LABORATORIJSKI NALAZI KOD SINDROMA LEŽEĆIH KRAVA
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1 Letopis naučnih radova Godina 38 (2014), Broj I, strana 190 UDK: Originalni naučni rad Original scientific paper LABORATORIJSKI NALAZI KOD SINDROMA LEŽEĆIH KRAVA Slaviša Đokić, Marko R. Cincović, Branislava Belić, Miodrag Radinović, Dragica Stojanović,Radojica Đoković 56 REZIME Sindrom ležećih krava nastaje najčešće u periodu posle teljenja, dakle tokom rane laktacije. Ovaj poremećaj se odlikuje prolongiranim ležanjem krava bez mogućnosti ustajanja, sa ili bez poremećaja homeotermije. Izuzetno je složen i nastaje kao posledica hipokalcemije, poremećaja fosfatemije, povrede nerava i mišića uz degeneraciju, jake inflamacije vimena i uterusa i masne infiltracije jetre. Brza dijagnostika oboljenja i sprovođenje brze terapije omogućuje da se krava sačuva od uginuća, ali i da se očuva njena buduća proizvodnja mleka i reproduktivna efikasnost. Cilj ovog rada je da se utvrdi mogućnost diferencijalne dijagnoze uzroka sindroma ležećih krava upotrebom laboratorijskog profila, koji će sadržati sve parametre koji ukazuju na neki od uzroka ove bolesti, te da se ispita potpuna specifičnost laboratorijskog nalaza ili postojanje preklapanja laboratorijskog nalaza, koje bi ukazalo na istovremeno delovanje više etioloških faktora i njihovu povezanost. U ogled je uključeno 30 krava. Na osnovu laboratorijskih rezultata može se zaključiti da postoje 4 glavna etiološka faktora, koji dovode do sindroma ležećih krava a to su: hipokalcemija (promena koncentracije Ca, P, Mg, K), sindrom masne jetre (promena koncentracije bilirubina, holesterola i aktivnosti AST), slabost mišića (visoka aktivnost CK i AST) i postojanje inflamacije (odnos neutrifila:limfocita veći od 1 i pad 56 DVM Slaviša Đokić; Dr Marko R. Cincović, docent; Dr Branislava Belić, vanr.prof.; Dr Miodrag Radinović, docent; Dr Dragica Stojanović, redovni profesor, Dr Radojica Đoković, redovni profesor; Departman za veterinarsku medicinu, Poljoprivredni fakultet Novi Sad; Agronomski fakultet Čačak. Ovaj rad je deo projekta TR31062, podržan od strane Ministarstva prosvete, nauke i tehnološkog razvoja R.Srbije. 190
2 koncentracije hemoglobina). U određenom broju slučajeva nađen je laboratorijski nalaz koji ukazuje na tzv. komplikovanu etiologiju, kada postoje i znaci hipokalcemije, masne jetre i inflamatorne reakcije. Kod većine ispitanih krava u metaboličkom profilu postoje odstupanja gotovo svih parametara u odnosu na referentne vrednosti. Odstupanje glavnih parametara koji ukazuju na neki od sindroma ležećih krava je veliko i iznosi 25 do preko 100% od referentne vrednosti. Postoji i odstupanje sporednih parametara, ali je ono u rasponu od 5-15%. Na osnovu laboratorijskih analiza moguće je detektovati uzrok sindroma ležećih krava i usmeriti dalju terapiju. Ključne reči: laboratorijski profil, sindrom ležećih krava. UVOD Sindrom ležanja kod krava posle teljenja je izuzetno čest problem na farmama krava. Dugotrajno ležanje posle partusa povezano je sa smanjenim unosom hrane, što dodatno povećava rizik za nastanak bolesti i poremećaja metabolizma i umanjuje proizvodnju mleka. Sledeći problem koji se javlja je taj što krave prilikom ležanja posle teljenja značajno slabije izbacuju lohije kada raste rizik za upalu materice, što može umanjiti reproduktivnu efikasnost krava. Zbog tih i drugih faktora brzo rešavanje problema ležećih krava je od prioriteta za veterinarsku struku. Uzroci koji mogu dovesti do sindroma ležanaj su različiti. Tradicionalno je poznato da hipokalcemija dovodi do sindroma ležećih krava (Goof, 2006). Ona je povezana sa povećanim gubitkom kalcijuma iz kostiju i povećanom potrošnju kalcijuma za proizvodnju mleka. Međutim, pored ovog uzroka postoje i drugi uzroci. Povreda nerava prilikom partusa može biti uzrok ležanja i pareze ztadnjeg dela tela kada dolazi do velikog pritiska na muskulaturu i propadanja mišića (Divers i Peek, 2008). Povećana potropnja lipida za potrebe energetskog metabolizma u cilju čuvanja glukoze za proizvodnju mleka, dovodi do povećane ketogeneze i nastanka masne jetre kod krava. Masna jetra vrlo brzo može da pređe u porođajnu jetrenu komu ili netipični simptom sa ležanjem krava i hipertermijom (Bobe i sar., 2004), kada klinički postoji sindrom ležećih krava. Svi navedeni uzroci ostavljaju specifičan metabolički profil u krvi. Cilj ovog rada je da se ispita da li se može vršiti diferencijalan dijagnostika sindroma ležanja krava posle partusa analizom metaboličkog profila krvi. 191
3 MATERIJAL I METODE RADA U ogled je uključeno 30 krava Holštajn-frizijske i Simentalske rase koje su pokazale sindrom ležanja u periodu rane laktacije (od prvog do desetog dana). Krv je uzimana iz v. jugularis ili v.coccigea u vakutajnere za odvajanje plazme i transportovana u laboratoriju. U laboratoriji je komercijalnim kitovima određena koncentracija glukoza, BHB, AST, ukupni bilirubin, holesterol, CK, Ca, P, Mg. Korišćeni su kitovi proizvođača Randoc i Point Scientific. Korišćen je spektrofotometar marke Rayto. Na hematološkom brojaču tipa Hemavet određena je koncentracija hemoglobina i odnos broja neutrofila i limfocita. Statistika: Ispitano je variranje svih laboratorijskih parametara u odnosu na fiziološki obim. Izvršeno je grupisanje krava prema dominantnom laboratorijskom nalazu na krave sa poremećajem metabolizma jona, jetre, mišića/nerava i inflamacije kao glavnog uzroka sindroma ležećih krava. Ispitano je u kojoj meri se prepliću laboratorijski nalazi. Ispitano je u kojoj se meri zajednički pojavljuju poremećaji u vrednostima pojedinih metabolita kod krava. REZULTATI ISTRAŽIVANJA I DISKUSIJA Rezultati ispitivanja pokazuju da u 30 slučajeva postoji značajno variranje odabranih biohemijskih i hematoloških parametara. Dobijeni rezultati ukazuju da krave koje u ranoj laktaciji boluju od produženog ležanja i nemogućnosti ustajanja pokazuju laboratorijsku sliku poremećaja metabolizma jona sa smanjenjem koncentracije Ca, P, Mg i K. U drugim slučajevima javlja se sindrom masne jetre koji se ogleda u hiperketonemiji i povećanoj koncentraciji ukupnog bilirubina i aktovnosti AST. Nađeni su i slučajevi kod kojih dominira mišićna degeneracija koja se odlikuje povišenim vrednostima CK i AST. Ovi nalazi mogu biti praćeni inflamacijom koja se ogleda u smanjenoj koncentraciji hemoglobina i povećanom odnosu neutrofili/limfociti, koji govori u prilog akutnoj stresnoj opterećenosti. U određenom broju slučajeva nađen je laboratorijski nalaz koji ukazuje na tzv. komplikovanu etiologiju kada postoje i znaci hipokalcemije, masne jerte i inflamatorne reakcije. Prilikom analize individualnih slučajeva u obzir treba uzeti i određene fiziološke varijacije metabolita u ranoj laktaciji. U tom smislu variranje enzima za 5 ili % iznad fiziološke vrednosti nema kliničkopatološki značaj, dok njihovo variranje za preko 50 ili čak više od 100% ne može biti zanemareno. Na osnovu toga koji parametar najviše odstupa od očekivane vrednosti krave su podeljene na četiri pomenute grupe, prema etiologiji pareze, a broj slučajeva i procentualna zastupljenost sa različitim laboratorijkim nalazima prikazan je na Grafikonu 1. Tipičan metabolički profil u navedenim slučajevima prikazani su na Grafikonima 2-5 (pokazani su tipični predstavnici iz 30 ispitanih slučajeva). 192
4 Diferencijalna laboratorijska dijagnostika se može postaviti ukoliko poznajemo u kom obimu se promenjene vrednosti pojedinih parametara javljaju zajednički i u kom obimu variraju vrednosti parametara u odnosu na priznate referentne vrednosti. Rezultati pokazuju da se u metaboličkom profilu najčešće javljaju istovremeno promene u vrednostima većeg broja parametara (Tabela 1), što posebno traži pažnju u procesu diferencijalne dijagnostike nalaza. Međutim, nisu sve promene jednako opasne. U procesu diferencijalne laboratorijske dijagnostike neophodno je ispitati u kom se rasponu kreću laboratorijski nalazi u odnosu na referentnu vrednost. Zbog velikog broja parametara i šarolokosti uzroka sindroma ležećih krava, jasno je da određeni parametri neće pokazati variranje izvan referentne vrednosti, pa je minimalno variranje za sve parametre na nivou 0. Maksimalno odstupanje vrednosti parametara karakteristično je za promene koje su navedene ka uzrok sindroma ležećih krava (Tabela 2). Zbog patofizioloških mehanizama i povezanosti laboratorijski nalaz kod krava je takav da pokazuje čitav niz metaboličkih adaptacija. Međutim, odstupanje parametara u odnosu na glavne parametre koji reprezentuju uzrok sindroma ležećih krava je nisko i na nivou 5-15%, što omogućuje jednostavnu diferencijalnu dijagnozu 4 glavna uzorka sindroma ležećih krava, koji pokazuju vrlo visoko variranje parametara u odnosu na referentu vrednosti, od 25 do preko 100%. Rezultati su prikazani u Tabeli 3. Grafikon 1. Broj i % krava sa laboratorijskim nalazom koji ukazuje na uzrok sindroma ležećih krava Graphic 1. Number and % of cows with diferent laboratory findings which represent etiology of downer cows syndrome Broj i % krava sa laboratorijskim nalazom koji ukazuje na uzrok sindroma ležećih krava 2; 7% 6; 20% 10; 33% Masna jetra Hipokalcemija 12; 40% Inflamacija Problemi sa mišićima 193
5 Grafikon 2-5. Karakterističan laboratorijski profil kod masne jetre, hipokalcemije, distrofije mišića i inflamacije kao uzroka sindroma ležećih krava Graphic 2-5. Characteristic laboratory profile in cows with fatty liver, hypocalcemia, muscular distrophy and inflamation as etiology of downer cows syndrome Odstupanje od referentne vrednosti (%) Odstupanje od referentne vrednosti (%) Glu BHB Ca P Mg K Tbil Hol AST CK N/L Hgb Slučaj br. 1 Glu BHB Ca P Mg K Tbil Hol AST CK N/L Hgb Slučaj br. 6 Odstupanje od referentne vrednosti (%) Glu BHB Ca P Mg K Tbil Hol AST CK N/L Hgb Slučaj br. 9 Odstupanje od referentne vrednosti (%) Glu BHB Ca P Mg K Tbil Hol AST CK N/L Hgb Slučaj br
6 Tabela 1. Učestalost pojave sporednih laboratorijskih nalaza kod krava sa različitim uzrocima sindroma ležanja (% od broja krava sa datim uzrokom) Table1. The incidence of secondary laboratory findings in cows with various etiology of the downer cows syndrome (% of the number of cows with a given etiology) 1* Jetra / / / Hipokalcemija / Mišići / / Inflamacija** x x x x x x x x x x x x / / *1-hipoglikemija, 2-hiperketonemija, 3-hipokalcemija, 4-hipofosfatemija, 5-hiperfosfatemija, 6- hipomagnezemija, 7-hipokaliemija, 8-hiperkaliemija, 9-hiperbilirubinemija, 10- hipoholesterolemija, 11-povećana aktivnost AST, 12-povećana aktivnost CK, 13- povećan odnos N/L, 14-pad vrednosti hemoglobina, **klinički beznačajno jer su samo dve krave u grupi. Tabela 2. Prosečno i maksimalno variranje (%) laboratorijskih parametara kod ispitanih krava u odnosu na poznate referentne vrednosti Table 2. Average and maximal variation (%) of laboratory parameters in relation with referent value Prosečno Maksimalno Glukoza BHB Ca P -3-7 Mg -4-6 K +/- 10 +/- 20 Tbil Hol AST CK N/L Hgb
7 Tabela 3. Prosečno odstupanje (%) glavnih (siva polja) i sporednih (bela polja) laboratorijskih nalaza kod 4 osnovna uzroka sindroma ležećih krava u odnosu na referentne vrednosti. Table 3. Average % deviation of main (gray field) and secondary (white field) laboraotri findings in four etiology of downer cow syndrome in relation with reference value. 1* Jetra Hipokalcemija Mišići Inflamacija *1-hipoglikemija, 2-hiperketonemija, 3-hipokalcemija, 4-hipofosfatemija, 5-hiperfosfatemija, 6- hipomagnezemija, 7-hipokaliemija, 8-hiperkaliemija, 9-hiperbilirubinemija, 10- hipoholesterolemija, 11-povećana aktivnost AST, 12-povećana aktivnost CK, 13- povećan odnos N/L, 14-pad vrednosti hemoglobina. U periodu posle teljenja prirodno dolazi do metaboličkih adaptacija koje imaju sledeće karakteistike: povećana koncentracija NEFA, BHB, bilirubina i AST, uz smanjenu koncentraciju glukoze, albumina i kalcijuma. Ove promene nastaju kao posledica adaptacije na laktaciju koja sledi i smanjen unos hrane koji se dešava u periodu oko teljenja. Međutim, razlika između prirodne metaboličke adaptacije i nastanka poremećaja zdravlja je često nejasno vidljiva, obzirom da se zna da krave sa izraženom mobilizacijom lipida i produkcijom ketona imaju mnogo veći rizik za nastankom različitih oboljenja, a često pokazuju i metabolički profil koji liči na profil krava koje pate od pojedinih oboljenja u ranoj laktaciji (Cincović i sar.,2012; Ospina i sar., 2010; Đoković i sar., 2013; González i sar.,2011). Hipokalcemija je najpoznatiji uzrok sindroma ležećih krava. Metabolizam kalcijuma u peripartalnom periodu je definisan različitim faktorima (Goof, 2006; Đoković i sar., 2014).Kako bi se sprečio pad koncentracije Ca kod početka laktacije, koji ima mnogo ozbiljnih posledica na životne procese (pored peripartalne pareze), krava mora gubitak Ca kroz mlečnu žlezdu nadoknaditi otpuštanjem Ca iz kostiju ili povećanjem apsorpcije hranom unetog Ca. Faktori koji narušavaju homeostazu kalcijuma i mogu biti u vezi sa nastankom hipokalcemije i sindroma ležećih krava su: metabolička alkaloza, hipomagnezemija i hiperfosfatemija. Metabolička alkaloza je predispozicija za nastanak mlečne groznice. Metabolička alkaloza smanjuje odgovor organizma na PTH. Hipomagnezemija utiče na metabolizam Ca na dva načina: (1) smanjuje lučenje PTH kao odgovor na hipokalcemiju i (2) smanjuje osetljivost tkiva na 196
8 PTH. PTH sekrecija je obično podstaknuta čak i blagim padom koncentracije Ca u krvi. Međutim, hipomagnezemija može da oslabi ovaj odgovor. Kada koncentracija P u krvi pređe gornju granicu, od oko 2 mmol/l, fosfat ima direktan inhibitorni efekat na bubrežni enzim koji konvertuje 1,25-hidroksivitamin D u 1,25-dihidroksivitamin D. Usled toga, čak i kada postoji normalna sekrecija PTH, a tkiva prepoznaju PTH, krava neće biti u mogućnosti da produkuje hormon, koji je neophodan za apsorpciju Ca u intestinalnom traktu i doći će do narušene homeostaze Ca. Kod sindroma masne jetre raste koncentracija bilirubina i aktivnost jetrinih enzima u krvi. Hepatični profil govori u prilog citotoksičnog oštećenja hepatocita. Oštećenje hepatocita dovodi do poremećaja u razvoju inflamatornog odgovora i klirensa toksina (Bobe i sar., 2004). Smanjen klirens endotoksina i povećana koncentracija proteina akutne faze-haptoglobina i serum amiloida A, koji se sintetišu u jetri ukazuju da akumulacija lipida u jetri može direktno da promeni sposobnost jetre da razgradi i sintetiše komponente imunog odgovora. Različiti vidovi imunog odgovora su suprimirani kod krava sa masnom jetrom. Niža ćelijska citotoksičnost i reaktivni kiseonik, stvaranje polimorfonukleara u krvi, ukazuju na smanjeni kapacitet fagocitoze polimorfonukleara i makrofaga kod krava sa masnom jetrom. Niže koncentracije IgG nakon imunizacije tetanusnim toksoidom i niža produkcija interferona u leukocitima ukazuju da krave sa masnom jetrom imaju smanjenu sposobnost otpuštanja određenih medijatora inflamacije. Oštećenje mišića i nerava čest je uzrok sindroma ležećih krava (Divers i Peek, 2008). Kod različite stoke i nejasnim slučajevima, serumska keratin kinaza (CK) i aspartat aminotransferaza (AST) traba da posluže za prognostičku pomoć. Najviše ležeće stoke ima povišene nivoe CK i AST, tako da blago povećanje nije najbolji dijagnostički pokazatelj ozbiljne miopatije. Relativni stepen povećanja nivoa CK i AST je važan dijagnostičko-prognostički indikator. Važno je znati da CK skače naglo usled povrede mišića i ima kratak (4-8h) poluživot u serumu, dok AST dostiže vrh sporije, ali može da ostane povišena i nekoliko dana. Tako da je CK bolji dijagnostički test za krave koje leže 24-48h, a krave koje su u ovakvom stanju oko nedelju dana će bolje odgovoriti na AST test, jer predpostavljamo da se oštećenje mišića dogodilo na početku perioda ležanja. CK koji dostiže više od IU/L je tipičan za akutnu miopatiju usled napora i često može da iznosi i preko IU/L ako je mišić teško oštećen. Ozbiljne miopatije mogu da uzrokuju porast AST na 3000 ili više, a to traje nekoliko dana nakon pika. Uopšteno, vrednosti CK preko IU/L i AST IU/L su veoma loši prognostički znaci. Ova stoka možda ima povišene nivoe kalijuma u serumu, u akutnoj fazi, kao rezultat pucanja mišićne ćelije, a nose rizik i za mioglobinsku nefrozu. Može da se javi metabolička acidoza usled povećanja nivoa mlečne kiseline i slabe perfuzije mišića. Kod krava sa drastičnom miopatijom, koja zahvata važne grupe mišića, stanje slično šoku i neurološki znaci mogu biti ispoljeni usled oslobađanja laktata, kalijuma i verovatno drugih produkata iz oštećenih mišića. Povrede peronealnog nerva su česte kod ležećih krava, uključujući i pacijente koji pate od mlečne groznice, jer je lokacija ovog nerva superficijalna i prelazi lateralnu površinu glave gastroknemiusa i fibule kako bi dospeo do kruralnih mišića. 197
9 ZAKLJUČAK Na osnovu laboratorijskih rezultata može se zaključiti da postoje 4 glavna etiološka faktora koja dovode do sindroma ležanja krava i to su: hipokalcemija (promena koncentracije Ca, P, Mg, K), sindrom masne jetre (promena koncentracije bilirubina, holesterola i aktivnosti AST), slabost mišića (visoka aktivnost CK i AST) i postojanje inflamacije (odnos neutrifili:limfociti veći od 1 i pad koncentracije hemoglobina). Na osnovu laboratorijskih analiza moguće je detektovati uzorok sindroma ležećih krava i usmeriti dalju terapiju. LITERATURA 1. Goof J.P. (2006): Macromineral physiology and application to the feeding of the dairy cow for prevention of milk fever and other periparturient mineral disorders. Animal Feed Science and Technology, 126: Bobe G., Young J.W., Beitz D.C. (2004): Invited Review: Pathology, Etiology, Prevention, and Treatment of Fatty Liver in Dairy Cows. J. Dairy Sci. 87: Đoković R.D., Cincović M.R., Belić B.M. (2014): Fiziologija i patofiziologija metabolizma krava u peripartalnom periodu. Departman za veterinarsku medicinu, poljoprivredni fakultet Novi Sad. 4. González F.D., Muiño R., Pereira V., Campos R., Benedito J.L.: Relationship among blood indicators of lipomobilization and hepatic function during early lactation in highyielding dairy cows. J. Vet. Sci., 12(3): , Cincović M.R., Belić B., Radojičić B., Hristov S., Đoković R Influence of lipolysis and ketogenesis to metabolic and hematological parameters in dairy cows during periparturient period. Acta veterinaria (Beograd), 62(4): Ospina P.A., Nydam D.V., Stokol T., Overton T.R Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. J. Dairy Sci., 93(4): Đoković R., Kurćubić V., Ilić Z., Cincović M., Fratrić N., Stanimirović Z., Petrović M.D., Petrović M.P Evaluation of the metabolic status of Simmental dairy cows in early and mid lactation. Animal Science Paper and Report, 32, 2, Divers T.J., Peek S.F. (2008): Rebhun s diseases of dairy cattle. Saunders Elsevier, St.louis, Missouri. 198
10 LABORATORY PROFILE IN DOWNER COW SYNDROME by Slaviša Đokić, Marko R. Cincović, Branislava Belić, Miodrag Radinović, Dragica Stojanović, Radojica Đoković SUMMARY Downer cow syndrome occurs most frequently in the period after calving, so during early lactation. This disorder is characterized by prolonged cow lying down without possibility of getting up, with or without homeothermy disorders. This disorder is very complex and is the result of hypocalcemia, phosphataemia disorders, injury to the nerves and muscles with degeneration, severe inflammation of the udder and uterus and fatty infiltration of the liver. Rapid diagnosis of diseases and rapid implementation of treatment allows to save cows from death, and to preserve her future milk production and reproductive efficiency. The aim of this study was to determine the possibility of differential diagnosis of causes syndrome recumbent cows using laboratory profile that will contain all the parameters that indicate some of the causes of this disease and to investigate whether the laboratory findings completely specific or there is an overlap in the laboratory findings such that shows the activity of etiologic factors and also how they are connected. The experiment involved 30 cows. Based on laboratory results, it can be concluded that there are four major etiological factors that lead to the downer cows syndrome: hypocalcemia (changing concentrations of Ca, P, Mg, K), fatty liver syndrome (changes in the concentration of bilirubin, cholesterol and AST) muscle weakness (high activity of CK and AST) and presence of inflammation (ratio neutrophyl:lymphocytes greater than 1 and decrease in hemoglobin concentration ). In some cases, it was found that the lab report indicates so complicated etiology when there are signs of hypocalcemia, fatty liver and inflammatory reactions. In most of the cows in the metabolic profile variations are almost all parameters compared to reference values. Deviation of the main parameters that indicate some of the recumbent cow syndrome is high and amounts to 25 to over 100 % of the reference value. The deviation is also a secondary parameter but is in the range of 5-15 %. Based on laboratory analysis it is possible to detect the apparent cause of the syndrome recumbent cow and direct further therapy. Key words:laboratory profile, downer cow syndrome. Primljeno: godine Prihvaćeno: godine 199
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