Krüptosporiidide levimus, seos üldise põletikuvastuse, rooja mikrobioota ja halofuginoon laktaadi raviga vasikatel

A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Sciences.Väitekiri filosoofiadoktori kraadi taotlemiseks loomaarstiteaduse erialal.Calf diarrhoea is a common problem in cattle herds, and it has many causative agents including the protozoan parasites of the genus Cryptosporidium. This parasite is detected worldwide and has clinical importance particularly in pre-weaned calves. The most common species in calves is Cryptosporidium parvum. In this thesis, the prevalence of Cryptosporidium spp. in Estonian calves was determined, which species of this parasite were shed with faeces, and which subtypes of C. parvum were shed. Additionally, associations of Cryptosporidium species, general inflammatory response, and faecal microbiota were studied. The effects of treatment with halofuginone lactate were evaluated. Cryptosporidium spp. are widely spread among Estonian calves during their first two months of life. In this thesis, three species were detected, and ten subtypes of C. parvum were described, one of them being novel. Shedding of Cryptosporidium spp. oocysts was most common during the second week of life. The general inflammatory response was measured through blood serum concentrations of acute phase proteins (specifically serum amyloid A and haptoglobin), that are synthesised in liver as a result of pathogen invasion or tissue damage. The more Cryptosporidium spp. oocysts the calves shed in their faeces, the higher the concentration of these proteins in the blood, and the lower the microbial diversity in faeces. Some bacterial genera in faeces were positively associated with the serum concentrations of the acute phase proteins (e.g., Fusobacterium), or negatively with cryptosporidiosis (e.g., Ruminiclostridium 9). Halofuginone lactate treatment decreased mortality of calves and delayed the oocyst shedding. Understanding the interactions of Cryptosporidium spp., host responses, and the microbiota of the calf can help to comprehend the development of the calf as a whole. Based on this knowledge, factors benefiting the welfare and performance of production animals could be found and possibly influenced.Vasikate kõhulahtisus on levinud probleem veisekarjades ja sellel on palju võimalikke põhjustajaid, sealhulgas perekond Cryptosporidium parasiidid. Need parasiidid on ülemaailmase levikuga ja nendel on kliiniline tähtsus, eriti võõrutamata vasikatel. Levinuim liik vasikatel on Cryptosporidium parvum. Käesolevas väitekirjas uuriti Cryptosporidium’i perekonna levimust Eesti vasikatel, milliseid liike eritatakse, ja milliseid C. parvum'i alamtüüpe neil esineb. Lisaks uuriti seoseid Cryptosporidium'i liikide, üldise põletikuvastuse ja rooja mikrobioota vahel. Hinnati halofuginoon laktaadi ravi mõju krüptosporidioosi korral. Cryptosporidium'i liigid on Eesti vasikate seas laialt levinud nende esimesel kahel elukuul. Selles väitekirjas tuvastati kolm liiki ja kirjeldati kümmet C. parvum’i alamtüüpi, millest üks oli uudne. Cryptosporidium'i ootsüste eritati kõige rohkem teisel elunädalal. Üldist põletikuvastust mõõdeti ägeda faasi valkude (täpsemalt seerumi amüloid A ja haptoglobiini) vere seerumi kontsentratsioonide mõõtmise kaudu, mida sünteesitakse maksas patogeeni invasiooni või koekahjustuse tulemusena. Mida rohkem Cryptosporidium'i ootsüste vasikad oma väljaheidetega eritasid, seda suurem oli nende valkude kontsentratsioon veres ja seda väiksem oli rooja mikrobioota mitmekesisus. Mõned väljaheidete bakteriperekonnad olid seotud ka ägeda faasi valkude kontsentratsiooniga (nt Fusobacterium, positiivne seos) või krüptosporidioosiga (nt Ruminiclostridium 9, negatiivne seos). Ravi halofuginoon laktaadiga vähendas vasikate suremust ja lükkas edasi ootsüstide eritamist. Cryptosporidium'i liikide, peremeesorganismi füsioloogiliste reaktsioonide ja mikrobioota koostoime mõistmine võib aidata mõista vasika arengut tervikuna. Nende teadmiste põhjal on võimalik leida ja ehk mõjutada produktiivloomade heaolu ja jõudlust soodustavaid tegureid.The publication of this dissertation is granted by the Doctoral School of Clinical Medicine, supported by the European Union, European Regional Development Fund (Estonian University of Life Sciences ASTRA project „Value-chain based bio-economy“) and by the Estonian University of Life Sciences

Biosafety and biosecurity manual

ManualThe aim of this manual is to provide biosafety and biosecurity instructions for the staff and students of the Institute of Veterinary Medicine and Animal Sciences (VLI) of Estonian University of Life Sciences (EMÜ). Standard operating procedures gathered in this manual are applied only in VLI. The goals of these procedures include minimizing the risk of nosocomial infections and minimizing the exposure to zoonotic disease agents. New staff members and students will be notified about this manual by his/her immediate supervisor. After reading this manual the employee will give his/her signature as a sign of having acquainted with the manual and understanding the procedures needed in his/her special line of work and/or studies. For students, this will be done during the course ‘Introduction to veterinary studies’. On institute level the staff member who is responsible for biosafety and biosecurity is appointed by the director of the Institute

Faecal microbiota in two-week-old female dairy calves during acute cryptosporidiosis outbreak-Association with systemic inflammatory response

In the present study, relationships between the intestinal microbiota and innate immunity response, acute cryptosporidiosis, and weight gain in female dairy calves were investigated. A total of 112 calves born during a natural outbreak of cryptosporidiosis on one dairy farm was included in the study. Microbiota composition was analysed by means of 16S ribosomal RNA gene amplicon sequencing from faecal samples collected during the second week of life, while the status of Cryptosporidium spp. infection was determined using immunofluorescence. Serum samples from the second week of life were colourimetrically analysed for the following markers of acute inflammation: acute-phase proteins (serum amyloid A and haptoglobin) and pro-inflammatory cytokines (interleukin-1 beta, interleukin-6, and tumour necrosis factor-alpha). Statistical analyses were performed using random forest analysis, variance-partitioning, and negative binomial regression. The faecal microbiota of the two-week old calves was composed of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria (in order of decreasing abundance). Microbial diversity, measured in terms of the Shannon index, increased with the age of the calves and decreased if a high count of Cryptosporidium spp. oocysts was found in the faeces. Fusobacterium was positively associated with Cryptosporidium spp. oocyst count and serum amyloid A concentration. Peptostreptococcus was positively associated with haptoglobin and serum amyloid A concentrations, and negatively associated with average daily weight gain at 9 months of age. The markers of innate immunity, in combination with age, explained 6% of the microbial variation. These results suggest that some components of the intestinal microbiota may have a long-lasting negative effect on animal growth through the stimulation of the systemic innate immune response.Peer reviewe

Bioohutuse ja bioturvalisuse juhend

JuhendJuhend on mõeldud Eesti Maaülikooli (EMÜ) veterinaarmeditsiini ja loomakasvatuse instituudi (VLI) töötajatele ja üliõpilastele bioohutuse ning bioturvalisuse tagamiseks. Juhendisse koondatud standardne töökord (STK) kehtib üksnes VLI-s. Standardse töökorra eesmärk on minimeerida haiglanakkuste riski ja kokkupuudet zoonootiliste mõjuritega. Uusi töötajaid ja üliõpilasi teavitab juhendist ning sellega tutvumise kohustusest nende vahetu töökorraldaja või juhendaja. Juhendiga tutvumise järel kinnitab töötaja/üliõpilane oma allkirjaga, et ta on teadlik STK-st, mis puudutab tööd/õpinguid tema valdkonnas/erialal. Üliõpilased tutvuvad STK-ga kursuse „Sissejuhatus loomaarstiõppesse“ raames. Instituudi tasandil määrab bioohutuse ja bioturvalisuse eest vastutava töötaja instituudi direktor

Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016

Introduction: This paper reviews the current knowledge and understanding of Cryptosporidium spp. an d Giardia spp. in humans, animals and the environment in 10 countries in the eastern part of Europe: Bosnia and Herzegovina, Croatia, Czech Republic, Estonia, Hungary, Latvia, Poland, Romania, Serbia and Slovenia. Methods: Published scientific papers and conference proceedings from the international and local literature, official national health service reports, national databases and doctoral theses in local languages were reviewed to provide an extensive overview on the epidemiology, diagnostics and research on these pathogens, as well as analyse knowledge gaps and areas for further research. Results: Cryptosporidium spp. and Giardia spp. were found to be common in eastern Europe, but the results from different countries are difficult to compare because of variations in reporting practices and detection methodologies used. Conclusion: Upgrading and making the diagnosis/detection procedures more uniform is recommended throughout the region. Public health authorities should actively work towards increasing reporting and standardising reporting practices as these prerequisites for the reported data to be valid and therefore necessary for appropriate control plans.Peer reviewe

Prevalence of Taenia saginata cysticercosis in Estonian cattle

Taenia saginata on zonootiline helmint, mille leviala hõlmab tervet maailma. Veistel põhjustavad selle parasiidi vastsed tsüstitserkoosi. Selle töö eesmärgiks oli uurida T. saginata tsüstitserkoosi levimust Eesti veistel. Uurimismeetodiks oli rutiinne lihainspektsioon tapamajades. Uuringu jaoks koguti proove veebruarist aprillini 2014 neljas erinevas Eesti tapamajas. Tsüstide leidmiseks kasutati rümpade visuaalset vaatlemist, palpeerimist ja sisselõigete tegemist eeliskohtadele. Eeliskohtadeks olid: välimine mälumislihas, keel, süda ja diafragma, lisaks veel sisemine pterygoidlihas ja söögitoru. Kokku uuriti 564 veist. Leiti kokku kaks Taenia-moodi tsüsti erinevate loomade rümpadelt: üks leiti 20-kuuselt pullilt, teine 32-kuuselt lehmal. Mõlemad tsüstid leiti keelest ja selle sidekoest. Tsüste leiti 0.36% (95% CL: 0.06-1.17) uuritud veistest. Leitud tsüstid hoiustati alkoholis enne DNA eraldamist ja cox1-geeni amplifitseerimist PCR-iga liigi täpsemaks väljaselgitamiseks perekonna tasandil. Kasutades PCR-i saavutasime geelis mitte väga selged, kuid umbes õige pikkusega (~550 bp) piirkonnad. Sellest tulenevalt usume, et leitud tsüstid kuuluvad Taenia perekonda. Oletades, et leitud tsüstid on liigilt T. saginata, on ilmne levimus 0.36% (95% CL; 0.06-1.17). Tõeline levimus aga oleks sellisel juhul >1% (95% CL; 1.40-1.43). Ainult mõningate tsüstide leidmine võib tuleneda valitud uurimismeetodi madalast sensitiivsusest. Arvestada tuleks, kas rutiinne lihainspektsioon on piisava sensitiivsusega et sellega suudaks leida kõiki nakatunud loomi.Taenia saginata is a zoonotic helminth with a worldwide distribution. In cattle the larval stage of this parasite can result in the diagnosis of cysticercosis. Our aim was to investigate the prevalence of T. saginata cysticercosis in Estonian domestic cattle using routine meat inspection in slaughterhouses. Sampling spanned from February to April 2014, and was conducted in four Estonian abattoirs. Cysts were searched for by visual inspection of carcasses, palpation and incisions at chosen predilection sites: external masseter, tongue, heart, and diaphragm, also the internal pterygoid muscle and esophagus. A total of 564 cattle were examined. Two Taenia-like cysts were found: one from a 20-month old bull and one from 32-month old cow. Both of these cysts were found in the tongue and its connective tissues. Cysts were found in 0.36% (95% CL: 0.06-1.17) of the investigated animals. All found cysts were stored in alcohol before DNA extraction and PCR amplification of the cox1-gene for species identification on genus level. Using PCR we acquired unclear, but about right sized (~550 bp) bands on the gel. From this we believe that the cysts are very likely to be Taenia spp. (T. saginata). We found the apparent prevalence to be 0.36% (95% CL; 0.06-1.17) and the true prevalence was calculated to >1% (95% CL; 1.40-1.43). Only few cysts were detected possibly due to low sensitivity of the applied inspection method used in abattoirs. It should be discussed if the current routine meat inspection is sensitive enough to find all the infected animals

Associations of neonatal acute phase response with first lactation performance in dairy cows

ABSTRACT: During the first 3 wk of life, the immune system of newborn ruminants starts to work, as indicated by fluctuations in the concentrations of proinflammatory cytokines and acute phase proteins (APP). They have been shown to be markers for short and long-term weight gain in ruminants. This observational study investigated these proteins as possible indicators of first lactation performance of dairy cows. A total of 117 dairy calves from a single farm were enrolled in the study. Serum and fecal samples were taken once a week for the first 3 wk of life. Cryptosporidium spp. infection and its treatment were monitored and accounted for in statistical analysis. The concentrations of the APP serum amyloid A (SAA) and haptoglobin (Hp), and the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were analyzed in serum. After the first lactation, health and performance data of the cows were retrieved, and associations between APP and cytokine concentrations with first lactation outcomes were investigated through linear and logistic regression. This study found a negative association between the concentration of Hp in the second week of life and average daily weight gain at one year. The SAA concentration measured during the second and third weeks of life was positively associated with age at first calving. IL-6, SAA, and Hp measured in the second week of life were positively associated with calving to conception interval. The concentrations of IL-6 and TNF-α during the first week of life were associated with higher odds of reproductive issues during the first lactation period. These markers can be used to help understand underlying processes that influence animal health and production. In conclusion, inflammatory responses during the first week of life are related to reproductive success, whereas the second and third weeks of life seem to influence the future productive performance in dairy cows

Associations between group sizes, serum protein levels, calf morbidity and growth in dairy-beef calves in a Finnish calf rearing unit

Efficient dairy-beef production relies on good quality of purchased calves, defined by breed, health, and growth characteristics. Several management factors, such as commingling of calves and large group size, predispose calves to diseases. Acute phase proteins are sensitive detectors of calf diseases. We studied the associations between group size, serum acute phase proteins, immunoglobulin G (IgG), calf morbidity and growth of dairy-beef calves in a random field trial in a calf-rearing unit in Finland. The randomized trial was carried out at a calf rearing unit, where approximately 80 dairy or crossbred calves were allocated either into a single group of 40 calves or into four groups of 10 on arrival at the calf-rearing unit (at age 24.1 SD +/- 9.2 days). The study was carried out on 6 arrival batches: 476 calves. Calves were clinically examined and blood sampled on arrival (day 0), and haptoglobin (Hp), serum amyloid A (SAA), albumin and IgG were determined. Calves were weighed on arrival (day 0, average age 24.1 days), at the end of the milk feed period (day 49), at approximately 200 days of age and at slaughter (carcass weight) at 15-18 months of age. During the rearing calves were observed by the farm workers and treated, if necessary, according to pre-determined instructions of the veterinary surgeon. All NSAID and antimicrobial treatments were recorded and used as morbidity indicators in statistical analysis. There were no differences in the numbers of antimicrobial treatments or growth among the groups. The majority (84.1%) of antimicrobial treatments were used against respiratory tract infections. Higher concentrations of albumin and IgG on arrival extended the time before the first and the second antimicrobial treatments. Complex relationships between group size, morbidity, concentrations of serum acute phase proteins and IgG at arrival, and growth of calves were explored. Group size of 10 calves did not protect calves from respiratory tract infections, when the small groups were sharing the air space with a large group. An increased SAA concentration on arrival was associated with poorer average daily gain at two rearing periods and with lower carcass weight at slaughter. Serum proteins could be valuable health indicators for purchased calves because they have numerous and variable associations with health and growth. The mechanisms that connect increased SAA concentration and poorer average daily gain over the long term remain unclear.Peer reviewe