LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY. Faculty of Veterinary Medicine. Arianna Saidi

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY Faculty of Veterinary Medicine Arianna Saidi GALVIJŲ ŪKIŲ TYRIMAS DĖL PARATUBERKULIOZĖS LIETUVOJE IR KONTROLĖS PLANO PARENGIMAS INVESTIGATION OF TWO DAIRY CATTLE FARMS IN LITHUANIA WITH SUSPECTED INFECTION OF PARATUBERCULOSIS FOR A DEVELOPMENT OF A CONTROL PLAN MASTER THESIS of Integrated Studies of Veterinary Medicine Supervisor: Ass. Prof. Alvydas Malakauskas

2 THE WORK WAS DONE IN THE DEPARTMENT OF PATHOBIOLOGY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK I confirm that the presented Master Thesis " INVESTIGATION OF TWO DAIRY CATTLE FARMS FOR PARATUBERCULOSIS AND DEVELOPMENT OF A CONTROL PLAN 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. (date) (author s name, surname) (signature) CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE LITHUANIAN LANGUAGE IN THE DONE WORK I confirm the correctness of the Lithuanian language in the done work. (date) (author s name, surname) (signature) CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES (date) (supervisor s name, surname) (signature) THE MASTER THESES HAVE BEEN APPROVED IN THE PATHOBIOLOGY DEPARTMENT (date of approbation) (name, surname of the manager of department/clinic) (signature) Reviewers of the Master Theses 1) 2) (name, surname) (signatures) (date) Evaluation of defence commission of the Master Theses: (name, surname of the secretary of the defence commission) (signature) 2

3 TABLE OF CONTENTS SUMMARY... 4 SANTRAUKA... 5 ABBREVIATIONS... 6 INTRODUCTION LITTERATURE REVIEW Paratuberculosis the rising problem in cattle Epidemiological situation a) Worldwide b) Europe Detection of paratuberculosis Control strategies METHODOLOGY Research workload, location and method Collection of samples Diagnostics RESULTS DISCUSSION OF RESULTS CONCLUSIONS ACKNOWLEGEMENTS REFERENCES

4 SUMMARY INVESTIGATION OF TWO DAIRY CATTLE FARMS IN LITHUANIA WITH SUSPECTED INFECTION OF PARATUBERCULOSIS FOR A DEVELOPMENT OF A CONTROL PLAN Arianna Saidi Master Thesis Paratuberculosis is a disease that is important in the cattle industry due to the huge economical losses and its chronic nature, which makes it very difficult to detect in time. The aim of this study was to obtain epidemiological data of paratuberculosis for a development of a control plan/strategy for each of the farms. This scientific work was based on the collection of blood samples obtained from two farms in Lithuania farm X and farm Y. Farm X with previous clinical cases of paratuberculosis and farm Y with no previous cases of the disease. Sampling was performed twice in farm X, May 2016 and October-November During the first sampling, blood was collected from 30 cows considered to be of a high-risk group and during the second sampling, blood was collected from 56 dry cows. In farm Y, a total of 28 blood samples were collected from cows of a high-risk group. The serum of the collected blood samples was analysed in an enzyme-linked immunosorbent assay to detect possible antibodies against Mycobacterium avium subsp. paratuberculosis. Obtained results from the testing on farm X gave us an apparent prevalence of 8.9% CI (95%, ). In farm Y, no seropositive animals were found. 4

5 SANTRAUKA GALVIJŲ ŪKIŲ TYRIMAS DĖL PARATUBERKULIOZĖS LIETUVOJE IR KONTROLĖS PLANO PARENGIMAS Arianna Saidi Master Thesis Paratuberkuliozė yra labai svarbi galvijininkystės problema sukelianti reikšmingus ekonominius nuostolius. Ši liga pasižymi lėtine eiga ir dėl to sudėtinga ligos sukėlėjo diagnostika ankstyvose ligos stadijose. Šio darbo tikslas atlikti klinikinius ir serologinius tyrimus galvijų ūkiuose ir remiantis gautais duomenimis parengti šios ligos kontrolės planus. Kraujo mėginiai buvo renkami dviejuose ūkiuose X ir Y, kurie sutiko dalyvauti tyrime. Ūkyje X iki tyrimų buvo pastebėti keli klinikiniai paratuberkuliozės atvejai, o ūkyje Y nebuvo. Ūkyje X mėginiai buvo rinkti du kartus 2016m. gegužės ir spalio mėn. Pirmąjį kartą 30 mėginių buvo paimta iš karvių pirklausančių rizikos grupei. Sekančio tyrimo metu buvo paimti 56 mėginiai iš užtrūkusių karvių. Ūkyje Y birželio mėnesį buvo surinkti 28 mėginiai iš rizikos grupės karvių. Mėginių rinkimom metu buvo atliekama klinikinė apžiūrą paratuberkuliozės atžvilgiu. Mėginiai tirti inmunofermentinės analizės metodu siekiant nustatyti antikūnus pries Mycobacterium avium por. paratuberculosis. Pirmajame ūkyje buvo nustatytos karvės turinčios antikūnus ir tai sudarė 8.9% CI (95%, ) galimą paplitimą bandoje. Antrajame ūkyje antikūnų nebuvo rasta. Ūkiuose įvertinta biosauga ir bandos valdymas paratuberkuliozės kontrolės atžvilgiu. Pasiūlyta strategija abiems ūkiams dėl paratuberkuliozės kontrolės ir prevencijos. 5

6 ABBREVIATIONS LUHS - Lithuanian University of Health Sciences MAP Mycobacterium avium subspecies paratuberculosis ELISA - Enzyme-linked immunosorbent assay PCR Polymerase chain reaction OIE World Organisation for Animal Health CVO Chief Veterinary Officer CI Confidence interval OD Optical density 6

7 INTRODUCTION Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis. It can occur in different animal species, both ruminants and non-ruminants, but the disease is mainly associated with the economic losses and adverse effects in dairy and beef-cattle industry [1]. The true herd prevalence among European countries varies a lot, for example, in the Netherlands 31-71% are affected, Denmark 47%, Belgium 18%, and 70% in the UK [2]. Many countries are nowadays participating in different types of control programs. Lithuania has unfortunately no official control program at the moment, but a general infectious disease program is available for breeding herds. This means that the farms that are dealing with breeding activities are encouraged to do regular MAPtesting. In Lithuania, the prevalence is considered low, but no survey is carried out to establish the exact prevalence percentage [3]. That is why I found it interesting to investigate dairy cattle farms in Lithuania. Control programs are mainly implemented for early detection of arising infections in a herd, but also to determine that a herd is free from the disease or to evaluate the prevalence of the infection in a herd. Detection of paratuberculosis is quite difficult because of the chronic nature of the disease and accurate tests do not exist due to the tedious pathogenesis. First of all, it is important to determine if the herd is infected or not. This can be performed by strategic sampling from either the environment or from a group of cows that is further analysed either through bacteriological sampling using culture or PCR or through immunological analysing. This type of testing gives an estimation of the herdlevel diagnosis, which indicates on whether the infection is present in the herd or not. When a herd is diagnosed or suspected to have the disease, bacteriological or immunological analysing can be performed at animal level. There are numerous tests available, and no testing strategy is considered to be better than the other. All kinds of testing methods might give false-positive results, this is really important to be aware of when dealing with individual testing. It is also important to decide the outcome of the testing before performing it, what actions are the farmers willing to take if the results will be positive? At first, paratuberculosis control programs were based on vaccinations, but nowadays they focus more on the biosecurity within the farm and restriction of movement of infected animals. When reading about control programs in different countries I noticed that they are designed differently depending on: 1) A lower priority in comparison to other, more significant infectious diseases 7

8 2) Economical limits within the country 3) Low prevalence of the disease The challenging part of controlling paratuberculosis is, as previous mentioned, the chronic nature of the infection. Not all animals are showing clinical signs during their lifetime even though they might be shredders. There is a lack of accurate testing methods to detect infection in its early stages. There is also a quite poor understanding and use of the diagnostic tests in a purposeful structure. Furthermore, the MAP-infection has a long survival in the environment and the motivation of the farmers is usually low due to whether the control plan is profitable for the farm or not [4]. The motivation for controlling the disease can be different depending on what the farmers find most important. Reduced animal welfare due to the illness such as diarrhoea and emaciation is one of the biggest concerns, as well as the economic losses due to the drop of milk yield of infected cows. One of the main factors that needs to be taken into account when following a control program is to decrease the transmission of MAP from infected to non-infected animals. Another important factor is to detect animals before they become clinically affected. It is also important to monitor the prevalence through annual testing, to easier control the disease. Therefore, the aim of the present study is to achieve knowledge about paratuberculosis and obtain epidemiological data for the specific farms that I have been working at for this thesis, to be able to develop a control plan. To achieve the aim of the study, the objectives are as follows: 1. To investigate two farms using clinical examination, collection of blood and perform serum-elisa in order to provide data on the prevalence of M.paratuberculosis 2. To investigate the present management system on the farms and to assess the critical control points for the transmission of M.paratuberculosis on the farms 3. Based on the obtained ELISA results and established critical control points, prepare a control plan for the investigated farms 8

9 1. LITTERATURE REVIEW 1.1 Paratuberculosis the rising problem in cattle Mycobacterium avium subsp. paratuberculosis is a small, Gram-positive, acid-fast and facultative bacterium that belongs to the Mycobacterium avium complex [5]. MAP causes chronic, contagious, granulomatous enteritis in ruminants, also called Johne's disease [6]. This may be confusing because of the word disease which indicates that the infection always causes a clinical disease. The clinical disease could for example be diarrhoea, but the MAP infection might exist in the herd at high rates and result in production losses without any cases of diarrhoea or other clinical signs [7]. It is widespread around the world in both domestic and wild animals, and is considered as one of the most serious diseases in dairy cattle. The infectious agent is excreted in a low amount through colostrum and milk and in much higher amounts in the faeces of affected animals. It is very resistant to factors of the environment and can survive on pasture and bedding for more than 1 year. MAP is also both highly resistant to heat treatment and chemical agents. The bacteria is commonly acquired in young cattle through ingestion of contaminated milk or through environmental contamination. Another common route of infection is through the placenta of an infected pregnant cow to the foetus. Shedding occur before the animal is showing any clinical signs of the disease [8, 9]. This means that a lot of animals have a subclinical infection, which results in adverse effects such as infertility or a lower milk yield than normal [10]. The animals are infected early in life, often directly after birth, but the clinical disease rarely occur in cattle less than 2 years old [6]. A Danish survey revealed that cows that are showing a clinical disease rise with age, and that the incubation period can vary from some months up to a lifetime of an animal [8]. Fig. 1 Pathogenesis of MAP-infection according to age [7]. Generally the symptoms appear in 3 to 5 year old animals, with the onset of diarrhoea, poor milk yield and weight loss. This is typically following calving or other stressful situations like sale, 9

10 transportation, etc. [11]. It is important to note that the first onset of clinical signs have been recognized in 6 months old animals as well as 15 year old animals [12]. It is possible to categorize the MAP-infection into 4 stages based on the symptoms. Animals that are infected with paratuberculosis who does not show any clinical signs and are undetectable through the diagnostic tests are in stage 1. Animals that show an immune response in the diagnostic testing and are shedding a low level of MAP, but enough to infect other animals are in stage 2. These animals can remain in this stage for the rest of their life, or develop to a stage 3 if the infection gets triggered of other factors such as stress that makes the bacteria to emerge. Stage 3 and 4 refer to those animals that develop the clinical disease with the common symptoms of diarrhoea and weight loss. These two stages involves only a minority of animals, the majority remain subclinical. The estimated amount of infected animals in stage 1 and 2 are 25 cows for every cow that is showing clinical signs, an event called the 'iceberg effect' [13, 14]. 1.2 Epidemiological situation a) Worldwide MAP is mainly affecting ruminants, such as cattle, sheep, goats or deer, both domestic and wild. Johne's disease is more common among dairy cattle than beef cattle. The higher the animal density is on the farm, the higher the amount of contact with the infectious agent and the ability to get infected [15]. Johne's disease have been reported in every country that has animal husbandry and laboratory diagnostic availability. It is estimated that approximately over 50 % of all dairy cattle herds in Europe and North America are infected with MAP [5]. Paratuberculosis disease is listed by the OIE, which means that it is a priority disease when talking about the international trade market. As this disease typically is introduced to a herd through animals with a subclinical infection that looks healthy for the producer, it is an important issue when purchasing new animals to a farm [6]. To assure that no infection will occur through trading, herd-level testing should be performed [4]. Year 1996, it was estimated that 3-10% of U.S. dairy cattle were infected with MAP. A similar estimation was performed year 2007 that show an increase to 68% [15]. In Japan, it was decided year 1998 that all dairy farms must participate in a compulsory testing program every 5 th year according to the Act on Domestic Animal Infectious Disease Control. Around 1000 cows of animals in total are diagnosed with paratuberculosis in Japan annually [16]. 10

11 Australia was one of the first countries to develop and apply a national control program initiated in 1996 against paratuberculosis. All herds are annually categorized by the Chief Veterinary Officer (CVO) into 4 different groups: 1) Non-assessed herd (NA), which is a herd with no previous history of MAP or with no suspicion of infection 2) Suspected herd (SU), a herd that is suspected for numerous reasons 3) Infected herd (IN), is a herd with infected animals 4) Restricted herd (RD), is a herd that previously was an IN-herd, but is presently participating in a control program under the supervision of the CVO Herds that are participating in the Australian MAP control program have increased a lot throughout the years. When the control program first was introduced, 180 herds were participating. In December 2003, as many as 1623 herds were registered in total [17]. In Canada, the seroprevalence at the animal level in dairy cattle farms ranged from 1.3% to 7.0%, and 9.8% to 40.0% at the herd level in 2006 [18]. The Canadian control program is divided by multiple provincial programs independent of one another. They include producer education and herd risk assessment/testing [19]. Fig. 2 Epidemiology of paratuberculosis worldwide [4]. b) Europe In Denmark, a voluntary control program was established in 2006 with the purpose of acting as a tool for farmers to control infections and as an intention to reduce the prevalence of the disease in the country. A minority of 23% of all dairy farmers participated in the program by the mid of The herds that were participating tested all lactating cows 4 times per year by using milk-elisa. The 11

12 obtained results of the testing are mainly used to assess the risk management of the infectious animals. The control program is intended to be followed for 6 to 8 years, and a surveillance component might be added at a later stage of the program [20, 21]. In Germany, the seroprevalence was estimated to 84,7% year All clinical cases in the country have to be reported and registered by the German authorities, without any consequences for the infected animals or herds. Guidelines have been released and suggests hygienic measures and control of paratuberculosis for reduction of the clinical cases and prevention of transmission [22]. In the Netherlands, it was estimated that there were around 4 million cattle in About 54% were considered positive for paratuberculosis. In 2006, a voluntary control program was introduced, which in 2008 was agreed to be paid by the Dutch Dairy Board. This resulted in 80% participating herds from the whole country [22]. In Norway, there are approximately cattle and an incidence of 10% of paratuberculosis positive animals. A national surveillance and control program was introduced in 1996 which includes all dairy cattle herds and beef herds that are receiving state support [22]. In Lithuania, the prevalence is considered low, but no survey is carried out to establish the exact prevalence. There is no official control program at the moment, but a general infectious disease program is available for breeding herds. This means that the farms that are dealing with breeding activities are encouraged to do regular MAP testing [3]. 1.3 Detection of paratuberculosis A combination of ELISA-analysis and faecal culturing is the most effective way of diagnosing MAPinfections. Testing is mainly performed of cattle older than 36 months of age [2]. The first approach is to detect cows with diarrhoea or other typical symptoms of infection by clinical observation and after that target all suspected animals. ELISA-analysis can thereafter be performed and show whether the diarrhoea is caused by MAP or not. A positive result is an indicator of infection. Decreased milk yield might also be a consequence of MAP, it is proved that cows that were ELISA-positive had a reduced milk production over a period of days [7]. The animals that are MAP-positive could be confirmed by faecal culture analysis. Faecal culture is not very ideal, firstly there is only a small amount of the positive animals that are actually shedding amounts of MAP large enough to detect trough faecal culture, secondly, the culturing takes around 2-3 months to perform because of the slow growth of the infectious agent [7]. The testing and analysing is giving us different categories of sick 12

13 animals that requires a system to easier interpret and maintain the classification in practice. Cows that are negative could for example be considered as green, low-risk cows, and the positive ones are considered as high-risk cows. They could further be classified into yellow or red depending on if they are high or low shredders. Cows that were tested and got positive results repeatedly are considered to be high-shredders and are therefore red. The yellow cows are on the other hand the cows that have been tested with a positive result recently but have not got positive results repeatedly as the red cows [7]. This type of classification strategy is very successful, it cannot eliminate the infection but it can reduce the prevalence of MAP, which is a very important key element of a control program. 1.4 Control strategies The control strategies of MAP-infection can be plenty, different countries have developed a lot of variations among the control programs. The only country in the world that is considered to be free from paratuberculosis is Sweden. The control program in Sweden was introduced year 1998 and was based on annual faecal sampling of all cattle older than 24 months. During the first year, the samples were cultured individually. The second year, samples were pooled three by three (except the samples from imported animals or new animals purchased to the herd). From the third year, samples were pooled five by five (except the samples from imported or new animals purchased to the herd). The herds that were participating in the program were only allowed to introduce new animals to their herds from other herds within the same category. The animals were divided in 3 different categories: A tested negative 5 times B tested negative 3-4 times C tested negative 1-2 times By the end of 2001, 774 herds were included in the program. A total of 189 herds were in category C, 485 herds had reached category B and only 100 were in category A. In 2007, only one herd was MAP-positive. In this herd, all animals were culled, the building was disinfected and specific restrictions decided by the responsible authorities were established for the farm [23]. Another control program that is more cost effective and easier to maintain than the Swedish, is the one designed by the Netherlands. It consists of 3 diagnostic screenings, the first screening is performed for the classification of the herd. The second screening is performed for surveillance of the 13

14 herds that were tested negative. And the third one is performed for the control of those herds that were tested positive. The classification is based on individual serum-elisa of all animals older than 3 years, or individual milk-elisa of all lactating animals, not depending on age [24]. The negative herds are monitored through individual serum-elisa every second year of all animals older than 3 years or individual milk-elisa of all lactating animals once every second year. The positive herds have 3 different options of continued testing: 1) Individual serum-elisa of all animals over 3 years performed annually 2) Individual milk-elisa of all lactating animals annually 3) Individual faecal culturing of all animals over 2 years once every second year When these strategies were introduced in 2005, it implied to result in a significant stabilization of the MAP-infection if combined together with other control measures such as closing the herd and culling sick cows together with their last born calves. A closed herd is the key component for this type of control program, which is relying a lot on how effective the herd management is. The Dutch control program costs around to per litre milk and is very successful if all parts are strictly followed [24]. Some other European paratuberculosis control programs that are interesting to mention is Denmark, Norway and Germany. In Denmark, the control program was introduced year 2006 when the prevalence of MAP was quite high in the country and a lot of farmers experienced economic losses due to this. The program was and still is voluntary. The only thing that is regulated is that the farms are not allowed to deliver milk from diseased cows, and trade cannot take place without testing for MAP-infection. The idea of Danish control program was inspired by the eradication plan for bovine tuberculosis, also known as the Bang Method. It is based on testing performed 4 times per year with milk-elisa where all animals with positive results are considered shredders of infection. Some farmers make a confirmatory testing with faecal culturing, but this is not obligatory. Farmers are recommended to cull those animals that have had repeatedly high test levels accompanying a clinical disease, or those animals that are a few positive among a herd of negative cows. Animals with positive results are divided into red and yellow cows, none of them are allowed to provide milk for calf feeding purposes. Red cows are not recommended to calve, yellow cows are allowed to calve but only if kept in a separate area from the green cows. The yellow cows calving area have to be a single pen that is cleaned and disinfected after every calving. Tested animals with negative results are considered to be green and are not infectious, these cows are good for production of colostrum in a colostrum bank. Green cows are only allowed to be kept together with other green 14

15 cows, calve and not have as strict biosecurity measures within their facility as the red and yellow cows [25]. Depending on what the testing results are, the animals are further divided into 6 different groups: 0 negative result 2 times or more 1 negative result once 2 the previous results were negative but the recently obtained result is positive (the animal is considered as/to have: infected, very infectious, affected, production losses) 3 the last 4 results were negative but the recently obtained result is positive (the animal is considered as: infected, infectious) 5 the previous results were positive, but the recently obtained result is negative (the animal is considered as: infected, infectious) 9 the previous 2 or more results are positive (the animal is considered as/to have: infected, very infectious, affected, production losses [25]) In Norway, a control program for MAP-infection was introduced in The first 2 years, testing was only performed of animals that were imported or had been in contact with imported animals. Interpretation of testing was carried out in various ways; serology, histopathology, faecal culturing or bacteriological sampling from target organs [26]. After 2 years it was decided to include animals that had not been in contact with imported animals as well. Diagnostic methods by serology were used to firstly detect the infectious status of herds and secondly, carry out a confirmatory test which then could show whether the animals with positive results were true-positive or false-positive due to environmental mycobacteria. This resulted in a decision of a randomized national survey based on serological analysis mainly due to the low chance of detection of an infected herd and the high amount of false-positive results. The herds that were suspected to have or were diagnosed with MAPinfection had to follow strict measurements regarding sale, common pasture and manure deposit. The present paratuberculosis surveillance program in Norway relies more on faecal culturing than serological analysing due to the false-positive results. Collection from the 5 oldest animals of a herd in 50 random herds are performed annually for faecal culturing. The infected animals are usually culled, but there are no rules regarding the culling [26]. 15

16 In Germany, the government published recommendations for paratuberculosis control in The guidelines were mainly introduced to reduce the amount of clinical cases of paratuberculosis and reduce the economic losses that occurred due to this. Guidelines are based on hygienic measures, monitoring measures and measures for the preparation of a nationwide control program [27]. Hygienic measures include most importantly handling of young animals, colostrum management and restocking/purchasing of new cattle. Monitoring measures includes clinical cases of infected animals, serology, faecal culturing and bacteriological sampling from target organs. The different states made different amount of effort for these sort of guidelines, several states made cross-section studies to get an overview of the present situation of MAP, some states put no effort into this at all, so the situation varies a lot within the country [27]. Although no nationwide control program has been set up yet, different attempts have been performed at a regional level for monitoring and controlling MAPinfection. The most important one is the control program of Lower Saxony which was initiated This program depends on screening of all animals older than 2 years with ELISA and a follow-up verification test with faecal culturing annually during 5 years. Measures that are obligatory to follow by the farmers are culling of positive animals, calf management, purchase of infectious-free animals and restricted movement of animals from herds with positive test results [27]. In countries that does not have any compulsory control programs, the control of the disease is quite costly, and it is often very difficult to convince farmers to participate. The incubation time of MAP is long and there are usually very few cases of clinical diseases in growing animals. There are some studies that indicates on that the animals infected with MAP are more prone to diseases like mastitis and infertility, but these evidences are not enough for the farmers [26]. Estimations have been made of the economical losses that can occur when a MAP-infection is present on the farm. One clinically infected animal costs over 1000 for the farmer (excluding the costs for sampling and culling). But unfortunately this is not convincing enough, since a control program for paratuberculosis takes time, effort and a lot of changes in the biosecurity and arrangements within the farm [28]. The benefits will not be visible until many years after the instalment of the control program measures. A farmers choice is preferably the current state of affairs, rather than losses that might occur due to changes on the farm, even though the losses might result in something more beneficial in the future for the farm itself. This way of thinking is a business-related psychological phenomenon called 'behavioural economics'. There has to be a professional type of communication between the veterinarian and the farmer, and the control program has to be affordable and have a great chance of success to attract and convince farmers to participate [28]. 16

17 2. METHODOLOGY 2.1 Research workload, location and method This thesis work was carried out in the Veterinary pathobiology department of LUHS in Kaunas, Lithuania. The farms that were included in this study had animals that were suspected to have an MAP-infection, no previous testing had been carried out on the farms before. Due to the low milk prices in 2016, only two farms agreed to collaborate. The first farm (X) had 560 milking cows and the second farm (Y) had an amount of 900 cows in total. The first sampling was performed in May 2016 at farm X where 30 samples were collected in total. Second sampling was performed in October-November 2016 when a total of 56 samples were collected. In farm Y, sampling was only performed once in May 2016, and included the collection of 28 blood samples. Both farm X and Y are keeping the animals in loose-housing conditions. On each of the farms data was obtained of the routine management of healthy and sick animals and biosecurity measures were controlled. 2.2 Collection of samples The most desirable option would have been to test all animals in both farms, but due to the difficult economic situation among dairy cattle farms none of them agreed to that. Therefore, first sampling on both farms was performed based on high-risk animals with one or more signs known as a possible risk factor for MAP-infection. The risk factors of animals infected with MAP could for example be: drop in milk yield, high somatic cell count, mastitis, wasting or diarrhoea. It was aimed to collect minimum 28 blood samples from each farm. Sample size was decided according to the least amount of samples required that needs to be obtained to get at least one positive result (if the prevalence of the disease is suspected to be around 10% on the farm) [30]. Blood samples were collected from high-risk cows older than 36 months. The second sampling in farm X did differ from the first since the strategy was changed to only collect blood from dry cows. Blood was obtained from each animal in the coccygeal vein using aseptic technique. Around 5-6 ml of blood was collected with a needle and a vacutainer into serum collecting tubes. The blood was coagulating for 3 hours before 10 min centrifugation at the speed of 3500x. Prepared serum was stored in a freezer at -20 o C until the ELISA-analysis could be performed. 17

18 Table 1. High-risk cows from farm X investigated in this research. Cow no. Clinical signs Age 339 High somatic cell count 3 years 370 High somatic cell count 4 years 573 High somatic cell count 4 years 20 High somatic cell count 5 years 48 High somatic cell count 3 years 57 High somatic cell count 5 years 93 High somatic cell count 3 years 106 High somatic cell count 5 years 108 High somatic cell count 3 years 129 High somatic cell count 3 years 2 High somatic cell count 4 years 171 High somatic cell count 4 years 210 High somatic cell count 4 years 254 High somatic cell count 4 years 270 High somatic cell count 3 years 411 High somatic cell count 3 years 530 High somatic cell count 4 years 632 High somatic cell count 3 years 672 High somatic cell count 3 years 653 Repeated problems with mastitis 4 years 173 Repeated problems with mastitis 3 years 140 Repeated problems with mastitis 4 years 43 Repeated problems with mastitis 4 years 78 Repeated problems with mastitis 3 years 121 Repeated problems with mastitis 3 years 134 General weakness 4 years 198 General weakness 4 years 642 General weakness 4 years 645 General weakness 3 years 18

19 Table 2. High-risk cows from farm Y investigated in this research. Cow no. Clinical signs Age 176 High somatic cell count 3 years 168 High somatic cell count 3 years 432 High somatic cell count 3 years 17 High somatic cell count 3 years 289 High somatic cell count 4 years 290 High somatic cell count 4 years 65 High somatic cell count 3 years 301 High somatic cell count 4 years 345 High somatic cell count 5 years 306 High somatic cell count 5 years 419 High somatic cell count 4 years 543 High somatic cell count 3 years 597 High somatic cell count 3 years 332 High somatic cell count 5 years 90 Repeated problems with mastitis 3 years 782 Repeated problems with mastitis 4 years 590 Repeated problems with mastitis 3 years 322 Repeated problems with mastitis 5 years 349 Repeated problems with mastitis 4 years 387 Repeated problems with mastitis 3 years 765 Repeated problems with mastitis 3 years 632 Repeated problems with mastitis 5 years 608 Repeated problems with mastitis 5 years 247 Repeated problems with mastitis 3 years 25 General weakness 3 years 6 General weakness 3 years 579 General weakness 4 years 19

20 2.3 Diagnostics The diagnostic test that we used to analyse the collected samples was IDEXX M. phlei screening ELISA serum paratuberculosis-kit, and composed of 7 steps: 1) The antigen was first added to the wells of the plate 2) The centrifuged serum from the collected blood samples was then added to the wells (primary antibody) 3) The wells were washed to remove excess antibodies and to prevent non-specific binding 4) The test antibody was then added to the wells (secondary antibody) 5) The wells were washed to remove the excess antibodies that remained unbound 6) A substrate was thereafter added to the wells, the substrate makes the colorimetric tag of the secondary antibody to change colour which indicates a positive result 7) The absorbance was then measured with a computer program that evaluated the optical density of the wells The sensitivity of the ELISA-test is 55%-78%, and the specificity is 99%. The test results are divided in 5 categories negative, suspicious, low positive, positive and high positive. Negative = 45% meaning that the antibodies for MAP were not detected and the animal is either not infected or is in an early undetectable stage of infection. Suspicious = > 45% to < 55% meaning that the amount of antibodies are above the normal background levels. This animal might be in the early stage of infection, and is 5-15 times more likely to be infected than the one with the negative test result. Positive = 55%, meaning that the animal has moderate serum level of antibodies against MAP [29]. Statistical analysis was performed using online epidemiological calculations: 20

21 3. RESULTS There were a total of 560 cows on farm X, of those were 240 heifers and 365 calves. Blood samples were collected from 30 cows in May The selected animals were considered as high-risk animals for MAP which means that they had to be between 3 to 7 years old, have had typical problems related to MAP-infection such as repeated problems of high somatic cell count or mastitis and drop in milk yield, were affected by lameness or were generally depressed. 19 from the highrisk group had problems with high SCC, 6 had repeated problems with mastitis and 5 were generally weak animals. Table 3. OD-values of the first testing in farm X. Cow number No. 632 No. 573 No. 645 OD 166,9 (positive) 52,3 (positive) 119,8 (positive) Age 3y 8m 4y 2m 3y 11m This gave an apparent prevalence of 10% CI (95%, ). Due to financial constrains, the farm refused to test all animals in the herd, instead the decision was made to test only dry cows before calving. During October-November, 56 dry cows were tested in farm X, 5 of them got a positive result and one of them suspicious. These results gave an apparent prevalence of 8.9% CI (95%, ) (table 2). Table 4. OD-values of the second testing in farm X. Cow No. 603 No. 326 No. 280 No. 681 No. 294 No. 241 number OD 200,96 (positive) 111,6 (positive) 62,36 (positive) 128,6 (positive) 161,55 (positive) 49,54 (suspicious) Age 5y 4y 6m 6y 2m 7y 3m 5y 1m 3y 4m 21

22 Total amount of animals Posi/ve animals 10 0 First tes/ng Second tes/ng Fig. 3 Diagram of the testing performed during the whole study period in farm X. During the study period a total of 86 animals were tested in farm X where 8 were found positive, giving us an apparent prevalence of 9.3% CI (95%, ). MAP prevalence on farm X Total amount of animals Posi/ve animals Fig. 4 The prevalence of MAP-infection on farm X. On farm Y, there were 890 cows, and 28 blood samples were collected from animals of a high-risk group. None of the 28 samples was positive giving an apparent prevalence of 0.0% and CI (95%, ). The management of the animals on the two different farms was pretty similar. Dry cows are kept in a pen until parturition and afterwards moved back to the milking cow pen directly. Sick animals with for example, lameness, mastitis, weakness etc., are separated from the healthy cows. All animals that are planned to be culled are placed in a separate pen. In those separate groups, the cows are free ranging and can walk around freely within the pen area. Animals are separated into different age categories in various pens that are long, narrowed and situated throughout the whole farm. The manure scraping collector goes throughout the whole farm facility. The pens have a lot of bedding 22

23 and are in a good condition. On farm X, there is a separate building away from the main farm facility where they keep young livestock, while on farm Y all animals are kept in the same territory. In both of the farms, distribution of feed is performed with the help of a tractor that is driving through the facilities once a day. According to the farm manager in farm X, the general hygiene rules such as cleaning of farm workers hands, calving pens and cows back and udder before calving or giving colostrum, are not strictly followed by the workers. There are no records on following of the biosecurity rules on the farm, which indicates that there is also no monitoring of the hygiene. Newborn calves are directly separated from the mother (within 30 min) and are given colostrum from the cows that are considered healthy and not having any symptoms of clinical paratuberculosis. The day after, calves are transported on a trailer to a pen where newborn calves are divided in groups of 4. They are kept like this until 3 months old, and fed with milk replacement. When the calves turn 3 months old they are moved to a pen where young calves are kept in a group of 20. All animals are kept indoors, but dry cows are sometimes grazing during spring/summer-season. Unfortunately, there is no specific pattern or schedule set for the grazing manner of the cows, some cows are just randomly let to graze while others are not. All sick cows are sold, and clinically infected cows with paratuberculosis are culled. One year ago was the last time new cows were purchased to the farm, the animals were kept in a quarantine-pen for one month. In farm Y, there are separate facilities for every pen and group of animals which also have its own manure scraping collector. The animals are categorized in the same manner as on farm X according to age, lactation stage and sickness. The newborn calves are moved directly after they are born by the farm workers by foot. No trailer is used for transportation. Calf cribs are located right outside the farm facility and all calves are separated and kept individually until one month old. When the calves turn one month they are moved to heifers pen where they are kept in a group of 20 animals. 23

24 4. DISCUSSION The obtained results from the first testing on farm X revealed that the prevalence on the farm was 10% CI (95%, ), the second testing gave us an apparent prevalence of 8.9% CI (95%, ). In the first investigation, the animals were selected according to the high-risk group and the second investigation was based on all dry cows on the farm. There has to be mentioned that there were some difficulties in the decision of which animals should be included in the so called high-risk group due to improper collection of records. Because testing only was performed on 30 animals the first time, and 56 the second time, it gave us a quite wide CI for the farm prevalence. If all animals on the farm were tested, it would give us the more correct percentage. The prevalence of % indicates that a subclinical infection of MAP is present and the need for control measures is necessary to implement for prevention of further spreading of the disease. The obtained results from farm Y gave us an apparent prevalence of 0% CI (95%, ). As we suspected the herd prevalence to be 10% we tested 28 cows of 890 in total which should have given us at least one positive animal [30]. We tested the animals considered to be of a high-risk group. No signs of clinical paratuberculosis were previously discovered by the vets or farm workers on farm Y. As no seropositive animals were found in this investigation, it is very likely that MAP-infection is very rare or absent in this herd [21]. In farm X, the major source of transmission and infectious route of the disease is the contamination from manure, which is a consequence of the pen architecture inside the farm. The faeces from possibly infected cows are contaminating environment in calving areas and infecting healthy individuals. Even though there is a lot of good-quality bedding, it is not enough to completely stop the transmission of MAP through contaminated manure from positive animals. Considering that faecal shedding may occur in animals long before clinical signs are appearing, this is of a high priority [10]. This specific route of transmission is important on the farm as the shedding of bacteria is high in manure and the manure might contaminate the feeding and drinking lots, which is the most common route for infecting healthy animals. If this risk factor will not be eliminated, transmission will continue to proceed and even though testing and other control measures will be taken, it will not be enough for controlling the spread of infection within the farm. The risk factor of spreading infection through manure is too high, as it is the most important source of transmission [21]. Dry cows that are grazing during spring/summer-season are considered to be in a risk of getting infected through the pasture which may be contaminated with MAP from previous years, since MAP has a long survival time in soil and soil-faeces mixture [12]. Therefore, it is necessary to have this in mind 24

25 and not let negative cows graze on the same pasture as previous non-tested animals that might have been infected with MAP have been grazing. Other important risk factor at farm X is that the calving area is not cleaned before calving, neither is the cows back or udder before she will give birth. The possible route of infection might be from the dirty pen that is contaminated from previous animals to the newborn calf. Another possible route of transmission of the bacteria is from the back of the cow that might contaminate the calf during parturition or from the teats of the cow that the calf might be suckling right after parturition [7]. Even though the calves are separated from their mother as fast as 30 min post-partum, the time is sufficient for the infection to take place as the calf is able to start suckling seconds after parturition or even already be infected by in utero [21]. According to the farm manager, the calves are only fed with colostrum from healthy cows, but not all of these animals that are considered to be healthy have been tested for paratuberculosis. Just because they are not showing any clinical signs of the disease, does not mean that they actually are healthy. So this should also be considered as a risk factor, as long as the calves are not only restrictedly fed with colostrum from cows with negative test results. The solution would be to test all animals on the farm and detect all the negative cows that could be considered as healthy and only feed calves with colostrum from these cows. Another major source of transmission that could be considered at farm X, is the transportation of newborn calves, since the trailer that is used for transportation is not cleaned and disinfected between transportation of different animals, both young and adult. This is considered to be a risk factor, as the transmission may simply occur from the bacteria that is present in the dirt left on the surfaces inside the vehicle. The animals immunity is weakened by the stress during transportation, a new environment and new herd mates, which makes them very susceptible for an infection. By preventing the young animals from becoming infected by decreasing their exposure to infectious manure, colostrum and milk, the disease would become easier to control, as the most common routes of infection would then be eliminated. The critical control point that is not maintained which I found relevant on farm Y is the hygiene of the farm workers apparel. The boots are not cleaned between entering the different facilities and clothes are not changed so possible contamination can occur through infected manure that is attached on boots/clothes and carried across the different facilities. It is difficult to eliminate some transmission factors on the farms, such as the method of feed distribution with a tractor. The tractor is driving through the whole farm facility and might contaminate areas with for example infectious slurry from the wheels. This is an impossible thing to solve as it would require too much time and effort to clean the wheels of the tractor between each and every pen. 25

26 The main steps for eliminating the transmission of the infection in farm X should be: 1) Arrange the manure scraping in a different manner so that the manure from adult cows does not contaminate calf and heifer pen 2) Clean and disinfect the vehicle used for transportation of new born calves 3) Cleaning of the calving area, as well as the cow s back and udder before parturition 4) Calves should only be fed with colostrum from cows with negative test results 5) Negative cows should not be kept on the same pasture as previously infected cows have been grazing on These are the optimal steps for eliminating the transmission of MAP according to the theory, but in practice it is unfortunately difficult to fulfil all the requirements, mainly due to economical issues. For example, to rearrange the whole farm and manure scraping collector would cost too much money and take too much effort, as the whole facility then would have to be rebuilt. The ideal, but quite costly control plan would be to test all animals on the farm at first, with serum-elisa, for a detection of high shredding animals and divide the cows into categories of for example green, yellow and red to easier maintain the disease on the farm [7]. The following testing could be repeated quarterly of all animals for detection of possible changes in the results, as there could be a slight possibility that non-detected animals become high-shredders. Interpretation of the animals with a positive result during both testing times would make it easier to detect the high-shredding individuals for further culling and prevention of the transmission. Testing could be performed quarterly until the prevalence percentage drops and the disease is at a lower prevalence. After this, testing of animals from high-risk group only could be performed annually. The latest strategy, which is based on ELISA-testing of all dry cows and allows detection of infected or infectious cows before calving makes it possible to eliminate the infected calves born to positive cows. This type of strategy also allows to minimise the risk of other cows getting infected by isolation of positive cows before parturition. The calves that are born to positive cows should then be reared for slaughtering and not kept on the farm together with the other animals that might be healthy [7]. Calves born to positive cows should be moved to an individual pen or facility where they cannot be in contact with the rest of the herd. This method will prevent the transmission of MAP with the help of elimination of possibly infected calves and also prevent spreading of the disease within the calving area. To test dry cows before calving every year and divide the tested animals into risk groups, as well as their calves after parturition is still an option. But it would still be necessary to test all animals on the farm at first, to get an overview of the current situation and further continue testing 26