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.

Determination of the effect of iatrogenic blood contamination on lactate dehydrogenase and creatine kinase activity in canine cerebrospinal fluid.

BACKGROUND: Lactate dehydrogenase (LDH) and creatine kinase (CK) have differential tissue activity and isoenzyme profiles. LDH and CK exist as 5 and 3 isoenzymes, respectively, in both serum and cerebrospinal fluid (CSF). Studies have demonstrated that measuring LDH, CK, and their isoenzymes in CSF has diagnostic and prognostic values for dogs and people with neurologic disorders. OBJECTIVES: Iatrogenic blood contamination can distort the results of CSF analysis. The purpose of this study was to determine allowable thresholds of blood contamination (RBC/µL) for accurate measurement of LDH, CK, and their isoenzymes in canine CSF. METHODS: Venous blood and CSF were collected from healthy dogs. Total LDH and CK activity were measured spectrophotometrically. Isoenzyme profiles were determined using gel electrophoresis and densitometric scanning. All samples were analyzed within 6 hours of collection. A nonlinear mixed effects regression model was used to estimate the allowable thresholds of blood contamination for accurate measurement of LDH, CK, and their isoenzymes in canine CSF. RESULTS: The threshold of iatrogenic blood contamination for total LDH and total CK in healthy dogs are 6696 RBC/µL (95% CI 3879-11 187) and 5961 RBC/µL (95% CI 2939-12 085), respectively. LDH-1 is the most sensitive isoenzyme to iatrogenic blood contamination, while LDH-4 is the least sensitive. CONCLUSIONS: These results are important for the interpretation of LDH, CK, and their isoenzymes in canine CSF. Additionally, our methodology is translatable for determining thresholds of acceptable iatrogenic blood contamination in CSF for other diagnostic and prognostic biomarkers of neurologic disease.

Increasing the data on elasmobranch plasma protein electrophoresis: electrophoretogram reference values determination in the undulate skate (Raja Undulata) and the nursehound shark (Scyliorhinus stellaris) maintained under human care.

BACKGROUND: This study determined plasma protein electrophoresis (PPE) reference intervals in two elasmobranch species: the undulate skate (Raja undulata) and the nursehound shark (Scyliorhinus stellaris), using a reference population of 48 undulate skates (27 males, 21 females) and 62 nursehounds (32 males, 30 females), considered to be clinically healthy. Plasma samples were analyzed using capillary zone electrophoresis (CZE). RESULTS: The undulate skate electrophoretogram resembled those previously reported in other batoids and could be divided into seven consistent fractions. No statistically significant differences were detected between sexes and developmental stages. The nursehound electrophoretogram was similar to that previously described in other shark species and could be divided into eight consistent fractions. Fraction 5% was significantly higher in juvenile nursehounds when compared to adults, while fraction 6 concentration and percentage were significantly higher in adults. Fraction 4% was higher in males than in females. Albumin band was not detected, and pre-albumin was negligible in both studied species. Alpha-globulins were predominant in the undulate skate, while beta-globulins were predominant in nursehounds. Statistically significant differences were found in all electrophoretogram fraction percentages and concentrations between the two species. CONCLUSION: To the authors knowledge, this is the first study reporting PPE values in undulate skates and nursehounds, and the first study using CZE in elasmobranch plasma. These findings can serve as a primary reference for health monitoring in both species and will add to the limited data available on PPE in elasmobranchs.

Age-related changes in the electrophoresis pattern of serum proteins in Korean indigenous calves.

This study aimed to investigate changes in the serum protein profiles of healthy newborn Korean calves and provide a reference index for these changes during growth. The serum protein composition of 15 healthy bovine calves aged 1 week to 4 months was analyzed using electrophoresis. The albumin to globulin (A/G) ratio increased until calves reached 2 months (1.3 ± 0.2) and then decreased until they reached 4 months (1.1 ± 0.1). Albumin concentrations increased up to month 2 (3.4 ± 0.1) and decreased until month 4 (3.2 ± 0.2). α-1 globulin concentrations decreased up to week 4 (0.4 ± 0.1) and then increased until month 2 (0.6 ± 0.1). γ globulin decreased until month 2 (0.7 ± 0.2) and then increased until month 4 (1.1 ± 0.3). These age-related changes in protein concentrations indicate the importance of considering age in calf blood tests. The γ globulin concentration was at its highest in the first week, due to colostrum intake, and decreased to reach its lowest at 2 months, when passive maternal immunity is also at its lowest. Therefore, provision of sufficient passive immunity through the colostrum in neonatal calves might enable healthy growth.

Cette étude visait à examiner les changements dans les profils de protéines sériques de veaux coréens nouveau-nés et de fournir un index de référence pour ces changements durant la croissance. La composition des protéines sériques de 15 veaux en santé âgés de 1 semaine à 4 mois a été analysée par électrophorèse. Le ratio albumine:globuline (A/G) a augmenté jusqu'à ce que les veaux atteignent l'âge de 2 mois (1,3 ± 0,2) et diminuait par la suite jusqu'à ce qu'ils aient 4 mois (1,1 ± 0,1). Les concentrations d'albumine augmentèrent jusqu'au mois 2 (3,4 ± 0,1) et diminuèrent ensuite jusqu'au mois 4 (3,2 ± 0,2). Les concentrations de α-1 globuline ont diminué jusqu'à la semaine 4 (0,4 ± 0,1) et ont par la suite augmenté jusqu'au mois 2 (0,6 ± 0,1). Les concentrations de γ globuline ont diminué jusqu'au mois 2 (0,7 ± 0,2) et ont par la suite augmenté jusqu'au mois 4 (1,1 ± 0,3). Ces changements dans les concentrations de protéines en fonction de l'âge démontrent l'importance de considérer l'âge lors des tests sanguins chez les veaux. La concentration de γ globuline était à son maximum durant la première semaine, due à l'ingestion de colostrum, et diminua par la suite pour atteindre un minimum à 2 mois, alors que l'immunité maternelle passive est à son plus bas. Ainsi, le fait de fournir suffisamment d'immunité passive via le colostrum chez les veaux nouveau-nés peut permettre une saine croissance.(Traduit par Docteur Serge Messier).

Serum protein electrophoresis in 26 dogs with chronic hepatitis.

Serum protein electrophoresis (SPE) shows the distribution of protein fractions, helping clinicians to characterize some pathologic processes. Information is lacking in the literature about SPE alterations in dogs with chronic hepatitis (CH). Our aim was to describe SPE alterations in canine CH, to compare SPE results to histologic scores, and to study SPE trends during follow-up. We reviewed retrospectively case data from dogs with a histologic diagnosis of CH. Only cases with SPE, CBC, and serum chemistry results available were included. Dogs were divided into subgroups based on histologic necroinflammatory activity (A) and fibrosis (F) scores (groups A0-1 and A>1; groups F<2 and F≥2). We included 26 dogs; 15 had follow-up SPE. The most common SPE alterations at admission were hypoalbuminemia (n = 16), increases in α1-globulins (n = 11), γ-globulins (n = 11), α2-globulins (n = 8), ß2-globulins (n = 7), and ß1-globulins (n = 6), and decreased albumin:globulin (A:G) ratios (n = 20). Four of 11 dogs had ß-γ bridging. Groups with higher A and F scores had higher ß2-globulins. Eleven of 15 dogs with a post-treatment SPE had a decrease in γ-globulins and increase in A:G ratio compared to their T0, although there was no statistically significant difference. Although further studies are warranted, SPE may be useful for monitoring canine CH.

Immune and Inflammatory Response in Horse Vaccinated Against Equine Herpesviruses 1 (EHV-1) and 4 (EHV-4) Assessed by Serum Protein Electrophoretic Pattern and Leukocyte Population.

Protection against infectious diseases can be obtained with vaccines generating immunogenic response through a combination of humoral and cellular immunity. In this study haematological and serum protein electrophoretic profiles of horses vaccinated against herpesvirus 1 (EHV-1) and 4 (EHV-4) were evaluated. Blood samples were collected from 16 horses before (T0), after 24h, 48h, 72h, 1st week, 2nd week and 3rd week (T1I, T2I, T3I, T7I, T14I and T21I) from the first EHV vaccine-dose administration as well as before (TPREII), and after 24h, 48h, 72h, 1st week, 2nd week, 3rd week and 4th week (T1II, T2II, T3II, T7II, T14II, T21II and T28II) from the EHV vaccine-booster. Total leukocyte values increased at T1I, T1II, T3II and T28II compared to T0 (P < .01). Higher lymphocytes and lower neutrophils values were found after first vaccine-dose and vaccine-booster administration compared to the T0 (P < .01). Monocytes showed higher values at T14II than T0 (P < .01). Higher serum values of total proteins, α1-, α-2-, ß1-, ß2- and γ-globulins were found in horses after first vaccine-dose and vaccine-booster administration (P < .01). Gathered results suggest that horses vaccinated against EHV1 and EHV-4 exhibited a dynamic change of WBC, lymphocytes, neutrophils and monocytes. Moreover, the analysis of serum electrophoresis pattern suggested that EHV vaccination induced the development of inflammation and antibody response in vaccinated horses as highlighted by the increase of α-, ß- and γ-globulin fractions. These changes probably reflect the systemic immunological adaptation of animals to EHV vaccine.

Establishment of reference intervals for plasma protein electrophoresis and comparison of biochemical and protein electrophoresis evaluation of albumin in central bearded dragons (Pogona vitticeps).

The aim of this study is to develop a reference interval for plasma protein electrophoresis for wild free-roaming central bearded dragons (Pogona vitticeps). This will assist the veterinarian in diagnosing and monitoring diseases in wild and captive animals. The study group consisted of 29 wild central bearded dragons of which 22 were males and 7 were females. The plasma samples were obtained from animals in a previous study and were stored frozen at -20°C for 2-3 years before analysis. Plasma protein electrophoresis was performed on 100 uL of plasma. The data were evaluated for the difference between reproductively active females and pooled males with non-reproductively active females as well as the difference between biochemical and protein electrophoresis albumin. Biochemical albumin was found to be significantly higher than electrophoresis albumin in all animals, with a mild proportional bias present. Plasma protein electrophoresis identified albumin, α - 1 and 2, ß - 1 and 2 and γ fractions. The reproductively active females in this study exhibited results that were distinct from pooled males and non-reproductively active females. This study provides a reference interval for plasma protein electrophoresis in central bearded dragons that can be used as a tool for health and disease monitoring and diagnosis. The importance of the use of method and sex/season appropriate reference intervals in reptile medicine is confirmed in this study.

Serum protein electrophoresis in Dirofilaria immitis naturally infected dogs: Latest news and a systematic literature review.

According to the main Guidelines on canine heartworm disease (HWD) by the American and European Societies (i.e., AHS, ESDA, and ESCCAP), a correct diagnosis of Dirofilaria immitis infection should include the detection of circulating microfilariae in the whole blood and the adult antigens in serum or plasma sample. So far, scant data are available on laboratory abnormalities in dogs affected by HWD, although techniques including serum protein electrophoresis (SPEP) have proved to be useful for the diagnosis and monitoring of other vector-borne diseases, such as the canine leishmaniosis. Therefore, this study aims to evaluate the SPEP pattern in dogs naturally infected by D. immitis. Furthermore, a systematic review of the literature on this topic was carried out. Medical records from heartworm-positive dogs, of any sex, age, and breed and with available clinical examination and laboratory test results (i.e., complete blood count, serum biochemical profile, and SPEP) were retrospectively collected. If available, laboratory results obtained from dogs after treatment for HWD were also evaluated. When compared with the reference intervals, out of 30 dogs infected by D. immitis and enrolled, 63.3% (n = 19) had a lower percentage of albumin, and 80.0% (n = 24) had higher percentages of beta globulins, with beta-2, and especially beta-3 globulins the most frequently altered fractions. In terms of absolute values (g/dL), the proportion of dogs with hypoalbuminemia, and increased total globulin, alpha, beta- and gamma globulins were 4/30 (13.3%), 6/30 (20.0%), 2/30 (6.7%), 16/30 (53.3%) and 8/30 (26.7%), respectively. For 7 dogs, SPEP results evaluated three and six months after treatment with doxycycline (10 mg/kg BID for 4 weeks) were available. In these dogs a significant post-treatment increase in the percentage of albumin, alpha-2 globulin, and albumin/globulins ratio was observed, as well as a significant decrease both in the percentage and in the absolute value of total-, beta-, and beta-3 globulins. The systematic review of literature databases yielded a total of three studies that were considered eligible and included in the qualitative synthesis. This study provides novel information on SPEP alterations in dogs naturally infected by D. immitis. The evaluation of serum proteins and their electrophoretic pattern may represent an important diagnostic tool for a prompt and accurate diagnosis (e.g., differentiating infections in dogs sharing similar clinical signs and endemic in the same geographical area) and monitoring of HWD.

Protein Electrophoresis of Serum and Heparinized Plasma in the Common Mynah (Acridotheres tristis).

Although serum protein electrophoresis is a diagnostic tool available through many veterinary laboratories, there currently are no reference intervals for protein fractions in healthy common mynahs (Acridotheres tristis). Therefore, electrophoretic patterns of proteins in serum and heparinized plasma of the common mynah were evaluated. Blood specimens were collected from 55 healthy adult common mynahs of unknown age (26 males and 29 females). The serum total protein and protein fractions were measured using the biuret method followed by cellulose acetate electrophoresis (CAE). The serum level of albumin was compared with bromocresol green (BCG) dye-binding and CAE methods. Four protein fractions, including albumin and α, ß, and γ globulins, were recorded in the electrophoretogram of serum specimens. Sex appeared to have no significant effect on the measured parameters. The serum BCG albumin fraction was significantly higher than the CAE albumin fraction (P = .01). Also, the comparison of total protein and protein fractions in serum and plasma specimens of 25 of the 55 birds sampled showed that total protein (Cohen index d = 0.66, P = .03), gamma globulin (d = 1.13, P = .00), and total globulin (d = 0.67, P = .00) in plasma samples were significantly higher than those in serum samples. The results of this study provide the specific reference intervals for total protein and protein fractions in common mynahs, which are essential for proper interpretation of laboratory results and also revealed that the albumin measurement by the BCG method yields unreliable results in common mynahs.

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation.

OBJECTIVE: In humans, misdiagnoses of monoclonal gammopathy after use of therapeutic monoclonal antibodies has been documented. This triggers concerns for similar misdiagnoses in animals treated with monoclonal antibodies. The aim of this study was to evaluate if lokivetmab interferes with serum protein electrophoresis and immunofixation electrophoresis in dogs. MATERIAL AND METHODS: Residual sera from 25 client-owned, healthy blood donor dogs from 2 veterinary hospitals in Germany were used. The residual sera were analysed with serum protein electrophoresis and immunofixation electrophoresis before and after being spiked with lokivetmab at a concentration of 10 µg/ml (corresponding to the mean peak serum concentration after a subcutaneous injection of 2 mg/kg lokivetmab). RESULTS: No monoclonal gammopathy was observed on serum protein electrophoresis and all proteins had a normal distribution pattern without any pathologic bands on immunofixation electrophoresis. The absolute γ-globulin values of spiked samples, however, were significantly higher than in the native sera although they remained within the reference interval. No other globulin fractions were significantly different. CONCLUSION AND CLINICAL RELEVANCE: This study suggests that lokivetmab at a dose of 2 mg/kg is not detected as a monoclonal peak on serum protein electrophoresis or immunofixation electrophoresis, and thus is unlikely to lead to a misdiagnosis of other diseases that are characterised by monoclonal gammopathies.

Proteome Analysis of Toxic Fractions of Iranian Cobra (Naja naja Oxiana) Snake Venom Using Two-Dimensional Electrophoresis and Mass Spectrometry.

Snake venoms are mostly composed of various proteins and peptides with toxicity and pharmacological effects depending on their geographical sources. Naja naja oxiana is one of the most medically important venomous snakes in Iran and Central Asia. The bite of this type of snake can cause severe pain and swelling, as well as neurotoxicity. Without medical treatment, symptoms quickly worsen and death can occur soon. A detailed understanding of venom components can provide new insight into the production of antivenom against toxic agents instead of crude venom. Specific antibodies against toxic fractions are of utmost importance in neutralizing crude venom. Therefore, the proteome profile of these fractions of Naja naja oxidana venom was analyzed using fractionation by gel filtration, two-dimensional electrophoresis, mass spectrometry, and data mining. Base on the results, in total, 32 spots were detected and categorized into three protein families, namely three-finger toxin (3FTx), phospholipase, and Cysteine-rich secretory proteins (CRISP). These proteins consist of more than 70% crude venom all with a molecular weight below 25 kDa. The 3FTx as a highly diverse constituent in the venom of Naja species was in large quantity in this district. Short-chain neurotoxins, including short neurotoxin, cytotoxin, and muscarinic toxin-like protein, were in abundance, respectively. In conclusion, the recognition of toxic fractions of Naja naja oxiana in this region could be of great help in the production of an effective antivenom against similar compositions. It can also help the medical care department to find out the clinical sign of cobra venom. To the best of our knowledge, this was the first study to report the proteomic of toxic fractions of Naja naja oxiana in Iran.

HEMATOLOGY, PLASMA BIOCHEMISTRY, AND PLASMA PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR BLUE IGUANAS (CYCLURA LEWISI) FROM GRAND CAYMAN ISLAND.

The blue iguana (Cyclura lewisi) is an endangered rock iguana species native to Grand Cayman, in the Cayman Islands. Health assessments were conducted on captive and free-roaming iguanas in 2001 and 2003-2014 and were performed in the summer wet season (June-July) of 2003-2004 and 2010-2014 and in the winter dry season (November-December) of 2001 and 2005-2009. Morphometric data were recorded from iguanas when blood samples were collected: 903 samples were collected and data from 890 samples from 775 iguanas were included. Samples were analyzed for hematology, plasma biochemistry, protein electrophoresis, mineral panels, 25-hydroxyvitamin D, and testosterone. Reference intervals were created for captive subadults, captive adults, and free-roaming adults when data were sufficient. Significant differences among these groups were described, as were differences on the basis of sex, season, and origin (captive vs free-roaming). In captive iguanas, most analytes were significantly different between subadults and adults, mature heterophils and copper were significantly higher in the dry season, zinc levels were significantly higher in the wet season, and cholesterol and triglycerides were significantly higher in adult females than adult males. Testosterone in adult males was significantly higher in the dry season. These results will aid in future health assessments and disease investigations in wild and captive populations of blue iguanas and are of comparative value for other Cyclura species that are free-roaming, captive, and, especially, in similar conservation release programs.

CLINICAL CHEMISTRY, VITAMIN, ELECTROPHORESIS, AND HEMATOLOGIC ANALYTES OF BLACK-HEADED IBIS (THRESKIORNIS MELANOCEPHALUS).

The evaluation of laboratory blood analytes is an important tool for health assessment in avian medicine. Unfortunately, there is a lack of suitable reference values for many zoo bird species. The goal of the present study was to establish reference intervals for a wide range of blood analytes in healthy black-headed ibis (Threskiornis melanocephalus) kept in a zoologic garden. Lithium heparinized blood samples from 15 individuals were collected, and 18 different clinical chemistry analytes were measured. New are especially the minimum and maximum values for bile acid (2.38-49.4 µmol/L), cholinesterase (439-1260 U/L), vitamin A (0.65-1.70 mg/L), vitamin E (26.3-52.5 mg/L), and capillary zone electrophoresis (prealbumin, 10.2-23.4%; albumin, 35.9-44.1%; A/G ratio, 1.07-1.69; α-, 15.7-20.0%; ß-, 13.5-19.1%; γ-globulin, 6.08-11.3%). The measured values for clinical chemistry, vitamin, electrophoresis, and hematologic analytes are a basis for further studies and for diagnostics and clinical treatment in this ibis species.

Changes in serum protein electrophoresis profiles and acute phase proteins in calves with diarrhea.

Calf diarrhea leads to substantial economic losses in the livestock industry worldwide due to medical treatment costs, retarded growth performance, and even death. The objective of this study was to investigate changes in serum protein profiles and acute phase proteins in calves with diarrhea and identify the association between these changes and diarrhea. A total of 185 Korean beef calves were used and divided into 3 groups by age: 1 to 10 days (n = 46), 11 to 20 days (n = 65), and 21 to 30 days (n = 74). Blood and fecal samples were collected from each calf. Serum concentrations of total protein, protein fractions (albumin, α1-globulin, α2-globulin, ß-globulin, and γ-globulin), haptoglobin (Hp), and serum amyloid A (SAA) were analyzed. Compared to calves without diarrhea, calves with diarrhea had significantly lower albumin concentrations at 11 to 20 days and 21 to 30 days of age (P = 0.017 and P = 0.000, respectively) and significantly higher α1-globulin fractions at 21 to 30 days of age (P = 0.01). Interestingly, α2-globulin fractions were significantly higher in diarrheic calves in all age groups, whereas γ-globulin fractions were significantly lower in calves with diarrhea aged 1 to 10 days, compared with normal animals. In calves with diarrhea, the concentration of Hp was significantly higher, whereas SAA levels were not different between normal and diarrheic calves. In addition, a positive correlation was found between α2-globulin and Hp (P = 0.0004). Taken together, these results provide useful information about the use of serum protein profiles and Hp as prognostic and diagnostic markers for animal health status.

La diarrhée des veaux entraîne des pertes économiques substantielles dans l'industrie de l'élevage dans le monde entier en raison des coûts des traitements médicaux, du retard de croissance et même de la mort. L'objectif de cette étude était d'étudier les changements dans les profils des protéines sériques et les protéines de la phase aiguë chez les veaux souffrant de diarrhée et d'identifier l'association entre ces changements et la diarrhée. Un total de 185 veaux de boucherie coréens ont été utilisés et répartis en trois groupes par âge : 1 à 10 jours (n = 46), 11 à 20 jours (n = 65) et 21 à 30 jours (n = 74). Des échantillons de sang et de matières fécales ont été prélevés sur chaque veau. Les concentrations sériques de protéines totales, les fractions protéiques (albumine, α1-globuline, α2-globuline, ß-globuline et γ-globuline), d'haptoglobine (Hp) et d'amyloïde sérique A (SAA) ont été analysées. Par rapport aux veaux sans diarrhée, les veaux souffrant de diarrhée avaient des concentrations d'albumine significativement plus faibles à 11 à 20 jours et 21 à 30 jours d'âge (P = 0,017 et P = 0,000, respectivement) et des fractions d'α1-globuline significativement plus élevées à 21 à 30 jours d'âge (P = 0,01). Il est intéressant de noter que les fractions d'α2-globuline étaient significativement plus élevées chez les veaux diarrhéiques de tous les groupes d'âge, tandis que les fractions de γ-globuline étaient significativement plus faibles chez les veaux souffrant de diarrhée âgés de 1 à 10 jours, par rapport aux animaux témoins. Chez les veaux souffrant de diarrhée, la concentration de Hp était significativement plus élevée, tandis que les niveaux de SAA n'étaient pas différents entre les veaux normaux et diarrhéiques. De plus, une corrélation positive a été trouvée entre l'α2-globuline et Hp (P = 0,0004). Pris ensemble, ces résultats fournissent des informations utiles sur l'utilisation des profils de protéines sériques et de Hp comme marqueurs pronostiques et diagnostiques de l'état de santé des animaux.(Traduit par Docteur Serge Messier).

The first analysis of Trichinella spiralis and Trichinella britovi adult worm excretory-secretory proteins by two-dimensional electrophoresis coupled with LC-MS/MS.

The two most common Trichinella species present in European countries are Trichinella spiralis and Trichinella britovi. The consumption of raw or undercooked meat with invasive larvae results in trichinellosis. Currently, the most commonly used sources for detecting specific anti-Trichinella antibodies is ELISA with the muscle larvae (ML) excretory-secretory (E-S) proteins. However, these serological methods cannot be efficiently applied at early stage of infection. The aim of the current study was to identify the common and species-specific E-S proteins of T. spiralis and T. britovi adult worms which could have potential for accurate diagnosis of Trichinella infection at an early stage of invasion. Different sets of immunoreactive proteins were identified in T. spiralis and T. britovi proteomes by a combination of two-dimensional electrophoresis (2-DE), immunoblot and LC-MS/MS analysis. Polyubiquitin-B, a possible enoyl-CoA hydratase/isomerase YngF or polyubiquitin-like protein was found to be common for both species; gene ontology analysis confirmed its involvement in proteolysis, oxidation-reduction and translation processes, as well as in molecular transport. These molecules, being secreted or excreted at an early stage of parasite development, may play a critical role in the processes occurring during the initial steps of the host invasion and hence be suitable for diagnostic test development.

Haematological, serum biochemical and electrophoretic data on healthy captive Egyptian fruit bats (Rousettus aegyptiacus).

Bats play a key role as reservoir hosts of many emerging viral diseases with zoonotic potential. However, little is known about the laboratory reference intervals (RIs) of bats, especially Egyptian fruit bats (Rousettus aegyptiacus). The aim of this study was to obtain haematological, biochemical and electrophoretic RIs from captive fruit bats. Blood was collected from 21 R. aegyptiacus (11 females and 10 males). Complete blood cell count was performed using an impedance cell counter followed by the morphologic analysis of blood smears. Clinical biochemistry was performed with an automated spectrophotometer and agarose gel electrophoresis was carried out with an automated instrument. Reference intervals were determined using the Reference Value Advisor V2.1, following the American Society for Veterinary Clinical Pathology guidelines. Possible differences related to sex or sexual maturity were also investigated. The RIs for most of the analytes investigated were similar to those of other types of bats and other mammalian species. Haematology revealed mild polychromasia and slightly lower haematocrit, haemoglobin, leukocyte and lymphocyte counts compared to other bats. Glucose levels varied possibly due to stress, the anaesthetic protocol and fasting time. Creatine kinase was higher, while triglycerides were lower compared with domestic mammals and other bats. No sex- or age-related differences were found. Serum protein electrophoresis showed five fractions (albumin, α-, ß1-, ß2- and γ-globulins). The values recorded in this study could be helpful as a reference biological dataset to monitor the health status of wild and captive R. aegyptiacus and, possibly, of other Chiroptera.

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors.

BACKGROUND: Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis. RESULTS: For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5-drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group. CONCLUSION: PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Electrophoretic analysis (sds-page) of canine urinary proteins according to the stage of chronic kidney disease / [Análise eletroforética (SDS-PAGE) de proteínas urinárias de cães de acordo com o estágio da doença renal crônica]

Glomerular proteinuria is characterized by the loss of high-molecular-weight proteins (HMWPs), while tubulointerstitial proteinuria is characterized by the loss of low-molecular-weight proteins (LMWPs). The objective was to assess the molecular weight of urinary proteins (MWUP) in dogs with naturally acquired CKD and determine the proportion of HMWPs and LMWPs according to CKD stage. Twenty-eight dogs with CKD were recruited and divided into 4 groups based on serum creatinine (Cr) levels (group1: Cr<1,4, n=8; group2: 1,45,0, n=5). The control group consisted of 5 healthy dogs. The MWUP was determined by SDS-PAGE. The urinary protein-to-creatinine ratio (UP/C) was used to quantitatively assess proteinuria. The electrophoresis pattern revealed a proportionally greater loss of HMWPthan of LMWP in all groups with CKD and an increased loss of LMWP in group 4 (P<0.05). These results suggest a predominance of glomerular injuries throughout all stages of CKD in these dogs and an increase in tubulointerstitial injury towards the end-stage of the disease. The results of the present study support the recommendation of SDS-PAGE as an effective technique for the qualitative assessment of proteinuria, as well as a method for assessing the severity and location of renal injury.(AU)

A proteinúria glomerular é caracterizada pela perda de proteínas de alto peso molecular (PAPM), enquanto a proteinúria tubulointersticial se caracteriza pela perda de proteínas de baixo peso molecular (PBPM). O objetivo do trabalho foi determinar o peso molecular das proteínas urinárias (PMPU) de cães com DRC naturalmente adquirida e a proporção de PAPM e PBPM de acordo com o estágio da DRC. Foram utilizados 28 cães com DRC, divididos em quatro grupos, de acordo com o nível sérico de creatinina (cr) (grupo 1: cr<1,4, n=8; grupo 2: 1,45,0, n=5). O grupo controle era composto por cinco cães saudáveis. O PMPU foi determinado por SDS-PAGE. A relação proteína:creatinina urinária (RPCU) foi utilizada como um método quantitativo de proteinúria. A eletroforese revelou uma perda proporcionalmente maior de PAPM, quando comparada às PBPM, em todos os grupos de DRC, bem como uma perda crescente de PBPM no grupo 4 (P<0,05). Esses resultados sugerem uma predominância de lesão glomerular em todos os estágios de DRC nesses cães e uma progressão crescente na lesão túbulo-intersticial no estágio terminal da doença. Os resultados deste estudo reafirmam a recomendação de que a eletroforese de proteínas urinárias é uma técnica qualitativa efetiva de avaliação da proteinúria, bem como um método que permite avaliar a extensão e a localização da lesão renal.(AU)

Modified comet assay with Giemsa staining: a suitable method for assessing genotoxicity in rats / Ensaio cometa modificado com coloração de Giemsa: um método adequado para avaliar a genotoxicidade em ratos

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)

O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)

Protein electrophoresis in Andean condors (Vultur gryphus): Reference values and differences between wild and rehabilitating individuals.

The study of wildlife health greatly contributes to understanding population dynamics and detecting conservation threats. The determination of the different fractions of plasma proteins (proteinogram) is an important laboratory tool to study wildlife health. The aim of this study was to characterize protein electrophoresis in wild Andean condors (Vultur gryphus) from north-western Patagonia and to evaluate differences according to age and sex classes. Once reference values of wild, apparently healthy individuals, were established, we compared these values to those of individuals received at the Buenos Aires Zoo in Argentina for rehabilitation due to various health problems. Reference proteinograms from wild Andean condors differed only in the α 1 and ß 2-fractions between sex categories. Males showed higher concentrations of these protein fractions than females. We found clear differences between wild birds and rehabilitating individuals. Total proteins, globulins, α 1-globulins, total α-globulins, ß 2-globulins, total ß-globulins, and γ-globulins were significantly higher in rehabilitating than in wild individuals, whereas albumin, α 2, and ß1-globulins were similar between these groups. The albumin/globulin ratio, as a general indicator of health, was significantly lower in rehabilitating than in wild individuals. The results indicate the effects on different protein fractions of pathologic processes occurring in individuals undergoing rehabilitation. Our results provide useful insights, contributing to improving diagnoses and prognoses in this species. This information may also be useful to assess the health status of Andean condors in studies of wild populations and for comparisons with other bird species.