1
LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
VETERINARY ACADEMY
Faculty of Veterinary Medicine
Sanna Eleonora Irene Ivarsson
ERKIŲ SUKELTŲ LIGŲ EIGOS IR PREVENCIJOS LIETUVOJE
IR ŠVEDIJOJE PALYGINIMAS
THE COMPARISON OF COURSES OF DISEASE AND
PREVENTION OF CANINE TICK-BORNE DISEASES IN
LITHUANIA AND SWEDEN
MASTER THESES
of Integrated Studies of Veterinary Medicine
Supervisor: Assoc. Prof. Dr. Vaida Andrulevičiūtė
2
THE WORK WAS DONE IN THE DEPARTMENT OF BIOCHEMSTRY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Theses “
THE COMPARISON OF COURSES OF
DISEASE AND PREVENTION OF CANINE TICK-BORNE DISEASES IN
LITHUANIA AND SWEDEN
”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.
2017-12-19 Sanna Ivarsson
(date) (author’s name, surname) (signature)
CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK
I confirm the correctness of English language in the done work.
2017-12-19 Sanna Ivarsson
(date) (author’s name, surname) (signature)
CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES
2017-12-19 Vaida Andrulevičiūtė
(date) (supervisor’s name, surname) (signature) THE MASTER THESES HAVE BEEN APPROVED IN THE BIOCHEMSTRY DEPARTMENT
(date of approbation) (name, surname of the manager of department/clinic)
(signature)
Reviewers of the Master Theses
1) 2)
(name, surname) (signatures)
Evaluation of defense commission of the Master Theses:
(date) (name, surname of the secretary of the defence commission)
3
TABLE OF CONTENTS
SUMMARY ... 5 SANTRAUKA ... 6 LIST OF ABBREVIATIONS ... 7 INTRODUCTION ... 8 1. REVIEW OF LITERATURE ... 101.1 Tick-borne diseases and their vectors in dogs ... 10
1.2 Babesiosis ... 12
1.3 Granulocytotropic anaplasmosis ... 14
1.4 Borreliosis ... 15
1.5 Borrelia and co-infection with Anaplasma and Babesia ... 17
1.6 Tick prevention ... 17
1.7 Acute phase response and proteins in TBD ... 18
1.8 CRP in TBD ... 19
2. RESEARCH METHODS AND MATERIAL ... 20
2.1 Sample collection ... 20
2.2 Diagnosis of TBD ... 22
2.3 Determination of haematological and biochemical parameters ... 22
2.4 Questionnaire and statistical analysis ... 23
3. RESEARCH RESULTS ... 24
3.1 The comparison of the main haematological and biochemical parameters ... 24
3.2 The analysation of CRP results ... 29
3.3 Clinical manifestation of TBD ... 32
3.4 The analysis of the questionnaire of dog owners about prevention from TBD... 34
4. DISCUSSION OF RESULTS ... 37
4
SUGGESTIONS/RECOMMENDATIONS ... 41
ACKNOWLEDGEMENT ... 42
LIST OF LITERATURE ... 43
5
SUMMARY
THE COMPARISON OF COURSES OF DISEASE AND PREVENTION OF CANINE TICK-BORNE DISEASES IN LITHUANIA AND SWEDEN
Sanna Ivarsson Master Thesis
The aim of this study was to compare canine TBD in Sweden and Lithuania and to found out the dog owners knowledge and awareness of canine TBD. This master thesis work is performed in the department of Biochemistry in of Lithuanian University of Health Sciences (LUHS) in Kaunas and at a Swedish veterinary clinic, Läckeby Djursjukhus (and its branches). A total of 41 blood samples from dogs infected with various TBD in Sweden and Lithuania were collected to investigate haematological and biochemical parameters. Some of the dogs (n=30) were collected from the clinics database. Information about gender, age and breed, clinical signs and body temperature was gathered. Eleven dogs were infected with canine granulocytic Anaplasmosis, ten with canine Lyme disease, twelve with coinfection (Anaplasma and Borrelia) and eight with canine Babesiosis. Diagnosis was made by blood picture, serology (IFA) or blood smear investigation. Apathy and fever were the most frequently observed clinical signs and polyarthritis was a frequent finding in Borreliosis and coinfection. The most important haematological parameters for TBD were RBC, LYMP and PLT. GLOB and CRP are the most important biochemical parameters for Anaplasma, Borrelia and Coinfection, while ALP, UREA and CRP are the most important for Babesia. This study showed that CRP may be used as an indicator of Canine Lyme disease. Dog owner in Lithuania and Sweden were aware of Canine Lyme disease and Babesiosis, while Anaplasmosis and tick-borne encephalitis were less known TBD. Dog owners in Lithuania used more tick-preventative drugs than dog owners in Sweden.
Key words: Canine tick-borne disease, Canine granulocytic anaplasmosis, Canine babesiosis, Canine lyme borreliosis, Coinfection, CRP
6
SANTRAUKA
ERKIŲ SUKELTŲ LIGŲ EIGOS IR PREVENCIJOS LIETUVOJE IR ŠVEDIJOJE PALYGINIMAS
Sanna Ivarsson Master thesis
Šio tyrimo tikslas buvo palyginti šunų erkių sukeltų ligų atvejus Švedijoje ir Lietuvoje bei įvertinti šunų savininkų žinias ir supratimą apie erkių sukeltas ligas. Šis magistro darbas buvo atliktas Kauno sveikatos mokslų universitete (LSMU) Biochemijos katedroje ir Švedijos veterinarijos klinikoje, Läckeby Djursjukhus (ir jos filialuose). Vertinti 41 kraujo mėginių, šunų, sergančių įvairiomis erkių sukeltomis ligomis, Švedijoje ir Lietuvoje, kraujo hematologiniai ir biocheminiai parametrai: 11 šunų – anaplazmoze, 10 – borelioze, 12 – koinfekcija (anaplazmoze ir borelioze) ir 8 – babezioze. Kai kurie šunų mėginiai (n = 30) buvo surinkti iš klinikų (Švedija) duomenų bazės. Surinkta informacija apie lytį, amžių ir veislę, klinikinius požymius ir kūno temperatūrą. Liga diagnozuota vertinant kraujo, serologijos ar kraujo tepinėlio tyrimo rezultatus.
Apatija ir karščiavimas buvo pagrindiniai stebimi klinikiniai požymiai, o boreliozės ir koinfekcijos atveju buvo nustatomas ir poliartritas. Svarbiausi erkių sukeltų ligų šunims hematologiniai parametrai buvo eritrocitai, limfocitai ir trombocitų skaičius. Globulinų kiekis ir C-reaktyvaus baltymo aktyvumas yra svarbiausi anaplazmozės, borelijos ir koinfekcijos biocheminiai parametrai, o karbamido kiekis kraujyje, šarminės fosfatazės ir C-reaktyvaus baltymo aktyvumas yra svarbiausi babeziozės atveju. Šis tyrimas parodė, kad C-reaktyvus baltymas gali būti naudojamas kaip šunų Laimo ligos rodiklis. Šunų šeimininkai Lietuvoje ir Švedijoje puikiai žinojo apie šunų Laimo ligą (boreliją) ir babeziozę, o anaplazmozė ir erkinis encefalitas buvo mažiau žinomi. Šunų savininkai Lietuvoje naudojo daugiau erkių prevencinių priemonių nei šunų savininkai Švedijoje.
Raktažodžiai: šunų erkių sukeltos ligos, šunų anaplazmozė, šunų babeziozė, šunų boreliozė, koinfekcija, CRP.
7
LIST OF ABBREVIATIONS
CRP C – Reactive protein
APR Acute phase response
APP Acute phase proteins
SAA Serum amyoloid A
RBC Red blood cell
PCR Polymerase chain reaction
ELISA Enzyme-linked immunosorbent assay AST Aspartate aminotransferase
ALT Alanine aminotransferase
IM Intramuscularly
SC Subcutaneously
TBD Tick borne diseases
CGA Canine granulocytic anaplasmosis PBS Phosphate-buffered saline
8
INTRODUCTION
Ticks are transmitters of many diseases and can transfer canine tick-borne diseases (TBD) such as anaplasmosis, borreliosis and babesiosis. The geographical distribution of canine TBD has expanded in recent years and multiple diseases can be transmitted during tick sucking resulting in coinfections. This in turn has resulted in the importance of owner’s awareness of canine TBD and knowledge about their prevention [30].
Ixodes Ricinus is the vector of borreliosis and anaplasmosis and is well distributed throw-out Europe. Dermacentor reticulatus is the vector of babesiosis and has become a frequent canine TBD in Lithuania [6,27].
Babesia is the most important blood parasite in dogs and it was first described in Europe in 1895 in Italy. It causes haemolytic disease in dogs that varies from subclinical to multi-organ failure, and may result in death of the dog. Canine granulocytic anaplasmosis first occurred in dogs in 1982 in California. Most dogs that are infected with Anaplasma phagocytophilum are clinically healthy, and there is no documented case of death due to anaplasmosis. Lyme disease is a zoonotic TBD. Its disease in dogs remains controversial since the diagnosis rarely has shown to demonstrate the presence of the bacteria. Diagnosis is based on a positive titer with or without clinical signs. Only as little as 5 % of dogs develop clinical signs. A common clinical sign is shifting lameness/polyarthritis, and in rare cases (<2%) the development of Lyme nephritis may occur which is progressive and fatal [9,20,23,27].
Anaplasmosis and coinfection with Borrelia may result in higher likeliness of development of clinical signs and in a more severe clinical presentation. Confection with Babesia spp. is rarely reported and is poorly documented. Coinfections in TBD are of clinical importance since they may be more difficult to diagnose, worsen the clinical presentation and prognosis [9,21].
An acute-phase response is seen in dogs infected with B. canis, resulting in an elevation in CRP concentration in the blood. CRP concentration has shown to be important in human granulocytic anaplasmosis but so far there is only one report about CRP and anaplasmosis in dogs. The CRP was elevated in three dogs infected with Anaplasma phagocytophilum and may be of value in diagnosing the disease. In a human study of CRP concentration in Lyme borreliosis, the serum concentration was elevated in 86 % of the patients. The concentration decreased in the blood in response to treatment in one patient and may be of value in monitoring of the disease [5,36,38].
9 Aim: to compare clinical biomarkers, prevention, and courses of disease of tick-borne diseases in Lithuania and Sweden.
Objectives:
1. To evaluate biochemical and morphological blood parameters significant for Canine Tick-borne Diseases.
2. To determine the amount of acute phase proteins in infected canine blood during the process. 3. To compare knowledge of dog owners in Sweden and Lithuania.
10
1. REVIEW OF LITERATURE
1.1 Tick-borne diseases and their vectors in dogs
Tick borne diseases are a global emerging problem for both animals and humans. This has presented a need for interaction between the human medicine and veterinary sector to benefit the health of humans and animals as well as the global environment. To achieve this goal there are many challenges that needs to be met; developing appropriate diagnostic tests and surveillance systems, research the connection between reservoirs and pet animals, making owners aware of TBD and their prevention, and to promote the detection of TBD in both humans and animals [14].
Ticks are vectors of many bacterial, protozoal and rickettsial diseases that can be transmitted to dogs. The main TBD in dogs that have been described more frequently during the last years are babesiosis, anaplasmosis, borreliosis and ehrlichiosis. Ticks can transmit single infections or multiple infections that leads to co-infections. The kind of infection that is transmitted during tick feeding depends on tick specie and geographical region [30].
Dermacentor reticulatus is a hard tick and is the vector off Babesia canis. The tick has a high adaptiveness to different climate conditions. Experimental studies show that the tick can survive 4 years without feeding and withstand temperature from – 10 °C for 150 days. D. reticulatus has a high rate of development and a wide variety of hosts [31].
Ixodes ricinus is a hard tick and vector of Borrelia burgdorferi and Anaplasma phagocytophilum. The tick requires a relative humidity of over 80 % to survive without a host, restricting it to geographical areas from moderate to high rainfall [34].
It takes between 24-48 hours for transmission of TBD to occur. According to studies of animal models, transmission of Lyme borreliosis has shown to occur in less than 16 hours [19, 21, 23, 30, 32].
In recent years TBD have been described more frequently. This is believed to be because of increased pet travel, better animal care from pet owners and increased interest of clinical practitioners to investigate unusual clinical signs [30].
Canine babesiosis has become a frequent TBD in Lithuania. Canine babesiosis was previously not considered endemic in this area. The reason for this is believed to be because of the spread of its vector. D. reticulatus has spread to northern Europe countries such as Belgium, Netherlands, Germany, and Poland. Countries that previously were considered too cold for D. reticulatus to become established. The global warming has been reported as the most frequent cause of the spread of the vector. The spread of D. reticulatus in Europe according European Centre for Disease Prevention and Control, up to April 2017 is shown in figure 1 [6, 31,39].
11 Fig. 1. Geographical distribution of D. reticulatus based on the European Centre for Disease Prevention and control (ECDC) up to April 2017 [39].
I. ricinus can withstand temperatures of -10 ◦C for a short period of time. Thirty days of exposure to temperatures of -10 ◦C, kills most of the ticks. This is believed to be the reason of the northern limit in the tick’s distribution. A follow-up questionnaire-based study from Sweden showed that during the past 30 years I. ricinus has expanded north ways. Leading to increased risk of Lyme borreliosis in north of Sweden. The spread of I. ricinus in Sweden is shown in figure 2 [32,33].
12 Fig. 2. Distribution of I. ricinus in the first part of the 1990s (left) and 2008 (right) in Sweden [33].
1.2 Babesiosis
Canine babesiosis is caused by a haemoprotozoan that causes haemolytic disease in mammals. Babesia species has a worldwide distribution in the canine population. Their difference in geographical ranges depends mainly on its relationship with the vector specie. Babesia species are divided into large (3-5 μm) and small (0.5-2.5 μm) form depending on their merozoit size. B. gibsoni which is seen to cause canine babesiosis in Europe is of small form. B. canis is categorized into the large form with three subspecies: B. canis canis, B. canis rossi and B. canis vogeli. These subspecies could be considered 3 separate species since they differ in geographical distribution, vector specificity, pathogenicity, and genotype. Based on molecular analysis B. canis is the main Babesia specie in Lithuania [5-8].
Transmission of canine babesiosis occurs mainly through tick vectors, other means of transmission are dog fighting, blood transfusion or via congenital trans-placental transmission [5,9]. Ticks infect dogs during the transfer of infected saliva; sporozoites are released into the blood stream and infect red blood cells (RBCs). Inside the RBCs sporozoites develops into piroplasms through multiplication, 2 or sometimes 4 daughter cells. When the piroplasm leaves the RBC, haemolysis occurs. The released piroplasm start to infect other RBCs, and multiplication continues until the dog dies or the immune system stops it [10].
Dogs are most frequently presented with weakness/lethargy and anorexia. A study in Zambia of naturally occurring canine babesiosis showed that the most common clinical signs were the
13 presence of ticks, fever, pallor, and lymphadenopathy. The most frequent laboratory findings were anaemia and nucleated erythrocytes [11].
Canine babesiosis is classified clinically as either uncomplicated or complicated disease. Clinical signs of uncomplicated disease are related to haemolytic anaemia and hypoxia, and is further divided into mild, moderate, or severe according to the degree of anaemia. Complicated babesiosis involves abnormalities of body systems and may result in acute kidney failure, hepatopathy, disseminated intravascular coagulation, immune-mediated haemolytic anaemia, pancreatitis, myocardial dysfunction, rhabdomyolysis, acute respiratory distress syndrome and cerebral babesiosis [3,12].
Anaemia and thrombocytopenia are the major haematological abnormalities. The first days following infection the anaemia is mild, normochromic, and normocytic. As the severity of the disease progresses the anaemia becomes hypochromic, macrocytic, and regenerative. The reticulocyte count increases as the anaemia worsens. Leukocyte abnormalities vary and may include leucocytosis, neutropenia, neutrophilia, eosinophilia, leukopenia, or lymphocytosis. Severe thrombocytopenia is an invariable finding in canine babesiosis [5,12-14].
Increased serum urea and creatinine is not an uncommon finding and is an indication of renal failure. Increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), together with hyperbilirubinemia and hypoalbuminemia are the most common biochemical abnormalities. Icterus is only present in severe cases of canine babesiosis. Urinalysis may indicate the presence of renal tubular epithelial cells and granular casts, bilirubinuria, proteinuria and haemoglobinuria [12-16].
Blood smear examination is the simplest way to diagnose canine babesiosis. A blood smear is obtained from the capillary blood of either the ear or nail, since the concentration of the parasite is greater in the peripheral blood. B. canis can be seen under light microscope as a large pyriform shaped parasite that appears single or in pairs. Blood smear examination should not be used alone as it is of relatively poor sensitivity. Polymerase chain reaction (PCR) allows for identification of specie and accuracy of the diagnosis. Serological testing using enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence (IFAT) can be used for diagnosing canine babesiosis. These are quantitative methods and determine the antibody levels in the blood. A positive coombs test and auto-agglutination are often present in dogs infected with canine babesiosis [5,7,9].
Treatment is directed towards eliminating the parasite, treating anaemia and supportive care of the complications. Imidocarb dipropionate, Trypan blue and Diminazene aceturate are effective drugs against B. canis. Imidocarb dipropionate at a dose of 7,5 mg/kg once or 7 mg/kg twice intramuscularly (IM) or subcutaneously (SC) with 14 days interval, eliminates the infection [5,9,16].
14 Prognosis is generally good with 85-90 % survival rate after initiation of treatment. In case of development of acute renal failure, cerebral babesiosis or acute respiratory distress syndrome prognosis is guarded, and mortality may be greater than 50 % and sometimes reaching up to 100 %, despite treatment [16].
1.3 Granulocytotropic anaplasmosis
Canine granulocytic anaplasmosis (CGA) is caused by an obligate intracellular gram-negative bacterium called Anaplasma phagocytophilum. A. phagocytophilum is predominantly found in neutrophils and rarely, in eosinophils. The bacteria are between 0.2-0.4 μm in size and can be found inside the cytoplasmic vacuoles, forming inclusion bodies called morulae [5,17,18].
Natural infections occur predominantly by the mean of tick transmission in dogs. Other ways of transmission that is not described in dogs are through blood transfusion, direct contact with respiratory secretions or infected blood in humans, contact with infected carcass and experimentally trans placental transmission in cattle [5,18,19].
The pathogenesis of CGA is poorly understood. A. phagocytophilum is transmitted to the host through tick bites. Studies on human neutrophils showed that the bacterium attaches to the surface of the neutrophil with the help of P-selectin and enters the cell through endocytosis. Inside phagosomes the bacteria multiply through binary fission, leading to the formation of the morulae. The multiplication of the bacteria eventually leads to cell rupture and A. phagocytophilum is released. Newly formed bacteria can now enter and infect new neutrophils, escaping the hosts’ immune system by delaying neutrophil apoptosis enough to develop morulae [19,20].
Most dogs that get infected by A. phagocytophilum are asymptomatic. Clinical signs are more likely to develop in coinfections with B. burgdorferi. Dogs who develop clinical disease of CGA are most commonly presented with fever. Lethargy, depression, and anorexia are present in over 75 % of clinically ill dogs, and musculoskeletal pain in over half of the cases. Less commonly observed clinical signs are gastrointestinal upset, lymphadenopathy, splenomegaly, polydipsia, epistaxis, and central nervous system involvement. There are no documented cases of death due to CGA infection in dogs [5,18-22].
The most common haematological laboratory abnormality is mild to severe thrombocytopenia. Other frequent abnormalities are lymphopenia, neutropenia, neutrophilia, monocytosis and eosinophilia. Mild non-regenerative normocytic normochromic anaemia is seen in some infected dogs and IMHA has been reported in one dog diagnosed with anaplasmosis [5,18,20,21].
Biochemistry profiles may show decreased albumins, potassium and urea and increased ALP and total bilirubin concentrations [5,18,20].
15 There is no standardized method of diagnosing A. phagocytophilum in dogs. In human’s diagnosis can be made in three ways: (a) through suspected laboratory evidence of infection without clinical information; (b) when clinical signs are event together with laboratory and serological evidence or presence of morulae in blood smear; (c) evidence of a fourfold serological IgG-specific antibody titer, PCR or microbiological culture of the bacteria from the blood. Diagnosis of CGA should be done in similar way as for human anaplasmosis with some limitations to certain diagnostic methods [5,20].
FASTest ANAPLASMA is a commercial serological snaptest to detect antibodies (IgG) against A. phagocytophilum and A. platys in serum, plasma, or whole blood. Identifying morulae in peripheral blood smear is a cost-effective way of diagnosing anaplasmosis in endemic areas. Morulae appear in neutrophils only 4-8 days during the first 2 weeks post-infection, making it suitable as an early diagnostic method. PCR and serology (ELISA, IFAT) are needed for a definitive diagnosis. PCR is an accurate and specific way of diagnosing acute CGA. It should be kept in mind that PCR may show positive results in clinically healthy dogs because of a previous exposure, or negative results in a clinically ill dog because of the variation of circulating bacteria. A fourfold rise in titer is required for diagnosing acute CGA using PCR. Serology could be used 8 days post-infection, but is best used after 2-3 weeks, when enough IgG has been produced [18-22].
Doxycycline 5 mg/kg per os every 12 hours for 14 days is the treatment of choice. After administration, dogs usually show improvement after 24-48 hours. Other antibiotics such as rifampicin and fluoroquinolones may also be sufficient [5,18-21].
Prognosis is good, and full resolution of clinical disease can be expected after initiation of treatment. If unresponsive to treatment, alternative diagnosis should be considered. Most dogs are asymptomatic and clear the infection themselves without any treatment. There is on-going research if dogs can have chronic infections, but the disease seems to be self-limitating [5,18, 20].
1.4 Borreliosis
Canine borreliosis is also called “Lyme disease” or “Lyme borreliosis”, and is caused by spirochete bacteria belonging to the Borrelia burgdorferi sensu lato complex. Several genospecies belongs to this complex, the most common specie to cause borreliosis in dogs in Europe is B. burgdorferi senu stricto [5,23,27].
Transmission occurs the most frequently through tick bites. Transmission by urine, blood, milk, semen or trans placentally are unlikely in natural infections [24,25,27].
According to experimental studies the incubation period of B. burgdorferi is between 2-6 months in dogs. The bacteria enter the host through the tick saliva during feeding. The bacteria express surface proteins that allow dissemination of the organism in the tissue and escape of the
16 hosts’ immunity. B. burgdorferi replicates at the site of tick bite and either migrate through the dog’s skin and connective tissue or disseminate via the blood. Later this leads to colonization of several tissues, like the joints [5,25,27].
Most dogs infected with borreliosis are asymptomatic, and only about 5 % of infected dogs develop clinical signs. The clinical signs are acute and unspecific; fever, apathy, anorexia, shifting leg lameness/polyarthritis and lymphadenopathy. In seropositive dogs, lameness has been reported in 9-28 %. The lameness is usually acute, at first affecting one limb for several days and then affecting another limb. Dogs develop a red, small lesion at the site of tick bite that disappears within a week. Lyme nephritis refers to a severe protein loosing, immune complex nephropathy and renal failure that can develop in severe disease and is usually fatal. Neurological and cardiac involvement has been reported in dogs with borreliosis but is rare [5,23-26].
Usually there are no haematological or biochemical abnormalities. In rare cases of development of Lyme nephritis haematological or biochemical abnormalities may show non-regenerative anaemia, thrombocytopenia, hypoalbuminemia, hyperphosphataemia, hypercholesterolemia and azotaemia. Urinalysis may show decreased ability to concentrate the urine, glycosuria, haemoglobinuria, bilirubinuria, haematuria, casts, and active sediment. Synovial fluid aspirate shows increased protein content and inflammatory cells, most commonly neutrophils, with negative bacterial culture [5,23,25,27].
Diagnosis is made from clinical signs, ruling out differential diagnosis, positive serological testing, or PCR. Since the bacterial concentration in tissue and fluids is low, bacterial culture is usually time consuming. Dark-field microscopy is required to visualize the organism. SNAP tests; SNAP-4Dx plus test from IDEXX are available for the detection of antibodies against B. burgdorferi. This test should not be used solely for the diagnosis of canine borreliosis since it only reveals exposure to the organism and not clinical disease. Both clinically healthy dogs and clinically ill dogs can be seropositive for B. burgdorferi [5,23-25,27].
Doxycycline or amoxicillin are the antibiotics of choice, and clinical improvement is seen after 24-48 hours. Many authors recommend treatment for at least 30 days, but based on research; clearance of B. burgdorferi from the body after 1 month of treatment is unlikely and relapse may occur. The disease is believed to be self-limiting in dogs and positive titer may remain for months to years. In experimental studies, chronic borreliosis showed persistence of clinical signs after treatment with antibiotics and another course of treatment was needed. Doxycycline is most
17 frequently used because of the high possibility of coinfections with other organisms (Anaplasma and Ehrlichia spp.). Treatment of seropositive dogs that are clinically healthy is controversial. An argument for treating seropositive dogs is to prevent clinical disease to occur, because of the long incubation period [5,24,25].
If the dog develops renal manifestation, prognosis is guarded, since the disease usually is progressive regardless of treatment [5].
1.5 Borrelia and co-infection with Anaplasma and Babesia
Ixodid ticks can in endemic areas carry both B. burgdorferi and A. phagocytophilum, and dogs can be infected simultaneously and produce antibody titers against both bacteria. Dogs that carry both infections are more likely to show clinical signs than single infected dogs. The consequence of coinfections in dogs are unknown, experimental studies on mices has shown more severe clinical signs and a more likelihood of expressing clinical disease, than with single infections. In humans, co-infections result in a more complex and severe illness. A study from 2001 showed that 40 % of dogs that were seropositive for B. burgdorferi where also positive for A. phagocytophilum [20,24,25,28,29].
Dogs get infected with B. canis and B. burgdorferi via multiple tick exposures. Co-infections with Babesia and other species are not well documented and rarely reported. Dogs that experience co-infections have a more complicated clinical picture, which makes diagnosis more difficult. A dog that is unresponsive to treatment, live in endemic area and show atypical clinical signs should be considered for co-infections with other infectious organisms. A thorough anamnesis, specific clinical signs and laboratory abnormalities are important for making a correct diagnosis [9,21,29].
1.6 Tick prevention
Prophylaxis is important to prevent transmission of disease and should begin before the tick season starts which is different in different regions depending on climate. There are 4 ways of prophylaxis; (1) tick repellents (acaracide products), (2) vaccinations, (3) chemoprophylaxis (drugs against the pathogen) and (4) behaviour prophylaxis, by avoiding high-risk areas [27].
Acaracide compounds have different properties; some has killing effect on ticks, some repellency or expellency, others have anti-feeding affect or disruption of the attachment of the vector. A study showed that the most efficient way to prevent TBD is to prevent its attachment. Vaccinations exist against borreliosis and babesiosis. Borrelia vaccination has been a subject of dispute over the last years since clinical borreliosis in dogs is rare. There are two types of vaccinations against babesiosis, one against B. canis (Pirodog®) and another against B. canis and B. rossi (Nobivac Piro®) [5,9,25-27].
18
1.7 Acute phase response and proteins in TBD
The innate immunity serves to protect the body and initiating the acquired immune response. The acute phase response (APR) is seen with all animal species. An APR is a complex systemic reaction to tissue damage in form of acute and chronic inflammation, infection, neoplasia, stress and/or trauma. In response to any of previous stimuli, damaged tissue and activated inflammatory cells releases pro-inflammatory cytokines, including TNF- α, IL-1 and IL-6. These cytokines play an important role in inducing an APR, by activating hepatocytic receptors. IL-6 is the most important cytokine in the hepatic activation and thus responsible for most of the APP production. TNF- α and IL-1 both have anti-inflammatory activity acting on muscle cells metabolism and affecting the amino acid uptake, together with glucocorticoids [1,2].
An APR results in the production of acute phase proteins (APP), some APP will increase in serum concentration, called positive APP, and others will decrease in serum concentration called negative APP [3].
A down-regulation of the APR is achieved by cytokine removal by the hepatocytes; kupffer cells secrete IL-10 that results in suppression of IL-6 [1].
APP are produced in response to different stimuli, and rises in the blood before clinical manifestation is noticed. APP are markers of inflammation and its magnitude correlates with the extent of the disease. They do not point to a specific disease but can help in patient monitoring as well as predicting patient mortality and morbidity. APP include complement proteins, coagulation proteins, protease inhibitors, transport proteins, ceruloplasmin and other proteins like Serum amyloid A (SAA), C- reactive protein (CRP), acid glycoprotein (AGP) and serum amyloid P (SAP), etc. There are major, moderate, and minor APP. Major APP, are positive APP that increase in plasma/serum concentration 10 to 100 folds within hours after primary stimuli. Moderate APP are those that increases 2-10 times their normal concentration and minor APP less than 2 times their normal value. Albumin is a major negative APP in all animals and it is especially significant for bacterial infections [3,4,35].
CRP and SAA are the major APP in dogs, whereas Fibrinogen, Haptoglobin and AGP are considered the moderate APP. CRP is synthesized in response to pro-inflammatory cytokines mainly by the hepatocytes, but also by other cells like monocytes, Kupffer cells, lymphocyte subsets and alveolar macrophages. During an APR, the main function of CRP is to recruit the mononuclear cells and complement system to the site of inflammation. It is a sensitive marker of inflammation in humans and increases rapidly, 100-1000-fold, within 24-48 hours post stimulation. As for humans, CRP increases in canine inflammatory conditions such as acute pancreatitis, babesiosis, pyometra, surgical trauma, immune mediated haemolytic anaemia, malignant tumours and more. CRP can be measured in whole blood, serum, saliva, and effusions. It can be measured
19 using different methods including ELISA, immunodiffusion, electro immunoassay, turbidimetric immunoassay and time-resolved immunofluorometric assay [4,35,37].
1.8 CRP in TBD
CRP is more specific and sensitive than the total white cell count (WBC). Serum concentration can rapidly increase from less than 1 mg/L to over 100 mg/L. CRP can be used to monitor the response to treatment, and is especially useful in diseases that are prone to relapse. In Trypanosoma brucei infection CRP decreases in response to treatment but serum levels increase again as the disease relapses. An infection that shows a significant rise in CRP is canine babesiosis and is one the diseases where an APR has been described. A study of dogs infected with B. canis showed that the plasma CRP concentration rises before clinical signs and before the detection of the parasite in the blood. The amount of days before the CRP could be detected in blood depended on the infectious dose; those that received the highest dose showed detectable levels 2 days post infection compared to the low dose that was detectable after 4 days post infection. Dogs with B. canis rossi infections have low total protein (TP), albumin, α –globulin and albumin/globulin ratio. Infections with B. canis also show elevated CRP, SAA, and AGP. A study on coinfections of B. canis and E. canis showed that hyperglobulineamia is more common in dual infections than in single infections alone. Dogs infected experimentally with B. canis showed a moderate increase in CRP together with moderate decrease in serum albumin concentrations. CRP has not shown to predict the outcome in dogs infected with B. canis rossi [3,4,5,13].
Three cases of CGA diagnosed by positive PCR together with morulae detection in blood smear, all showed elevated levels of CRP. CRP can increase ten-folds in acute anaplasmosis and may be of value in making a diagnosis [27,36].
CRP in Lyme borreliosis is not well described in dogs. In humans a study was made of 14 patients suffering from borreliosis, and in 86 % of the patients the CRP was abnormally high [38].
20
2. RESEARCH METHODS AND MATERIAL
2.1 Sample collection
Haematological, biochemical, and serological results where collected from dogs (n=33) infected with anaplasmosis, borreliosis or both at a Swedish veterinary clinic, Läckeby Djursjukhus (and its branches) between 2013-05-13 and 2017-07-26. Ten dogs where diagnosed with borreliosis, eleven with anaplasmosis and twelve dogs with coinfection. From three of the dogs result from serial CRP concentration where collected. Some of the dogs (n=30) were collected from the clinics database.
Eight dogs that where presented with clinical signs of babesiosis at a veterinary clinic in Lithuania, Dr. L Kriaučeliūnas Small Animal Clinic during spring 2017 were involved in the investigation. Blood samples for haematology, biochemstry and a blood smear were collected from these dogs.
Clinical examination and patient history was collected from patients that arrived at the clinics. More focus was directed towards the most commonly observed clinical signs and temperature of the animal.
Dogs that came to the clinic infected with TBD had a wide range of age from 1-11 years old. The most frequent age of dogs were four years (n=8) old followed by seven, three and nine. There where no difference between the number of males and females. 19 males (four neutered) and 22 females (three neutered) where presented to the clinics. The most commonly presented dog breed was crossbreed (n=10), followed by Bernese mountain dog (n=5) and Golden retriever (n=5). Dogs used in the study are presented in table 1.
21
No. Disease Gender Age
A1. Anaplasma M 5 A2. Anaplasma M 3 A3. Anaplasma FN 7 A4. Anaplasma F 10 A5. Anaplasma M 5 A6. Anaplasma M 8 A7. Anaplasma MN 3 A8. Anaplasma F 3 A9. Anaplasma F 6 A10. Anaplasma F 2 A11. Anaplasma M 7 B12. Borrelia FN 9 B13. Borrelia F 4 B14. Borrelia F 8 B15. Borrelia MN 4 B16. Borrelia M 6 B17. Borrelia F 9 B18. Borrelia M 5 B19. Borrelia F 3 B20. Borrelia MN 8 B21. Borrelia F 2 C22. Coinfection F 4 C23. Coinfection F 7 C24. Coinfection F 6 C25. Coinfection F 7 C26. Coinfection M 9 C27. Coinfection FN 11 C28. Coinfection MN 4 C29. Coinfection M 10 C30. Coinfection M 4 C31. Coinfection M 9 C32. Coinfection M 1 C33. Coinfection F 4 B34. Babesia F 9 B35. Babesia M 1 B36. Babesia M 7 B37. Babesia F 1 B38. Babesia F 3 B39. Babesia F 7 B40. Babesia F 4 B41. Babesia M 4
Table 1. Dogs used in the study. M= Male, MN= Male Neutered, F= Female, FN= Female Neutered.
22
2.2 Diagnosis of TBD
Indirect immunofluorescence (IFA) was used to diagnose anaplasmosis and borreliosis. Serum was collected from dogs infected with anaplasmosis and borreliosis or both. The titer starts at 1:80 and ends at 1: 1 280, a final titer was made on the owner’s request. Serum was kept in the fridge until time of analysis. IFA slides (MegaScreen Fluoanaplasma ph. and MegaScreen Fluoborrelia from MegaCor) specific for A. phagocytophilum and B. burgdoreferi sensu lato are kept in the freezer and warmed until room temperature before analysis. Phosphate-buffered saline (PBS) and FITC anti-dog IgG conjugate is prepared and stored in the fridge.
Dilutions are prepared with dog serum and PBS (1:40, 1:80, 1:160, 1:320, 1:640 and 1:1 280). Commercial positive and negative controls are available and 1 drop (20 μl) of each are placed gently on the slides. 20 μl of the dilutions are each placed on a separate antigen wall. The slide is incubated at 37 °C for 30 min. Next the slide is carefully washed in PBS solution and left in a PBS- bath for 5 minutes. The slides are rinsed with deionized water and left to dry. 20 μl of the conjugate is added to each antigen wall and incubated at 37°C for 30 min,2 and rinsed as previously described. Some drops of Mounting medium are placed onto the slide and is evaluated under a Leitz fluorescence microscope using 400 x magnification. The results were positive if a clear green fluorescence were observed [40].
Babesiosis was diagnosed using blood smear analysis. Capillary blood was collected from the ear pinna. The blood was directly placed onto a glass slide and spread with a second glass slide. The blood smear was left to air dry and stained with Quickdiff. Diagnosis was made while observing the intra-erythrocytic piroplasm with light microscope at 100x.
Anaplasmosis was diagnosed using blood smear analysis (n=3). EDTA- blood was used to make a blood smear. The blood smear was stained with Hemacolor® and analysed under light microscope using 100x. Diagnosis was made observing morulae inside granulocytes (neutrophils, eosinophils).
2.3 Determination of haematological and biochemical parameters
Haematology of babesiosis was determined in the clinic in Lithuania using IDEXX LaserCyte Dx Haematology Analyser. Blood from the cephalic vein and collected into EDTA tubes. The sample was analysed within 10 min. Biochemical analysis of babesiosis was analysed in the clinic for four cases of Babesia using Spotchem EZ SP-4430. CRP from all dogs infected with Babesia and biochemical parameters from four dogs was analysed in the Biochemistry department of LSHU. Samples were collected from the cephalic vein, between 1-5 ml of whole blood into serum tubes. The serum sample was left to coagulate at room temperature for at least 30 min, before it was spun in the centrifuge at 3500 for 10 minutes. The serum was separated from the blood and collected into
23 plain tubes that were frozen down to -18 oC until sample analysis. The serum was analysed using
Dialab automatic biochemistry analyser.
Haematology of anaplasmosis and borreliosis was determined using different analysers like IDEXX Procyte Dx Haematology Analyser and Medonic. Blood was collected from the cephalic vein and collected into EDTA tubes. Biochemical analysis was analysed from plasma, blood collected from the cephalic vein was collected into heparin tubes and spanned in the centrifuge to separate the plasma from the blood. The analysers used were Cobas c111, IDEXX Catalyst Dx Chemistry Analyser and Fuji. CRP was determined using LifeAssays® Canine CRP Test or Cobas c111 analyser, using plasma and Dialab automatic biochemistry analyser using serum.
2.4 Questionnaire and statistical analysis
The questionnaire was conducted in April-June 2017 and can be seen in Annex 1. Questionnaire of Survey. Dog owners from Sweden and Lithuania were asked 11 questions, 4 basic questions and 7 specialized. The reason for limited questions was to encourage dog owners to participate. The questionnaire was made using eSurvey Creator and posted into several dog groups on the Internet. In Lithuania 99 dog owners participated and in Sweden 377 dog owners. Dog owners had the opportunity to skip questions they did not want to answer to or choose several answers. Answers were collected and analysed using Microsoft Excel 2011.
The statistical analysis was made using Microsoft Excel 2011. Median, maximum, minimum and average values were calculated. Diseases were compared to each other and ANOVA single factor was used to calculate P-Value. P-value was considered statistically significant <0.05. Correlation was calculated to evaluate the relationship between the parameters. Standard deviation was calculated for some of the parameters.
24
3. RESEARCH RESULTS
3.1 The comparison of the main haematological and biochemical parameters
Haematological and biochemical parameters were measured at admission for all dogs with TBD. Their median, maximum and minimum results are presented in the table 2. The importance of the parameters is explained in the right paragraph. These are the most important haematological parameters for the described diseases.Anaemia (a red blood cell count below reference values) is present in Anaplasmosis and babesiosis. Anaemia was present in 62.5 % of dogs infected with Babesia and in 27 % of the cases of anaplasmosis. Coinfection with Borrelia did not cause anaemia in any of the cases. 55% of the cases of anaplasmosis had lymphopenia and 33 % of coinfections. Thrombocytopenia was another frequent laboratory finding and is explained in fig 7.
Biochemical parameters were compared in various TBD and are shown in table. 3. ALP was above reference values for Anaplasma in 18 %, Borrelia in 20 % and Coinfection in 8 %. ALP was increased in 50 % of the cases of babesiosis. ALT was within normal values in all cases of coinfection and anaplasmosis. It was above reference values in 10 % of borreliosis and in 12.5 % of babesiosis. UREA was within normal values in all cases of anaplasmosis and borreliosis and above reference values in 75 % of the cases of Babesia and 8 % of coinfection. CREA was below reference values in 8 % of coinfection. CREA was within reference values in all cases of anaplasmosis and borreliosis. CREA was above reference values in 14 % of babesiosis and 8 % of coinfection. CREA was below reference value in 8 % of coinfection. Hyperglobulinemia was a frequent laboratory finding in anaplasmosis, borreliosis and coinfection and was present in 58 % of the cases.
25 25 TBD/ Parameter Reference values Anaplasma (n=11) Borrelia (n=10) Coinfection (n=12) Babesia (n=8) Importance RBC (x1012/L) 5.65-8.87 5.50-8.5 6.81 (5.54-8.91) 5.08 (2.53-6.18) 6.93 (5.68-7.28) 6.39 (6.09-6.98) 6.63 (5.69-7.51) 6.34 (5.74-6.75) 5.26 (4.06-9.47) Babesia, n=5 Anaplasma, n=3 HCT (%) 37.3-61.7 37-55 43.5 (36.6-56.7) 35.35 (15.4-39.3) 44.1 (36.4-47.4) 44.7 (39.4-49.4) 45.6 (36.8-49) 41.05 (33.5 – 43.3) 33.7 (26.4-71.3) Reticulocytes (K/uL) 10.0-110.0 35.9 (8.3-130.1) 26.9 32.05 (15.4 – 60) 47.4 (23.8 – 116) WBC (x109/L) 5.05-16.76 5.5-16.9 6-17 6.51 (5.21-11.74) 11.67 (5.98 -17.36) 6.9 (5.9-7.9) 10.45 (7.16-18.02) 10.94 5.6-19.24) 11.9 (6.93- 21.32) 8.22 (6.07 – 10.37) 12.25 (10.1 – 15.8) 9.13 (3.14 – 34) NEU (x109/L) 2.95-11.64 2-12 5.09 (4.16.8.7) 7.43 (2.88-11.97) 7.81 (4.21-16.06) 8.05 (6.91 -16.33 5.6 (2.8-17.21) 5.18 (4.43-5.92) 2.73 (1.75 – 15.4) Lymph (x109/L) 1.05-5.10 1.2-5.0 0.5-4.9 0.72(0.46-2.37) 0.45 (0.4-0.5) 2.5 (2.4-2.59) 1.8 (1.19-2.26) 1.75 (1.5-2.22) 1.92 1.05 (0.8-1.5) 1.46 (0.68-2.24) 1.04 (0.37-7.2) Anaplasma, n=6 Coinfection, n=4 Eos (x109/L) 0.06-1.23 0.1-1.49 0.08 (0-1.21) 0.805 (0.31-1.3) 0.28 (0.02-0.4) 0.28 (0.1-0.71) 0.42 (0.02-1.55) 0.57 (0.35 – 0.79) 0.15 (0.1 – 1.94) PLT (k/ul) 148.0-484.0 200-500 175-500 27 (0-252) 65 (33-97) 260 (191-330) 223 (16-436) 275 (136-415) 303.5 (136-415) 182 (17-285) 239 (4-318) 300.5 (263-338) 61 (7-715) Anaplasma, n= 6 Babesia, n=5
Table 2. Median haematological results from various TBD. Maximum and minimum results in brackets of each parameter. Reference values according reference laboratory.
26 26 TBD/ Parameter Reference values Anaplasma (n=11) Borrelia (n=10) Coinfection (n=12) Babesia (n=8) Importance ALP (U/L) 3.0-88.0 23.0-212.0 1-114 * 60(32.1-125) 114.5 (59-189) 56.9 (9.9-146.6) 155 (49-192) 32.4 (15-101.3) 87 (59-98) 117 (21.5-804) Babesia, n=4 ALT (U/L) 9.0-96.0 10.0-125.0 10.0-100.0 40.4 (37-78.4) 33 (21-58) 75 44 (30-95.4) 119 (106-132) 53.5 (39-68) 27.3 (18.9-90.2) 72.5 (29-116) 43 (34-46) 54.9 (37.5-172) Urea (mmol/L, g/L, mg/dl) 2.7–8.7 mmol/L 2.5–9,6 g/L 8–28 mg/dl * 5.23 (3.28-8.56) 4.2 (3.1-5.4) 5.26 (3.79-5.38) 4.45 (2.5-7.9) 4.32 (2.42-8.87) 3.5 (2.8-6.2) 41.45 (32.2-335) Babesia, n=7 Creatinine (μMOL/L) 42-110 44-159 70 (50-106.1) 95.5 (81-129) 78.5 (57.5-92.3) 119 (76-153) 69.6 (37-104.2) 97 (62-174) 97 (83-765) TP (G/L) 49.0-71.0 52.0-82.0 54-75* 67 (60.4-72.5) 67.5 (64-69) 65.35 (60.2-76.3) 75 (69-78) 67.8 (56-76.3) 71 (67-79) 60.2 (53.9-88) Albumin (G/L) 30-45 23.0-40.0 22-39 34 (27-36.9) 30.5 (29-32) 37.75 (33.2-42.4) 29.5 (29-30) 30.5 (28-33) 33.2 (18.5-39.6) 33 (30-36) 32 (29-32) Globulin (G/L) 19.0-26.0 25.0-45.0 33 (30.1-36) 37.5 (36-40) 26.15 (22.3-42.4) 50 34.2 (25.7-42.7) 39 (31-49) CRP (mg/L) 0-10 81.5 (19-177) 63.3 (05-202) 59 (14-210) 27.3 (0-31.1) Anaplasma, n=11, Borrelia, n=5, Coinfection, n=12, Babesia, n=5
Table 3. Median biochemical results from various TBD. Maximum and minimum results in brackets of each parameter. Reference values according reference laboratory. * [42].
27 Anaplasmosis is known to cause thrombocytopenia and lymphopenia in dogs. The comparison of the incidence of thrombocytopenia and lymphopenia in anaplasmosis and in coinfection with Borrelia is shown in fig. 5. Thrombocytopenia and lymphopenia was present in 16.7 % of the cases in coinfection and in 55 % of the cases in anaplasmosis.
Fig 5. Incidence of lymphopenia and thrombocytopenia in dogs infected with coinfection (left) and Anaplasma (right). Reference value PLT < 148 K/uL, reference value lymphocytes < 1.05 x 109/L. (Coinfection: P>0.05, correlation 0.3. Anaplasma: P<0.05, correlation 0.9).
Globulins were measured in nine dogs infected with Anaplasma, seven dogs with Borrelia and eight dogs with coinfection. Results of all dogs are shown in fig. 6. Globulins were measured with two different analysers and their reference values are according reference laboratory. Globulins were above reference values in 58 % of the cases. In anaplasmosis globulins were above reference values in 56 % of the cases, compared to Borrelia, which was increased in 57 % of the cases. In coinfection globulins were increased in 63 % of the cases.
28 Fig. 6. Globulin concentrations in dogs infected with Anaplasma (A), Borrelia (B) and Coinfection (C). Reference value (blue) = 19-26 g/L, reference value (red) = 25-45 g/L
Platelet count of seven dogs infected with Anaplasma, six dogs with Borrelia, six dogs with coinfection and eight dogs with Babesia were compared. Their median, maximum and minimum platelet count is shown in Fig. 7. Anaplasma has a low median platelet count while coinfection with Borrelia is unlikely to cause thrombocytopenia (in 25% of cases). Babesia caused thrombocytopenia in 75 % of the cases and Anaplasma in 55%. Borrelia caused thrombocytopenia in 30% of the cases.
29 Fig. 7. Median PLT count in dogs infected with various TBD. Anaplasma and Borrelia reference value = 148.0-484.0 K/uL, Coinfection reference value = 200-500 K/uL, Babesia reference value = 175.0-500.0 K/uL.
3.2 The analysation of CRP results
CRP was measured in 39/41 dogs infected with TBD. The changes of CRP concentration (mg/L) for a period of time are shown in fig. 8. The CRP of the dog B15 infected with Borrelia had been measured at the 1st, 2nd, and 3rd and the 5th day. Treatment against borreliosis was given at day two and response to treatment is seen at day five when the concentration has dropped to 21.9 mg/L. The CRP concentrations of the dog A1 infected with Anaplasma were measured through three continuous days. Treatment against anaplasmosis was given at day three. The CRP concentrations of the Dog A4 infected with Anaplasma were investigated at day 1,3 and 20.
Treatment was initiated at day three when the CRP concentration was >210 mg/L. A follow-up test was performed at day 20 and concentrations where found to be within normal limits. Anaplasma shows higher peaks of CRP concentration compared to Borrelia, also the plasma concentration of CRP is higher in anaplasmosis than in borreliosis.
30 Fig. 8. Concentration of CRP during a serial of days in one dog infected with Borrelia and two dogs infected with Anaplasma. CRP reference value = 0-10 mg/L
Median, maximum, and minimum concentrations of CRP in dogs’ blood infected with TBD are shown in fig 9. Anaplasma has the highest median and minimum concentration of CRP, all cases where above the reference values (0-10 mg/L). Infection with Borrelia showed a wide range of CRP concentration in the blood. CRP was below the reference values in 40 % of the cases. Coinfections showed an increased median, maximum and minimum CRP concentration. Coinfection had the highest CRP concentrations of all TBD. Babesiosis shows an increased median CRP concentration. Babesia had the lowest median, average and maximum concentration of CRP. 29 % of the dogs had a CRP concentration below the reference values.
31 Fig. 9. Average concentration of CRP in dogs with various TBD. CRP concentration in dog’s blood with reference value 0-10 mg/L. (p<0.05) a, and b, are statistically significant from each other.
Lymphopenia was a frequent finding in dogs infected with Anaplasma. The correlation between CRP and lymphocytes is shown in fig. 10a. In all the cases lymphopenia was accompanied by an increased CRP concentration. Anaemia was a frequent finding in babesiosis and the correlation between. The correlation between CRP and RBC count is shown in fig. 10b. Anaemia together with increased CRP was present in 43 % of the cases.
Fig 10. The correlation between CRP and lymphocytes in eleven dogs infected with Anaplasma
(a). Reference interval CRP= 0-10mg/L and Lymphocytes < 1.05 x 109/L. R2= 0.44(left).
Correlation between RBC count and CRP in seven dogs infected with Babesia (b). Reference
interval CRP= 0-10mg/L and Lymphocytes < 1.05 x 109/L. RBC 5.5-8.5 x10^12/L. Anaplasma:
32 Correlation between temperature and CRP of dogs (n=31) infected with various TBD is shown in fig11. Fever is defined as a rectal temperature > 39.2 °C. As shown by the figure the more the temperature increases, as does the CRP concentration. In two cases the dogs had fever, but the CRP concentration was within normal limits.
Fig. 11. The correlation between CRP concentration and temperature in dogs infected with various TBD. CRP reference value = 0-10 mg/L. Temperature reference value = 37.8-39.2 °C. P<0.05, correlation 0.4.
3.3 Clinical manifestation of TBD
Data about clinical signs and clinical parameters where collected from dogs infected with TBD. The most commonly observed clinical signs at admission are seen in fig. 12. Apathy was seen in all cases of anaplasmosis, babesiosis and coinfection but only in half of the cases of Borrelia. Anorexia is seen most frequently in babesiosis and anaplasmosis.
Polyarthritis was seen in 70 % of dogs infected with Borrelia and in 67 % of the dogs with coinfection. Apathy seen in coinfection is likely because of infection with Anaplasma and polyarthritis because of Borreliosis. Most frequent presentation is polyarthritis in dogs infected with Borrelia. Vomiting and diarrhoea is an uncommon clinical sign of TBD, although most frequently seen with dogs infected with babesiosis.
33 Fig. 12. Most commonly observed clinical signs in various TBD. V/D = Vomiting and
diarrhoea.
Temperature was measured in 30/41 dogs and their average, maximum and minimum temperature can be seen in Fig. 13. Dogs infected with anaplasmosis have the highest average, maximum and minimum temperature at admission. Dogs infected with Borrelia have the widest range of temperature from 38.0-40.5 °C. Normal rectal temperature of the dog is 37.8-39.2 °C. Dogs infected with babesiosis and anaplasmosis on an average has fever on admission to the hospital. Coinfection is unlikely to cause fever in dogs with 92 % of the dogs having a rectal temperature < 39.2 °C.
34 Fig. 13. Average, maximum, and minimum temperature at admission in various TBD. n=30. Normal rectal temperature of the dog is 37.8-39.2 °C. (P<0.05)
Diagnosis of Borrelia was established with serology in all ten cases. Nine dogs had titer of >1280, and one of 320. One dog had an end titer of 2560. Anaplasma was diagnosed with serology in 7/11 cases, four dogs had titers >1280 and three dogs had a titer of 640. Three dogs were diagnosed using blood smear identification and one dog was diagnosed by the blood picture (thrombocytopenia, lymphocytopenia, eosinophilia, increased ALP and CRP and response to treatment). Coinfections where all diagnosed by serology. 7/12 cases had titers of >1280 of both Anaplasma and Borrelia, two cases had titers of Anaplasma of 640 and >1280 for Borrelia. One dog had a titer of 320 for Anaplasma and >1280 for Borrelia. One dog had a titer of 160 for Anaplasma and >1280 for Borrelia, while another dog had a titer of 160 for Borrelia and >1280 for Anaplasma. Babesiosis where all diagnosed using blood smears.
3.4 The analysis of the questionnaire of dog owners about prevention from TBD
The questionnaire was an electronically based survey with 11 questions to dog owners in Sweden and Lithuania. The aim of the questionnaire was to compare the knowledge of dog owners in Sweden and Lithuania, to find out the owner’s awareness of TBD, their knowledge about the vector, their usage of preventative drugs and the most common way of administration of tick-preventative drugs. All answers to the questionnaire can be seen in Annex 2. Answers to questionnaire.Amount of people that use tick prevention in Sweden and Lithuania is shown in Fig. 14. 99 dog owners answered the question in Lithuania and 376 in Sweden. More dog owners in
35 Lithuanian use tick prevention compared to Sweden. Owners were asked what kind of tick preventative drug they used, and the answer is shown in fig X. In Sweden dog owners used tablets the most and in Lithuania spot-on solutions. In Sweden 295 dog owners answered the question and in Lithuania 89.
Dog owners in Sweden and Lithuania were asked what kind of TBD they knew about and the results are shown in fig. 15. In Sweden most dog owners know Borrelia while in Lithuania most knows Babesia. In Sweden 371 dog owners answered the question and 97 in Lithuania.
Fig. 14. Amount of people (%) that use tick preventative drugs in Sweden and Lithuania (left), type of tick preventative drug used in Sweden and Lithuania (right). (p<0.05)
36 Figure 16 shows the relationship between age and usage of tick preventative drugs in Sweden and Lithuania. 377 dog owners participated in the question in Sweden and 98 in Lithuania. In Lithuania age 41-50 used the most tick preventative drugs while age 51-61 in Sweden. In Lithuania 51-61 was the age group that used the least tick preventative drugs while in Sweden it was age <20.
Figure 16. The relationship between age of owner and the usage of tick preventative drugs in Sweden and Lithuania. (p<0.05)
37
4. DISCUSSION OF RESULTS
There was no difference between the amount of females and males infected with TBD in this study. There were more intact females and males than neutered dogs. There has been shown that neutered dogs were less likely to be infected with B. rossi than intact dogs [9].
Dogs infected with babesiosis are most commonly presented with fever, apathy, and anorexia. The dogs infected with Babesia in this study had an average rectal temperature of 39.6 degrees, which is the same as the average rectal temperature of dogs (n=363) in a study on babesiosis in dogs infected in Zambia. All dogs where diagnosed with blood smear examination and it is an easy way to diagnose babesiosis. Serology together with PCR is a good tool for diagnosing infection. PCR is helpful in confirming diagnosis when blood smear is not sensitive enough [6,7,11].
The most common haematological abnormalities in dogs infected with Babesia were anaemia and thrombocytopenia, which in both cases was present in 62.5 % of the dogs in this study. The most frequent biochemical abnormalities were elevated ALP, urea, and CRP. Mildly elevated liver enzymes may be found in dogs infected with babesiosis together with an increased CRP. An elevation in urea is not an uncommon finding on dogs infected with babesiosis. CRP was increased in 71.4 % of the dogs infected with babesiosis, and the CRP concentration was significantly (P<0.05) lower compared to dogs infected with Anaplasma, Borrelia or both. CRP levels are shown to be higher in dogs with complicated babesiosis than in uncomplicated, and the elevation might be related to the severity of the disease. The results showed that there was no statistical significance (P>0.05) or correlation between RBC and the amount of CRP in the blood in dogs infected with babesiosis. Anaemia together with an increased CRP concentration was present in 43 % of the cases in this study. The severity of the disease is related to the amount of haemolysis due to parasitic replication inside the RBC. As both the amount of anaemia and amount of CRP is related to the severity of the disease, this may indicate that the digs in the study was not suffering from severe babesiosis [5,7,12,16,43].
Dogs infected with anaplasmosis all showed lethargy and fever at admission. Other common clinical signs were anorexia and gastrointestinal signs (9%). Anaplasmosis had statistically significantly (P<0.05) higher body temperature (median, minimum and maximum) than the other TBD. The most commonly observed haematological abnormalities were low RBC, lymphocytes and PLT. Thrombocytopenia was a common laboratory finding in was present in 54.5 % of the dogs. It was more common in anaplasmosis than in any of the other TBD (P<0.05).
38 Thrombocytopenia and lymphopenia was present in 55 % of the dogs (P<0.05). While in coinfection with Borrelia it was present in 16.7 % of the cases (p>0.05). There was no statistically significance of thrombocytopenia and lymphopenia in coinfections. There was a strong correlation (r=0.9) between thrombocytopenia and lymphopenia in anaplasmosis. In all cases where lymphopenia was present, CRP concentration in the blood was elevated (P<0.05). In a study of three dogs infected with anaplasmosis all showed lymphopenia together with a CRP concentration above reference value. In two of the dog’s thrombocytopenia and lymphopenia was present and in the other dog the PLT count was unevaluable. Further studies with larger sample sizes are required to evaluate the importance. All dogs in this study were diagnosed using serology (IFA) and a positive titer was considered as infected. In the same study all three dogs where diagnosed using blood smear, PCR, and serology. They were all positive for blood smear and PCR but only one dog had a positive titer on serology. Suggesting some dogs might go under diagnosed with the use of serology solely [19,21,27].
The most commonly observed clinical sign in dogs infected with borreliosis was polyarthritis (70%). Usually polyarthritis is reported in 9-28 % of the seropositive dogs. Other common clinical signs were fever and apathy. In this study there was no specific haematological or biochemical parameters, although CRP was increased in 50 % of the dogs. Since the lack of studies on CRP in canine Lyme disease this might be used as an additional tool in diagnosis [23].
Dogs with coinfection are more likely to develop polyarthritis than in single infection with Borrelia. In this study there where almost as many dogs in coinfection as in borreliosis that developed polyarthritis. Dogs were less likely to have fever in coinfection than in single infections alone. The most important haematological parameter for coinfection was lymphopenia (33 %). When dogs that has a titer for Borrelia and shows thrombocytopenia and/or lymphopenia they are most likely caused by coinfection with Anaplasma. Important biochemical parameters were increased CRP concentration (100%) and hyperglobulinemia. Dogs with coinfection had a lower average plasma concentration than dogs with single infections (p<0.05) [5,22].
Hyperglobulinemia is a frequent finding in dogs infected with anaplasmosis. Hyperglobulinemia was also present in borreliosis [5,19].
Serial CRP was measured in two dogs with anaplasmosis. In one dog (A4) response to treatment can be seen at day 20. In another dog infected with Borrelia (B1), response to treatment can be seen at day 5. Treatment was given at day two, a rise in CRP can be seen the day after treatment and may be due to that response to antibiotics can be seen in 24-48 hours. In dogs infected with Trypanasoma brucei and Leishmania infantum CRP decreased in dog’s blood in response to treatment and rises again when relapse occur [3].
39 In one dog infected with Anaplasma (A1) a drop in CRP concentration is seen the day before initiation of treatment and may suggest the clearance by the dogs’ own immune system due to the self-limitation of the disease [20].
There was no correlation between CRP and body temperature in TBD (r=0.4). When comparing the CRP concentration and degree of temperature there was a higher CRP concentration in dogs with fever (P<0.05) [37].
There were more dog owners in Lithuania that used tick-preventative drugs that in Sweden (P<0.05). When dog owners in Sweden were asked why they did not use tick-preventative drugs they said that they were afraid it would hurt their dogs. While Lithuanian dog owners answered other reason, followed by that their dogs never get ticks. The most commonly way to administer tick-preventative drugs in Sweden were tablets and in Lithuania it was spot-on solution (P<0.05). Dog owners were asked who recommended them this treatment and dog owner in both countries answered the Veterinarian. Concluding that Veterinarians have influence on dog owners in their choice of prevention in Sweden and Lithuania. Lyme disease is a well-known TBD is Sweden while Babesiosis is a well-known disease in Lithuania. Few dog owners new about babesiosis in Sweden, since the disease not yet exists among the dog population. Few dog owners in Lithuania knows about anaplasmosis. In Sweden people aged 51-61 used the most tick-preventative drugs, which was the age that used the least tick-preventative drugs in Sweden. In Lithuania age 41-50 used the most tick preventative drugs and in Sweden age 20-30 used the least tick-preventative drugs.
40
CONCLUSIONS
1. Apathy is the most commonly observed clinical signs in TBD. Polyarthritis is a frequent observed clinical sign in Borrelia as in coinfection with Anaplasma. Fever is a common finding in Borrelia, Anaplasma and Babesia.
2. RBC, LYMP and PLT are the most important haematological parameters in TBD. GLOB and CRP are the most important biochemical parameters for Anaplasma, Borrelia and Coinfection, while ALP, UREA and CRP are the most important for Babesia. CRP may be used as an indicator of Borrelia.
3. Lithuania uses more tick-preventative drugs than Sweden. Spot-on is the most popular drug administration in Lithuania while in Sweden it is tablets. Dog owners are well aware of Borrelia and Babesia, while Anaplasma and TBE are less known TBD in dogs.
41
SUGGESTIONS/RECOMMENDATIONS
There are several suggestions that could be made to this research to improve the reliability of the results. One of the most important factors would be the incidence of concurrent diseases of dogs infected with TBD, which may alter the blood results and physical exam.
The amount of dogs used in each TBD is unequal which makes it more difficult to compare them. With an equal number of each group of disease and a higher number of patients the results would be easier to compare.
Haematological and biochemical parameters were made with different analysers since tests were performed in different clinics. This makes the diseases more difficult to compare, the same analyser should be used for all blood samples using the same reference values.
Different veterinarians have diagnosed the disease and performed clinical examination together with a thorough anamnesis. This may alter the details of recording the clinical signs and physical examination.
Methods for diagnosing TBD influence the likelihood of disease. A high titer with serology just indicates exposure of the organism; it does not mean that the dog has the organism present in the body. PCR together or alone would be a more accurate diagnosis of TBD.
42
ACKNOWLEDGEMENT
I would like to express my deepest gratitude to everyone who has made this work possible. To the Swedish small animal hospital Läckeby Djursjukhus for letting me collect data about TBD and to Dr. L Kriaučeliūnas Small Animal Clinic for letting me collect blood from dogs infected with babesiosis.
I am very thankful to all people who took their time to answer the questionnaire about TBD in Sweden and Lithuania.
Furthermore, I would like to devote my gratitude to my supervisor Assoc. Prof. Vaida Andrulevičiūtė for all the guidance and suggestions during this research work. To the
Biochemistry department at the Veterinary Academy of Lithuanian University of Health Sciences for letting me use their laboratory and using necessary material and analyzers.
