LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Faculty of Veterinary Medicine
Linnea Svelander
ŪMINĖS FAZĖS AKTYVIŲJŲ BALTYMŲ VERTINIMAS ARKLIŲ KRAUJYJE
ANALYSIS OF ACTIVATED PROTEINS OF ACUTE PHASE RESPONSE IN HORSES BLOOD
MASTER THESIS
of Integrated Studies of Veterinary Medicine
Supervisor: Assoc. Prof. Vaida Andrulevičiūtė
2 THE WORK WAS DONE IN THE DEPARTMENT OF BIOCHEMISTRY
CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Theses "ANALYSIS OF ACTIVATED PROTEINS OF ACUTE
PHASE RESPONSE IN HORSES BLOOD”
1. has been done by me;
2. has not been used in any other Lithuanian or foreign university;
3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.
2016-12-16 Linnea Svelander
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CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE LITHUANIAN LANGUAGE IN THE DONE WORK
I confirm the correctness of the English language in the done work.
2016-12-16 Linnea Svelander
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CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES
2016-12.16 Vaida Andrulevičiūtė
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THE MASTER THESES HAVE BEEN APPROVED IN THE BIOCHEMISTRY DEPARTMENT
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Reviewers of the Master Theses
1) 2)
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Evaluation of defence commission of the Master Theses:
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3
TABLE OF CONTENTS
SUMMARY ... 4 SANTRAUKA ... 5 ABBREVIATIONS ... 6 INTRODUCTION ... 7 1. LITTERATURE REVIEW ... 91.1 Acute phase response ... 9
1.2 Acute phase proteins ... 12
1.3 Application in veterinary diagnosis ... 15
1.4 Acute phase reaction in horses ... 16
1.5 The behaviour and symptoms of stressed horses ... 18
2. METHODOLOGY ... 20
2.1 Research workload, location and method ... 20
2.2 Research objectives ... 20
2.3 Collection of samples ... 21
2.4 Determination of acute phase proteins ... 22
2.5 Statistical data ... 22
3. RESULTS ... 23
3.1 The horses presented in this research... 23
3.2 Dynamics in serum of SAA, haptoglobin, CRP and iron in horses ... 23
3.3 Comparison of parameter concentrations in different diseases and control group ... 27
4. DISCUSSION OF RESULTS ... 30
CONCLUSIONS ... 35
ACKNOWLEGEMENT ... 36
4
SUMMARY
ANALYSIS OF ACTIVATED PROTEINS OF ACUTE PHASE RESPONSE IN HORSES BLOOD
Linnea Svelander Master Thesis
The aim of this scientific work is analysis of activated proteins in the acute phase reaction in horses’ blood with different clinical health statuses. This master thesis work is performed in the department of Biochemistry in of Lithuanian University of health Sciences (LUHS) in Kaunas. The horses involved has different health statuses with the common feature – stress. Total amount of samples collected were 24 , from these samples 4 were taken from healthy horses as a control group and 12 samples from sick horses. Three of the sick ones had follow up samples taken to check the dynamics of the parameters analysed. The values of the proteins C-reactive protein, serum amyloid A, haptoglobin and the value of the negative indicator iron were analysed.
The report is introduced by other scientific works in the field so that the results of this work can be compared with other similar researches, but also to get an introduction in the field for the reader. The values of acute phase proteins and iron in this research are within following ranges: 1.0-5.0 mg/L for C-reactive protein, 3.8-7.4 mg/L for serum amyloid A, 160-1000 mg/L for haptoglobin and 6.9-42.6 µmol/L for iron. Horses with a present disease might still have values within the previous set normal standards but would still change in accordance with an improved state and with treatment course. Haptoglobin and serum amyloid A is generally deceasing as a response to treatment as iron is increasing. The C-reactive protein level is not changing significantly. The haptoglobin average level is higher in sick horses than clinically healthy horses, especially high in infection diseases. Therefore this can be a good indicator in diagnostics of horses.
5
SANTRAUKA
ŪMINĖS FAZĖS AKTYVIŲJŲ BALTYMŲ VERTINIMAS ARKLIŲ KRAUJYJE
Linnea Svelander Master Thesis
Šio darbo tikslas buvo į vertinti ūminės fazes aktyviųjų baltymų kiekius arklių, turinčių į vairius susirgimus, kraujyje. Darbas atliktas LSMU Biochemijos katedroje.Tirti įvairiomis ligomis sergantys arkliai, kurių bendras bruožas – stresinė savijauta. Kaip kontrolinė grupė pasirinkti arkliai neturintys ligos ir streso požymiai. Ištirti 24 arklių kraujo serumai, kurių 4 buvo sveiki (kontrolinė grupė). Vertinta C-reaktyvaus baltymo, haptoglobino ir serumo amiloido A kiekiai beitų kiekių kitimai ligos gydymo metu. Papildomas rodiklis – geležies kiekis kraujyje. Atlikta mokslinės literatūros analizė, siekiant palyginti gautus rezultatus su jaužinomais.
Nustatyta, kad C-raktyvaus baltymo kiekiai svyravo nuo1.0 iki 5.0 mg/l, serumo amiloido A nuo 3.8 iki 7.4 mg/L, haptoglobinonuo 160 iki 1000 mg/l ir geležieskiekis nuo 6.9 iki 42.6 µmol/l. Nors visos gautos reikšmė sati tiko literatūros randamas normas, jos kito gydymo eigoje.Tai reiškia, kad ūminės fazės baltymai žymiai padidėja labai sunkios arba lėtinės ligos atveju. Sekant ūminės fazės baltymų ir geležies dinamiką gydymo procese pastebėta, kad haptoglobino ir serumo amiloidas A kiekia mažėja, o geležies auga, tuo tarpu C-reaktyvaus baltymo kiekiai kinta labai nereikšmingai. Kadangi haptoglobino kiekiai sergančių arklių kraujo serume buvo žymiai didesni nei sveikų, jis yra geras arklių ligos indikatorius.
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ABBREVIATIONS
LUHS - Lithuanian University of Health Sciences CRP - C-reactive protein
SAA - Serum amyloid A APR - Acute phase response IL - Interleukin
TNF - Tumour necrosis factor NK - Natural killer cell IFN - Interferon
DIC - Disseminated intravascular coagulation ELISA - Enzyme-linked immunosorbent assay OCD - Osteochondritis dissecans
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INTRODUCTION
Acute phase proteins and iron can be found in the blood of various animal species when an acute phase reaction is present in the body due to some disease or stress. These proteins can be measured and depending on the amount in blood of the animal, it is possible to establish if for example trauma, infection, stress, neoplasia or inflammation is present in the body and the severity of it. After treatment there might be a significant change in the acute phase protein levels and iron whereas the other levels remains the same, making it hard to estimate a successful treatment [1]. But the reactive proteins are only to inform about present disease, not specific diagnosis [2].
It has been shown that during gastrointestinal diseases of horses, the concentrations of serum amyloid A, haptoglobin and fibrinogen are increased in the horses serum [3]. It has also been proven that clinically healthy horses performing hard exercise will feel stress which might increase level of acute phase proteins in blood [4]. Finally, it has been established that when a surgery is performed in horses as a treatment method, iron levels and serum amyloid A are depending on the intensity of the surgery rather than the primary state of the horse while white blood cells and fibrinogen does not change with intensity of surgery and due to post-operative condition [5].
The investigations of acute phase protein in horses' blood performed previously includes research work in specific conditions such as in surgery treatment [5] or in gastrointestinal disease [3] or arthritis [6]. The main focus of this research is stressed horses with different diseases. Investigation includes the evaluation of the values of the acute phase proteins as C-reactive protein, serum amyloid A and haptoglobin in horses blood. The negative indicator iron is also investigated. These parameters are compared to find out which acute phase protein is the best indicator in horse and what kind of disease that has biggest influence on them. Iron is known as “negative acute phase reactant” [5]. The evaluation of treatment and healing period of three different horses is also presented, to evaluate the influence of the disease state and severity on the different acute phase proteins and iron to see the dynamics of them over time.
Aim: To evaluate depending factors of activated proteins of acute phase response in the blood of various stressed horses.
Objectives:
1. To determine the values of specific activated proteins in horses' blood, both clinically healthy and sick horses.
8 2. To compare the values over time of specific activated proteins in some selected horses' blood. 3. To evaluate depending factors of activated proteins of acute phase response.
4. To establish which of the investigated parameters are good indicators of health state in horses and how they can be used.
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1. LITTERATURE REVIEW
1.1 Acute phase response
The acute phase response (APR) is an early defence mechanism of the body's immune system that can be triggered by a lot of factors. This can be any kind of stimuli including trauma, infection, stress, neoplasia, and inflammation [1]. But also from genes in different breeds, dietary pattern, obesity or specific living patterns [7]. Acute phase proteins are a part of this mechanism and the goal of the APR is to re-establish homeostasis in the body and promote healing[1].
The APR is activated by pro-inflammatory cytokines like interleukin-1 (IL-1), tumour necrosis factor α (TNF-α), and interleukin 6 (IL-6), secreted from macrophages and monocytes at site of inflammatory lesion or infection [8]. These cytokines are themselves activated by the innate immunity of the body which together with the activating of cytokines also includes physical barriers, phagocytes, complement system, and toll-like receptors which are giving an inflammatory process. The Fig. 1. illustrates the body's immune response [1].
All pro-inflammatory cytokines have multiple sources, multiple targets and multiple functions and they can be found in a large number of animal species. However, the major ones mentioned earlier has some functions that they are mainly related to. These are: IL-1 has the
10 common function to induce hepatic acute response, IL-6 induces fever and TNF-α activates T-, B-, and NK cells and induce IL-2 in T-cells [2].
It has also been shown that some acute phase cytokines like IL-1β and TNF-α are present in the mammalian brain to activate the pituitary-adrenal-corticosteroid axis in response to acute inflammation. This shows the involvement in the area of neuroimmunology and thereby an example of additional involvements of the cytokines in the body. Where there are more areas to be discovered [9].
In addition to the general response, at the site of action of foreign injury by any kind, the tissue itself will initiate a number of responses. Pro-inflammatory cytokines are released, inflammatory cells and the vascular system are activated. These in turn gives production of more cytokines and other inflammatory mediators which diffuse to extracellular fluid compartments and circulate in blood [10]. The expression of IFN-β or IL-6 proteins is enhanced in response to other cytokines like IL-1, TNF and other IFN. Bacterial products such as endotoxin also strongly enhance expression and secretion of IFN-β and IL-6 by monocytes and fibroblasts [11].
Cytokines are activating receptors which are located on target cells, and these can then activate the hypothalamic-pituitary-adrenal axis. They also reduce growth hormone secretion and gives physical changes like fever, anorexia, negative nitrogen balance and catabolism in muscle cells [10]. When making laboratory testing during these symptoms, there are also some changes shown in the results, these are: decrease of blood plasma low and high density lipoprotein-bound cholesterol and decrease of leukocyte numbers in blood. There will also be an increase in adenocorticotrophic hormone and glucocorticoids. In addition there will be an activation of complement system and blood coagulation system, also a decrease in serum levels of calcium, zinc, iron, vitamin A and a change in plasma proteins and acute phase proteins which is due to changed hepatic metabolism. This acute response can then become chronic in case of repeated acute responses [10].
After some time of introduction of inflammatory reaction, the metabolism in liver of proteins will change. This will cause an increase of some proteins in serum and a decrease of others. The acute phase proteins will increase, these are: C-reactive protein, serum amyloid A, haptoglobin, which are released by hepatocytes in the liver from cytokine stimulation [10]. The Fig. 2 is showing the response by an illustration.
11 The acute phase response with its changes in plasma is thought to help by making prevention of microbial growth and helps to restore homeostasis. Some acute phase proteins makes pathogens more susceptible to the body's defence system and some activate complement system and some neutralizes enzymes [10].
Acute inflammation may alter drug disposition in domestic animals, while chronic diseases does not alter drug disposition. The effect on drug disposition and plasma protein binding is thought to be responsible for altered drug disposition during febrile stage of disease. This could explain the relatively more common adverse effects reported at this time [12].
There are evidence that cytokines and their receptors are present in the neuroendocrine system and brain. It has been shown in researches with laboratory animals that IL-1, IL-6 and TNF-α are present to modulate metabolism of carbohydrates, fats and proteins substrates, they
Fig. 2 Induction and regulation of acute phase protein
12 also regulate the hypothalamic-pituitary outflow and act in the brain to reduce food intake [13]. They might also affect the process of bone growth when there is an acute phase response and they are released and activated. Therefore poorly nutritional young animals might have problems with growth in case of long-term infection and can then get life-long problems with the bone structures [10].
There are three groups of cytokines divided according to their action, these are: Cytokines acting as negative or positive growth factors, cytokines with pro-inflammatory properties, and cytokines with anti-inflammatory activity. The pro-inflammatory ones are responsible for making the symptoms and reactions of the body to help fight the infection. They also activate other cytokines and cells which all together starts the inflammatory reaction mainly from the liver. After the release of acute phase proteins from the liver, the acute phase reaction is suppressed by the cytokines with anti-inflammatory effect [10].
There are some more effects of the pro-inflammatory cytokines, these are induction of heat shock proteins [14] and metallothionein synthesis [15]. This has a large impact on the metabolism and toxicity of various chemical compounds and drugs. Stress proteins (heat shock proteins) are helping with covering damaged cellular molecules. Activated metallothionein synthesis is increasing the hepatic resistance against metal toxicity and enhances the metal ion binding capacity. When this together with the decreased hepatocytic secretion of albumin is happening, there is a decrease in serum zinc and iron in the body and since iron is essential for microbial growth, this will aid in the defence mechanism [10].
The sickness behaviour of decreased appetite and anorexia is caused by prostaglandins which are formed by the cause of cytokines. Immunological stress also induces release of adrenal gland medullary hormone which causes re-distribution of blood flow to brain and muscles instead of viscera and causes intestinal villus atrophy which reduces absorption. This can then cause diarrhoea and dehydration. This gives a negative energy balance. Plasma viscosity also increases due to an increase in total protein concentration. There is a significant increase in the level of fibrinogen which can be used as an indicator of inflammation. Unfortunately the increase rate is very slow so this would therefore be a late indicator of disease [10].
1.2 Acute phase proteins
There are in general two classes of acute phase proteins, these are negative and positive proteins. These are showing a decrease and an increase in levels respectively, but only in response to challenge. The group of negative acute phase proteins are albumin and transferrin. The positive
13 ones are glycoproteins, and these are released by hepatocytes from stimulation of pro-inflammatory cytokines. These includes C-reactive protein, haptoglobin, serum amyloid A, fibrinogen and alpha 1-acid glycoprotein. There have been a lot of studies showing that stress elevates the plasma levels of acute phase proteins [1,2,10,16,5], parturition of healthy animals have also shown an increase in these proteins [16].
Glucocorticoids are known to suppress pro-inflammatory cytokines and increase the anti-inflammatory cytokines that will decrease level of acute phase proteins. But this is working in a complicated pattern were the cytokines of different kinds are in a ongoing competition of expression during the inflammatory process. This complicated network is important in defence mechanisms and kills infectious microbes, repairs tissue damage and restores a healthy state [16].
Function of the different acute phase proteins are different, and the amount excreted during an acute phase response is differing a lot between them. The alpha 1-acid glycoprotein have different functions locally and systemically. Locally it maintains homeostasis by reducing the tissue damage from the inflammatory process. The systemic kind has two functions; drug-binding and immunomodulation. Fibrinogen is produced in the liver and is involved in homeostasis by providing a substrate for fibrin formation and tissue repair. It also triggers a cascade of intracellular signals that lead to enhancement of degranulation, phagocytosis and delay of apoptosis [16]. High fibrinogen levels in serum together with low iron levels can be a good indicator for inflammation, but in some cases not as good as the other indicators since fibrinogen levels rise the first 24 hours but just reach peak levels after 2-3 days [17]. Hypofibrinogen is also possible and can happen in accordance with disseminated intravascular coagulation (DIC) and is due to the higher consumption of fibrinogen during this condition. But most commonly an elevated level is seen, in case of stress, pregnancy or disease. After a tissue damage, highest levels of fibrinogen can be seen at day 3-4 [18].
C-reactive protein got its name from the capability to bind C-polysaccharide of Streptococcus pneumonia [19,20]. It plays an important role in protection of autoimmunization and regulation of the inflammatory response [16]. This by interacting with ligands, active classical complement pathway, stimulate phagocytosis and by binding to immunoglobulin receptors. This is enabled by the specific structure of five identical protomers arranged symmetrically around a central pore, "pentraxins" is used to describe these kinds of proteins. Each protomer has a binding site consisting of two calcium ions adjacent to a hydrophobic pocket. This site has a phosphocholine recognition face making binging possible. The opposite site of the pentamer is the
14 effector face, where components of the complement system binds and in that way can trigger the complement system to start [7].
CRP can bind with a variety of pathogenic bacteria or intracellular antigens of damaged cells and by that recognize foreign molecules. It can also activate complement pathway, mediate phagocytosis by interaction with specific receptors and induce production of anti-inflammatory cytokines. C-reactive proteins also inhibits chemotaxis and respiratory burst of neutrophils [16]. In studies in humans, it has been shown that constant higher levels of CRP due to stress or obesity give an increased risk of cardiovascular disease later in life, which probably also apply to animal species [21].
Haptoglobin binds with free haemoglobin in plasma and reduces oxidative damage associated with haemolysis. This complex can be found on the surface receptors of macrophages and scavenged by phagocytes. A variety of immunomodulatory effects are associated with haptoglobin. It also has an inhibitory effect on granulocyte chemotaxis, phagocytosis and bactericidal activity. Haptoglobin may inhibit mast cell proliferation to a certain level and prevent spontaneous maturation of epidermal Langerhans cells. These proteins can also suppress T-cell proliferation [16]. Normally the haptoglobin level is between 200-1000 mg/l in horses and during inflammation it increases to 400-2700 after 12-24 hours [18].
Serum amyloid-A, like C-reactive protein is thought to have a role in host defence during inflammation, but is not fully diagnosed. It is detoxifying endotoxins, inhibits lymphocyte and endothelial cell proliferation, inhibits platelet aggregation and inhibits T lymphocyte adhesion to extracellular matrix proteins. Serum amyloid-A is also involved in dispersing inflammatory cells to the site of infection. It is involved in local defence mechanism of gut to endotoxins [16]. Serum amyloid A is the major protein involved in secondary amyloidosis. The concentrations correlates well with the c-reactive protein concentration in an acute phase response, but increases more dramatically from several hundred to one thousand times in inflammatory diseases [22]. Normal value of SAA in horse is everything under 5 mg/L. By this the measure for a light inflammation is 50-250 mg/L and more severe inflammation everything over 250 mg/L [18]. It has been shown that mares in perinatal period and clinically healthy horses have no increase in serum amyloid A concentrations. But in case of clinically sick horses with inflammation or induced inflammation the concentration is increasing markedly [22].
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1.3 Application in veterinary diagnosis
Before the use of acute phase protein as an assay in estimating inflammatory processes, the albumin and globulin ratio was a standard used in veterinary medicine. In the beginning, only protein electrophoresis was used, then technology got better and specific assays of protein was possible. This could then offer a more detailed examination of acute phase proteins and how they change during a disease progression. There are several conditions that alter the level of acute phase proteins in animals, and of what these measures then can be used. There are triggering events like inflammation, infection, trauma, neoplasia and stress [1]. For checking the quantity of C-reactive protein in horses, the double antibody sandwich ELISA can be used. The CRP present in the samples will react with the anti-CRP antibodies which have been absorbed to the wells. Then anti-CRP antibodies are added, these enzyme-labelled antibodies form complexes with the already bound CRP [19,20].
During infection in horses there is a significant increase of serum amyloid A and during inflammation from for example joint disease and after surgery. Meaning this is the main protein during an inflammatory response even though there are always several involved. The evaluation of different slow and rapid acting proteins makes a more specific analysis of the inflammation state that is not possible to measure as precise with other blood analyses and complete blood count. These measures has less sensitivity than the proteins, in for example testing treatment methods [1]. After treatment there might be a significant change in the protein levels whereas the other levels remains the same, making it hard to estimate a successful treatment. It is also well used in diagnosing cause of colic in horses and gives a good help in finding the cause and also the treatment for a colic patient. Since there is a more significant increase in proteins when there is an infection colic in comparison to colic that has to be treated with surgery, this makes it possible to fast make a decision if surgery or medical treatment is required [1].
But the reactive proteins are only objective information about ongoing lesions of an animal and cannot be used for a specific diagnosis. It can also be used as a fast tracking of spread of a disease, because of its fast measurement and activation in body, it's a good tool to see spread in a herd. This to see severity of contamination of a specific agent with unknown morbidity rate. It is also important to consider the environmental factors influencing the result, for example in stressful situations [2].
The C-reactive protein got its name from the ability to bind Streptococcal C-polysaccharide, as mentioned earlier. Therefore a high number of this protein usually indicate bacterial of viral
16 infection of lungs causing pneumonia. In early stage of inflammation from infection there is a significant increase in this protein, before the rectal temperature increases, therefore it's a good indication of disease in early state [2]. But of course, CRP values cannot be diagnostic on their own, it can only be interpreted on the side of clinical and pathological results. But it can be very helpful in early diagnostics [23]. In horses specifically, it has been shown that there is a huge increase of this protein during carbohydrate induced laminitis, but also in horses with pneumonia, enteritis, arthritis and after castration [2].
Serum amyloid A is usually found in a complex with lipoproteins. There are two isoforms of this protein fund in horses. The levels of serum amyloid A in horses increases rapidly during tissue injury, infection or inflammation. In foals there is high levels of this protein until one or two weeks of age. In mares there is peak levels of serum amyloid A three days before foaling and persist until approximately one month post partum. Increased levels in horses has been seen in surgical trauma, induced inflammation and in infections. In foals with bacterial infections there is an increase as well and in case of septicaemia and focal infections. Serum amyloid A correlates well with C-reactive protein in horses with clinical signs of inflammation, fever and clinical signs of respiratory disease [2].
1.4 Acute phase reaction in horses
The concentration of CRP for healthy horses are in the interval from 5.0-9.6 mg/L [24]. Already 3-4 days after introduction of inflammation the level is 5-6 times higher the normal [18]. When performing a surgery in horses it is important to find markers that will show the intensity of trauma from surgery, meaning not only that there is a trauma caused by the surgery, but also how big the trauma is. The response to surgical stress is a cooperation between hormonal, metabolic and inflammatory reactions directed to make the body adapt to the newly injured tissues and to start the healing process. By identifying the causes to a bigger trauma to the body from surgery, it is also possible to improve the surgical work and minimize trauma and thereby shorten the time of convalescence. When comparing the results of white blood cells and acute phase proteins in the sense of measuring the intensity of trauma, the proteins are actually increasing with increasing intensity, while white blood cells are not, they only show that there is a trauma present. General anaesthesia also has no effect on the acute phase proteins making them a good marker for surgery performance [5]. But it is important to remember that an acute phase response has influence on the hepatic drug-metabolizing system and thereby alter the drug disposition in horses. This information is showing that not only disease-triggered acute phase response will alter the drug
17 disposition in horses, but also stress-triggered, making medical treatment a bit more complicated in stressed horses than in calm ones [25].
During gastrointestinal diseases of horses, giving colic, the concentrations of serum amyloid A, haptoglobin and fibrinogen are increased in the horses blood. This can be a helpful diagnostic tool in many cases. Increased haptoglobin and fibrinogen concentrations in peritoneal fluid can also be seen during colic. Changes in protein concentration in peritoneal fluid is more pronounced and occurs earlier than in serum. This can give faster diagnosis and thereby also more chance of saving more horses. It has been shown that these proteins are changing in concentration according to the duration of colic, meaning that according to the concentration it is possible to count how long the horse have been going with colic because the concentrations will increase with the duration. It is also possible to measure the severity of disease according to concentration of these acute phase proteins. This due to amount of tissue involved and the severity of disease of tissue. For example obstruction, strangulation and inflammatory disease. This will give possibility to diagnose approximately how much tissue is involved and thereby what kind of disease there is. In horses with peritonitis there will be more tissue involved than in a simple obstruction [3].
In case of clinically healthy horses performing acute exercise there will be induced stressed to the horse which might be thought to cause acute phase response and thereby increase in the acute phase proteins. But from researches it has been shown that the main acute phase proteins remain unchanged. There will however be a decrease in iron concentrations and increase in hemoglobin levels [4]. The change in iron levels during exercise to either increased or decreased levels is due to antioxidant defence mechanism. Sequestration of iron minimise formation of OH−
ions, which is a major antioxidant defence mechanism. But high intensity training may cause intravascular haemolysis and increase levels of loosely bound iron in tissues. Reactive oxygen species secreted during exercise-induced tissue damage and muscle fatigue can cause release of iron from normally unavailable storage sites. This will show free iron in sweat during exercise. Shorter exercises has less effect on iron levels while longer exercises will changed it more significantly [26].
When talking about serum Amyloid A concentration during exercise, it can be said as before that race training of experienced horses show no change in serum concentration. But for horses beginning their endurance training there is an increase in serum amyloid A levels in comparison to other levels. Therefore serum amyloid A may be useless as an indicator for race training but
18 helpful in endurance training. As the horses get more use to the training and more experienced there should be less increased levels of serum amyloid A during training [27].
Arthritis and joint problems in general is a very common problem in horses. Even when there is no infection present, such as in non-infectious arthritis, there will be an acute phase response triggered. This will change biochemical, haematological as well as clinical parameters. Inflammatory response in synovial joint will activate synovial cells and chondrocytes to produce and secrete cytokines that will give a local inflammatory response in joint that further will produce an acute phase response. The proteins involved in this response reaction are C-reactive protein, haptoglobin, serum amyloid A and fibrinogen. Serum amyloid A is the main one and will increase thousand-fold after tissue damage and the other ones 2-10 times. The proteins will stay elevated for days, but the values will go back to normal level after some time even though arthritis is still present. This because the inflammatory activity will reduce after time but may become more and less in waves during chronic cases and thereby giving a new response with elevated concentrations [6].
Iron is a negative acute phase reactant, meaning that it will decrease when inflammation is present because of being sequestered by mononuclear phagocytes to limit iron available for microbial growth. Therefore it is evaluated different from the acute phase proteins; iron levels in serum will decrease with disease and increase as treatment is performed. In people, iron have been shown to be a very sensitive indicator for inflammation [5,17]. When talking about performing surgery in horses as a treatment method, iron levels are depending on the intensity of the surgery rather than the primary state of the horse in the diseased state before the surgery. Iron levels also depends on age, the higher age the higher iron levels. Therefore the iron levels are turning back to normal levels fast when there is no postoperative inflammation and there have been an uncomplicated surgery performed. Iron is from this information a good indicator to establish the intensity of the inflammatory state as it is not influenced by the baseline levels of the specific horse, but rather on the intensity state of inflammation in that specific moment. Serum amyloid A can also be used for this purpose, while fibrinogen and white blood cells are influenced by the state of the horse preoperatively and not the intensity of inflammation and therefore the levels postoperatively cannot be used as a safe marker of severity of disease [5].
1.5 The behaviour and symptoms of stressed horses
The behaviour of horses during stressed conditions is a very objective indicator and relies almost only on the observer and what skills that person possess. This is also according to the
19 welfare of animals, how their normal behaviour should be and how they behave when feeling stressed. For making an easier estimation of the stress level of a horse, a behavioural score can be used divided as follow; no stress (factor 1), low stress (factor 3) and medium stress (factor 2). This would be given in accordance to the type of behaviour and physiological measures. Indicators that can be used to simplify the evaluation of the stress level could be the heart rate, respiratory rate, abnormal behaviours examined and physiological data such as cortisol levels in blood. The different features can then be compared with each other in the different cases to establish the right behavioural score [28].
During stressed conditions, the hypothalamo-pituitary-adrenocortical axis is activated due to stimuli which will elevate the plasma cortisol level. The heart rate and heart rate variability are representing the sympatho-adrenomedullar axis during stress. For these parameters to get activated they need stimuli, which would be the stress symptoms from a stress event which could be anything abnormal happening as horses are flight animals and therefore want to flight from this abnormal event [29].
The behavioural signs of stress varies depending on the event causing the stress and the experience to the event happening. For example during hyperthermic conditions and during fever as a result of a disease, the horse will most often demonstrate widening of nostrils, increased respiratory rate and heart rate, head movement, increased breathing depth and apathy. For other events or disease states there might be other signs displayed [30].
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2. METHODOLOGY
2.1 Research workload, location and method
This research work was carried out in the Biochemistry department of LUHS in Lithuania, Kaunas. The test objects was 16 selected horses from different places of Lithuania. Blood samples were taken during the research time from horses in the large animal clinic of LUHS and from surrounding stables in Kaunas in the period of 16 months.
Sampling started in December 2014 and continued randomly during the seasons until April 2016 as horses were available for collection. When some horses were staying in the clinic for a longer treatment procedure, several samples were collected to see changes during treatment time, either medically or surgically to make a dynamic analysis of these horses.
2.2 Research objectives
Twenty-four collected samples were selected for investigations: four samples from healthy horses (control group), and 20 samples from 12 sick or stressed horses were three of these were followed and had several samples taken during treatment either medically or surgically (Table 1). Horses with different genders, ages, breeds, sizes, colours and diseases were involved in studies. Sick horses were horses with a diagnosed clinical disease such as colic, arthritis, Osteochondrosis dissecans (OCD) and internal and superficial wounds. Stressed horses were the ones on treatment schedule for a disease, but stressed from staying in the clinic and changing environment. A horse was counted to be in a stressed state by the behaviour in the stable, by the reaction to the staff when performing procedures and treatment and from the general condition influenced by the disease. Different traits, feeding and environment were not included as influencing factor in the survey, but only diseased compared with clinically healthy animals. Body conditions were varying among the tested horses depending on disease, feeding and environment. This was also not involved in survey.
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Table 1. Horses investigated in this research.
Horse no. Name Sickness Dates of sampling Determined parameters
1 Lordas Lameness 2016.01.11, 2016.01.13, 2016.01.18, 2016.01.20, 2016.02.04 Fe, CRP, Hapto 2 Connect Infection 2016.01.13, 2016.01.18, 2016.02.03
Fe, CRP, Hapto, SAA
3 Ertina Infection
2016.04.11, 2016.04.15, 2016.04.18
Fe, CRP, Hapto, SAA
4 Chigoni Clinically Healthy 2016.03.06 Fe, CRP, Hapto
5 Ivevan Gastric Problems 2015.01.07 Fe, CRP, Hapto
6 Viva Gastric Problems 2016.02.03 Fe, CRP, Hapto
7 Kvebas Eye Problems 2014.12.02 Fe, CRP, Hapto
8 Amūras Eye Problem 2015.09.23 Fe, CRP, Hapto
9 Demokratia Clinically Healthy 2016.03.06 Fe, CRP, Hapto
10 Faustas Lameness 2014.12.02 Fe, CRP, Hapto
11 Komaro Infection 2016.02.03 Fe, CRP, Hapto
12 Pella Clinically Healthy 2016.02.05 Fe, CRP, Hapto
13 Favoritas Gastric Problems 2014.12.16 Fe, CRP, Hapto
14 Test Gastric Problems 2016.01.11 Fe, CRP, Hapto
15 Sadokan Gastric Problems 2015.09.23 Fe, CRP, Hapto
16 Revo Clinically Healthy 2016.02.05 Fe, CRP, Hapto
2.3 Collection of samples
Blood samples were obtained by jugular vein, with aseptic technique (using alcohol solution for cleaning). The place of sampling at jugular vein (Vena Jugularis) were cleaned, plastic gloves were used and 7 ml of blood were collected into serum (red top) tubes.
Blood was collected in tubes (Venosafe blood collection system, TERUMO® Europe, Belgium) containing no additive for preparation of serum. Serum was prepared by letting blood samples coagulate for 2-3 hours before centrifugation at 3500xg for 10 minutes. Serum analyses
22 were stored at -20oC until analysis. CRP, serum amyloid A, haptoglobin and iron of each sample were analysed in Medicinių tyrimų laboratoija, UAB „Baltic Medics“.
2.4 Determination of acute phase proteins
Determination of acute phase proteins (C-reactive protein, haptoglobin and serum amyloid A) and iron was all carried out in JSC “XX”, Kaunas. Iron and CRP were determined by biochemical analyzer Olumys AU 640. The analyzer is normally used for determination of CRP of human, but as it has been shown, the structure and amino acid composition of human and horses are very similar [24], so CRP kits used for humans determination could be used for horse too [31].
Haptoglobin was determined by immunoturbidimetric procedure using ABBOTT LABORATORIES kit for determination of haptoglobin. Serum amyloid A was determined by Siemens Latex A kit.
2.5 Statistical data
The programme Microsoft office excel 2007 was used to calculate the statistical result of the survey. So the results from the blood samples evaluation was used in the statistical analysis. The function used for establishing the P-value of all statistical analyses was the ANOVA single factor. Results are considered reliable when P<0.05 and unreliable when P>0.05. However, P>0.05 analyses was still used in presentation of results, but critically discussed in the result discussion part. Other values presented by the ANOVA system was count, sum, average and variance. These are not presented, but used to a certain extent for evaluation of results during discussion.
23
3. RESULTS
3.1 The horses presented in this research
The horses investigated in this research work were all from Lithuania and most of the presented ones were stabled at the large animal clinic at LUHS for treatment program at the time of sampling. The main information about horses, their diseases, collection time and the parameters analyzed in each horse (iron and acute phase proteins; C-reactive protein (CRP), haptoglobin (Hapto) and Serum amyloid A (SAA)) are presented in Table 1. The diseases were split in the major groups Gastric Problems, Eye Problems, Lameness, Infection and Clinically healthy (for example "Lameness" means all horses with diseases causing lameness and "Eye Problems" involves all kinds of eye diseases or traumas).
The horses in the investigation were all in some kind of diseased state (except the control group) and they display a stressed condition due to their state of health, change in environment and the different kinds of treatments performed to them. The control group of horses were all in a clinically healthy condition, but could still experience a stress stimuli from the collection of blood samples due discomfort or suspiciousness. However, the clinically healthy horses in this study were all expressing calmness during the collection procedure. The horses in the clinic were all showing signs of stress during the collection of blood samples as they were experiencing discomfort previously every time an unknown person were entering their stable places. This was shown by signs such as sounds, dilated nostrils, moving away, kicking and head movements.
3.2 Dynamics in serum of SAA, haptoglobin, CRP and iron in horses
The three first horses mention in Table 1 had several samples taken for making a follow-up study of them to see how the concentration of the acute phase proteins and iron changed with the disease and treatment (these values are presented in Fig. 3, 4 and 5). Samples from each of them were taken at different times which was due to their visit in the large animal clinic and due to the kind of sickness and what kind of treatment was used.The first horse, Lordas, was followed during a 25 day period which started from the moment he arrived to the clinic in a sick state (day 1) until after a surgery was made (day 25) (Fig. 3). Lordas was diagnosed with Osteochondritis dissecans (OCD) and needed arthroscopic surgery which was made after the fourth sampling, more exactly at day 16. Lordas was in severe pain before surgery and had a total recover after the operation was made.
24 Fig. 3 represents the changes of iron and acute phase proteins concentration in blood of Lordas during disease and treatment time. The iron level in serum is first increasing (from 8.9 to 22.3 µmol/L) and then decreasing till 20.2 µmol/L in the end, but the changes are not very prominent. The C-reactive protein concentration is almost the same for all the period (from 3 mg/L at the 1st day to 2 mg/L at the 25th day with slight increase at the 2nd day). The haptoglobin concentration is decreasing with time of analysing (from 340 mg/L at day 1 to 310 mg/L at day 10). When comparing the levels of haptoglobin and iron concentrations in this case, it is possible to see that they are changing in an opposite manner, iron is getting higher as haptoglobin is getting lower. It is important to take into account that a medical treatment against pain and inflammation was given as the horse arrived to the clinic even though the surgery was just performed at day 16. This means that medication treatment was actually started after the 1st sample.
Fig. 3 Change in iron and acute phase proteins concentrations over time for Lordas,
(P<0.05).
Sampling of Connect, which had gone through a trauma and had to be sutured at the clinic, started the day after Connect was sutured together (Fig. 4). But because of infection he had to stay in large animal clinic for a longer time on medication of non-steroidal anti-inflammatory drugs (NSAID's) and antibiotics. So sampling was made during this period of treatment. The last sample
0 10 20 30 1 2 7 10 25 µ m ol /L Days
Iron
0 2 4 6 1 2 7 10 25 m g/ L DaysCRP
250 300 350 400 1 2 7 10 25 m g/ L DaysHapto
25 (day 23) was taken when Connect was not entirely clinically healthy but with signs of improved health status and got fully recovered a short time after that.
Connects changes of iron and acute phase proteins concentrations in blood over a 23-day period is presented in Fig.4. The first sample taken at day 1 was when Connect was already in the clinic for a traumatic wound and was sutured together and medication had just started against infection. As shown in the first chart, the iron levels are in the beginning higher and are decreasing with time (from 36.6 to 13.9 µmol/L) . The C-reactive protein concentration is decreasing during the analysed time, but not significantly (from 2 mg/L to 1 mg/L). The haptoglobin concentration in the blood is first increasing (to 1070 mg/L) and then decreases (to 670 mg/L). It is also a fact in this case that the haptoglobin concentration is changing in an opposite manner to iron concentration. As the haptglobin level is increasing, the iron level is decreasing. The serum amyloid A concentration is decreasing with time in a steady pace (from 11.3 to <0.8mg/L). It is important to mention that the horse was not diagnosed clinically healthy on the last collection time, but a health improvement was seen.
Fig. 4 Change in iron and acute phase proteins concentrations over time for Connect,
(P<0,05).
The horse Ertina had a traumatic wound that had to be sutured. The first sample (day 1) was taken after Ertina was sutured and one day after treatment of non-steroidal anti-inflammatory
0 20 40 1 5 23 µ m ol /L Days
Iron
0 1 2 3 1 5 23 m g/ L DaysCRP
0 500 1000 1500 1 5 23 m g/ L DaysHapto
0 5 10 15 1 5 23 m g/ L DaysSAA
26 drugs (NSAID's) and antibiotics had started (Fig. 5). Ertina was stabled in the clinic during the treatment period and therefore a similar case to Connect. Last sample (day 7) was taken when Ertina was not fully recovered but in a significant improved health state, she fully recovered a short time after that.
So as mentioned, 1st sample of Ertina was after suture and in the beginning of medical treatment against pain and inflammation. She had to be stabled for a period of time to be treated medically against infection and inflammation as the wound covered a very big area. The iron concentration in blood is first rising and then declining at the third sampling (from 6.9 to 26.6 to 17 µmol/L). The C-reactive protein concentration stays the same during the sampling (2 mg/L). The haptoglobin concentration is increasing with time, without fluctuations (from 320 to 680 mg/L). The serum amyloid A is also increasing with time (from 3.8 mg/L), but is a bit higher on second (7.4 mg/L) sampling than third (7.0 mg/L). But still the levels in the first sample is significantly lower than last sampling. It is also in this case important to take into account that the last sampling was not made on a clinically healthy horse, but when the health status of the horse was increased. The horse got diagnosed healthy a short time after last sampling. This case is very similar to the previous in the aspect of disease condition and treatment method.
Fig. 5 Change in iron and acute phase proteins concentrations over time for Ertina
(P<0.05). 0 10 20 30 1 4 7 µ m ol /L Days
Iron
0 1 2 3 1 4 7 m g/ L DaysCRP
0 200 400 600 800 1 4 7 m g/ L DaysHapto
0 2 4 6 8 1 4 7 m g/ L DaysSAA
27 131514 6 5 7 8 10 1 211 3 4 9 1216 0 5 10 15 20 25 30 35 40 45
Gastric Problems Eye Problems Lameness Infection Clinically healthy
µ
m
ol
/L
Each horse number
Iron
3.3 Comparison of parameter concentrations in different diseases and
control group
The clinically sick horses used for this survey were randomly picked from the large animal clinic as they arrived and were hospitalized there. Therefore the disease state and severity was different among all horses and it was just established that the horses was sick (or still sick) when collection was made. Also the treatment plan was different in all cases and in most of the samples from sick horses the treatment had already started at time of collection. The clinically healthy horses were collected also randomly from horses whose state was without abnormality and no signs of disease present in the nearest time.
The Fig. 6 shows the iron concentration in serum of horses in groups according to kind of disease and compares them with the concentration in a group of healthy horses functioning as a control group. The numbers below each column correspond with the horses listed in table 1. The concentration of iron is in general quite high in all groups (from 6.9 to 42.6 µmol/L). Almost all of these values meet the normal level of iron in blood (8.955-365.05µmol/L) [32].
Fig. 6 Comparison of iron concentration between horses with different diseases and
clinically healthy horses, (P>0,05).
Fig. 7 represents the results of the CRP concentration in blood in the different groups of sick and clinically healthy horses for comparison. In this chart it is possible to see that in general diseased animals have a higher CRP than the healthy control samples. Highest values are seen in
28 the horses with gastric problems (from 5 to 1 mg/L) and eye problems (From 5 to 2 mg/L). The concentration of CRP for healthy horses are in the interval from 5.0-9.6 mg/L [24].
Fig.7 Comparison of C-reactive protein concentration between horses with different
diseases and clinically healthy horses, (P>0,05).
Fig. 8 shows the comparison of haptoglobin in blood of the selected horses. The significant difference of haptoglobin between healthy and sick animals can be observed. The concentrations in the horses of the healthy control group are from 160 to 380 mg/L, but in the general picture the values are higher in the sick horses. The diseases with the highest concentrations screened in this survey are those with either infection (from 320 to 1000 mg/L) or gastric problems (from 120 to 700 mg/L). The absolute highest numbers (320 to1000 mg/L) are seen in the infection group of horses. The obtained concentrations of haptoglobin contribute the values (290-2260 mg/L) of others researches [33]. 1315146 5 7 8 10 1 2113 4 91216 0 1 2 3 4 5 6
Gastric Problems Eye Problems Lameness Infection Clinically healthy
m
g/
L
Each horse number
CRP
29
Fig. 8 Comparison of haptoglobin concentration between horses with different diseases and
clinically healthy horses (P>0,05).
1315146 5 7 8 101 2113 4 91216 0 200 400 600 800 1000 1200
Gastric Problems Eye Problems Lameness Infection Clinically healthy
m
g/
L
Each horse number
Haptoglobin
30
4. DISCUSSION OF RESULTS
It has earlier been established that the levels for the acute phase proteins are increasing due to disease and are decreasing significantly after a treatment program such as surgery. There is a strong relationship between the parameters and how they act together, as if a disease is present and shows an increase in the acute phase proteins concentration in serum, the concentration of iron will decrease. This because iron is a negative indicator and will just increase when animal is healthy. Iron and serum amyloid A are also more sensitive indicators in horse serum than C-reactive protein and these are not dependent on previous values but change according the current state and can therefore give more specific information about severity of disease [5,17].
The follow up samples for dynamics of the acute phase proteins and iron were taken according availability and course of disease and treatment. The first horse, Lordas, had 5 samples taken, were only the last sample was after surgery was performed. But medical treatment of anti-inflammatory drugs and pain medication was given during the whole disease course from the very beginning. The values of iron is getting bigger with time (from 8.9 to 20.2 µmol/L), meaning that the treatment is working as iron levels should be higher in healthy horses. This is due to the fact that during an inflammatory event, the iron levels are getting decreased to prevent microbial growth, this is accomplished by an activation of mononuclear phagocytes by haptoglobin to sequester iron within the cells and thereby decrease the concentration of iron in blood [5]. Another reason for the shown improvement from the iron level could be the less stress levels from getting used to the new environment and not getting transported which can cause elevated the stress level. The levels are not differing very much in this case during the whole sampling time.
When checking the CRP levels it is possible to see that the highest levels are the day two (4 mg/L), but otherwise the levels are very low within normal values (5.0-9.6 mg/L) [24], showing that in this case the CRP value is not a good indicator of disease as this horse had a very severe disease requiring surgery and still had low levels. CRP should get activated during an inflammatory reaction to protect against infection, clear away damaged tissue and to prevent autoimmunization. The CRP molecule binds with pathogens and can activate pathogenesis to stop infection [16]. Since there was no diagnosis of an infection in this case but rather an inflammatory reaction due to the disease (OCD) this an additional reason the low levels of CRP. There might also be some influence from the medication given from the beginning of sampling period which should be taken into account.
31 The haptoglobin values are showing a better curve of the disease course which is more according to prediction. Even though the values are within normal range for horse (290-2260 mg/L) [33], the curve is showing a decrease with time and the lowest value can be seen at day 10 (310 mg/L) and at the sample taken after surgery, the haptoglobin level was not readable. This shows a perfect response to treatment. As it has been shown that haptoglobin elevates in response to inflammations in horses and especially in non-infectious arthritis [2], which is a condition close to this case, this also shows that a healing process and response to treatment is present as the levels are decreasing with time. In case of a worsen condition, the haptoglobin levels would continue to be high or become higher.
With this statistical analysis it is possible to say that disease state has an influence on the levels of these acute phase proteins and iron, even though a longer follow up period and with more samples taken the curve could be even more clear and informative. It is still hard to make a conclusion of how the disease state and treatment is influencing the parameters with just this case. It can also be stated that since all these values are counted in the "normal" category for horses, but the horse is still clinically sick, it might be a good recommendation to take several samples during treatment to see an improvement curve rather than just taking one sample that shows levels of healthy horses even though disease is indicated.
The next follow up case for checking dynamics was Connect. He came to the clinic at day 1, got sutured that day and medical treatment was started with anti-inflammatory drugs with pain relieve properties and antibiotics. When over-viewing all charts together, the general appearance is that the values are decreasing with time. But as iron is a negative inflammatory indicator, the value should increase as the other values are decreasing. According to the iron value in this cart, the disease state is getting worse by time during the measured time period as the level is getting lower (from 36.6 to 13.9 µmol/L). This could be explained by a blood loss from the traumatic wound which can decrease the iron levels. When looking at the C-reactive protein value, the parameter is staying within a normal range, but is decreasing with time (from 2 to 1 mg/L), which indicates that the treatment is working and the horse state is getting better.
The haptoglobin value is first increasing and then decreasing, indicating that the treatment is working, and since the horse was sutured at day 1 and medical treatment was started then, it is just in the last sampling in day 23 where an improvement is seen. The Serum amyloid A levels are, in the same way as CRP, decreasing with time (from 11.3 to 0.8 mg/L) indicating an improvement of the horse state and therefore a positive reaction to the treatment. With these
32 results in mind, it is possible to say that according the acute phase proteins, there is an improvement of health sate, while iron is indicating a worsen health state. Therefore a longer follow up period is probably required to get a more accurate and reliable result showing if these parameters are trustworthy for diagnosing severity of disease.
The last dynamic case checked was Ertina, which similar to Connect had a wound that was sutured together and she was given anti-inflammatory medication and antibiotics from day 1. The iron value is increasing and then decreasing again, but with a general overview the value is higher in the end than in the very beginning (from 6.9 to 17 µmol/L). This indicates an improvement of health state, which differs from Connect with the same kind of disease and treatment. The C-reactive protein value is staying the same during the whole sampling time (2 mg/L), meaning a false indication of no change in the inflammatory state. The haptoglobin level is increasing with time (from 320 to 680 mg/L) which differs from Connect where it was decreasing. This suggests that the healing did not yet start at the period time of sampling, therefore it is not showing a decrease in concentrations because the inflammatory reaction was still present. But it could also be due to the stress from transportation to a new environment and from handling and treatment of veterinarian. Serum amyloid A is also following these patterns and is getting higher (from 3.8 to 7 mg/L) is also indicating that the inflammatory reaction is still high in the body and the healing haven't started yet. But is still within normal values (light inflammation at 50-250mg/L [18]).
From a general overview it would also in this case be better with a longer follow-up period to see a better dynamic curve which is more predictable to the previous information that we have got from earlier sources saying that iron should increase and the acute phase proteins should decrease with time as treatment is lowering the severity of inflammation in the body. When just looking at the values in these charts they are still in the normal ranges and therefore also this suggests that the inflammatory state is not strong enough to give a higher and more prominent increase of the values. A more severe disease like for example colic might get a higher elevation than just an external wound as in these cases. These statements can be strengthen by previous studies done in different ways. One example of investigation was done by following different horses from the day of surgery and several days after surgery rather than before the surgical procedure, to see how the acute phase proteins are changing in concentrations during time of healing. This kind of research shows better dynamics in the acute phase proteins making it possible to establish that SAA and iron is better indicators in horses to establish the intensity of trauma than WBC's and fibrinogen which depends more on the previous state of the horse [5]. Or in another case were horses with colic was tested over time of acute disease. This showed that
33 SAA and haptoglobin were both increasing to over 1000 mg/L and over 4000 mg/L respectively when reaching over 24 hours of acute disease duration [3]. As mentioned earlier, a conclusion from this could be to make it a standard to always take several samples during the disease treatment so see an improvement rather than just taking one sample that might show a false normal value within ranges even though the horse display symptoms of disease. It is also possible and most probably a fact that all horses have an individual standard value of concentration of each parameter in a healthy state and therefore to know if it is increased, a dynamic curve should be done at beginning of disease and during the treatment to see if there is improvement.
When looking at the overview of the iron values of all horses involved in this research, it is possible to see that in general the clinically healthy horses have a higher value of iron than the sick horses (average of healthy: 28.7 µmol/L and average of sick: 21.6 µmol/L). This is emphasizing the previous researches that implies that iron is a negative indicator and therefore the value is getting lower in case of presence of disease [5,17]. In this statistical analysis, as in the rest of the analysis done on all horses, the P-value is more than 0.05. This probably because the numbers of horses tested in each group differed a lot, the number of healthy horses was 4 while number of sick horses was 12. But this amount of horses was enough to show that the iron values are lower in sick horses than in clinically healthy ones. But of course a bigger number of samples from different horses would make a safer statement.
The next value compared between sick and clinically healthy horses was C-reactive protein. By viewing just the charts it is hard to say the difference between sick and healthy horses as they are very much similar, varying in the same manner. When calculating the average of these groups the number is the same; 2.5 mg/L. This means that from the samples taken in this research, the CRP value is not differing between sick and healthy horses. Maybe if a bigger sample amount would be obtained from more severe diseased horses, the numbers would differ more. But as also stated in earlier in analyses, the CRP is not as good of an indicator as iron, haptoglobin and serum amyloid A in horses [5]. Therefore from this analysis, it can be said that C-reactive protein is not a good indicator in horses. The P-value is here also higher than 0.05, probably for the same reason as mentioned earlier.
The last value compared between all horses tested in this research was haptoglobin. In the charts it is a more prominent increase in the infection group and the gastric problems group. In general the values of the sick horses is higher than the clinically healthy ones (average of healthy horses: 260 mg/L and average of sick horses: 450 mg/L). This shows that haptoglobin is a good
34 indicator of inflammation state in horses and may be used as a diagnosing tool. But since the values are still in the normal range, a sample from one of these sick horses would still indicate that inflammation is not present or at least not severe. The reason might be that the inflammatory state have to be more severe or longer gone to see a more prominent increase in this value. The P-value as is higher than 0.05, probably for the same reason as before mentioned.
When putting this information together it is possible to say that according to the values from these acute phase proteins and iron, the values are too low to prove that inflammatory disease is present by just looking at the samples separately. But when comparing them all it shows that iron is lower in case of disease, CRP is not changing and haptoglobin and SAA are changing in the same way; higher in case of inflammation. According to dynamics the values are changing as the inflammatory state is changing, but the samples used in this analysis was too few to fully interpret the result and to conclude a general course of how they are changing with treatment and healing process.
It can also be said as earlier mentioned, that when symptoms of a disease is displayed in a horse, it could be a recommended standard to always take several samples during the treatment to see if the levels improves with time rather than relying on one sample taken that shows a values within normal values even though the horse is sick. Since the ranges of normal values are very wide of most acute phase proteins and iron, it might still be a very diffuse subject, and not really stated what are the real normal values. In addition to this, it is probably very individual what the normal value is for each horse at healthy condition. Therefore a quite high level of any acute phase protein during disease may be a very high increase in some horses whereas a small increase in others, as well as a small decrease in iron for one individual horse can be a large decrease for another horse.
35
CONCLUSIONS
1. The values of acute phase proteins and iron in this research were within the following ranges both of clinically healthy and sick horses: 1.0-5.0 mg/L for C-reactive protein, 3.8-7.4 mg/L for serum amyloid A, 160-1000 mg/L for haptoglobin and 6,9-42.6 µmol/L for iron.
2. The dynamics of the acute phase proteins in a treatment course over time shows that haptoglobin is generally deceasing as a response to treatment as iron is increasing. The C-reactive protein level is not changing significantly in any case and serum amyloid A is responding in a similar manner as the haptoglobin, declining with treatment.
3. Iron is shown to have an average higher in healthy horses than horses diagnosed with a disease, this because it is a so-called negative indicator. C-reactive protein average level is not differing much between healthy and sick horses. The haptoglobin average level is higher in sick horses than clinically healthy horses, especially high in infection diseases.
4. The best parameters for indication of health state in horses are iron, serum amyloid A and haptoglobin. The best usage of these diagnostic measurements is by checking dynamic over treatment time to see how the health state is changing. A single collection and testing time during course of disease might make a false result not reflecting the presence of disease and the severity of it.
36
ACKNOWLEGEMENT
I would hereby like to express my deepest appreciation to the people that have given me the support, help and possibility to complete this master thesis. I would like to devote a special gratitude to my supervisor, Assoc. Prof. Vaida Andrulevičiūtė, for contributing with her encouragement, suggestions and valuable aid during the whole master thesis research period.
Furthermore, I would like to acknowledge with appreciation a special person that made the collection of samples for the statistical analysis possible by instructing and by providing patients for testing, thank you Indrė Stasiulevičiūtė.
Finally, I would like to devote my last acknowledgement of appreciation to Veterinary Academy of Lithuanian University of Health Sciences for supplying with all necessary instruments and collection equipment, without which it wouldn't be possible to accomplish this master thesis. Nevertheless, a tremendous appreciation also to last mentioned for providing all necessary education, information and supplement to give me the potential to finish this master thesis.
