Identification of serum biomarkers in dogs naturally infected with <i>Babesia canis canis</i> using a proteomic approach

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Canine babesiosis is a tick-borne disease that is caused by the haemoprotozoan parasites of the genus Babesia. There are limited data on serum proteomics in dogs, and none of the effect of babesiosis on the serum proteome. The aim of this study was to identify the potential serum biomarkers of babesiosis using proteomic techniques in order to increase our understanding about disease pathogenesis.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Results&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Serum samples were collected from 25 dogs of various breeds and sex with naturally occurring babesiosis caused by B. canis canis. Blood was collected on the day of admission (day 0), and subsequently on the 1st and 6th day of treatment.&lt;p&gt;&lt;/p&gt; Two-dimensional electrophoresis (2DE) of pooled serum samples of dogs with naturally occurring babesiosis (day 0, day 1 and day 6) and healthy dogs were run in triplicate. 2DE image analysis showed 64 differentially expressed spots with p ≤ 0.05 and 49 spots with fold change ≥2. Six selected spots were excised manually and subjected to trypsin digest prior to identification by electrospray ionisation mass spectrometry on an Amazon ion trap tandem mass spectrometry (MS/MS). Mass spectrometry data was processed using Data Analysis software and the automated Matrix Science Mascot Daemon server. Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI Genbank database.&lt;p&gt;&lt;/p&gt; A number of differentially expressed serum proteins involved in inflammation mediated acute phase response, complement and coagulation cascades, apolipoproteins and vitamin D metabolism pathway were identified in dogs with babesiosis.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our findings confirmed two dominant pathogenic mechanisms of babesiosis, haemolysis and acute phase response. These results may provide possible serum biomarker candidates for clinical monitoring of babesiosis and this study could serve as the basis for further proteomic investigations in canine babesiosis

Blood markers of fibrinolysis and endothelial activation in canine babesiosis

Background: Canine babesiosis is a tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. The disease can be clinically classified into uncomplicated and complicated forms. The aim of this study was to assess the level of endothelial activation and alterations in the fibrinolytic pathway during canine babesiosis.Results: Blood samples were collected on the day of admission and on the 6th day after treatment with imidocarb propionate, from 30 dogs of various breeds and of both sexes with naturally occurring babesiosis caused by B. canis. In this prospective study, plasminogen activity was assessed using a chromogenic assay, and concentrations of high mobility group box-1 protein (HMGB-1), intercellular adhesive molecule-1 (ICAM-1), vascular adhesive molecule-1 (VCAM-1), soluble urokinase receptor of plasminogen activator (suPAR), thrombin activatable fibrinolysis inhibitor (TAFI), soluble thrombomodulin (TM) and plasminogen activator inhibitor-1 (PAI-1) were determined using a canine specific ELISA. Concentrations of TM, HMGB-1, VCAM-1 and suPAR were increased in dogs with babesiosis at admission compared to healthy dogs. After treatment, concentrations of TM were lower in infected dogs compared to healthy dogs. Dogs with babesiosis also had increased concentrations of TM, ICAM-1 and HMGB-1 and decreased plasminogen and PAI-1 at presentation compared to day 6 after treatment. Dogs with complicated babesiosis had higher concentrations of TM, HMGB1 and TAFI at admission compared to the 6th day.Conclusions: Biomarkers of endothelial activation and fibrinolysis were altered in dogs with babesiosis. Further studies into their usefulness as biomarkers of disease severity or prognosis is warranted

Integrated dataset on acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin

Data herein describe the quantitative changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system obtained by shotgun quantitative proteomic tandem mass tags approach using high-resolution Orbitrap technology. Statistical and bioinformatic analyses were performed to specify the effect of bacterial endotoxin. Plasma from chicken (N=6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post injection along with plasma from a control group (N=6) challenged with sterile saline. Protein identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by the Cytoscape application ClueGO based on Gallus gallus GO Biological Process database, and refined by REVIGO. Absolute quantification of several acute phase proteins, e.g. alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and ovotrensferrin (OVT) was performed by immunoassays to validate the LC-MS results. The data contained within this article are directly related to our research article”Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin” [1]. The raw mass spectrometric data generated in this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009399 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD009399)

Pokazatelji upale i fibrinolize u pasa oboljelih od limfoma

Canine lymphoma is one of the most common haematological neoplasms. Results of a numerous studies indicate the important role of chronic inflammation in the development of cancer. The aim of this study was to determine the concentrations of novel inflammatory biomarkers in canine lymphoma. Fifteen dogs with lymphoma and 15 healthy dogs (control group) participated in this study. The plasma levels of high sensitivity C reactive protein (hs CRP), interleukin-6 (IL-6), high mobility group box-1 (HMGB-1), soluble intercellular adhesive molecule -1 (sICAM-1), plasminogen activator inhibitor -1 (PAI-1) and soluble urokinase receptor of plasminogen activator (suPAR) were measured using canine specific enzyme-linked immunosorbent assay (ELISA). Lactate dehydrogenase activity (LDH) was measured using a spectrophotometric method. Dogs with lymphoma had significantly increased plasma hsCRP, IL-6, HMBG-1, sICAM-1, and LDH compared with the control group. In addition, hsCRP, IL-6, HMBG-1, and suPAR concentrations and LDH activity showed a significant strong positive correlation in the lymphoma group. The data obtained support the role of cytokine-mediated inflammation in canine lymphoma, and suggest the possibility that anti-inflammatory therapies might provide a supportive effect. The significant and strong positive correlation between the established (hsCRP, IL-6) and novel (HMGB-1, suPAR) inflammatory biomarkers indicate their potential usefulness in canine lymphoma diagnostics.Limfom je najčešći hematopoetski tumor u pasa. Brojna istraživanja potvrđuju važnu ulogu upale, napose kronične, u razvoju ove, ali i drugih malignih bolesti te je cilj ovoga rada bio odrediti koncentraciju novijih markera upale u plazmi pasa oboljelih od limfoma. U istraživanje je bilo uključeno 15 oboljelih pasa i 15 pasa koji su činili kontrolnu skupinu. Određena je koncentracija C-reaktivnog proteina (hsCRP), interleukina 6 (IL-6), proteina visoke pokretljivosti grupe 1 (HMGB-1), solubilne intercelularne adhezijske molekule 1 (sICAM-1), inhibitora aktivatora plazminogena 1 (PAI-1), solubilnog receptora urokinaznog aktivatora plazminogena (suPAR) i aktivnosti laktat-dehidrogenaze (LDH). Pretrage za sve navedene pokazatelje učinjene su imunoenzimnim testom, osim laktat-dehidrogenaze čija je aktivnost određena spektrofotometrijski. Utvrđeno je da psi oboljeli od limfoma imaju statistički značajno povišenu koncentraciju hsCRP-a, IL-6, HMBG-1, sICAM-1 te LDH u plazmi u odnosu na kontrolnu skupinu. Rezultati potvrđuju ulogu upale posredovane citokinima u patogenezi limfoma u pasa, a to zatim upućuje na mogućnost uključivanja protuupalnih lijekova u terapijske protokole kod ove bolesti. Nadalje, jaka pozitivna korelacija između CRP-a i IL-6 te HMGB-1 i suPAR kao novih pokazatelja upućuje na potencijalnu mogućnost njihova korištenja u dijagnostičke svrhe

Prospektivna hematološka i biokemijska evaluacija pasa sa spontano stečenom prevelikom tjelesnom težinom i pretilosti.

Across species overweight and obesity are associated with numerous disorders. To evaluate the occurrence of disturbances in terms of canine overweight and obesity, the authors undertook a prospective examination of hematological and biochemical parameters in 45 overweight and obese dogs, and compared the data with those obtained in 28 healthy normal-weight dogs. Neutrophil count, total cholesterol concentration and alkaline phosphatase activity were increased in the total group of overweight and obese dogs (P<0.01; P<0.007; P<0.000007), while concentrations of blood urea nitrogen decreased (P<0.007). Overweight was characterized by increased neutrophil count, cholesterol and triglycerides (P<0.03; P<0.00005; P<0.02), while obesity resulted in an increase in glucose, alkaline phosphatase and total cholesterol (P<0.02; P<0.002; P<0.0004), and a decrease in blood urea nitrogen and creatinine (P<0.0004; P<0.004). The obtained results support the idea that neutrophils could be involved in canine obesity-related pathology. Overweight and obesity were characterized by disturbances in the glucose metabolism and dyslipidemia. One of the interesting aspects of our examination was that obesity in dogs was related to a decrease in urea and creatinine concentrations in the circulation, which may be a consequence of increased glomerular filtration or hyperlipidemia. This study implies that canine obesity significantly affects alkaline phosphatase activity. A favorable circumstance related to the epidemic proportions of obesity in dogs is that most related potential health problems can be successfully prevented by maintaining a normal body weight.Prevelika tjelesna težina i pretilost povezane su s mnogobrojnim poremećajima u mnogih vrsta životinja. Da bi istražili promjene do kojih dolazi kod povećane tjelesne težine i pretilosti pasa, autori su prospektivno istražili hematološke i biokemijske pokazatelje u 45 pasa s povećanom tjelesnom težinom i pretilošću i usporedili rezultate dobivene u 28 zdravih pasa s normalnom tjelesnom masom. Broj neutrofila, koncentracija ukupnog kolesterola i aktivnost alkalne fosfataze bili su povećani u skupini pasa s prevelikom tjelesnom masom i skupini pretilih pasa (P<0,01; P<0,007; P<0,000007), dok je koncentracija ureje bila snižena (P<0,007). Povećana tjelesna težina pasa bila je popraćena povećanim brojem neutrofila, povećanom koncentracijom kolesterola i trigliceridima (P<0,03; P<0,00005; P<0,02), a pretilost je rezultirala povećanjem glukoze, alkalne fosfataze i kolesterola (P<0,02; P<0,002; P<0,0004) i snižavanjem ureje i kreatinina (P<0,0004; P<0,004). Dobiveni rezultati idu u prilog mišljenju da neutrofili mogu imati ulogu u patološkim procesima uzrokovanim pretilošću. Povećana tjelesna težina i pretilost očitovali su se poremećajima u metabolizmu glukoze i dislipidemijom. Jedan od zanimljivih rezultata ovog istraživanja je povezanost pretilosti i smanjenja koncentracije ureje i kreatinina u krvotoku, što može biti posljedica povećane glomerularne filtracije ili hiperlipidemije. Istraživanje ukazuje da pretilost pasa značajno utječe na aktivnost alkalne fosfataze. Većina zdravstvenih problema u pasa može se uspješno spriječiti održavanjem normalne tjelesne težine

Analiza koncentracije minerala u plazmi mladih i odraslih jelena (Cervus elaphus).

Eastern Croatia (Kopački rit) is one of the most famous habitat of red deer (Cervus elaphus). Knowledge of normal electrolyte and mineral concentrations, together with other hematological and biochemical values, are essential for appropriate diagnosis of diseases and health control. The aim of this study was to determine mineral and electrolyte concentrations in young red deer population and compare them with adults. Serum total calcium (tCa), inorganic phosphorus (iP), total magnesium (tMg), total protein and albumin were determined by spectrophotometric analysis; and ionized calcium(Ca2+), magnesium (Mg2+), sodium (Na+), potassium (K+) and chloride (Cl-) concentrations were determined by ion-selective electrodes (ISE). Young animals showed significantly higher values of K+, tCa, Ca2+, Mg2+, iP and albumin, while Na+, Cland protein concentrations were lower. There was no significant difference in the tMg concentration and Ca2+/Mg2+ ratio in relation to age. In wild animals, these laboratory analyses are still in their very beginning due to difficulties in collecting data and lack of equipment in veterinary practice. Electrolyte and mineral values determined in this study can be considered reference data for health control and disease diagnosisIstočni dio Hrvatske (osobito područje Parka prirode Kopački rit) značajno je stanište jelena običnog (Cervus elaphus). Utvrđivanje referentnih vrijednosti za laboratorijske pokazatelje kod brojnih vrsta divljih životinja, pa tako i ove, nije još u potpunosti provedeno. Razlog tome su poteškoće u prikupljanju uzoraka i relativno pomanjkanje opreme u područnim laboratorijima. Utvrđivanje referentnih raspona za serumske odnosno plazmatske koncentracije minerala i elektrolita, uz ostale hematološke i biokemijske pokazatelje predstavlja temelj za objektivno procjenjivanje zdravstvenog stanja. Pri tome je posebno važno precizno utvrditi referentne vrijednosti za svaku pojedinu dobnu kategoriju, kako zbog nepoznavanja fizioloških razlika ne bi došlo do pogrešnog tumačenja laboratorijskih nalaza. Cilj ovog istraživanja bio je odrediti i usporediti serumsku odnosno plazmatsku koncentraciju minerala i elektrolita kod mladih i odraslih jedinki u običnog jelena te utvrditi postoje li značajne razlike ili se mogu koristiti iste referentne vrijednosti za ove pokazatelje u obje dobne skupine. Serumska razina ukupnog kalcija (tCa), anorganskog fosfora (iP), ukupnog magnezija (tMg) te albumina i ukupnih proteina određena je spektrofotometrijski, a plazmatska koncentracija ioniziranog kalcija (Ca2+), magnezija (Mg2+), natrija (Na+), kalija (K+) i klorida (Cl-)određena ion-selektivnim elektrodama (ISE). U mladih jedinki nađene su signifikantno više vrijednosti K+, tCa, Ca2+, Mg2+, iP i albumina, dok su vrijednosti Na+, Cl- i proteina bile niže u odnosu na odrasle jelene. Za tMg i omjer Ca2+/Mg2+ nije ustanovljena značajna razlika kod različitih dobnih kategorija. Dobiveni rezultati mogu se smatrati referentnim za koncentraciju istraživanih minerala i elektrolita u jelena običnog (Cervus elaphus) te biti oslonac u procjeni i praćenju zdravstvenog stanja ove divljači. Pri tome treba istaknuti da je za neke pokazatelje utvrđena fiziološka razlika kod različitih dobnih kategorija pa je za pravilno tumačenje nalaza od najvećeg značenja rabiti referentne vrijednosti dobivene od primjerene dobne skupine

Acido-bazni status u pasa oboljelih od babezioze uzrokovane vrstom Babesia canis

Acid-base disturbances have been reported in severe canine babesiosis caused by Babesia rossi (B. rossi), but they have not been studied in babesiosis caused by B. canis. The objective of this study was to determine the acid-base status, blood gases and electrolyte concentrations in naturally occurring canine babesiosis caused by B. canis, and to compare the results to those in healthy dogs. Two groups of animals were used: group 1 consisted of 10 healthy dogs, and group 2 consisted of 14 dogs naturally infected with B. canis. The following acid-base disturbances occurred in the dogs with naturally occurring babesiosis: half of the dogs had a mixed acid-base disorder, and the other half a simple acid-base disorder. The most common mixed disorder was metabolic acidosis with metabolic alkalosis. It may be said that a variety of acid-base disorders occurs in canine babesiosis. The dogs in the present study had metabolic acidosis due to hyperlactemia and hyperchloremia, metabolic alkalosis due to hypochloremia and hypoalbuminemia, and respiratory alkalosis due to hypoxemia. With the use of the strong-ion difference approach clearer recognition of mixed acid-base disorders and their better understanding is possible.Poremećaji acido-baznog statusa opisani su u pasa oboljelih od babezioze uzrokovane vrstom Babesia rossi (B. rossi), no nisu istraživani u slučajevima babezioze u pasa uzrokovane vrstom B. canis. Cilj je istraživanja bio utvrditi acido-bazni status u pasa oboljelih od babezioze uzrokovane vrstom B. canis. U istraživanju su korištene dvije skupine životinja: prvu skupinu činilo je deset zdravih pasa, dok je drugu skupino činilo 14 pasa oboljelih od babezioze. U pasa prirodno inficiranih vrstom B. canis pojavili su se sljedeći poremećaji acido-bazne ravnoteže: polovica pasa imala je mješoviti poremećaj acido-bazne ravnoteže, dok je druga polovica imala jednostavni poremećaj acido-bazne ravnoteže. Najčešći mješoviti poremećaj bila je metabolička acidoza s metaboličkom alkalozom. Uzroci metaboličke acidoze bili su hiperlaktemija i hiperkloremija, metaboličke alkaloze hipokloremija i hipoalbuminemija, a respiratorne alkaloze hipoksemija. Zaključno se može reći da psi oboljeli od babezioze uzrokovane vrstom B. canis razvijaju poremećaj acido-baznog statusa te da se na temelju razlike jakih iona mješoviti poremećaji acido-bazne ravnoteže mogu jasnije razlikovati i bolje razumjeti

Application of proteomics in research of parasitic diseases of wildlife

Točna identifikacija parazitskih vrsta i dijagnostika invazijskih bolesti temelj je znanstvenog razvrstavanja, ali i liječenja pojedinih bolesti. Identifikacija temeljena na morfološkim značajkama parazita i nadalje je osnova dijagnostičkih metoda. Iako je ova metoda vrlo pouzdana, razvoj molekularnih metoda pruža nove mogućnosti za kombiniranje više metoda radi točnije dijagnostike i izbjegavanja mogućih poteškoća uvjetovanih razlikama u uzorkovanju te pripremi i pohrani parazita, uvjetima u kojima je lešina boravila do dopreme na analizu i sl. Osim u navedenim slučajevima, kombinacija metoda također se posebno preporučuje u slučaju visoke srodnosti parazita. Osim toga uvođenje proteomike otvara nove mogućnosti u istraživanju parazita divljih životinja s posebnim naglaskom na potencijalnu primjenu u liječenju, razvoju mjera kontrole i dizajnu cjepiva.Accurate identification of parasitic species and disease diagnostics is a prerequisite for species systematics and treatment of potential diseases. Diagnosis based on analysis of morphological keys is still a fundamental method in parasitology. Despite its reliability, development of molecular methods offers new possibilities in combining these methods with the aim of more accurate diagnostics and avoidance of potential problems caused by variations in sampling and parasite preparation/fixation, the conditions in which carcasses were stored before analysis, etc. Apart from these cases, method combination is highly recommendable in closely related species. Additionally, introduction of proteomics in this field opens new areas in the research of wildlife parasitic diseases, with special emphasis on potential applications in treatment, development of disease control strategies, and vaccine design

Vrijednosti acidobaznih pokazatelja i elektrolita u arterijskoj krvi pasa: tradicionalni pristup i pristup „jakih iona“.

Arterial blood gas analysis is an essential part of diagnosing and managing an animal’s oxygenation and acid-base status. The aim of this study was to establish reference intervals for acid-base values and blood gases in clinically healthy dogs of different ages and sex using Stat Profi le Critical Care Xpress (CCX), and to provide an insight into the most appropriate approach to analyze complex acid-base abnormalities. Whole-blood arterial samples were obtained from 40 clinically healthy adult dogs of various breeds. Parameters of quantitative acidbase analysis (SIDCLIN, SIDAPP, SIDEFF, SIG) were calculated using the Stewart-Figge approach. Reference ranges were: pH 7.37 - 7.44, pCO2 22.62 - 34.52 mmHg, pO2 91.35 - 106.05 mmHg, A 106.33 - 120.12 mmHg, a/A 0.80 - 0.90 mmHg, AaDO2 -0.72 - 32.90 mmHg, HCO3 - 14.06 - 22.10 mmol/L, BEecf -12. 05 - 1. 61 mmol/L, BEb -9.7 - 0.46 mmol/L, SO2 94.31 - 100 %, pO2/FiO2 437.00 -507.45 mmHg, AG 5.49 - 19.67 mmol/L, ATOT 11.29 - 12.06 mmol/L, SIDCLIN 27.94 - 31.41 mmol/L, SIDAPP 26.00 - 31.60 mmol/L, SIDEFF 29.27 - 36.21 mmol/L, SIG 2.32 - 7.46 mmol/L. Reference values obtained on the CCX analyzer provide valuable baseline information for assessing new acid-base parameters and for interlaboratory comparisons. Differences compared with previously published reference values may be attributed largely to differences in methodology and sampling. The acid-base values determined in this study may be considered to be reference data for health control and disease diagnosis.Analiza plinova u arterijskoj krvi ključni je dio dijagnostike i praćenja oksigenacije organizma te acidobazičnog statusa. Svrha ovog istraživanja bila je utvrditi referentne vrijednosti za acidobazne pokazatelje i plinove u krvi klinički zdravih pasa različitog spola i dobi pomoću analizatora Stat Profile Critical Care Xpress (CCX) i omogućiti uvid u najprikladniji način analiziranja kompleksnih acidobaznih poremećaja. Arterijski uzorci pune krvi uzeti su od 40 klinički zdravih odraslih pasa različitih pasmina. Pokazatelji kvantitativnoga acidobaznog statusa (SIDCLIN, SIDAPP, SIDEFF, SIG) izračunani su Stewart-Figge-ovim pristupom. Referentni rasponi bili su: pH 7,37-7,44, pCO2 22,62-34,52 mm Hg, pO2 91,35-106,05 mm Hg, A 106,33-120,12 mm Hg, a/A 0,80-0,90 mm Hg, AaDO2 -0,72-32,90 mm Hg, HCO3 - 14,06-22,10 mmol/L, BEecf -12,05-1,61 mmol/L, BEb -9,7-0,46 mmol/L, SO2 94,31-100%, pO2/FiO2 437,00-507,45 mm Hg, AG 5,49-19,67 mmol/L, ATOT 11,29-12,06 mmol/L, SIDCLIN 27,94-31,41 mmol/L, SIDAPP 26,00-31,60 mmol/L, SIDEFF 29,27-36,21 mmol/L, SIG 2,32-7,46 mmol/L. Referentne vrijednosti dobivene na analizatoru CCX pružaju vrijedne informacije za postavljanje novih acidobaznih pokazatelja i za njihovu usporedbu između laboratorija. Razlike u odnosu na prethodno objavljene referentne vrijednosti mogu se većim dijelom pripisati razlikama u metodologiji i uzimanju uzoraka. Dobivene vrijednosti acidobaznih pokazatelja iz ovog istraživanja mogu se smatrati referentnim vrijednostima za kontrolu zdravlja i dijagnostiku bolesti