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Evidence of Hemolysis in Pigs Infected with Highly Virulent African Swine Fever Virus

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Evidence of Hemolysis in Pigs Infected with Highly Virulent African Swine Fever Virus Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.9/December-2016/13.pdf Open Access Evidence of hemolysis in pigs infected with highly virulent African swine fever virus Zaven Karalyan1, Hovakim Zakaryan1, Elina Arakelova2, Violeta Aivazyan2, Marina Tatoyan1, Armen Kotsinyan1, Roza Izmailyan1 and Elena Karalova1 1. Laboratory of Cell Biology and Virology, Institute of Molecular Biology of NAS RA, 7 Hasratyan Street, 0014 Yerevan, Armenia; 2. Laboratory of Human Genomics and Immunomics, Institute of Molecular Biology of NAS RA, 7 Hasratyan Street, 0014 Yerevan, Armenia. Corresponding author: Zaven Karalyan, e-mail: [email protected], HZ: [email protected], EA: [email protected], VA: [email protected], MT: [email protected], AK: [email protected], RI: [email protected], EK: [email protected] Received: 29-08-2016, Accepted: 12-11-2016, Published online: 14-12-2016 doi: 10.14202/vetworld.2016.1413-1419 How to cite this article: Karalyan Z, Zakaryan H, Arakelova E, Aivazyan V, Tatoyan M, Kotsinyan A, Izmailyan R, Karalova E (2016) Evidence of hemolysis in pigs infected with highly virulent African swine fever virus, Veterinary World, 9(12): 1413-1419. Abstract Aim: The research was conducted to understand more profoundly the pathogenetic aspects of the acute form of the African swine fever (ASF). Materials and Methods: A total of 10 pigs were inoculated with ASF virus (ASFV) (genotype II) in the study of the red blood cells (RBCs), blood and urine biochemistry in the dynamics of disease. Results: The major hematological differences observed in ASFV infected pigs were that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrits were significantly decreased compared to controls, and the levels of erythropoietin were significantly increased. Also were detected the trends of decrease in RBC count at terminal stages of ASF. Analysis of blood biochemistry revealed that during ASF development, besides bilirubinemia significantly elevated levels of lactate dehydrogenase, and aspartate aminotransferase were detected. Analysis of urine biochemistry revealed the presence of bilirubinuria, proteinuria during ASF development. Proteinuria, especially at late stages of the disease reflects a severe kidney damage possible glomerulonefritis. Conclusion: The results of this study indicate the characteristics of developing hemolytic anemia observed in acute ASF (genotype II). Keywords: African swine fever virus, bilirubinemia, bilirubinuria, erythropoietin, hemolysis, proteinuria. Introduction They are widely used by biochemists and clinicians Since its introduction into Georgia, African swine because of their great convenience and informa- fever virus (ASFV) has rapidly spread into Armenia, vast tiveness. Hematological and biochemical profiles areas of western and southern Russia, and also Ukraine are commonly performed in veterinarian medicine for a variety of reasons: As screening tests on clin- and Belarus [1]. ASF is a highly lethal disease of domestic ically healthy animals, to diagnose a disease and to pigs caused by the only known DNA arbovirus. In pigs, assess its severity and consequences, to formulate a ASFV replicates in cells of the mononuclear phagocyte prognosis and to monitor the response of therapy or system, predominantly monocytes, and macrophages. the progression of the disease. Despite their clinical Several other cell types can be infected, especially in the importance, interpretation is sometimes difficult as later stages of the disease. Many scientists consider that many factors can significantly modify the experi- the massive destruction of macrophages plays a major mental parameters, such as gender, age, breed, diet, role in pathogenesis, particularly in the impaired hemo- environmental conditions, samples management, stasis due to the release of active substances such as cyto- laboratorial procedures, health status, and subclini- kines and complement factors however despite intensive cal diseases [3]. research efforts, most of these pathogenetical aspects are In the view of the mentioned points, the pur- still far from being understood [2]. pose of this study is to determine what pathological Serum biochemistry standards in veterinary changes occur in red blood cell (RBC), blood, serum, are now available from many commercial sources. and urine clinical biochemistry as well as urine sedi- ment analyses in pigs with acute ASF. Another objec- Copyright: Karalyan, et al. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 tive is the interpretation of the laboratorial data in gen- International License (http://creativecommons.org/licenses/ eral pathology of acute ASF. by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit Materials and Methods to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ethical approval The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data All procedures of sampling collection were per- made available in this article, unless otherwise stated. formed strictly as specified by Independent Ethics, Veterinary World, EISSN: 2231-0916 1413 Available at www.veterinaryworld.org/Vol.9/December-2016/13.pdf Committee of the Institute of Molecular Biology of Serum biochemistry NAS, IRB00004079 with minimal stress to animals. For clinical chemistry, whole blood samples without additives were allowed to clot at room tem- Virus perature for several hours and centrifuged for 5 min at Infections were carried out using ASFV (gen- 1500 rpm. The levels of bilirubin in serum were mea- otype II) distributed in Republic of Armenia and sured using a biochemical analyzer (COBAS Integra Republic of Georgia [4]. The titer of ASFV for each 400 analyzer) with standard methods. Enzymatic intramuscular injection was 104 hemadsorbing doses abnormalities were evaluated by measurement of (HAD50)/ml. Virus titration was done as described gamma-glutamyltransferase, lactate dehydrogenase previously and expressed as Log 10 HAD50/ml for (LDH) (U/L), alanine aminotransferase (ALT), and non-adapted cells [5]. aspartate aminotransferase (AST). Animals In our study, 10 healthy pigs (Landrace breed) Urine chemistry Urine chemistry was evaluated using Test Strips of the same age (3-month-old) and weight (30-33 kg) from Roche Diagnostics (on Urisys-1100 analyzer) were used for infection and two for control. and freshly expressed urine. Preinoculated blood samples were taken from all pigs to obtain control values. Considerable care was Tissue samples taken in the collection of the blood samples to avoid Samples from livers were fixed in 10% buffered hemolysis and tissue contamination. The blood sam- formalin solution (pH 7.2) for 24 h. After fixation, the ples were obtained from the anterior vena cava. samples were dehydrated through a graded series of About 10 pigs were infected by intramuscu- alcohols, washed with xylol and embedded in paraffin lar injection, and two pigs (intramuscular injection wax by a routine technique for light microscopy. For of physiological solution – 1.0 ml) were used as structural analysis, wax-embedded samples were cut an uninfected control. Animal care and euthanasia (Microm HM 355, 5 Lm) and stained with hematox- were done according to the AVMA Guidelines on ylin and eosin in accordance with the manufacturer’s Euthanasia, and Local Guideline for Animal Care and protocol (Sigma-Aldrich, Germany). The histolog- use (Institutional Review Board/Independent Ethics ical examination was implemented using a light Committee of the Institute of Molecular Biology of microscope. NAS, IRB00004079). Carbon dioxide inhalation (75- Statistical analysis 80% carbon dioxide for 20 min) was used to euthana- Statistical analyses were performed using the tize infected and control animals after 7 days post-in- Student’s t-test and Mann–Whitney U-test. SPSS fection (DPI). version 17.0 software package (SPSS Inc., Chicago, Analyses of peripheral blood Illinois, USA). RBC count, hematocrit (Hct), mean corpuscular Results volume (MCV), mean corpuscular hemoglobin (Hb) Experimental infection (MCH), and Hb concentration was prepared in eth- The clinical signs of experimental infection ylenediaminetetraacetic acid. Whole-blood samples have some differences from those in cited researches were measured using an automatic blood cell counter, with Malawi’ 83 ASFV isolate [9] and our previous Sysmex XS 1000i. For fine Hb measurements, the study [10]. specimens were analyzed on an automated micro- The first clinical signs were observed at 2-3 DPI scope system employing television scanning accord- when all infected pigs demonstrated loss of appetite ing to the Soret technique [6,7]. and depression. From 2 to 4 DPI, infected animals Blood collection and enzyme-linked immunosorbent displayed hyperthermia with body temperature more assay (ELISA) than 40°C. Simultaneously, decreased activity in From 1 to 7 DPI, blood samples were collected behavior, difficulties in breathing and reddening of from the swine ophthalmic venous sinus and for the skin were detected. Bloody diarrhea (present only controls all samples were collected at day 0 prior to in 20% cases) and lethargy were seen at 5-6 DPI, and virus inoculation. The levels of erythropoietin (Epo) therefore, all infected animals were sacrificed accord- in
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