LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Faculty of Veterinary Medicine
Sara Madelene Johansson
Epidemiological features of bovine viral
diarrhoea virus infection in cattle herds in
Lithuania
Galvijų virusinės diarėjos virusų infekcijos
paplitimas ir epidemiologiniai ypatumai
Lietuvoje
MASTER THESESof Integrated Studies of Veterinary Medicine
Supervisor: Assoc. Prof. Dr. Algirdas Šalomskas
THE WORK WAS DONE IN THE DEPARTMENT OF VETERINARY PATHOBIOLOGY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Theses “
Epidemiological features of bovine viral
diarrhoea virus infection in cattle herds in Lithuania
”.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.
Sara Madelene Johansson
(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. Sara Madelene Johansson
(date) (author’s name, surname) (signature)
CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES
Prof. Dr. Algirdas Šalomskas
(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
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TABLE OF CONTENTS
SUMMARY ... 4 SANTRAUKA ... 5 ABBREVIATIONS ... 6 INTRODUCTION ... 7 1. LITERATURE REVIEW ... 8
1.1 BVDV – THE SILENT INFECTION OF CATTLE ... 8
1.1.2 PESTIVIRUS & BVDV ... 11 1.2 EPIDEMIOLOGY ... 14 1.2.1 EUROPE ... 14 1.3 PREVENTATIVE MEASURES ... 15 1.3.1 VACCINATION ... 15 1.3.2 HERD MANAGEMENT ... 16
1.3.3 ALTERNATIVE TREATMENT & PREVENTION ... 18
2. METHODOLOGY ... 20
3. RESULTS ... 24
3.1 EPIDEMIOLOGY ... 24
3.2.1 PLANT EXTRACT ... 29
3.2.2 BLOCKING ELISA TEST OF SERA AND SALIVA SPECIMENS ... 31
4. DISCUSSION ... 32 5. CONCLUSIONS ... 36 6. RECOMMENDATIONS ... 37 ACKNOWLEDGEMENTS ... 38 REFERENCES ... 39 ANNEX 1. ... 43 ANNEX 2. ... 45
SUMMARY
Epidemiological features of bovine viral diarrhoea virus infection in cattle herds
in Lithuania
Sara Madelene Johansson
Master thesis
BVDV is a disease that causes huge economical losses for cattle farmers around the world. Because of the virus nature this is a very hard disease to control. The aim of my study was to analyse epidemiological data of BVDV in Lithuania, and to test one alternative treatment and investigate a non-invasive diagnostic test. The scientific work was based on laboratory work with MDBK cells and BVDV virus (NADL strain); with adding of 16 different plant extracts and then evaluate the antiviral activity of them. The non-invasive diagnostic test was based on sera and saliva samples from cattle, and trying to detect antibodies in sera and saliva. Epidemiological data was from the year 2017, 1389 cattle were tested in 32 different regions around Lithuania. The prevalence was calculated and compared, and the results show that BVDV is prevalent in Lithuania.
SANTRAUKA
Galvijų virusinės diarėjos virusų infekcijos paplitimas ir epidemiologiniai
ypatumai Lietuvoje
Sara Madelene Johansson
Master thesis
Galvijų virusinė diarėja yra liga, kuri sukelia didžiulius ekonominius nuostolius galvijų augintojams daugelyje pasaulio šalių. Dėl ypatingų viruso savybių šią ligą labai sunku kontroliuoti. Mano tyrimų tikslas buvo išanalizuoti galvijų virusinės diarėjos epidemiologinius duomenis Lietuvoje, išbandyti alternatyvius virusų kontrolės metodus bei ištirti neinvazinio diagnostinio metodo taikymo galimybes. Mokslinis darbas buvo pagrįstas laboratorijos darbu naudojant MDBK ląsteles ir GVD virusų NADL padermę. Tokiu budu buvo ištirta 16 vaistinių augalų ekstraktų, įvertinant šių preparatų antivirusinį veikimą. Neinvazinis diagnostinis testas buvo pagrįstas galvijų serumų ir seilių tyrimais bandant aptikti specifinius antikūnius šiuose mėginiuose. Epidemiologiniai GVD virusų infekcijos duomenys buvo rinkti 2017 metais. Iš viso buvo ištirti 1389 galvijai iš 32 Lietuvos rajonų. Duomenų analizė parodė, kad GVDV paplitimui didžiausią įtaką turėjo galvijų amžius, o ši infekcinė liga yra endeminė Lietuvoje.
ABBREVIATIONS
LUHS – Lithuanian university of health sciences BVDV – Bovine viral diarrhea virus
RT-PCR – Reverse transcription polymerase chain reaction ELISA - Enzyme-Linked ImmunoSorbent Assay
CP - Cytopathogenic NCP – Non-cytopathogenic PI – Persistently infected IHC – Immunohistochemistry CSF- Classical swine fever BDV- Border disease virus DMSO – dimethyl sulfoxide
BVDV-1 – Bovine viral diarrhea type 1 BVDV-2 – Bovine viral diarrhea type 2 RS – Respiratory Syncytial virus
Pi3 – Parainfluenza 3 OD – Optical density HCV – Hepatitis C virus Ab – Antibody
INTRODUCTION
BVDV is a viral and infectious disease among cattle. The virus primary infect cattle worldwide [1]. The BVDV virus infection cause significant economical loss around the world, and this has a huge effect on the cattle industry [2]. Research on the epidemiology and aetiology of the virus is made, to find better ways to minimize the losses. Also to find a better understanding and improve control programs. Nowadays is the most common control program is to detect persistently infected cattle and cull them. These findings are made by antigen capture ELISA or RT-PCR. This has been very efficient control programme but it is hard to detect and eradicate this disease that is because of the nature of the virus. PI cattle are infected by the non-cytopathogenic strain and act as a carrier of the virus. Cattle can go for years with the subclinical disease and by then have calves that will be PI since they get infected through the placenta.
Another control programme that is very common is vaccination, but it adds costs to farmers and they do not really understand the importance of proper use which often leads to the control programme is not used right or used more then once [3]. Also cattle farmers do not pay much attention to biosafety, so the prevention of introduction the virus to the herd is not avoided [4].
For BVDV infections there is no specific treatment and there is studies made on natural products. Plant extracts and essential oil of plants have been proved to have antiviral activity on viral infections [5]. Which made it interesting for me to see if these alternative treatments had any significance in control of BVDV.
Therefore, the aim of the present study is to achieve knowledge about BVDV, acquire epidemiological data about BVDV in Lithuania and investigation of possibilities in alternative BVDV infection control measures.
To acquire the aim of the study, the objectives are:
1. To evaluate the BVDV infection prevalence and analyse epidemiological data of BVDV in Lithuania
2. To perform in vitro testing of the antiviral effectiveness on BVDV using medicinal plant extracts
3. To evaluate saliva samples as alternative non-invasive specimens for detection of antibodies against BVDV
1. LITERATURE REVIEW
1.1 BVDV – the silent infection of cattle
BVDV infection in cattle is a very hard disease to detect and this is because the virus have a wide set of ways to express clinical signs in the host. So it is incredibly significant to have information and understanding about the pathogenesis of BVDV. What may be confusing for people that are not experienced with this virus. They might think that the main clinical sign is diarrhea. Although it is not, the clinical signs of the disease differ a lot depending on the situation. There can be cattle that are persistently infected for years before expressing clinical signs. Then these infected cows have spread the virus to their offspring, mainly because the virus transmits through the placenta. But it is not the only way the virus can spread.
In acute infections of non-cytopathogenic strain, non-pregnant cattle with no immunity start to be viraemic. It is transmitted through direct contact, aerosols or sexual contact with PI cattle [6]. In fetal infection, the age of the calf in the uterus has a major role when the BVDV virus occurred. If the infection would to occur in the uterus, it is usually after 18 days. This is because of the fetus is not attached to the uterine wall and then the virus cannot penetrate the zona pellucida. Between days 29 to day 41, if the fetus gets infected it usually results in embryonic death. After day 30, if infection happens this will often result in birth of PI calf [7]. In calves that are born with the virus, usually get infected as early as day 18 or latest day 125 of gestation. They will then be born with the virus and shed in all kinds of body fluid after birth [7]. Another development of PI cattle is mucosal disease. This is believed being caused by cytopathogenic BVDV that mutated from non-cytopathogenic BVDV [7]. In some cattle they appear to be free from BVDV, but can still give birth to calves that will be infected. These are called Trojan cows and this cause an enormous epidemiological risk due to that these cows are very hard to detect in the herd [7]
Fig. 1 Pathogenesis of BVDV [8,7]
Because of the many pathways this virus can take, there are different approaches for making a diagnosis in cattle. The most reliable one for detecting BVDV in cattle is antibody titer testing, specific for Ab and Ag [9]. Together with this antibody testing, Immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) & enzyme-linked immunosorbent assay (ELISA) is very popular. To determine a correct diagnosis these tests may be combined. Another popular testing method in calves is ear notching; it is a simple skin biopsy from the ear, which is later tested with IHC, RT-PCR or ELISA [10].
Acute infections
In acute infection the clinical symptoms are mild. Therefore the diagnosis should have some basis in if the (1) female is pregnant and will deliver a PI calf, (2) If reproductive failure is because of BVDV infection, (3) if another infection is present because of immunosuppression of BVDV. RT-PCR has a high sensitivity for detecting low virus levels in the blood, together with Ag ELISA. But relying on one test is not enough for making a correct diagnosis in acute infection of BVDV. Therefore a number of samples should be analysed if the cattle show increased levels of BVDV in the blood. With rising Ab titre there can be a suggestions of an acute infection have occurred during the last 10-12 weeks [7].
Fetal malformations
Some calves can be born with malformations, if it is not aborted by the BVDV infection. The malformations vary and depend on what part or tissue that is mostly affected. Diagnosis demonstration of testing these calves, Ag ELISA or PCR testing can be used. The test is made on fetal fluid or skin. If infection has occurred during gestation in the 150-180 days, the fetus will demonstrate an immune response and will be born Ab positive and Ag negative. [11].
Abortion & reproductive failure
When reproductive failure occurs because if BVDV, a testing of pregnant female early is crucial. Blood samples should be taken with 4 to 6 weeks in between. Using Ab ELISA for detecting a rise in titre after infection, neutralization of the virus is a test that also can be used. A rise would demonstrate a positive result of BVDV infection, but a decrease in the titre would demonstrate abortion. This makes it difficult to analyse the titres [7].
PI cattle
There are high levels of virus in PI cattle, therefore testing with IHC, RT-PCR, or Ag ELISA is the best tests for diagnosis in these individuals. However Ag ELISA is to be preferred because it is cheaper when testing just a few individuals. But when testing calves that were colostrum fed and born from a PI female the Ag ELISA test sensitivity is not the best choice, both positive and negative results have been demonstrated [12]. Then the RT-PCR is the best choice in these cases. If Calves are suspected and up for testing, they should be tested before they been fed colostrum, or after when the colostrum effect is lower.
Mucosal disease
For determining mucosal disease in cattle, there need to be presence of cytopathogenic and non-cytopathogenic in the affected female. But pathological findings of mucosal disease characteristic are also good for determining the presence of BVDV in PI cattle [7].
Trojan cow
Trojan cows are very hard to diagnose due to the fact that they are known to be virus negative, and Ab postive. Therefore they cannot be detected during testing and a diagnosis cannot be made. There
1.1.2 Pestivirus & BVDV
Pestiviruses infect both domesticated and wild animals, and one of their mechanisms that are highly successful is to infect the host in a way so they will persist. The best example of this persistent infection is in BVDV of cattle. Other diseases of the pestivirus genus are classical swine fever (CSF) and border disease virus (BDV). The pestivirus are very diverse, but have the same genome. The one thing that makes them diverse is the nucleotide sequence. This extends the antigenic properties and gives them a wide display to infect the hosts. Hosts of this genus are cattle, sheep, goats, pigs, camelids also exotic animals like the giraffe. Different virus has different strategies, but can be characterised into two different strategies. First strategy is to “infect and run” and the other strategy is to “infect and persist” [14]. However pestivirus express both strategies, for example in BVDV.
Fig. 2 Phylogenetic analysis and classification of pestiviruses based on Npro gene [15].
The most common route of transmission of pestivirus is oronasal, and then the virus replicate in the cavity of the nose before it starts to go to the other organs. The virus spread in all body secretions and excretions. But even if pestivirus infections transmit in the same way they have a different way of expressing clinical signs. In example, CSF was believed to be acute or per acute disease and the animal would die within 2-5 days after having high fever. Now the CSF is more believed to be chronic or having late onset of infection. For BDV in sheep and cattle, the clinical signs where not expressed or very mild with a low grade fever [14]. This is because the biology of the virulence
determinants depend on certain viral genes gets deleted or amino acids in the genes become mutated. In cattle, the BVDV infection can take certain pathways. The uncommon one is mucosal disease, but to show the best example on how pestiviurses depend on changes, then mucosal disease is the best example. In Mucosal disease, the ncp or cp form of the virus is present. Then it either develops from ncp to cp, or it stays at ncp and then the cow will be PI instead. A study was made on cultured cells to demonstrate the difference. Cells that were infected with cp BVDV strain, showed that their non-structural protein NS3 start to accumulate in the replication cycle, and when cells were infected with ncp the uncleaved non structural protein NS23 took over the early stage of replication cycle [16]. It is also known that pestiviurses infect naïve animals that have some sort of immunosuppression. That’s why pestiviruses are so successful in spreading and keep infecting animals worldwide. It can be silent infection and it can be very well disguised under the various symptoms that are expressed in the infected animal.
Bovine viral diarrhea virus
Bovine viral diarrhea virus is a member of the pestivirus genus, and belongs to the flaviviridae family. The virus have an envelope, single stranded RNA, spherical shape and is 40 to 60 nm in diameter [17]. The genome of the virus is single, linear, positive-sense, single-stranded RNA molecule of approximately 12.3 kb [17]. BVDV are usually divided into BVDV-1 or BVDV-2, and in those divisions there is also subtypes of them. BVDV-1 has 16 subtypes, and BVDV-2 have 3 subtypes. After first division there is one more way to classify the biotypes of BVDV. The virus can either be cytopathogenic, or non-cytopathogenic.
Fig. 3 Phylogenetic analysis and classification of BVDV based on Npro [15].
Cytopathogenic BVDV forms because of a mutation of the cytopathogenic form. The non-mutated virus will cause a persistent infection of cattle; with subclinical signs and the non-mutated one will express clinical signs of BVDV. Typically the ncp strain of BVDV will cause PI cow, and fetus might develop the disease or be born healthy. Most cattle that are PI will deliver calves that are born with the disease or they can develop mucosal disease which is very fatal.
Mostly BVDV is associated with cattle, but it can also cause disease in other domestic animals and wild animals similar to reindeer species. The virus can express different clinical signs in hosts with various immune statuses. In cattle it is not only the immune status that plays a role, but also which strain and what type of virulence. Clinical signs of the disease will differ depending on the development of the ncp to cp takes or vice versa. (1) ncp become cp, causing mucosal disease. The clinical signs are diarrhea, and erosions in the mucous, and death. (2) Infection in the first days of gestations, between day 18 to day 29 the fetus may abort, also the infection can occur later in the gestation and then a PI calf will be born or be born with malformations. (3) There is a chance that the calf can be born without the virus, and this is if the calf has been introduced to the virus when it already developed some immunity in the uterus. (4) Acute infection of animals, that are immunologically naïve usually gets mild clinical signs, diarrhea, fever and leukopenia.
1.2 Epidemiology
BVDV is one of the diseases that are economically important for cattle farmers. According to research done around the world, BVDV-1 is the most prevalent, compared to BVDV-2 [18]. Research also showed that different subgenotypes vary among the countries, for example in Europe there is BVDV-1 predominating with following subgenotypes 1b, 1c, and 1a. Another research was made, comparing all BVDV tests in different countries and continents, this concluded that BVDV-1 is more prevalent with 6117 isolates between 1962-2012 and BVDV-2 with 882 isolates the same years [18]. But it is to hard to develop a system to calculate exactly how prevalent BVDV actually is worldwide. So far subgenotyping is a way that gives us a lot of information what type of BVDV is present. If a subgenotype is predominate or a new genotype is introduced because of cattle movement. However surveys show that BVDV is endemic, reaching a level of 1-2% of the herd being PI and 60-85% of the cattle being antibody positive [19]
1.2.1 Europe
Studies on prevalence in Europe have been made for about 48 years until today. The studies have shown that BVDV infections are endemic in Europe and either no control programmes have been used or there is sporadic use of control programmes. Under these years, approximately 50% of the herds have PI cattle, and 90% will be exposed to BVDV during their lifetime [20]. There is lack of information to say exactly how prevalent BVDV is in Europe. There are a few countries that do not have any information or reports, and from some there are incidence reports. The incidence reports comes from countries that established good eradication programmes and they also claim to be free from BVDV. The countries that claim to be free of BVDV infection are Switzerland, Sweden, Denmark and Norway. In Lithuania there is no recent publications of the epidemiological situation of BVDV. The last one published is from 2002, and at the time they did an investigation of 147 herds from different regions [38]. In total there was 3798 animals examined within the time spawn 1997-2001 [38].
1.3 Preventative measures
1.3.1 Vaccination
Upon today BVDV infections are considered to cause huge economical losses for cattle farmers worldwide and in Europe. Today the knowledge about BVDV-1 and BVDV-2 is very wide and they show that they can be ncp and cp. In the early times there was no existing vaccine but today certain vaccines have been developed and used to try to prevent infections of BVDV.
The aim of the vaccines is to prevent infections by BVDV-1 and BVDV-2, also to increase cross immunity against these types. The types of vaccines developed are both live attenuated and inactivated types. This is because of the antigenic diversity BVDV virus express, and then one type is not enough [21]. It is important for farmers today to have a vaccination programme individually for their herd, in order to actually use and trust the vaccines. There are different types commercially available for cattle farmers, but there have to be some understanding in how BVDV act as a disease. The BVDV virus is more complicated then other infectious disease’s, which makes it harder to choose the best vaccine to the herd. The choice is either to use modified-live viral vaccines or inactivated vaccine [22]. The cytopathic biotype usually is included in the vaccines due to that this development of the virus is not causing PI cattle. But it is the non-cytopathogenic type that most farmers want to vaccinate against since it is proven to be more prevalent in herds [22]. The choice and timing of vaccinations must be considered thoroughly in order to improve the herd’s health and to function in the plan of the farm.
Inactivated vs modified-live viral vaccines
Why the use of MLV vaccines is because they are considered safe to the fetus when you vaccinate cattle that are pregnant [23, 24]. Inactivated vaccines have viral antigens, but the virus cannot replicate. That’s why they are called inactivated. The negative aspect of using inactivated vaccines is that they usually demand more dozes to be given, in order to get desired immunity levels [22]. Also it takes time before the immunity reach desired level of immunity. There is a common failure with these vaccines and it is that the vaccination protocol is not followed or understood therefore resulting in incomplete immunity and the herd can still get infected. MLV vaccines are not inactivated and they have proven to stimulate higher levels cell mediated immunity and antibodies. Leading to less time waiting for the desired immunity levels [28]. Therefore the use of MLV vaccines is considered to give a fast protection against BVDV after one vaccination time.
Monovalent vs. multivalent vaccines
BVDV is divided into genotypes and several subtypes, this is based on the genetic differences in the virus. Vaccines have been made with BVDV1a isolates, which is necessary to get protection against BVDV2 and BVDV1b. Neutralizing antibodies is produced to get heterologous viral strains, together with monovalent vaccines. But it has been proven that protection against homologous instead of heterologous viral strains is better [23, 24]. Studies recommend that multivalent vaccines are better to aid protection from strains in the field [25, 26].
Vaccinations of BVDV have been on the market for numerous years, and the popular one is multivalent vaccine that gives protection against BVDV1 and BVDV2. These multivalent vaccines do not only give protection against BVDV, but also strains from respiratory viruses. Studies that have been made on the efficacy of using vaccination programmes conclude that it is important to involve in the management of controlling BVDV infections [27]. Unfortunately the studies did not conclude that using only vaccination, in the aim to eradicate BVDV is proven to be not successful.
1.3.2 Herd management
Management of cattle herds is a very important subject, especially when dealing with infectious diseases and other health problems related to the herd. There are several infectious diseases that have been managed to control and eradicate. This has been done by the old school way of test and cull, or control programmes that involves vaccination, biosecurity, knowledge and legislations. Some countries in Europe have managed to claim them self free from BVDV infections by successfully using good control programmes. However not all the countries in Europe have applied these programmes due to economics or to less of knowledge.
Control programmes in the Nordic countries
Countries in Scandinavia claim to be free from BVDV infections and have less than 2 incidences per year. However a study that been made showed that the reindeers in the Nordic countries actually are capable of being infected. The study concluded that the seroprevalence with 95% confidence interval for Iceland was 8% (0- 18.6%), for Norway 43.4% (30.8-55.8%) and for Sweden 49.3% (37.7-60.9%). For Finland, no seroprevalence was found, a one-sided 97.5% confidence interval
Table 1. Control programme example, summarized by Torstein Sandvik, DVM, PhD [30].
Level Investigation Diagnosis Action
1. Herd and antibody screening,
bulk milk AbELISA spot test AbELISA
A) Negative
B) Positive
Inform how to avoid infections
Further sampling at level 2 to test for active infection 2. Active infection? PC milk AbELISA
young stock AbELISA
A) Negative
B) Positive
Follow-up serology at level 1. Eventually lift restrictions.
Impose movement restrictions. Go to level 3a.
3. Herd clear-out
a. Ab and virus testing of
individual blood samples A) Viremic B) Ab positive Verify persistent infection; cull. Maintain restrictions. Double-check age >3 months; keep. Continue at level 3b. b. follow-up sampling of
calves born to Ab+ dams A) Viremic B) BVDV Free Verify persistent infection; cull. Maintain restrictions. Keep. When all calves tested, go to level 2.
The first level is aiming to sort out cattle infected of BVDV and to keep them free from infection. Another part of the first level is bulk milk sampling and it is aiming to test antibodies in the milk against BVDV. In this way they get epidemiological data and quality checks in the same time. In Scandinavia these two tests are repeated every year, to maintain herds being free from BVDV and detect PI cattle or Trojan cows. The second level includes identification of cattle, which are suspected of being infected. Third level, main task is to really identify PI cattle in the herd, which also have an active infection. This step also includes testing of all herds in the farm, and thorough
testing of calves born from detected PI cattle. After these steps, PI cattle are culled and control program is going back to level 1 screening.
Control program in European countries
In Europe there are no control programs that are used as successfully as the Scandinavian countries are doing. But there is interest in trying to adapt into them. The voluntary programs exist, and were developed in Austria 1997 [30]. As described above, vaccines are not very commonly used, and testing is done by herd screening, bulk testing of milk for Ab ELISA. The program in Scandinavia is detected PI animals are culled, and animals that are positive are not allowed in the pastures. Austria is trying to develop legislation for Europe, which will aim to not move or have strict boundaries of moving cattle that is unidentified for BVDV.
When the BVDV vaccines are used, the control program in Scandinavia gets contraindicated and not reliable [30]. They have tried to use serum antibody testing instead but it is not favorable. Another aspect is the herd structure that differs a lot between the European countries. The differ in the sense of cattle density within the country, herd size, management and legislation of controlling infectious diseases – not only BVDV. That makes it complicated to develop a plan for only BVDV infections.
The antigenic diversity of BVDV also affects what kind of control program a country should use. In the countries where both types are prevalent, then it is important that the diagnostic tests used detect both types. In countries where prevalence of both types is common, the diagnostic tests are recommended to be based on genetic and antigenic properties. If only serology is the base, back up assays should be considered [30].
1.3.3 Alternative treatment & prevention
Studies have been made on alternative treatments against enveloped viruses, however their significance in actually treating the disease itself is questionable. The main goal of using alternative treatment is not to treat the disease, rather to prevent. It is in questions since bovines as a host, makes it hard to apply antivirals. One study with Mexican oregano oil showed that the efficacy on BVDV was lower compared to other enveloped viruses in the study. But when the oil was added
however there has been research with help of the BVDV virus since it is not infecting humans. It is considered safe to work with and in an investigation made on cytotoxicity, antiviral and antitrypanosomal screening of 82 herbal drugs [33]. BVDV showed efficacy in antiviral activity with low cytotoxicity on 10 plant extracts. The plants that were used was Panax ginseng, Cassia tora, Ginkgo biloba and Viola yezoensis, Celosia cristata, Ophioglossum vulgatum, Houttuynia cordata, Selaginella tamariscina, Alpinia galanga and Alpinia oxyphyll [33]. The research suggests that there is need for more investigation of different plant extracts to make a sustainable conclusion [33]. Also there is no publication of medicinal plants grown in Lithuania for this kind of research.
2. METHODOLOGY
Material and methods
Sampling of animals. The blood sera samples for this study were selected among samples submitted to the laboratory at Department of Virus Research, National Food and Veterinary Risk Assessment Institute, Lithuania during the year 2017. All samples had been submitted by practicing veterinarians from different sized cattle herds either due to clinical signs or due to general interest in the occurrence of BVDV infection in the herd. The large herd consisted of cattle breeding companies whereas the small herds were family farms. The cattle herds came from 32 Lithuanian regions. A total of 1389 blood sera samples obtained from animals of different age and sex were investigated.
Saliva was used as an alternative non-invasive specimen for detection of antibodies to BVDV. The saliva was sampled with sterile nylon FLOQSwabs™ (COPAN, Italy). The sterile swabs were kept under the animals tongue for 1min. Later they were put into sterile tubes that were closed, marked and placed into a container for storing at minus 20°C. Simultaneously the blood samples were taken. A total of 25 saliva and blood sera samples were obtained from cattle housed in one farm.
Testing of sera and saliva
The distribution of BVDV was evaluated by the number of seropositive animals. The commercial competition enzyme linked immunosorbent assay (ELISA) kits from institute Pourquer (France) was used for serological tests. All testing were completed according manufacturer’s instruction. The results were analysed by spectrophotometrical measuring of the optical density at the wavelength λ=450 nm. The validation criterion and percentage of blocking was calculated.
Testing of saliva
Saliva samples were tested by the same method as the sera testing. Samples were tested in 1:3 and 1:10 dilutions and optical density (OD) measured and compared.
Plant extracts. Sixteen 40 % (vol.) ethanol extracts of 15 medicinal plants were prepared. The plants were grown in Kaunas Botanical Garden of Vytautas Magnus University (Lithuania).
Table 2. Plant extracts
Nr. Latin name English name Lithuanian name Family Material 1 Satureja Montana L. Winter savory
Kalninis dašis Lamiaceae Herb
2 Chamaemelum nobile L. Chamomille Kilnioji blezdingūnė Asteraceae Herb 3 Perilla fruescens
Perilla Krūmine perilė Lamiaceae Herb
4 Agastache foeniculum Blue giant hyssop Pankolinė kinmėtė Lamiaceae Herb 5 Origanum vulgare Oregano Paprastis raudonėlis Lamiaceae Herb
6 Mentha piperita Peppermint Pipirmėtė Lamiaceae Herb
7 Geranium macrorrhizum Bigroot geranium Stambiašaknis snaputis Geraniaceae Herb 8 Melissa officinalis
Lemon balm Vaistinė melisa Lamiaceae Herb
9 Angelica archangelica L. Garden angelica Vaistinė šventagaršvė Apiaceae Leaf 10 Angelica archangelica L. Garden Angelica Vaistinė šventagaršvė Apiaceae Roots 11 Thymus vulgaris L. Common thyme Vaistinis čiobrelis Lamiaceae Herb 12 Hyssopus officnialis L.
Hyssop Vaistinis isopas Lamiaceae Herb
13 Nepeta cataria
L. Catnip Paprastoji katžolė Lamiaceae Herb
14 Echinacea purpurea L. Eastern purple coneflower Rausvažiedė ežiuolė Asteraceae Herb 15 Salvia officinalis L.
common sage Vaistinis šalavijas Lamiaceae Herb 16 Desmodium canadence L. canada tickclover Kanadinė jakšūnė Fabaceae Herb
Preparation of plant extracts. The solvent ethanol was diluted with sterile distilled water to 40% (vol.) concentration. Dried material of every plant (0.5 g) was extracted with 10 ml solvent. The extraction was done in an orbital shaker for 24 hours at room temperature (20 °C). Each extract was filtrated using paper filter and then polyvinyl difluoride membrane filter with 0.22 µm pore size. The concentration of the extracts was 50 mg/ml, with reference to the starting material. All the prepared plant extracts were stored at 4 ºC in a refrigerator (till the experiment).
Cells and virus. Madin Darby bovine kidney (MDBK) cells were kindly provided by Dr. I. Jacevičienė, Department of Virus Research, National Food and Veterinary Risk Assessment Institute, Lithuania. The bovine viral diarrhea virus (BVDV) NADL strain was generously provided by prof. dr. M. Polak, National Veterinary Research Institute, Pulawy, Poland. The cells were grown in Glasgow's MEM developed by Ian McPherson and Michael Stoker as a modification of Eagle's Minimal Essential medium (GMEM BHK-21) supplemented with 10 % free from BVDV Ab fetal bovine serum (FBS, Biochrom GmbH, Germany) at 37°C in the presence of 5% CO2. Penicillin (100 U/ml) and streptomycin (100 µg/ml) were used for the prevention of
microbial contamination.
Virus titration. Determination of TCID50 of control virus and treated ones was performed in
96-well plates with MDBK cells. Serial dilutions of BVDV in 10 fold steps were prepared. Every sample was tested in sextuplicates. CPE was evaluated after 96 hours. Virus titres and standard deviations were calculated using Kärber method (Kärber, 1931) and virus reduction factor was evaluated (Ruppach, 2014).
Cytotoxicity assay. The cytotoxic concentration (CC50) was determined for each extract
on MDBK cells using MTT assay (Mosmann T., 1983). At first, cells were seeded at a concentration of 1 × 104 cells/well in a 96-well plate and grown at 37 ºC for 1 day. The assay was performed in octuplicate wells for each extract.
Essential oil assay. Plate of 12-well plate was prepared as described above. Then essential oil was prepared with 20µl of essential oil of Hyssopus officinalis and 20µl DMSO and mixed. Hyssopus officinalis essential oil was selected because it was the only plant extract showing any potential of reducing antiviral activity. After we prepared 900µl of GMEM BHK-21 medium, 2%FBS, 100µl virus and 20µl of oil mix. The other was prepared 900µl of GMEM BHK-21 medium, 2%FBS, 100µl virus and 10µl of oil mix. Both prepared samples were in contact for 1 hour at 20ºC. Virus control preparation was made with 900µl of GMEM BHK-21 medium, 2% FBS, 100µl virus and contact in 1hour.
Screening of virucidal activity. Testing solutions were prepared by mixing of BVDV suspensions in GMEM BHK21 containing 2% of FBS (average 108.5TCID50/ml) with previously established noncytotoxic extracts volume (table 3). Virus and extracts mixtures were incubated at 20°C for 1 hour and titrated as described above. Controls of cells, virus, and extracts were included. After 96 h of incubation, the plates were examined using an inverted microscope (Olympus, Japan) to detect the CPE. Sodium hypochlorite (Sigma Aldrich, USA) at the final concentration of 0.1% (1000 ppm) was used as positive control. Sodium hypochlorite was diluted in water of standard hardness to a stock concentration. Sodium thiosulphate (Sigma Aldrich, USA) in distilled water was made to neutralize the activity of sodium hypochlorite.
Ethical consideration. The research was conducted following the requirements of the legislation of the Republic of Lithuania (Law on welfare and protection of animals, Valstybės žinios, 2012, N. 122, 11-2271).
Statistical analysis. The confidence interval (CI) and statistical significance of the differences between prevalence percentages were calculated at 95% probability following the standard methods.
3. RESULTS
3.1 Epidemiology
Fig 4. Dynamics of BVDV Ab per region
Eleven out of 31 (35.5%) regions in Lithuania during 2017 showed positive results against BVDV Ab. In Marijampole region the prevalence of Ab positive animals was 48% CI 95% (0.4527 - 0.5073), Sakiu was 50% CI 95% (0.4727 - 0.5273), Kalvarijos was 92% CI 95% (0.9052 - 0.9348), Kaunas was 46% CI 95% (0.4328 - 0.4872), Lazdiju was 52% CI 95% (0.4927 - 0.5473). In Kelme, Jonavos, Raseiniai and other regions the Ab prevalence was lower than 20% (fig. 4)
48% 50% 92% 6% 16% 10% 46% 52% 18% 3% 9% 25% 6% 1% 19% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Fig 5. Prevalence of BVDV ELISA Ab
In fig 5, a total of 1287 cows were tested for antibodies against BVDV in Lithuania during the year 2017. The prevalence of positive cattle was 14.2% CI 95% (0.1277 - 0.1663) and negative cattle was 85.7% with CI 95% (0.8379 - 0.8761).
Fig 6. Dynamics of antibodies against BVDV prevalence
The seroprevalence in different age groups significantly diminished from 26.6% to 6.4% in two years and in older than 24 months animals’ seroprevalence was significantly higher (30.1%; CI95% 0.2759 - 0.3261, fig 6). 14,2% 85,7% BVDV ELISA Ab Positive BVDV ELISA Ab Negative 26% 73% 9,75% 90,5% 6,4% 93,5% 30,1% 69,8% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% <6m Ab
Positive Negative <6m Ab 6-‐12m Ab Positive 6-‐12m Ab Negative 12-‐24m Ab Positive 12-‐24m Ab Negative
> 24m Ab
Fig 7. Difference of presence of BVDV antibodies between genders
There was no significant difference between genders. The seroprevalence in females was 13.9% with CI 95% (0.1201 - 0.1579) and in males was 16.05% with CI 95% (0.1404 - 0.1806; fig7).
Fig 8. Distribution of BVDV Ag positive animals per region
Three regions out of 26 (11.5 %) showed positive results to BVDV Ag in Lithuania during year 2017. Sakiu was 2% CI 95% (0.1404 - 0.1806) Sauliai was 1% CI 95% (0.0043 - 0.0157). The prevalence of Ag positive animals in Telsiai region was significantly higher (7,9%; CI 95% 0.0636
13,9% 86,1% 16,05% 83,9% 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% BVDV ELISA Ab
Positive female Negative Female BVDV ELISA Ab BVDV ELISA Ab Positive Male BVDV ELISA Ab Negative Male
2% 1% 7% 1% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Fig 9. Prevalence of BVDV Ag positive animals in Lithuania in 2017
In fig 9, the mean prevalence in Lithuania during 2017 of Ag positive cattle is 2.45% CI 95% (0.0157 - 0.0333) and negative cattle is 97.5% with CI 95% (0.966 - 0.984).
Fig 10. BVDV antigen positive cattle dynamics in different age groups
The Ag prevalence in the young <6 m animal group was 4.8% CI 95% (0.0358 - 0.0602) which was significantly higher than in the other groups. The Ag prevalence gradually decreased to 1.0% in > 24 months group (CI 95% 0.0043 - 0.0157) which is significantly lower than in the first group (P<0.01, fig10). 2,45% 97,5% BVDV ELISA Ag positive BVDV ELISA Ag negative 4,8% 95,2% 2,9% 97,1% 2% 98% 1% 99% 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% <6m Ag
positive negative < 6m Ag 6-‐12m Ag positive 6-‐12m Ag negative 12-‐24m Ag positive
12-‐24m Ag negative
>24m Ag
Fig 11. Presence of BVDV antigen between genders
The Ag prevalence in female cattle was 2.6% CI 95% (0.0169 - 0.0351) and in male cattle group there was 0.85% CI 95% (0.0033 - 0.0137). These differences were not significant (P>0.01, fig. 11). 2,6% 97,4% 0,85% 99,15% 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% BVDV ELISA Ag
3.2 Alternative treatment
3.2.1 Plant extract
Fig 12. In vitro screening of virucidal activity of 16 plant extracts
The primary screening for BVDV was done for extracts prepared from 16 plants grown in Lithuania (Fig. 12; Annex 1). Screening was done for active extracts at the highest nontoxic concentrations ranging from 6 to 74 µl/ml and we identified ten plants extracts with potent anti BVDV activity with virus reduction factor > 1 log10. Only Hyssopus officinalis extract showed moderate to
significant (1.83±0.255 log10) results according to EMA guidelines used for evaluating reduction
factor.
Fig 13. Kinetics of inactivation of BVDV 1 measured by counting viruses by the TCID50 assay
In fig 13, shows the results of inactivating BVDV with Hyssopus officinalis and sodium hypochlorite. The significant virucidal activity of Hyssopus officnialis extract was detected after 45
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 0 2 4 6 8 10 0 15 30 45 60 Tit er l og 10 Time min
Inactivation of BVDV
minutes of contact (p<0.01). Sodium hypochlorite showed a rapid effect after being added to the viral solution and then the virus titer gradually dropped from 4.5 to 2.5 log10 in 45 minutes (fig 13).
3.2.2 Blocking ELISA test of sera and saliva specimens
Fig 14. Blocking ELISA result of sera and saliva testing. PC – positive control; NC negative
control
Comparison of testing saliva and serum for detection of antibodies to BVDV is shown in fig 14. All sera samples were positive to antibodies while saliva was negative. OD of negative controls compared to saliva 1:30 was not significantly different. The OD of 1:3 diluted saliva samples was significantly lower than OD of saliva 1:30 and negative controls (P<0.01).
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
PC NC Serum Saliva 1:3 Saliva 1:30
O p ti ca l d en si ty
4. DISCUSSION
In this research, an epidemiological investigation in how prevalent BVDV is in Lithuania. The test was made during 2017, and for antibody ELISA 1287 cows were tested. For antigen 1185 cows were tested. Total number of cattle tested for BVDV Ab or Ag was 1521. The investigation was made to get an overview on the epidemiological situation in Lithuania. The disease is known to cause extreme economical losses for cattle farmers [2].
The prevalence of BVDV Ab during 2017 was 14.2% positive CI 95% (0.1277 - 0.1663). During the time of the tests there could be an error when estimating the prevalence of BVDV. This means that at the time of testing the disease didn’t affect the animals yet. Less then 5% suggest that there might be a small error of false-positive result. Hence we must take into reason that there are a larger percentage of cattle with antibodies against BVDV compared with the antigen testing. The ELISA Ab test doesn’t diagnose the disease but shows that there is presence of antibodies against BVDV, which is a response to viral infection [7]. But taking these test we must know or think about when the animals were vaccinated since vaccination are causing an immunity response, which can lead to false-positive results. This leads to the antibody blood work in young stock. Our results show that in the younger animals there is no significant difference in age groups. But when compared with the oldest age group >24m there is a significant difference. These results are telling us that there has been or is an active state of exposure to BVDV [7]. But also the larger percentage is in the age group of 30.1%(>24m). So this might mean that we have a reason to suspect that the healthy middle-aged animals can fairly deal with this virus, while the main population that suffers from the infection might be immunocompromised or infected. When sorting the tested animals into gender, the female gender showed 13.9% of positive results, and male 16.05% and there is not significant difference. This suggest that gender does not play a role in determining the risk factor to acquire this disease.
Antigen prevalence results gave us 2.45% positive with CI 95% (0.0157 - 0.0333). A positive result of antigens means that there is an active state of infection, and that the animals are spreading the virus [7]. But the main problem with this test is that we cannot draw a straight conclusion if the animals are PI or in the transient state of infection. Usually there will be a retesting after three
this we can suspect a recent exposure to the BVDV virus or an ongoing situation with PI cattle that deliver PI calves and there might be a possible chance of an outbreak in one of the regions with 7.9% prevalence of antigen against BVDV.
Comparison with other countries should be taken with caution when compared to our study. In England and Wales the prevalence from Ab titer of bulk milk gave 65%, [35] in Switzerland 121 dairy farms were investigated and they got a 15% of prevalence, [35] and in Estonia they found three herds with 46%, 16% and 18% prevalence of BVDV [35]. All these results suggest that there is either an active state of exposure or PI animals in the herd. Compared to these countries Lithuania do not have a significant high number, but they also belong to the countries that have prevalence to BVDV. Also when comparing prevalence of a disease with another country, we have to have in mind that not everyone use the same methods and that why we can get a large difference in percentage of seropositive animals. During 1997-2001, there was an investigation in Lithuania on cattle herds. The research showed there was no positive correlation between genders for this disease [38]. Between 2001-2003 another research was done on control measures against BVDV. The seroprevalence was 23.5%-47.6% [39]. The highest percentage of investigated sera samples was in Klaipeda with 55.9% of positive Ab results. Also they found 24 Ag positive cattle’s against BVDV but only 6 were persistently infected [39]. If we compare these results with ours, we can suggest that we still have a problem with BVDV in Lithuania.
Furthermore it is important to note that there were some limitations in this research. One of the limitations is that not all cattle were tested continually for both antibody and antigen. Also in some regions cattle were not tested at all which can suggest that there is a dark number of PI animals or active infections of BVDV.
Another important aspect for discussion is what kind of influence this disease has on the cattle farmers in Lithuania. Highly virulent infection with BVDV causes a severe onset of the disease, which ends in death, and that is one of the main points of economical losses [34]. This research also suggests that economical losses due to low or high virulent BVDV strain vary because of circumstances [34]. The circumstances involve what kind of strain, onset of acute infection and control programs [34]. Therefore there is importance on how to develop substantial and affordable control programs. In the Nordic countries they developed detect and cull program, which have showed huge progress in becoming BVDV free as they claim. As a third aspect for this discussion, we want to mention the damages of having undetected animals in the herd. For examples in a farm were they have 600 dairy cows, were 20% go undetected for BVDV. They will deliver one calve for approximately 5 years and some of these calves survive or are born as PI cattle. This is a problem
once the PI animals develop the disease, because there will be a huge dark number of PI cattle or there can also be Trojan cattle. Final aspect we want to focus on delivery of sick cattle, as mentioned above they can either die young or be PI for a few years before developing clinical signs. The calves from a mother that is infected with BVDV will transfer the virus through the placenta. Also a mother can be infected during the gestation period and either deliver a dead, malformed or PI calf. Delivery of PI calves is very bad for the cattle industry, since they affect the economic, go to human consumption and can cause unnecessary suffering of the animals. All those aspects that were mentioned are indirect repercussion of this disease, therefore we can see the broad spectrum of influence that this disease have on the cattle industry in Lithuania.
The other part of our research was to test how different plant extracts act on the viral replication of BVDV, NADL strain. In total, 16 plants extracts were tested and the results only gave one plant extract satisfying results. According to the EMA guidelines that were used for evaluation of the results, only Hyssopus officinalis had moderately satisfying results. According to other research done with Hyssopus officinalis, it have shown some results that this plant extract have bioactive substances that are of medicinal interests [36]. Therefore we added an inactivation test with sodium hypochlorite, to evaluate the time it took to inactivate the virus. Hyssopus offcinalis showed activity of inactivating the virus after 45 minutes of exposure. Also another study was done with BVDV virus but with Ocimum basilicum as an essential oil instead of plant extract. The study revealed that there can be activity against BVDV, but the results are pretty vauge [36]. The study also used monoterpenes, which had much greater result then Ocimum basilicum against BVDV and HCV. Furthermore when it comes to research about different plant extract used against viral activity, is that there should be more research done and more conclusive results. We are still in the stage were we test a huge amount of different extracts, but the conclusions always suggest the same problem.
Since the results of the plant extract testing were not satisfying we added another test with ELISA blocking test of sera and saliva. High optical density in this test means that the results are negative which further means that there is no infection of the virus. Low optical density in this test means that the results are positive, which is equal to an active infection. In this test all sera samples were positive, but all saliva were negative. We didn’t find antibodies in saliva because the concentration of antibodies in saliva is much lower. But it does not conclude that there might not be any
that all PI cattle are constantly shedding the virus into the environment, and get healthy cattle infected [37]. Today there are several different methods do detect virus not only from blood but also from milk, semen, and saliva. Some research that used this method suggest that there is interest of developing saliva sampling as a test method, but there is discussion if the sample of saliva should be pure or diluted [37]. In our test we used diluted saliva, and all sera samples were positive while all saliva samples were negative. This can be because the quantity of antibodies in saliva was too low [37] for blocking the antigen.
5. CONCLUSIONS
1. Seroprevalence of antibodies against BVDV in Lithuania during 2017 was 14.2% positive. This is important to know in order to develop and fund sustainable solutions in how to eradicate and control this disease. There were no significant results if BVDV affect genders, but it does affect different age groups.
2. Prevalence of antigen positive cattle BVDV in Lithuania during 2017 was 2.45%. The region with the highest percentage of antigen positive animals was, Telsiai. This means that there can be a beginning of a possible outbreak or PI cattle in the region. Most of seropositive animals were found in the age group younger than 6 months.
3. Plant extract of Hyssopus officinalis was the only plant that showed any result of antiviral activity. There is a need for further investigation with natural sources in order to make a sustainable conclusion.
4. According to manufacturer description the saliva test was carried out. The results we got was that all sera samples were positive, and all saliva samples were negative. For using this kind of testing to detect antibodies to BVDV there should be more investigations done on the optical density threshold determination.
6. RECOMMENDATIONS
Our studies on the prevalence and epidemiology of BVDV showed that this infection is endemic in Lithuania. BVDV remains endemic because there is not any established control programmes in Lithuania and little information to the farmers how to prevent this disease. Therefore our suggestion is to prepare national BVDV control programme and start to implement information activities for farmers and veterinary practitioners.
ACKNOWLEDGEMENTS
This document contains my master thesis, the final document for my Veterinary Medicine Doctor degree at Lithuanian University of Health Sciences. It describes the results of my research on BVDV in Lithuania and investigation of alternative diagnostic and treatment.
I would like to thank Dr. Ingrida Jaceviciene Department of Virus Research, National Food and Veterinary Risk Assessment Institute of Lithuania for help with the data collection for epidemiology and the data of sera and saliva samples.
Prof. dr. Ona Ragažinskienė for support with medicinal plant extracts. Department of Biology, Vytautas Magnus University
Prof. habil. dr. Audrius Sigitas Maruška for consultation on plant extracts properties research, Fitokontrole project leader, Department of Biology, Vytautas Magnus University
Assistance in the laboratory, Dr. Raimundas Lelesius, LUHS Institute of Microbiology and Virology.
Dr. Dainius Zienius for the assistance in master thesis writing, and at last I want to thank my supervisor Prof. Dr. Algirdas Šalomskas for sharing his knowledge and helping me finish my work.
Also the research was partially supported by Research Council of Lithuania, Fitokontrole project, No. MIP-065/2015.
Finally, I would like to express my appreciation to Lithuanian University of Health Sciences for providing with all necessary equipment and tools for collection and testing of samples that were used during this research work.
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ANNEX 1.
Nr . Plant Name. Mat erial. Vol of extra ct µl Titer control virus, log10 Titer after treatment, log10 t/P1 log10 reduction factor The virus reduction potential2 1 Saturera Montana L. Winter Savory Herb 74 8.33 ± 0.27 7.75 ± 0.25 3.152/ 0.01 0.58 ± 0.26 not significant 2 Chamaeme lum nobile L. All. Chamomill e Herb 16 8.33 ± 0.27 7.67 ± 0.27 4.231/ <0.00 1 0.66 ± 0.27 not significant 3 Perilla fruescens L. Britton Perilla Herb 76 8.33 ± 0.27 7.17 ± 0.21 8.286/ <0.00 1 1.16 ± 0.242 Indicative 4 Agastache foeniculum (pursh) Kuntze Blue giant hyssop Herb 6 8.33 ± 0.27 6.83 ± 0.21 10.714/<0.0 01 1.5± 0.24 Indicative 5 Origanum vulgare L. Oregano Herb 52 8.33 ± 0.27 8.25 ± 0.25 0.435/ 0.339 0.08 ± 0.26 not significant 6 Mentha piperita L. PeppermintHerb 27 8.33 ± 0.27 8.75 ± 0.38 1.803/0.061 0.42 ± 0.33 not significant
7 Geranium macrorrhiz um L. Bigroot geranium Herb 18 8.33 ± 0.27 7.33 ± 0.17 7.694/ <0.00 1 1.00 ± 0.22 not significant 8 Melissa officinalis L. Lemon balm Herb 58 8.33 ± 0.27 7.25 ± 0.25 5.87/<0.001 1.08 ± 0.26 Indicative 9 Angelica archangeli ca L. Norweigan Angelica Leaf s 24 8.33 ± 0.27 6.67 ± 0.17 12.67 9/<0.0 01 1.66 ± 0.22 Indicative
10 Angelica archangeli ca L. Garden Angelica Root s 32 8.33 ± 0.27 8.00 ± 0.27 2.115/ 0.03 0.33 ± 0.27 not significant 11 Thymus vulgaris L. Common thyme Herb 63 8.33 ± 0.27 7.25 ± 0.25 5.87/<0.001 1.08 ± 0.26 Indicative 12 Hyssopus officnialis L. Hyssop Herb 63 8.33 ± 0.27 6.50 ± 0.24 12.449/<0.0 01 1.83 ± 0.255 Indicative/mo derate 13 Nepeta cataria L. Catnip Herb 31 8.33 ± 0.27 6.83 ± 0.28 9.434/ <0.00 1 1.5 ± 0.27 Indicative 14 Echinacea purpurea L. purple coneflower Herb 49 8.33 ± 0.27 6.67 ± 0.17 12.67 9/<0.0 01 1.66 ± 0.226 Indicative 15 Salvia officinalis L. common sage Herb 11 8.33 ± 0.27 6.67 ± 0.17 12.679/<0.0 01 1.66 ± 0.226 Indicative 16 Desmodiu m canadence L. canada tickclover Herb 29 8.33 ± 0.27 7.25 ± 0.25 5.87/<0.001 1.08± 0.26 Indicative
