrophoresis in European mink (Mustela lutreola): reference intervals and comparison of agarose gel electrophoresis and capillary zone electrophoresis.

BACKGROUND: Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species. MATERIAL AND METHODS: Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, ß, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin's concordance correlation. RESULTS: A significant bias was found between methods for α1, α2, and γ-globulin. Lin's concordance correlation was considered unacceptable for α1, α2, and ß-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower. CONCLUSION: Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

An improved multiple-locus variable-number of tandem repeat analysis (MLVA) for the fish pathogen Francisella noatunensis using capillary electrophoresis.

BACKGROUND: Francisellosis, caused by the bacterium Francisella noatunensis subsp. noatunensis, remains a serious threat to Atlantic cod (Gadhus morhua) farming in Norway and potentially in other countries. As outbreak strains appear clonal in population structure, access to highly discriminatory typing tools is critical for understanding the epidemiology of francisellosis infections in aquaculture. In this study, a simplified multiple-locus variable-number of tandem repeat analysis (MLVA) targeting five highly polymorphic variable number of tandem repeat (VNTR) loci in a single multiplex PCR was developed to rapidly discriminate between outbreak strains. RESULTS: The assay resulted in identification of at least 13 different allelic profiles or subpopulations among 91 F. noatunensis isolates from farmed cod in Norway. The VNTR loci appear relatively stable, with isolates originating from individual outbreaks showing identical MLVA profiles following repeated passage. MLVA displayed greater discriminatory power than pulse-field gel electrophoresis (PFGE). Both MLVA and PFGE show good epidemiological concordance by their abilities to separate outbreak strains from epidemiologically unrelated isolates. CONCLUSIONS: The MLVA method presented here is robust, easy to perform and provides a good alternative to other typing systems for F. noatunensis subsp. noatunensis and epidemiological study of francisellosis in cod.

Evaluation of hippuric acid content in goat milk as a marker of feeding regimen.

Organic producers, traders, and consumers must address 2 issues related to milk: authentication of the production system and nutritional differentiation. The presence of hippuric acid (HA) in goat milk samples has been proposed as a possible marker to differentiate the feeding regimen of goats. The objective of this work is to check the hypothesis that HA could be a marker for the type of feeding regimen of goats by studying the influence of production system (conventional or organic) and feeding regimen (with or without grazing fodder). With this purpose, commercial cow and goat milk samples (n=27) and raw goat milk samples (n=185; collected from different breeds, localizations, and dates) were analyzed. Samples were grouped according to breed, feeding regimen, production system, and origin to compare HA content by ANOVA and honestly significant difference Tukey test at a confidence level of ≥95%. Hippuric acid content was obtained by analyzing milk samples with capillary electrophoresis. This method was validated by analyzing part of the samples with HPLC as a reference technique. Sixty-nine raw goat milk samples (of the total 158 samples analyzed in this work) were quantified by capillary electrophoresis. In these samples, the lowest average content for HA was 7±3 mg/L. This value corresponds to a group of conventional raw milk samples from goats fed with compound feed. The highest value of this group was 28±10 mg/L, corresponding to goats fed compound feed plus grass. Conversely, for organic raw goat milk samples, the highest concentration was 67±14 mg/L, which corresponds to goats fed grass. By contrast, the lowest value of this organic group was 26±10 mg/L, which belongs to goats fed organic compounds. Notice that the highest HA average content was found in samples from grazing animals corresponding to the organic group. This result suggests that HA is a good marker to determine the type of goats feeding regimen; a high content of HA represents a diet based mainly or exclusively on eating green grass (grazing), independently of the production system. Hence, this marker would not be useful for the actual organic policies to distinguish organic milk under the current regulations, because organic dairy ruminants can be fed organic compound feed and conserved fodder without grazing at all.

Spurious capillary zone electrophoresis pattern in hypercholesterolemic dogs.

Capillary zone electrophoresis (CZE) is a relatively new serum protein electrophoresis method with higher resolution than other electrophoretic techniques. Hypercholesterolemic dogs exhibit a peculiar CZE pattern. Specifically, they have a shoulder or peak immediately next to the albumin peak. We investigated the prevalence of this spurious peak in hypercholesterolemic dogs and its correlation with the serum cholesterol concentration. Moreover, possible discrepancies between the CZE and spectrophotometric (bromocresol green [BCG] method) albumin concentrations in those animals were evaluated, as well as the accuracy in measuring albumin by a different CZE fractionation system. We retrospectively enrolled 500 hypercholesterolemic and normotriglyceridemic dogs. Each electrophoretic curve was inspected visually to identify a spurious peak (prevalence of 68.8%). We chose 120 dogs to further investigate the albumin concentration; CZE albumin was significantly higher than measured using the BCG method. A weak but significant correlation (r = 0.412; p <0.0001) was observed between the magnitude of the spurious peak and the serum cholesterol concentration. Finally, the significant difference between CZE and BCG albumin measurement disappeared (p = 0.92) when the spurious peak was considered as α1-globulins instead of albumin.