LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Faculty of Veterinary Medicine
Isabell Josefsson
IMUNOSTIMULIACIJOS ĮTAKA KAI KURIOMS ŠVIEŽIAPIENIŲ KARVIŲ SUSIRGIMAMS, REPRODUKCIJAI IR PRODUKCIJAI
THE EFFECT OF AN IMMUNE STIMULANT ON SOME
PERIPARTURIENT DISEASES AND PERFORMANCE IN DAIRY COWS
MASTER THESIS
of Integrated Studies of Veterinary Medicine
Supervisor: Lector PhD. Danielius Starevičius
KAUNAS 2019
2 THE WORK WAS DONE IN THE DEPARTMENT OF LARGE ANIMAL CLINIC
CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Thesis “The effect of an immune stimulant on some periparturient diseases and performance in dairy cows”
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.
2018-12-13 Isabell Josefsson
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CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK
I confirm the correctness of the English language in the done work.
2018-12-13 Isabell Josefsson
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CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESIS
2018-12-13 Lector PhD. Danielius Starevičius
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THE MASTER THESES HAVE BEEN APPROVED IN THE LARGE ANIMAL CLINIC
2018-12-19 PhD Arūnas Rutkauskas
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Reviewers of the Master Theses 1)
2)
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Evaluation of defence commission of the Master Thesis:
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3
TABLE OF CONTENT
SUMMARY ... 5
SANTRAUKA ... 6
ABBREVIATIONS ... 7
INTRODUCTION ... 8
1. LITTERATURE REVIEW ... 10
1.1 Immune dysfunction in the periparturient cow ... 10
1.1.1 Nutritional influences on the immune system ... 11
1.1.2 Endocrine influences on the immune system ... 12
1.2 Associated diseases ... 13
1.2.1 Mastitis ... 13
1.2.2 Retained placenta ... 14
1.2.3 Metritis ... 15
1.3 Economic impact ... 16
1.4 Immunomodulation ... 17
1.4.1 Pegbovigrastim ... 18
1.4.2 Imrestor ... 18
2. METHODOLGY ... 19
2.1 Research workload, location and method ... 19
2.2 Study design ... 19
2.3 Grouping ... 21
2.4 Collection of samples ... 21
2.5 Determination of white blood cells count ... 22
2.6 Postpartum diseases ... 22
2.7 Collection of data ... 22
2.8 Statistical analysis ... 22
4
3. RESULTS ... 23
3.1 Effect of pegbovigrastim on hemogram parameters ... 23
3.2 Effect of pegbovigrastim on some periparturient diseases ... 26
3.3 Effect of pegbovigrastim on milk production ... 29
3.4 Effect of pegbovigrastim on reproduction ... 30
3.5 Culling rate ... 32
4. DISCUSSION ... 34
CONCLUSIONS ... 37
AKNOWLEDGEMENT ... 38
5
SUMMARY
THE EFFECT OF AN IMMUNE STIMULANT ON SOME PERIPARTURIENT DISEASES AND PERFORMANCE IN DAIRY COWS
Isabell Josefsson Master Thesis
The aim of this study was to evaluate the impact of pegbovigrastim administration in dairy cows on the white blood cells, postpartum diseases, production and reproduction performance. This master thesis was prepared in the department of the Large Animal Clinic of Lithuanian University of Health Sciences (LUHS) in Kaunas, Lithuania.
Two large scale dairy farms with a loose holding system from the southern part of Lithuania were included in the study. A number of totally 156 cows were included in the study, the included cows were examined before and after parturition in different ways. The 156 cows were divided into three different groups, treated in different ways pegbovigrastim was administered one alternatively two times and for comparison purposes a non-treated control group was included for examination.
Blood samples were collected one week prepartum, the day following parturition and one week postpartum. The blood parameters were evaluated by focusing on the distribution and changes in concentration of WBC´s divided into GRAN, LYMPH and MONO. The postpartum related diseases were followed during the first 30 days in milk (DIM) measured by the incidence rate of mastitis, metritis and retained placenta. The production was studied in terms of milk yield during the first 100 DIM, conception rate and days open. The culling rate in the different groups was also studied.
The study revealed that the cows receiving pegbovigrastim developed a lower incidence rate of mastitis and retained placenta contrary a higher incidence rate of metritis compared to non-treated cows. Cows treated with pegbovigrastim produced more milk during the first 100 DIM compared to non-treated cows. The study also revealed that the reproduction improved with decreased days open and increased conception rates. The culling rate was lower in treated cows compared to non-treated cows. Notable is that from treated cows the one that got culled did so in an earlier phase compared to the ones in the non-treated group. Comparing the groups that received one respectively two injections of pegbovigrastim it was seen for the majority of parameters investigated that it was more beneficial to receive two injections.
Key words: Pegbovigrastim, immunosuppression, postpartum diseases, milk production,
conception rate
6
SANTRAUKA
IMUNOSTIMULIACIJOS ĮTAKA KAI KURIOMS ŠVIEŽIAPIENIŲ KARVIŲ SUSIRGIMAMS, REPRODUKCIJAI IR PRODUKCIJAI
Isabell Josefsson Magistro baigiamasis darbas
Šio tyrimo tikslas - įvertinti pegbovigrastimo poveikį melžiamų karvių baltųjų kraujo kūnelių kiekiui, šviežiapienių karvių ligoms, pieno kiekiui ir reprodukcijai. Šis magistro darbas parengtas Lietuvos sveikatos mokslų universiteto (LSMU) Stambių gyvūnų klinikoje, Kaunas, Lietuva.
Į tyrimą buvo įtraukti du stambūs, palaido karvių laikymo tipo pieno ūkiai, iš pietų Lietuvos.
Tirtos buvo 156 karvės prieš ir po apsiveršiavimo. Tiriami gyvūnai suskirstyti į tris grupes: karvės gydytos pegbovigrastimu du kartus, karvės gydytos – vieną kartą ir negydytos karvės. Kraujo mėginiai buvo imti savaitę prieš veršiavimą, pirmą parą po atsivedimo ir dar po savaitės. Buvo vertinami kraujo morfologiniai rodikliai: baltieji kraujo kūneliai, granuliocitai, limfocitai ir monocitai. Ligos po atsivedimo – mastitas, metritas, ir nuovalų susilaikymas buvo stebėtos per pirmąsias 30 dienų. Vertinta pieno primilžis per pirmąsias 100 laktacijos dienų, apsivaisinimas ir Servis periodas. Taip pat buvo tiriamas skirtingų grupių gyvulių brokavimas.
Tyrimo metu nustatyta, kad karvės, gydytos pegbovigrastimu, rečiau sirgo mastitu, rečiau susilaikė nuovalos, tačiau dažniau sirgo metritu, palyginti su negydytomis karvėmis.
Pegbovigrastimu gydytos karvės per pirmąsias 100 dienų DIM davė daugiau pieno, nei negydytos.
Karvės gydytos preparatu ankščiau apsivaisino, pagerėjo sėklinimo indeksas. Mažiau gydytų gyvulių buvo išbrokuota, tačiau jos buvo ankstesnėje laktacijos stadijoje. Karvėms suleidus preparatą du kartus daugelis tirtų rodikliu buvo šiek tiek geresni nei gydžius vieną kartą.
Raktažodžiai: Pegbovigrastimas, imunosupresija, ligos po atvedimo, pieno primilžis,
apvaisinimo indeksas
7
ABBREVIATIONS
bG-CSF Bovine granulocyte colony-stimulating factor
DIM Days in milk
G-CSF Granulocyte colony-stimulating factor GRAN Granulocytes
hG-CSF Human granulocyte colony-stimulating factor LUHS Lithuanian University of Health Sciences LYMPH Lymphocytes
NEB Negative energy balance NEFA Non-esterified fatty acids
MONO Monocytes
PEG Polyethylene glycol
PMN Polymorphonuclear neutrophils
RP Retained placenta
SEM Standard error of mean
WBC White blood cells
8
INTRODUCTION
The time around parturition, especially the conversion from late pregnancy to early lactation is a major challenge for most mammals, which may jeopardize both health and welfare if the challenges exceeds the coping mechanisms of the animal. [1] This have contributed to substantial research of dairy cows in the periparturient period. Where various authors have demonstrated that dairy cows suffer from sub-optimal immune responses. [2-4] The transition period is a critical moment that defines the future productive performance, particularly in dairy cows of high production. This is because they are subject to intensive farming, where economic performance and market demands are fundamental to the success of producers. [5]
The transition period or the periparturient period occurs three weeks before parturition and extends to three weeks after parturition. It is marked by several changes in the endocrine and immune system of the cow. These changes and implicit demands of the cow are necessary to occur for fetal development, parturition and lactogenesis. [6,7]
Among the diseases with the greatest impact in the transition period and with greater association to immunosuppression are mastitis, metritis and retained placenta. [6,8-10] These generate direct costs such as the cost of the veterinarian, treatment or even the cost associated with the death of the animal. But also, indirect costs, such as reproductive efficiency, reduced milk yield, fertility and the possibility of involuntary culling. [8]
Due to the higher incidence of diseases in the periparturient cow, efforts have been made to understand the changes that occur and what factors are involved in these changes. Many are the authors who look for concrete answers, however a single cause has not yet been found. Instead a culmination of events that may simultaneously or as a cause-effect is probable to increase the likelihood of contracting the disease during this period. [10]
Immunosuppression in the periparturient period is described as multifactorial, however, it is
thought that there is a relation between decreased immune function and the appearance of clinical
diseases. Therefore, to understand the reasons for the impaired immune function it is important to
know the normal function of the cow, from its behaviour, feeding, metabolism, physiology and
immune system. [8,10,11]
9 Recently an innovative solution that aims to restore the immune system was placed on the market. Pegbovigrastim is an immunomodulatory drug compromising the PEGylated form of the cytokine Granulocyte Colony Stimulating Factor, which triggers the production of bone marrow leukocytes, especially neutrophils. [12,13]
The effect of pegbovigrastim demonstrated in periparturient dairy cows is to restore the activity of mature neutrophils and increase their number. It is believed to reduce the impact of immunosuppression and consequently the incidence of diseases in this period. [12,13] According to a study of Kimura et al. cows receiving G-CSF covalently bound to polyethylene glycol during the peripartum demonstrated an increase in the number and function of neutrophils. [12] Another research, developed by Hassfurther et al. demonstrated a reduction in the incidence of clinical mastitis, comparing the effect of the same compound from the previous study, on cows housed with few hygiene conditions. [14]
The aim of the study: To evaluate the impact of pegbovigrastim administration in dairy cows on the white blood cells, postpartum diseases, production and reproduction performance.
Objectives:
1. To evaluate the effect of pegbovigrastim on circulating white blood cells.
2. To evaluate the effect of pegbovigrastim on mastitis, metritis and retained placenta.
3. To evaluate the effect of pegbovigrastim on production performance.
4. To evaluate the effect of pegbovigrastim on reproductive performance.
10
1. LITTERATURE REVIEW
1.1 Immune dysfunction in the periparturient cow
The biggest challenge facing the dairy producers is the successful management of cows in the periparturient period. [13] A frictionless transition from gestation to lactation is important to ensure high production and reproductive performance in the subsequent lactation. [15] The periparturient period reaches from the last 1 to 2 months of gestation to the first few months in lactation. Within this timeframe is the delicate transition period, typically defined as 3 weeks prepartum to 3 weeks postpartum. [1]
The transition period is a critical time for a dairy cow, when she is facing enormous endogenous and exogenous changes. As a consequence, the majority of cows experience reduced immune function during this time, but in some individuals the immune dysfunction is more pronounced. The impairment of the immune system in the given period is accompanied with a high incidence of diseases, both infectious and non-infectious. Common diseases observed in the transition period is mastitis, hypocalcaemia, ketosis, fatty liver disease, retained placenta, metritis and abomasal displacement. Mastitis, metritis and retained placenta are diseases which are highly associated with decreased immune responses and more specifically to the neutrophil dysfunction. [7,12,16] These diseases not only compromise the welfare of the cow, they also cause economic losses. Studies shows that nearly 75% of all diseases in dairy cows occurs within the first month of lactation. Diseases acquired in the early lactation is particularly problematic for the reason that they significantly affect the productive efficiency in the following lactation. [17]
Not only are cows in the transition period more prone to develop diseases when in contact with
infectious agents, but the severity of infections is also aggravated. Dysfunctional inflammatory
responses seen both at the local and systemic level are particularly challenging due to the direct impact
on the pathogenesis of diseases in the periparturient cows. Impairment of the inflammatory reaction
can cause a hyporesponsive state which is characterized by a delay in migration of immune
components, especially fully functional neutrophils in the early state of the disease. Numerous former
studies describe decreased functions of neutrophils in the transition period. Such impairments of the
neutrophilic function are derived to impaired chemotaxis, phagocytosis, reactive oxygen production
and myeloperoxidase activity. [18] Features which all play important roles in the neutrophil-mediated
antimicrobial activities. [19] The reduction has been observed as early as 2-3 week before parturition,
and was greatest in cows that developed mastitis and metritis after parturition compared to healthy
11 herd mates. On the other hand, major host tissue damage can be a result of an excessively strong or chronic inflammatory response due to an inappropriate balance among initiation and resolution of inflammation. [18]
It is well documented that the immune system of cattle dramatically changes in efficacy around the time of calving. Several studies have found significant changes in both the innate and acquired immune responses that can greatly affect the susceptibility of transitioning cows to acquire new diseases. However, the underlying cause is complex and have not been fully elucidated. [11] Reasons suggested to contribute to the immune dysfunctions are metabolic and endocrine factors such as negative energy balance, the stress of parturition (glucocorticoids) and lactogenesis. [7,20]
1.1.1 Nutritional influences on the immune system
Metabolic changes in the transition period pose great challenges to maintain the physiological balance of the cow. The nutritional requirements in the final stages of fetal development and milk production are enormous for the cow, sometimes exceeding the feed sources. As an aggravating factor, there is about a 20% decrease in dry matter intake, beginning two weeks before delivery and reaches its minimum at delivery. [1,21]
Due to the lack of amino acids and glucose and the reduction of dry matter intake, a negative energy balance (NEB) inevitably occurs, which all cows experience to a certain degree. [6,21] This NEB results in the mobilization of adipose tissue to try to overcome the energy deficit, causing in the process, the release of non-esterified fatty acids (NEFA). The NEFA can be oxidized or esterified in the liver, depending on the glucose level. When moderate levels of glucose exist, the esterification of NEFA occurs, resulting in fatty liver disease if excessive mobilization of fatty acids occurs. With the increase in the concentrations of NEFA an accumulation of triglycerides occurs in the hepatocytes, which together with a decrease in the available glucose can impair the functioning of the liver, which is fundamental in the maintenance of glycogenesis. Consequently, the oxidation of NEFA and in turn, the production of acetyl-CoA and excess ketone bodies can lead to ketosis. [1]
According to LeBlanc et al., the mobilization of adipose tissue and an increased NEB may be
associated with a decreased bactericidal capacity of neutrophils. Studies have shown that the increase
of the NEFA is associated with high risk of clinical disease, namely metritis, retained placenta and
displacement of the abomasum. The high concentrations have a direct influence on the cells of the
immune system, as a reduced myeloperoxidase activity. [22] Other investigations have associated the
12 increase of NEFA to the high concentration of glucocorticoids in the circulation and the stimulation of pro-inflammatory mediators that supress the immune function. [7]
Many types of immune cells are affected in an environment where there are low concentrations of glucose and high concentrations of ketone bodies and NEFA, thus these metabolic mechanisms are interconnected to peripartum immunosuppression. All of these changes related have been seen to lead to a high risk of mastitis, evidencing an association of the negative energy balance with metabolic alterations and infectious diseases. [7,23]
1.1.2 Endocrine influences on the immune system
Changes in stress hormones of the cow associated with pregnancy and parturition show a negative impact on the function of neutrophils and lymphocytes, such as changes in progesterone, estradiol, type 1 insulin-like growth factor and growth hormone. [24] The concentration of estradiol increases just prior to delivery and is related to a suppression of neutrophil function. Progesterone maintains pregnancy, being elevated in plasma during the dry period, however a rapid decrease occurs two days before delivery, coinciding with the increase of prostaglandin F2α uterine and regression of the corpus luteum. This regression has a direct influence on the lymphocytes, macrophages and cytokines, with a recruitment and proliferation of immune cells at this site. Estrogen increases in the last trimester of gestation and decreases shortly after delivery known as a potent immunosuppressant.
[8]
Changes from stress can activate the hypothalamic-pituitary-adrenocortical axis, which
increases the concentration of plasma glucocorticoids, especially plasma cortisol. Cortisol at delivery
increases 3 to 4 times its normal baseline plasma concentration (5 ng/ml), and may even increase its
concentration by up to 7 times if associated metabolic disorders occur. Cortisol is known to be an
immunosuppressive agent that causes depression in leukocyte proliferation and function. [25,26] In
addition, it decreases the production of prostaglandins, such as prostaglandin F2α, which stimulates
phagocytosis and chemotaxis of neutrophils but also the production of cytokines. Cows with
imbalances in prostaglandin F2α concentrations demonstrated a higher risk of developing retained
placenta (RP). The ability of expression of major histocompatibility complex molecules, which
coordinate maternal immunological recognition of fetal antigens and are necessary for efficient
expulsion of the placenta soon after delivery, is also affected by increased cortisol. [26] Cortisol also
has an influence on the loss of expression of L-selectin (protein expressed on the surface of immune
cells) that allows the passage of neutrophils through the endothelium. [8] Another consequence of
this stress is an activation in the production of catecholamines (e.g. adrenaline) which may cause a
13 hypomotility of the uterus that affects the expulsion of the fetus, where placental detachment and secondarily uterine infection are likely to occur. [26]
Metabolic and hormonal stress, characteristic of the peripartum, is currently considered one of the most important factors of immunosuppression. This stress can be triggered by simple environmental changes such as heat, transfer of pen, mixing with unknown animals or intervention of the veterinarian at birth; but also by hormonal and metabolic disorders such as hypocalcaemia. The consequences can be diverse, especially the increase in plasma glucocorticoid concentration and catecholamines, consequently with a negative effect on the cells of the immune system. [7,25,26]
1.2 Associated diseases
1.2.1 Mastitis
Mastitis is characterized by an inflammatory reaction of the mammary gland that is usually caused by microorganisms. [27] Pathogens most commonly encountered responsible for mastitis infection is Streptococcus spp. Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae.
[28] Already 30 years ago, researchers started to document that cows in the early postpartum period was especially susceptible to intramammary infections. Today it is believed that a contributing factor to the higher incidence of mastitis at this crucial time can be linked between impaired immune function and the opportunistic nature of mastitis pathogens. [5]
Defence mechanisms of the mammary gland consists of both anatomic and cellular barriers that act against invading microorganisms. The teat sphincter together with the keratin layer of the teat canal makes up the first natural anatomic defence of the mammary gland. Beside the natural anatomic barrier that the teat compose a complex of innate and adapted immune responses is required to protect the mammary gland from microbial invasion. [14]
Under conditions of prompt recognition of invading bacteria and an adequate inflammatory
reaction, the immune system of the mammary gland is thought to work optimally. This results in a
rapid elimination of the infection and allows for a quick restoration of the mammary gland to normal
function, possibly without any noticeably clinical signs. Contrary, if the immune system of the
mammary gland is impaired or suboptimal the bacteria can damage the mammary tissue which may
lead to a state of acute inflammation or chronic mastitis. Which in turn has a negative effect of the
quality and quantity of the milk. [27]
14 The majority of intra-mammary infections arises from bacteria that overcomes the anatomical- physical barrier of the teat canal, due to the dilated teat canal in the beginning of the lactation. Once the bacteria have gained access to the teat cistern they can multiply and establish in the tissue of the mammary gland if the innate immune response is insufficient. The somatic cells are the most important effector cells in the mammary immune system. With varying amounts recruited from the blood steam depending on the type of pathogen involved. [29]
In cows with mastitis, neutrophils are the principal cells in the defence of the mammary gland, constituting about 90% of the total leukocyte population. On the other hand, in healthy cows the dominant cells in the mammary gland are macrophages, but in the inflammatory phase these tend to decrease their activity compared to neutrophils. [24] In addition, the number of neutrophils is also decreased in the peripartum, as well as its bactericidal activity, migration and chemotaxis. [12] The concentration of IgG2 is reduced during delivery, which is related to the incidence of mastitis in previous studies. With regard to complement proteins, they are elevated in colostrum, mastitic milk and mammary secretions, and plays its main role as a pro-inflammatory mediator during mastitis.
[24]
The mammary glands capacity to recruit neutrophils and the outcome of the intramammary infection is seen to have a strong inverse relationship. Cows with rapid and massive recruitment of milk neutrophil responses can undergo spontaneous cures, whereas cows with slow or no neutrophil response at all often suffers from severe or sub-acute forms of mastitis. [30]
Despite efforts to prevent and control mastitis, it continues to have a major impact on milk production, standing out as the disease with the highest associated costs, both direct and indirect costs.
[11,31] In the direct costs, we can enumerate the discarded milk, the time spent, the necessary treatments and even the possible death of the animal. In relation to indirect costs (which are often not accounted for but suffer large losses), milk production and quality decline, early drying, reduced animal welfare, increased involuntary culling and other health problems related costs. [11,31] The reproductive system may be affected, as several studies indicate, with an increase in the number of non-pregnant days, low conception rates, increased risk of pregnancy loss and hormonal changes that result in alterations in estrus. [32]
1.2.2 Retained placenta
Uterine diseases such as retained placenta (RP) and uterine infections is another major problem
of the transitioning cow and her further productive performance. The level of immune function in the
15 transition period is largely responsible for the occurrence of these diseases. It has been shown that cows with RP or metritis have a more profound immune dysfunction, that starts long before the disease appear. [33]
Retained placenta is defined as the failure to expel the foetal membranes within 24h after parturition. On average if RP occurs, a retention of the foetal membranes for 7 days is usual. RP is unwanted in the dairy production, as it may increase the risk of bacterial contamination and delay involution of the uterus. The incidence of RP is between 5-10% of all calving’s and greatly enhances the risk to develop metritis and endometritis. The most important aspect in the pathogenesis of RP is failure of the rapid breakdown of the cotyledon-caruncle attachment after the expulsion of the calf.
The lack of success to breakdown the placental attachment appears to be highly arbitrated by the impairment of the immune system to successfully degrade the placentomes after the delivery. It has been laid down that cows with RP have significantly lower leukocyte oxidative burst and chemotactic activity mainly derived from neutrophils, and interleukin (IL-8) concentrations in comparison to cows without RP. Studies show that these differences can be evident from as early as 2 weeks before parturition as well as at the time of parturition. [33]
The impact of RP is linked with the risk of delay in uterine involution, metritis, endometritis and associated economic costs. Reproductive performance and losses in milk production only seem to be affected when metritis or endometritis develops secondary to RP. [33] However, some authors disagree with this idea, directly associating cows with RP to a decrease in milk production as well as an increase in the calving interval, the calving to conception interval, and the number of services per conception. [21]
1.2.3 Metritis
When reading the literature many various definitions of uterine infections occurs, depending on the author. Here the definitions of metritis are mentioned for consistency. Puerperal metritis is defined as a cow with fetid watery red-brown uterine discharges and an abnormally enlarged uterus in addition to systemic signs of illness such as fever <39,5, dullness and reduced milk yield within 21 days postpartum. In contrast, the definition of clinical metritis is a cow with an enlarged uterus and purulent uterine discharge, but without systemic signs of illness within 21 days postpartum. [34,35]
Both of these definitions will be mentioned jointly as metritis in the further text.
The largest risk factor for uterine infections is RP followed by dystocia, twin births and the
calving environment. Other factors contributing to uterine infections are conditions that may impair
immune function and feed intake. [33,34]
16 Most of the inflammatory conditions of the uterus after parturition are initiated by microbial contamination of the uterine lumen. The uterus of cows after parturition is commonly contaminated with a variety of different bacteria, but this is not consistently linked with clinical disease. The most frequently isolated pathogens of the uterus causing disease are Arcanobacterium pyogenes and Escherichia coli. Uterine disease is followed by mucosal adherence of pathogenic organisms, epithelial colonization or penetration and/or bacterial toxin release. The bacterial load and species as well as the response of the immune system are determinants for the development of uterine diseases.
[35]
The uterus is capable of rapid and efficient clearance of bacterial infection in a healthy state. In experimental studies with intrauterine infusion of A. pyogenes scientists found it hard to produce chronic uterine infections in vivo. The incidence of cows with uterine infections is 25-40% within the first 14 days, and approximately 20% of the infections persists after this period. The most important defence of the uterus once bacteria is present in the lumen is the non-specific phagocytosis of neutrophils. [33,34]
The effects of metritis on cow health are detrimental at various levels: in reproductive performance (deteriorating the uterine environment, reducing conception rate and increasing embryonic loss), in the production of milk (with lower amount produced, lower protein composition and elimination of milk not suitable for human consumption) and premature culling. This disease generates high economic losses, an increase in the use of antibiotics and an increase in the number of treatments required, as well as consequences on animal welfare, generating intense pain in the affected cows, leading to research on new strategies to minimize their incidence. [35-37]
1.3 Economic impact
The periparturient period is of major concern to the dairy industry, as the impaired immune system and high prevalence of disease in this time negatively affect the health and production.
Not only has the immune dysfunction and high incidence of disease an impact on the welfare
of the cow, it also affects the economic profitability for the dairy farmer. The economic profitability
is reduced in terms of treatment and management costs, decreased production and reproductive
performance. [8,38] This is the costs in a general perspective, indirect and directly associated costs
are discussed further in relation to its disease or life happening.
17
1.4 Immunomodulation
Biotherapeutics have long been in the field of research to improve health remedies of various diseases. The frequent use of antimicrobials is an emerging threat to the health and welfare with increasing pathogen resistance. Which requires development of novel approaches for the treatment of infectious diseases. Immune modulators are one type of biotherapeutics that can be administrated to reduce or prevent symptoms of diseases caused by a range of bacterial and viral pathogens.
Immunomodulatory therapies are intended to interfere with the immune system of the host instead of the pathogen. The goal of immunomodulatory therapies is to provide the host with an increased level of immune defence during a period where the host experiences decreased immune function. [11]
Several researchers have investigated various cytokines that are part of the cow’s normal immune system, in an attempt to outline methods to improve the understanding of the immunosuppression in the periparturient period. A specific group of compounds that have been examined for its potential biotherapeutic value is cytokines. The use of recombinant cytokines to modulate the immune function in immunocompromised hosts is assumed to prevent microbial infections. [11,39]
The cytokine: “granulocyte colony stimulating factor (G-CSF) is an endogenous hematopoietic growth factor that stimulates the production and differentiation of neutrophils by progenitor cells in the bone morrow.” [14,19,40] Prophylactic treatment with recombinant human G- CSF (hG-CSF) started in human medicine, used for patients that go through myelosuppressive chemotherapy for the reason to improve their ability to combat infections. The G-CSF increases the number of the polymorphonuclear neutrophils (PMNs), which have shown a reduction in the incidence of microbial infections and total time spent in the healthcare unit. [12] M.E Kehrli et al.
carried out a study 20 years ago, investigating the efficiency of hG-CSF for periparturient cows
infected with Escherichia coli. hG-CSF was seen to reduce the number of cows exhibiting new
infections of clinical mastitis, increasing the rate of bacterial clearance and reducing the clinical
severity of the infections. [41] The same author a few years earlier investigated the bovine
counterpart, recombinant bovine G-CSF (bG-CSF) which was seen to induce a marked neutrophilia
and an increased activity of the neutrophils phagocytic and cytotoxic effects. This also formed the
hypothesis of that CSF could be advantageous for cows in their periparturient period to prevent
uterine diseases. [42]
18 1.4.1 Pegbovigrastim
Recently there has been launched a commercial form of the recombinant bG-CSF, pegbovigrastim. Which have been seen to increase the concentration of neutrophils in blood circulation and to improve their myeloperoxidase activity. [12,43] Specifically this study focuses on the recently launched product “Imrestor” produced by the company “Elanco Animal Health” which have shown beneficial effects in the periparturient period including the development of accompanied diseases and economic factors.
To prolong the duration of activity the native protein is modified by a covalent bound to a polymer, such as polyethylene glycol. The pegylation increases the hemodynamic volume, reduces first-pass renal clearance and reduces the proteolytic degradation of the protein. [13,14]
1.4.2 Imrestor
Pegbovigrastim (Imrestor, Elanco Animal Health, Basingstoke, UK) is marketed to reduce the incidence of clinical mastitis for periparturient dairy cows and heifers in the first 30 days of lactation.
The product is supplied in a 2,7 ml prefilled single dose syringe containing 15 mg of pegylated
recombinant bovine granulocyte colony stimulating factor (PEG bG-CSF) and excipients. The
treatment regimen constitutes of two doses. The first dose is to be administered subcutaneously
7 days (4-10) before anticipated calving. The second dose is to be administered subcutaneously within
24h after calving. The minimum interval between the two dosages should not be less than 3 days and
not longer than 17 days. [44]
19
2. METHODOLGY
2.1 Research workload, location and method
This research work was carried out over a 2-year period, between November 2016 and November 2018 in the department of the Large Animal Clinic of LUHS in Kaunas, Lithuania. Two dairy farms from the southern part of Lithuania were included in the study. The first one is located in the region of Marijampole (Farm X) and the second is located in the region of Alytus (Farm Y).
The herd of Farm X consisted of ~1000 dairy cows kept in a free stall system. Cows were foraged with a total mixed ration for ad libitum intake to meet their nutrient requirements, had unlimited access to water, and were milked thrice daily in a rotating milking parlour. Dry cows were kept in a separate barn, on deep litter and grouped based on their expected delivery date. After parturition cows were moved to a smaller pen with a milking facility and kept here for 3-4 days before released into the milking barn together with other fresh cows.
The herd of Farm Y consisted of ~500 dairy cows kept in a free stall system. Cows were foraged with a total mixed ration for ad libitum intake to meet their nutrient requirements, had unlimited access to water, and were milked thrice daily in a herringbone milking parlour. Also, here dry cows were kept in a separate barn, on deep litter and grouped based on their expected delivery date. After parturition cows were milked in the dry cow barn for 3-4 days and then moved to the dairy stall were they were kept in a group of early lactating cows (≤150 DIM).
A total number of 156 dairy cows from Farm X (n=41) and Farm Y (n= 115) were included in this research.
2.2 Study design
A total number of 156 dairy cows from Farm X (n=41) and Farm Y (n= 115) were included in this research. All cows were in the last semester of gestation.
The study was divided into two separate parts. The first part of the study was to investigate how
pegbovigrastim affects circulating white blood cells and the disease incidence including mastitis,
metritis and retained placenta. The research was performed on 27 dairy cows, were we injected
pegbovigrastim, analysed blood samples and recorded the disease incidence. Only a subset of cows
was analysed, due to the large workload and high costs. The selected cows were in their last weeks
of gestation and clinically healthy. Cows were divided into three groups. The first group received 2
injections of pegbovigrastim (PEG2), the second group received one injection of pegbovigrastim
20 (PEG1), finally the control group did not receive any treatment neither placebo (CTR), seen in figure 1. Animals was enrolled weekly approximately 7 days before their anticipated calving date. This was also the day where treated cows received their first injection. Cows that calved more than four days after their first injection received a second injection within 24h after parturition and was therefore assigned to the PEG2 group. Cows that calved less than three days after their first injection did not receive a second injection according to the product label, and was so assigned to the PEG1 group.
Blood samples were collected 3 times from each cow: first sample taken two or seven days prepartum, before treatment, second sample taken one day postpartum and the third sample taken one week postpartum.
The incidence of mastitis, metritis and retained placenta was observed for the first 30 days in milk (DIM). Cows with mastitis were diagnosed based on the visual examination of the milk including clots, flakes and watery appearance as well as swollen udder. Cows that developed mastitis were treated with both broad spectrum systemic antibiotics enrofloxacin and a dosage of intramammary suspension containing a complex of benzyl-penicillin, streptomycin, neomycin and prednisolone. Cows with metritis were diagnosed based on fetid watery or purulent uterine discharge with or without signs of systemic illness (fever, decreased milk yield or inappetence). Cows that developed metritis were treated with systemic antibiotics (ceftiofur). Cows with retained placenta were diagnosed based on failure to expel foetal membranes within 24h after parturition. Cows with retained placenta were treated with prostaglandin F2α.
The second part of the study was to investigate how pegbovigrastim affected the production
and reproduction performance of cows. For this part of the research, data were collected from already
treated cows. A total number of 156 dairy cows were enrolled in this part of the research, were 33 of
the cows were excluded due to culling in the follow up period when production and reproduction data
was supposed to be collected. The grouping of animals followed the same treatment as previously
described and seen in figure 2. The parameters were measured by measuring average milk yield for
100 DIM, average days open, conception rate, first service pregnancy rate and culling rate.
21
2.3 Grouping
Fig. 1 Distribution of the number of cows in the analysis of hemogram parameters and disease incidence
Fig. 2 Distribution of the number of cows in the analysis of production and reproduction parameters
2.4 Collection of samples
Blood samples were obtained by coccygeal venipuncture, by vacutainer blood collection system with an aseptic technique. 10 ml of blood were drawn into blood collection tubes containing the EDTA anticoagulant (Venosafe, TERUMO® Europe, Belgium). Samples were used for complete blood count determination within 3 hours after collection.
Blood samples for the subset of cows were taken at seven days before expected parturition, just before the treatment, within 24 h after parturition after the second treatment injection, and seven
Analysis of impact of PEG on hemogram parameters and
disease incidence No. 27
2 injections of PEG No. 10
1 injection of PEG No. 8
Control group No. 9
Analysis of impact of PEG on production and reproduction parameters
No. 123
2 injections of PEG No. 51
1 injection of PEG No. 38
Control group
No. 36
22 days postpartum. For cows that calved less than three days after first injection were assigned to PEG1 group, the average blood sample day of the first blood sample was two days before calving just before the first treatment, the following two samples were taken by the same intervals as previously described.
2.5 Determination of white blood cells count
Whole blood collected in EDTA tubes where analysed for complete blood counts. By using haematology analyser (Abacus Junior Vet, Hungary) in the laboratory of the Large Animal Clinic of LSMU. The analyse was used to determine the amount of total white blood cells divided into granulocytes, lymphocytes and monocytes in the collected blood samples.
2.6 Postpartum diseases
Disease incidence of mastitis, metritis and retained placenta was recorded for cows from 0 to 30 DIM. The postpartum diseases were recorded into the same groups as the treatment was divided.
Cows with mastitis was defined by clinical findings such as symptoms including milk abnormalities and systemic affects. Cows with metritis was defined by clinical findings such as abnormal postpartum related discharges from the uterus. Cows with retained placenta was defined as fetal membranes not expelled within 24 h after parturition.
2.7 Collection of data
Production and reproduction data from the two farms were collected from their individual herd management system. Both farms used herd management system GEA dairy plan. Parameters such as;
milk yield for 100 DIM, days open and number of inseminations was recorded for each cow.
2.8 Statistical analysis
Microsoft Office Excel 2016 was used for organization of all collected data. It was also used to
calculate the descriptive statistics with functions like; average, sum and count. Further statistical
analysis was carried out with SPSS for Windows. The statistical significance was calculated by using
mean comparison between groups, ANOVA post-hoc test LSD. The data was statistically significant
when P ≤ 0,05. However, data with P > 0,05 was still used in the presentation of results.
23
3. RESULTS
3.1 Effect of pegbovigrastim on hemogram parameters
Fig. 3 Effect of pegbovigrastim on white blood cell count at days (-7;-2 1 7) relative to parturition
0 = represents statistical significance (P<0,05) to CTR group at the same sampling point. 1= represents statistical significance (P<0,05) to PEG1 group at the same sampling point.
In Fig. 3 changes of total circulating white blood cells over time, for pegbovigrastim treated and control cows are seen. Pegbovigrastim is seen to increase the white blood cells over the 14-days period, while the white blood cells of control cows are seen to steadily decrease during the same period. The total number of circulating WBC´s for cows in the PEG2 group are seen to increase dramatically from 8,99 one week prepartum to 28,38 one day after parturition, and then slightly diminish to 24,01 x 10 9 /L one week postpartum. For cows in the group of PEG1 which received their first injection approximately two days prepartum the WBC increased from 11,47 to 18,93 the day after parturition, and in consistence with PEG2 declined slowly to 13,02 x 10 9 /L one week postpartum. The total number of circulating WBC´s for cows in the CTR group was seen to gradually decrease throughout the investigated period from 15,99 seven days before parturition to 10,77 one day after parturition, and then a further decrease to 8,61 x 10 9 /L was seen seven days after parturition.
The results show that after two injections of pegbovigrastim the WBC´s was three times higher at the day after parturition the same increase was seen one week postpartum, compared with the control group. A correspondingly high increase was not seen for the cows treated with one injection of pegbovigrastim. However, the WBC’s in the group treated with one injection of pegbovigrastim
0,1
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PEG2 PEG1 CTR
24 still increased 2 times at the day after parturition and with almost 0,5 time one week after parturition compared with the control group.
Fig. 4 Effect of pegbovigrastim on granulocyte cell count at days (-7;-2;1;7) relative to parturition
0 = represents statistical significance (P<0,05) to CTR group at the same sampling point. 1= represents statistical significance (P<0,05) to PEG1 group at the same sampling point.
The largest effect of pegbovigrastim on individual white blood cells was seen for granulocytes (Fig. 4). In the study the total granulocytes was counted even though no difference is seen in the concentration of distribution of the various cells including neutrophils, eosinophils and basophils.
Even though it is well known that the granulocytes consist of 90 % neutrophils. [45] Cows in the group of PEG2 was seen to have the highest increase of GRA, increasing from 3,48 one week before parturition to 17,89 the day after parturition and then regress to 14,67 x 10 9 /L one week after parturition. Cows in the group of PEG1 was also seen to increase, however not as much as cows in the group of PEG2. GRA for PEG1 increased from 3,23 one week before parturition to 10,11 one day after parturition, then in consistence with PEG2 a slight regress to 5,31 x 10 9 /L was seen one week after parturition. Granulocytes for the CTR group was seen to gradually decline from 5,34 one week before parturition to 3,23 one day after parturition and then a further decline to 1,70 x 10 9 /L was observed one week after parturition.
The results show that granulocytes of in the PEG2 group increased by 6 times at one day after parturition and almost 9 times one week after parturition compared with the CTR group. An increase of the granulocytes was also seen for the PEG1 group even not so high as PEG2, PEG1 increased with 3 times one day as well as seven days after parturition in the concentration of granulocytes.
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PEG2 PEG1 CTR
25 Fig. 5 Effect of pegbovigrastim on lymphocyte cell count at days (-7;-2;1;7) relative to
parturition
0 = represents statistical significance (P<0,05) to CTR group at the same sampling point. 1= represents statistical significance (P<0,05) to PEG1 group at the same sampling point.
The number of LYM was seen to differ greatly between pegbovigrastim treated (PEG2, PEG1) cows and untreated (CTR) cows before parturition (Fig. 5). However, the trend and proportions was similar at day 1 and day 7. The number of lymphocytes for cows in the PEG2 group are seen to increase from 4,81 one week prepartum to 9,49 one day after parturition, and then slightly diminish to 8,11 x 10 9 /L one week postpartum. For cows in the group of PEG1 which received their first injection approximately two days prepartum the lymphocytes increased from 6,95 to 7,90 the day after parturition, and in consistence with PEG2 declined slowly to 6,92 x 10 9 /L one week postpartum.
The number of lymphocytes for cows in the CTR group was seen to markedly decrease from 9,56 seven days before parturition to 6,73 x 10 9 /L one day after parturition. Then the concentration was stable throughout the investigated period.
0
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-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7
Ce lls x 10^ 9/L
Days relative to parturition
PEG2 PEG1 CTR
26 Fig. 6 Effect of pegbovigrastim on monocyte cell count at days (-7;-2;1;7) relative to
parturition
0 = represents statistical significance (P<0,05) to CTR group at the same sampling point. 1= represents statistical significance (P<0,05) to PEG1 group at the same sampling point.
The MONO for cows in the group treated with two injections of pegbovigrastim was seen to steadily increase throughout the investigated period (Fig. 6). Starting on 0,71 one week before parturition increasing to 1,02 at the day after parturition and further increase to 1,24 x 10 9 /L at one week postpartum. The MONO for cows in the group treated with one injection of pegbovigrastim started on 0,79 2 days before parturition and increased to 0,92 one day after parturition and they almost returned to the starting value 0,80 x 10 9 /L at one week postpartum. The MONO for the group of non-treated cows decreased throughout the investigated period. Starting from 1,09 one week before parturition to 0,82 the day after parturition, then continuing to decrease until one week postpartum, to a level of 0,44 x 10 9 /L.
3.2 Effect of pegbovigrastim on some periparturient diseases
Almost all cows (n=21/27) developed at least one of the diseases within 30 days after parturition. There were six cows in total which did not develop any of the diseases (PEG2 =3, PEG1=2, CTR=1), however one cow in the PEG2 group developed all the diseases. The most common combination of diseases was mastitis together with metritis (n=5).
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Ce lls x 10^ 9/L
Days relative to parturition
PEG2 PEG1 CTR
27 Fig. 7 Incidence of mastitis between groups
Disease incidence rate of mastitis for cows 30 DIM was seen to decrease for pegbovigrastim treated cows compared with cows in the control group (Fig. 7). The highest occurrence of mastitis was seen in the CTR group with 90% of the cows affected. The occurrence of mastitis for the pegbovigrastim treated cows were much lower, with 20% of the cows in the PEG2 group and 13% of the cows in the PEG1 group affected.
Pegbovigrastim was seen to reduce the mastitis incidence for cows in the PEG2 group with 77%
compared to the CTR group. While the largest reduction of mastitis could be seen for cows in the PEG1 group with 85% compared to the CTR group.
Fig. 8 Incidence of metritis between groups
20%
13%
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PEG2 PEG1 CTR
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28 Disease incidence rate of metritis within the first 30 days was seen to increase for pegbovigrastim treated cows compared with cows in the control group (Fig. 8). The highest incidence of metritis was seen in PEG2 and PEG1 group with 70% and 63% of the cows developing metritis respectively. While the CTR group had the lowest incidence, with 40% of the cows developing metritis.
The study revealed that pegbovigrastim increased the incidence of metritis with 75% for cows in the PEG2 group and 58% for cows in the PEG1 group compared with the CTR group.
Fig. 9 Incidence of retained placenta between groups
Fig. 9 shows the disease incidence of retained placenta between groups. 20% of the PEG2, 13%
of PEG1 and 20% of CTR cows was diagnosed with retained placenta. Pegbovigrastim was not seen to significantly affect the incidence of retained placenta. The incidence of retained placenta was equal for PEG2 and CTR group, while a 35% reduction was seen between PEG1 and CTR cows.
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29
3.3 Effect of pegbovigrastim on milk production
Fig. 10 Average milk yield for I00 DIM, litres
The average milk yield for cows 100 DIM is seen in Fig. 10, for the groups PEG2, PEG1 and CTR. Highest average milk yield was seen in PEG1, 4416 ± 70l. Second largest average milk yield was seen in PEG2, 4388 ± 83l, close to PEG1. Lowest average milk yield is seen in the CTR, 4097 ± 108l. Also seen in Fig. 10 minimum and maximum values for each group of cows. PEG2 varies from 3306 l to 5616 l, 100 DIM. PEG1 varies from 3396 l to 5359 l. The CTR group has the largest variation of the three groups and varies from 2916 l to 5652 l. Results are presented as mean ± standard error of mean (MEAN ± SEM).
The average milk yield for cows 100 DIM in was seen to be significantly higher (P <0,05) in in the groups with treated cows (PEG2, PEG1) compared to the cows in the control group (CRT). The largest increase, with 7,8% was seen between the PEG1 and CTR group, however it did not differ much from the increase of the PEG2 group which was 7,1% higher compared to the CTR group. The milk yield for PEG2 group was slightly lover, 0,6% compared to PEG1 group.
0 1000 2000 3000 4000 5000 6000
PEG2 PEG1 CTR
M ilk yi e ld , L
Group
30
3.4 Effect of pegbovigrastim on reproduction
Fig. 11 Average days open
Fig. 11 shows the average days open, which is the period in between calving and next successful insemination. Cows in the PEG2 group had the shortest interval, with 98 ±7 days open.
Cows in the PEG1 group had a little longer interval, with 108 ± 6 days open. While cows in the CTR group had the longest interval, with 123 ± 10 days open. Results are presented as MEAN ± SEM.
In the PEG2 group the days open was reduced by 20% and in the PEG1 group the days open was reduced by 12% compared to CTR group. The study shows that cows treated with two injections had a significantly (P<0,05) shorter calving-to-conception interval compared to those that received one injection and non-treated cows.
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31 Fig. 1 Conception rate
Fig. 12 graphically shows the efficiency of inseminations within each group. The results show that 55% of the inseminations were successful in the group of PEG2. Lower results were observed in the group of PEG1 were 47% of the inseminations were successful. The lowest result was observed for cows in the CTR group were only 43% of the inseminations were successful.
The results of the study show that the group of PEG2 had 17% higher conception rate compared to PEG1 and 28% higher conception rate compared to CTR cows. The results also show that cows in the PEG1 group had 9% higher conception rate compared to cows in the CTR group.
Table 1. Conception rate at each insemination
PEG2 PEG1 CTR
Pe rc en tage p re gn an t at e ac h se rvice
1 49% 39% 36%
2 27% 29% 31%
3 18% 16% 16%
4 6% 11% 8%
5 - 5% 3%
>5 - - 6%
Services per
conception 1,8 2,13 2,33
Table 1. shows services per conception and the distribution of cows becoming pregnant at each service. The result show that cows in PEG2 required 1,8 inseminations to conceive. This number was slightly higher for cows in PEG1 that required 2,13 inseminations to conceive. The highest
55%
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Per ce n tage
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32 number was seen for cows in CTR which required 2,33 inseminations to conceive. The number of services per conception in PEG2 group was significantly lower (P<0,05) than services per conception for the CTR group. It was seen that PEG2 cows required 0,55 (21%) less inseminations to conceive compared to CTR cows.
The highest frequency of pregnancy was seen at the first insemination independently of administration of pegbovigrastim (Tab. 1). The highest number of inseminations for cows in the PEG2 group was 4, which only 6% of the cows required. The same number for cows treated with only one injection of pegbovigrastim was 5, which 5% required. Lastly 6% of the untreated cows required more than 5 inseminations to conceive.
Fig. 13 First service pregnancy rate
Fig. 13 shows the percentage of how many cows that got pregnant at the first insemination.
The best result can be observed in the PEG2 group were 49% of the cows became pregnant at the first insemination. The second-best result was observed in the PEG1 group were 39% of the cows got pregnant at the first insemination. The lowest result was seen in CTR group were 36% of the cows became pregnant after the first insemination.
3.5 Culling rate
In total 156 cows were enrolled in the study, out of these 33 cows were excluded from the study due to euthanasia or culling (PEG2=11, PEG1=9 and CTR=13). The culling rate was calculated from the excluded cows in respective group.
49%
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Per ce n tage
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33
.
