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  • Orro, Toomas (Helsingin yliopisto, 2008)
    The early protection mechanism of the host against infection, trauma or other tissue damage comprises a set of reactions known as the acute phase response (APR). During APR, circulating concentrations of acute phase proteins (APPs) change. These proteins can serve as indicators of host response during various inflammatory conditions. In this thesis, APR in reindeer was investigated for the first time. Systemic concentrations of APPs during the neonatal period were studied in reindeer and cattle. APPs were also investigated during spontaneous bovine respiratory disease (BRD) in dairy calves. Escherichia coli endotoxin challenge in adult reindeer increased concentrations of serum amyloid A (SAA) in all animals. Haptoglobin (Hp) showed a less pronounced increase. SAA and Hp were concluded to be acute phase reactants in reindeer. In reindeer calves SAA concentrations increased during the first 2 weeks of life and decreased afterwards. Serum Hp concentrations increased throughout the first month after birth. In dairy calves SAA and lipopolysaccharide binding protein (LBP) concentrations changed similarly during first month of age as in reindeer calves. However, Hp concentrations generally remained low after birth. SAA rise in calves were not derived from colostrum as mammary specific SAA isoforms were not found from calves serum samples. Results of these two studies indicated that newborn reindeer and dairy calves have an inflammatory response during the first weeks of life and the age of young animals should by considered when interpreting APP concentrations. Very similar SAA changes in the two ruminant species also suggest that this inflammatory response may have role in the adaptation process of newborns to extrauterine life. The effect of different bovine respiratory pathogens on concentrations of APPs (SAA, LBP, Hp, alpha1-acid glycoprotein and fibrinogen) was studied in calves. Isolation of Pasteurella multocida was associated with increased concentrations of all APPs tested. In another study, concentrations of APPs were investigated in dairy calves during an outbreak of BRD. Initial cause for BRD outbreak was bovine respiratory syncytial virus (BRSV) infection. Concentrations of SAA and LBP increased in parallel with clinical symptoms at week 1 and peaked at week 3 of outbreak. Some calves had high Hp concentrations at week 3. Higher SAA, LBP and Hp concentrations at a later stage of BRD (week 3) were associated with lower BRSV-specific IgG1 production, suggesting that these calves had enhanced inflammatory response to secondary bacterial infection. In conclusion, APPs proved to be useful in exploring host response in bovine respiratory infections.
  • Airas, Niina (Helsingin yliopisto, 2014)
    Trichinella nativa is a member of the genus Trichinella, which includes nine different species and three genotypes. Trichinella spp. are spread worldwide and they can cause a disease called trichinellosis in both animals and humans. In this thesis, the epidemiology of T. nativa and other Trichinella species in a boreal environment was investigated. Unusual T. nativa infection in a domestic cat with clinical signs was reported. Host responses induced by T. nativa and Trichinella spiralis were compared in a selective host (rat) to identify the phase of the life cycle in which the selective responses take place. Of the 2483 carnivorous samples collected from Finland, 617 (24.8%) were positive for Trichinella spp. with higher prevalence in the North compared to southern part of the country. Four endemic Trichinella species were identified using multiplex PCR: T. spiralis, T. nativa, Trichinella britovi, and Trichinella pseudospiralis. Trichinella nativa was shown to be the predominant Trichinella species (80.1%) in all investigated wild host species. Red fox and raccoon dog were the most important reservoir animals when population size, estimated prevalence of Trichinella infection, and infection intensity were taken into account. Trichinella infection can cause clinical manifestations; a domestic cat had an ulcer below the eyelid and a skin biopsy revealed one Trichinella sp. larva surrounded by inflammatory reaction and granulation tissue. Enzyme-linked immunosorbent assay and Western blotting showed a seropositive reaction against Trichinella spp. antigens in the cat s serum. The Trichinella sp. larva was identified as T. nativa by multiplex PCR. During the 1-year follow-up, a subcutaneous mass started to cover the previous surgical site. A biopsy sample taken from that area showed inflammatory cells and fibroblasts, with some fibrodysplasia. To identify the phase of the life cycle in which the defense against Trichinella takes place, rats were infected both per orally (p.o.) and intravenously (i.v.) with T. nativa and T. spiralis larvae. After i.v. injection, 1.7% of the T. nativa NBL and 20% of the T. spiralis NBL reached the muscle tissue of the rat (p less than 0.05). These results showed that the defense against Trichinella did not solely localize to the enteral or parenteral phase. The different infectivity of the two Trichinella species can also be partly explained by difference in reproductivity; T. nativa females isolated from a mouse released more NBL than those isolated from a rat. In contrast, T. spiralis females isolated from a mouse produced fewer NBL than those isolated from a rat. Trichinella nativa and T. spiralis induced similar gene expression changes in the intestinal tissue of a selective host studied with whole-genome microarray on day five post infection (p.i)., even though the parasite burden caused by T. spiralis was significantly higher than that caused by T. nativa. When the two Trichinella-infected groups were pooled and compared with control animals, microarray data of the infected animals indicated nonspecific damage and an inflammatory response in the jejunal mucosa. Histopathological changes supported the microarray data. Trichinella spp. is highly prevalent in Finland; it is a risk for domestic animals and humans. Even low infection intensity can cause clinical signs or symptoms.
  • Kassinen, Anna (Helsingin yliopisto, 2009)
    Irritable bowel syndrome (IBS) is a functional bowel disorder, most likely with multiple interacting factors contributing to its aetiology. The intestinal microbiota has been proposed as one of these factors. The human intestinal microbiota is a rich and dynamic microbial community inhabited by 1014 microbial cells, most of which are uncultivable. Therefore, the use of molecular methods capable of detecting the uncultivable microbes is essential. Real-time polymerase chain reaction (real-time PCR) technology was assessed for the quantification of bacteria from faecal samples, and 43 assays were designed for quantifying resident, pathogenic, probiotic or IBS-related bacteria or bacterial phylotypes. With real-time PCR, a 0.01% subpopulation could be quantified from mixed faecal deoxyribonucleic acid (DNA) samples, with a linear range of five orders of magnitude. The method proved to be sensitive and accurate also with intact bacterial cells spiked to faecal samples. The intestinal microbiota of subjects suffering from IBS was then compared with that of healthy controls. For comparing the microbiotas on a scale covering the entire community, genomic microbial DNA extracted from faecal samples was pooled according to symptom subtype and percent guanine plus cytosine (G+C) profiled. The three most diverging %G+C fractions were analysed by cloning and partial sequencing of the 16S ribosomal ribonucleic acid (rRNA) gene. The clone libraries on the whole diverged from each other, and several differences were detected in the abundances of certain phylotypes. The genera Coriobacterium and Collinsella within the phylum Actinobacteria were considerably more abundant in the pooled healthy control sample. To analyse the quantities of putatively IBS-associated phylotypes within individual samples, real-time PCR was applied. Several significant differences were detected, including a novel clostridial 16S rRNA gene phylotype associated with mixed-subtype IBS and healthy controls and a Ruminococcus torques resembling phylotype associated with diarrhoea-predominant IBS. Diarrhoea-predominant IBS patients diverged from constipation-predominant and mixed-subtype IBS patients and the healthy controls, in a multivariate analysis of 14 phylotypes. It should be noted, however, that these results give no indication as to whether the observed alterations are a causative agent in IBS aetiology or merely a result of the altered environment in the disturbed gut. These results support the hypothesis of intestinal bacteria having a role in IBS, and the specific phylotype-level differences detected warrant further studies for their potential use in IBS diagnostics, therapeutic trial follow-up and host-microbe interactions.
  • Kirk, David (Helsingin yliopisto, 2015)
    Clostridium botulinum presents a risk to food safety through the production of endospores. These spores are highly heat-resistant and may withstand temperatures used in food processing. Despite this, the process of spore formation is poorly understood in C. botulinum. This study aimed to analyse in Group I C. botulinum ATCC 3502 the role of sigma (σ) factors σF, σE, σG, and σK. The role of these σ factors is well known in other spore formers, activating in an ordered cascade to regulate gene transcription during sporulation. To study gene expression during sporulation in C. botulinum ATCC 3502, we identified a suitable normalisation reference gene for reverse-transcription real-time PCR (RT-qPCR). Mutants of sigF, sigE, sigG, and sigK were examined on the transcriptional level during sporulation, and each strain was characterised for growth and spore formation. Furthermore, the role of σK in stress tolerance was investigated under cold, NaCl, and pH stresses. Transcriptional analysis, from exponential to stationary phases of growth, of eight candidate reference genes was performed. The candidate genes were 16S ribosomal RNA (rrn), the ATP metabolism enzymes adenosine kinase (adK) and glutamate dehydrogenase (gluD), the DNA-binding protein gyrase (gyrA), and ribosome-related proteins alanyl-tRNA synthetase (alaS), GTP-binding Era (era), RNA polymerase β subunit (rpoC) and 30S ribosomal protein S10 (rpsJ). Of these candidates, only 16S rrn was stable during the study period. 16S rrn was used as the normalisation reference gene for RT-qPCR analysis of spo0A, sigF, sigE, sigG, and sigK expression during the same growth period. Expression of spo0A was highest during exponential growth, suggesting a role in early sporulation. Induction of sigF, sigE, and sigG expression occurred on entry into stationary growth, indicating a role in sporulation. Expression of sigK appeared biphasic, being expressed in both exponential and stationary phases, suggesting σK may play a dual role in sporulation. The genes of σF, σE, σG, and σK were mutated using the ClosTron tool. RT-qPCR analysis of the sigF and sigE sense mutants suggested that the sporulation pathway was disrupted in the early stages. This was confirmed by electron microscopy, which showed that all sigF and sigE mutants were unable to form spores. They halted sporulation after asymmetric cell division, stage II of the seven-stage sporulation cycle. The sigG sense mutant showed delayed transcription of the sporulation pathway and both sigG mutants possessed a thin spore coat but no cortex. This indicated that σG may be responsible for cortex, but not coat, formation in C. botulinum. The sigK sense mutant did not express the early-sporulation genes spo0A and sigF. Both sigK mutants appeared to halt sporulation early. Sporulation was restored by complementing the sigK mutation in trans. These results suggested that σK plays an essential role in early sporulation of C. botulinum ATCC 3502, and adds further weight to the possibility of a dual role in sporulation overall in this strain. Expression of sigK was assessed in C. botulinum ATCC 3502 after cold, osmotic (NaCl), and acidic shock. After cold and osmotic shock, expression of sigK was induced. Both sense and antisense sigK mutants were then grown under stress conditions of low temperature, high NaCl, and low pH. Under low temperature and high NaCl conditions, but not in low pH, growth of the mutant strains was negatively affected compared to parent strain growth, suggesting that σK may play a role in tolerance to low temperature and high salinity stress conditions.
  • Keto-Timonen, Riikka (Helsingin yliopisto, 2008)
    In epidemiological studies, techniques that effectively discriminate between individual bacterial strains are essential. Recent developments in molecular techniques necessitate an ongoing need to tailor new genotyping methods for optimal characterization of different bacterial species and to evaluate their performance and suitability for research purposes. In this thesis amplified fragment length polymorphism (AFLP) analysis was tailored for optimal characterization of Listeria monocytogenes and Clostridium botulinum. The suitability of the developed AFLP protocol to type L. monocytogenes, C. botulinum and Clostridium perfringens at strain level was evaluated. AFLP proved to be a highly reproducible, easy-to-use, relatively fast and highly discriminative approach. When AFLP was applied to L. monocytogenes strains, its discriminatory power was shown to equal that of PFGE, which is considered the current gold standard for molecular fingerprinting of L. monocytogenes. These features make AFLP analysis a useful alternative to other genotyping methods in, for example, outbreak investigations and contamination route studies. Since phenotypic identification of Clostridium isolates is laborious, the suitability of AFLP for genomic species identification was assessed. The AFLP technique was applied to 129 strains representing 24 different Clostridium species. AFLP differentiated all species tested, except for Clostridium ramosum and Clostridium limosum. AFLP also differentiated between six different Listeria species. If AFLP profiles of well-defined strains are collected in identification libraries, the database can be a valuable additional tool for identification of Clostridium and Listeria species. Due to high throughput of samples, AFLP proved to be especially suitable for screening large numbers of isolates. AFLP was also used to trace contamination routes of L. monocytogenes in a chilled food processing plant producing ready-to-eat and ready-to-reheat meals during an 8-year period. Cleaning routines, product type and degree of compartmentalization seemed to have an influence on the contamination status in compartments that produced cooked meals. In addition, raw materials were shown to cause contamination of uncooked meals. Thus, special attention should be paid to quality control of raw ingredients when uncooked ready-to-eat meals are produced. This work also demonstrated that structural adjustments of a production line may facilitate the eradication of L. monocytogenes from the food processing environment. AFLP and PFGE analysis of sporadic L. monocytogenes strains and strains that cause persistent plant contamination revealed that persistent strains differ from sporadic strains. However, no specific evolutionary lineage of persistent strains was observed.
  • Herva, Tuomas (Helsingin yliopisto, 2015)
    Animal welfare (AW) is an issue of growing concern in Finland as well as in other developed countries. A public debate has focused on the potential AW problems resulting from current production systems. Possibilities to find mutual benefit for animals, farmers, industry and society have received less attention. According to the reviewed literature the inconsistency of determination and perception of AW appeared to be a major barrier to enhance AW. Farmers should be confident that their measures to promote AW satisfy public opinion and are ecomically sustainable. The main objective of the study was a thorough understanding of relationships between AW and beef production economics to find barriers and opportunities for enhanced AW. A version of the Animal Needs Index (ANI/TGI 35L), modified for Finnish beef production, called A-index was used for AW assessments. The A-Index was modified and evaluated based on Test Theory. On-field associations between A-index and production parameters were determined on 180 farms and over 12 000 bulls using statistical multilevel models. Economic evaluation of AW was based on comparison between cold and warm housing using the confirmed association between AW and production results. AW was associated with good production results. A-Index and the best subset of items used as welfare score (WFS) were covering different aspects of AW. The association between the used measures and production results, reflecting AW in certain degree, can be considered as a proof of the criterion validity of A-Index and WFS. Cold housing with enhanced welfare and bedding based on own straw at a reasonable price was economically favourable. Profitability of cold housing was sensitive to fluctuation in bedding price. Developing a reasonably priced market for bedding material would be a major way to enhance AW. Rubber covered slats were found to be a profitable way to enhance AW in warm housing. A reform of the subsidy system was suggested to be needed to fulfil the aims of the subsidy regime to support AW.
  • Kaikkonen, Matti (Helsingin yliopisto, 2006)
    The aim of this thesis was to develop a new simple method for measuring added caesium (Cs) in blood plasma and to use it in the study of short-time plasma kinetics. The new method, based on isotopic dilution, is analogous to radioimmunoassay. The dispersive complex salt ammonium-iron(III)-hexacyanoferrate(II) (AFCF) has a very high affinity towards Cs+. In a mixture of plasma, AFCF and added radioactive Cs tracer (134Cs), the amount of AFCF-bound activity depends o n the amount of stable Cs in plasma. This bound activity is separated from soluble activity by co-precipitating AFCF with plasma proteins using trichloroacetic acid and measured using a standard gamma counter. Using standard samples prepared from plasma collected prior to an intravenously given Cs dose, the Cs concentration in plasma samples after the dose can be determined. The qualitative detection limit for the method is around 0.2 mol l-1, and the practical limit for quantitative results around 1 mol l-1. Using this method, short-term plasma kinetics after an intravenous dose of Cs was measured in both goats and horses. The values measured for goats were very similar to those using 134Cs as a tracer in another study. The rate constant for the removal of Cs from the bloodstream was initially above 0.1 min-1, but decreased within 40 min to a value below 0.02 min-1. From 2 to 40 min, the plasma concentration can be approximated with a biphasic exponential decay curve. Exercise speeds up the rate of Cs removal from blood. Between 30 and 40 min after the start of exercise, the rate of Cs removal was twice as high in exercising individuals compared to resting individuals (0.06 min-1 vs. 0.03 min-1). Stimulation of muscle sodium-potassium pumps (Na, K-ATPase) is a plausible explanation for the increased removal of Cs from blood during exertion. At 30 min after dosing, the tissues with the highest 134Cs content were the gastrointestinal tract (22% of original dose), skeletal muscle (14% of dose) and the kidney (13%). A likely location for the large unrecovered portion of the original dose (38%) was connective tissue. Incorporation in one or several of these tissues probably explains the rapid initial removal of Cs from circulation.
  • Rantala, Merja (Helsingin yliopisto, 2009)
    Aims: This thesis investigated the prevalence of and trends in antimicrobial resistance in pneumococci in Finland, determined the genetic basis of macrolide resistance and evaluated the level of telithromycin nonsusceptibility prior to its widespread usage. In addition, the clonality of telithromycin-resistant and penicillin-resistant isolates was examined. Results: Of the 1007 pneumococci collected in 2002, 21.5%, 12.1%, and 14.4% were non-susceptible to erythromycin, penicillin and tetracycline, respectively. Multiresistance was detected in 10.5% of the isolates. Only 0.1% of the isolates were non-susceptible to ceftriaxone (non-meningitis breakpoint) and <1.5% to fluoroquinolones. Two isolates were nonsusceptible to linezolide. Among invasive pneumococci (n=3571) collected in 2002-2006, erythromycin resistance increased from 16% (2002) to 28% (2006) (Poisson regression, p < 0.0001), penicillin non-susceptibility from 8% to 16% (< 0.0001) and penicillin resistance from 0.8% to 3.7% (p = 0.03). Tetracycline resistance remained stable (~10%), as did the proportion of multiresistant isolates (~5%). Levofloxacin and ceftriaxone resistance was rare. In both sets of collections of pneumococci, the highest prevalence of erythromycin resistance was among isolates derived from 0- to 2-year-old children: in 2006, 45.8% of isolates were resistant to erythromycin in this age group. In 2002, disk diffusion testing revealed 26/1007 (2.6%) pneumococcal isolates that produced one to several clearly visible colonies inside the growth inhibition zone, indicating heterogeneous resistance to telithromycin. This type of telithromycin resistance has not been previously described. All such isolates were erm(B) positive, but the exact underlying mechanism of telithromycin resistance remained unresolved. Telithromycin resistant isolates had seven distinct sequence types, of which ST193 was the most frequent (n = 19). ST193 isolates were all 19A serotype variants of the PMEN clone Greece21-30. Among penicillin resistant isolates in 2002-2006, a total of 25 sequence types were found that distributed into ten clonal lineages. The most common clonal complex was CC156, accounting for 61% of all penicillin resistant isolates. The majority of the penicillin-resistant pneumococci were representatives of single to triple locus variants of the following PMEN clones: Spain9V ST156, Taiwan19F ST236, Spain23F ST81, and England14 ST9. In 2002, the most frequent macrolide resistance gene was the mef gene (49%), followed by erm(B) (41%). A double mechanism [mef(E)+erm(B)] was detected in 5 (2.3%) isolates. Of the mef genes, 89% had the mef(E) subclass and 11% had mef(A). Mutation was detected in 16 isolates, of which 14 isolates (6.4%) had no other known resistance factor. In 2002-2006 the macrolide gene distribution was the following: 56% of the macrolide resistant isolates had mef gene and 31% had erm(B). Only two isolates had both mef(E) and erm(B). mef(E) was the most common mef subtype (72%). Conclusions: The main observation of this thesis was the presence ofheterogeneous telithromycin resistance among pneumococci carrying erm(B). Such isolates harbour a minor population of bacterial cells capable of expressing high level telithromycin resistance in vitro, which may be clinically significant. Because dilution methods do not favour the detection of this resistance type, the disk diffusion susceptibility testing of erm(B)-positive pneumococci is recommended. Due to the high prevalence of resistance, macrolides cannot be recommended as the first line drugs for the treatment of pneumococcal infections. Apart from macrolide resistance, the proportion of penicillin non-susceptible isolates is rising.
  • Koskenniemi, Kerttu (Helsingin yliopisto, 2012)
    Lactobacillus rhamnosus GG is a probiotic bacterium that is known worldwide. Since its discovery in 1985, the health effects and biology of this health-promoting strain have been researched at an increasing rate. However, knowledge of the molecular biology responsible for these health effects is limited, even though research in this area has continued to grow since the publication of the whole genome sequence of L. rhamnosus GG in 2009. In this thesis, the molecular biology of L. rhamnosus GG was explored by mapping the changes in protein levels in response to diverse stress factors and environmental conditions. The proteomics data were supplemented with transcriptome level mapping of gene expression. The harsh conditions of the gastro-intestinal tract, which involve acidic conditions and detergent-like bile acids, are a notable challenge to the survival of probiotic bacteria. To simulate these conditions, L. rhamnosus GG was exposed to a sudden bile stress, and several stress response mechanisms were revealed, among others various changes in the cell envelope properties. L. rhamnosus GG also responded in various ways to mild acid stress, which probiotic bacteria may face in dairy fermentations and product formulations. The acid stress response of L. rhamnosus GG included changes in central metabolism and specific responses related to the control of intracellular pH. Altogether, L. rhamnosus GG was shown to possess a large repertoire of mechanisms for responding to stress conditions, which is a beneficial character of a probiotic organism. Adaptation to different growth conditions was studied by comparing the proteome level responses of L. rhamnosus GG to divergent growth media and to different phases of growth. Comparing different growth phases revealed that the metabolism of L. rhamnosus GG is modified markedly during shift from the exponential to the stationary phase of growth. These changes were seen both at proteome and transcriptome levels and in various different cellular functions. When the growth of L. rhamnosus GG in a rich laboratory medium and in an industrial whey-based medium was compared, various differences in metabolism and in factors affecting the cell surface properties could be seen. These results led us to recommend that the industrial-type media should be used in laboratory studies of L. rhamnosus GG and other probiotic bacteria to achieve a similar physiological state for the bacteria as that found in industrial products, which would thus yield more relevant information about the bacteria. In addition, an interesting phenomenon of protein phosphorylation was observed in L. rhamnosus GG. Phosphorylation of several proteins of L. rhamnosus GG was detected, and there were hints that the degree of phosphorylation may be dependent on the growth pH.
  • Hielm-Björkman, Anna (Helsingin yliopisto, 2007)
    The series of investigations presented in this thesis examined different methods of assessing chronic pain in dogs suffering from osteoarthritis (OA) and compared the effects of three different treatments. Data were obtained from two cohorts; 41 dogs with OA due to canine hip dysplasia (CHD) (I,III) and 61 dogs with OA due to CHD or elbow dysplasia (II,IV,V). Questionnaires, veterinary evaluations, visual analog scales (VAS), plasma hormones, radiographs, and force plate evaluations were assessed as OA treatment outcome measures and/or measurements of chronic pain. The results indicated that the multidimensional pain scale including 11 questions with 5-point scale responses was a valid and reliable tool for evaluating chronic pain. This Helsinki chronic pain index (HCPI) can be applied as an outcome measure in clinical trials where chronic pain is evaluated by owners. Of the evaluated complementary therapies for chronic pain due to OA, all three indicated a positive treatment outcome. In the first trial, gold bead implants resulted in a significant positive treatment outcome for the treatment group. However, the placebo group in this study also improved significantly. A positive effect was seen in 65% of the placebo dogs and this exceptionally high incidence of amelioration suggests that the placebo group may have got an effect of unintentional needle acupuncture. The results of this study are therefore controversial and treatment guidelines based on these findings cannot be given. The second trial tested two ingestible OA remedies, green lipped mussel and a homeopathic low-dose combination preparation. Both treatments resulted in statistically significant positive treatment outcomes compared with placebo, but with the positive control (carprofen) being more effective than either of them. The results suggest that both tested treatments may be beneficial for chronic OA. To establish the true role of all these three treatments in outcome-based animal analgesia, more clinical trials, using larger cohorts, should be conducted. Possible mechanisms of action should also be studied.
  • Junnila, Matti (Helsingin yliopisto, 2000)
  • Liljavirta, Jenni (Helsingin yliopisto, 2014)
    The majority of research studies on immunological mechanisms have been conducted in human or rodent models and the results are generalized in respect of other vertebrates. However, the generation of B cells varies considerably between species. B cells are produced in the bone marrow of rodents and humans throughout life. The initial antibody diversity is produced by the random assembly of a great variety of antibody encoding gene segments. The repertoire of these gene segments is limited in many domestic species such as cattle, sheep, chicken, rabbit and pig and de novo B lymphopoiesis takes place only during fetal and/ or neonatal life. This results in a basal B cell population but one that is capable of being further expanded by other mechanisms. In this thesis, the specific diversification mechanisms in the bovine are explored. Knowledge of immunoglobulin genes is essential in order to distinguish various biological mechanisms, which are involved in antibody repertoire diversification. After the bovine genome was sequenced in 2009, the characterization of the immunoglobulin loci became possible. Previously, the bovine immunoglobulin light chain loci were characterized by only a small number of functional gene segments that had been found. Subsequently, we were able to analyse the heavy chain locus and found a total number of 62 heavy chain variable gene segments, of which 10-20 were verified as functional genes. Many livestock species and chicken rely on gut-associated lymphoid tissue for further expanding their fetal/neonatal antibody repertoire to compensate for the limited effective recombinatorial diversity. In cattle, the ileal Peyer s patch (IPP) is considered to be the major organ for B cell proliferation. In this research, the fingerprints of somatic hypermutation (SHM) in the IPP were analysed. SHM is dependent on activation-induced cytidine deaminase (AID), which contributes to modifying the immunoglobulin variable regions. SHM is conventionally considered to be a secondary diversification mechanism, which is activated with external antigens. AID-mediated SHM was shown to diversify the antibody repertoire in the fetal IPP, before the exposure of exogenous antigen encounters. Junctional diversity is produced by the random additions and excisions of nucleotides between immunoglobulin gene segments that occur during the somatic recombination. Non-templated nucleotides are added by terminal deoxynucleotidyl transferase (TdT). Sequence investigation in this study indicated that TdT-mediated junctional diversity contributes to the diversification of the antibody repertoire in bovine fetuses. Extensive junctional diversity mainly in the heavy chain sequences in bone marrow, ileum and spleen but also to a lesser extent in the light chains was detected in this study. The following model for bovine preimmune repertoire diversification was suggested in this study. First, the restricted immunoglobulin germline repertoire is diversified by junctional diversity in fetal bone marrow. Second, a small population of the B cell clones migrates to the fetal/neonatal IPP. In the IPP, further diversification by AID-mediated SHM takes place, which is associated with extensive proliferation. Third, these clones migrate to other peripheral organs where they are subjected to secondary, antigen-induced modifications. The IPP starts to involute in young animals and the animal survives for the rest of its life by proliferating and differentiating its B cell clones from the peripheral repertoire.
  • Ekman, Anna (Helsingin yliopisto, 2012)
    Immunological research is dominated by studies on man and mouse, however, only some aspects of this field are universal among vertebrates. Whilst the production of T cells is universal, B cell production cannot be extrapolated from one species to another. As such, ruminant B cell biology has distinctive features not similar to the conventional man or mouse based models. Cattle are large long-lived ruminants, of major significance globally. The bovine ileal Peyer s patch, an organ of B cell follicles along the gut, is where V(D)J immunoglobulin gene rearranged B cells proliferate, however it is not known where and how the DNA rearrangements take place before the B cells enter these follicles. Furthermore, it is unclear whether post-recombinatorial modifications, such as somatic hypermutation or gene conversion, facilitate the generation of more antibody specificities of the immunoglobulin genes in these follicles. Herein, the bovine immunoglobulin light chain genomic locus was characterized and only a moderate number of functional gene segments that cause low combinatorial antibody diversity were found. The lambda locus is the larger of the two light chain loci, containing 25 functional variable gene segments, compared to the kappa locus, which contains only eight. Functional genes comprise less than half of all the variable genes in both loci, the remainder representing unfunctional pseudogenes. The immunoglobulin genes of the fetal ruminant ileal Peyer s patch can possibly be further modified. Accordingly, the expression of activation-induced cytidine deaminase (AID), a mutator protein, was demonstrated here in fetal cattle ileal Peyer s patch. Sequencing of expressed heavy chain variable genes in these follicles showed ongoing hypermutation. The mutations were concentrated on the complementarity determining regions (CDR) of the variable genes, and on the hotspot target sequences of AID. AID-dependent mutations have usually been ascribed to antigen dependent affinity maturation, but this work demonstrates mutations in fetal immunoglobulin genes before exposure to external antigen. Bovine B lymphopoiesis is studied here, its localization in the fetal cattle in particular. By analyzing the expression of RAG1 and RAG2, which take part in the rearrangement of immunoglobulin genes, active B lymphopoiesis was demonstrated in fetal bone marrow and lymph node. The expression of surrogate light chain genes VPREB1 and IGLL1 was also shown in these same tissues. The expression of these genes implicated that a pre-B cell stage exists in cattle. This was further confirmed by the presence of a phenotypic pre-B cell population in fetal bone marrow and lymph node. VPREB2 and VPREB3 were expressed differently from the other surrogate light chain genes, which indicate that their function in cattle might not be related to pre-B cells. Overall, B lymphopoiesis was shown to take place in fetal, but not in adult, bovine tissues. Pre-B cell related genes RAGs, VPREB1 and IGLL1 were not expressed in adult tissues. Further, adult bone marrow cells were not able to differentiate into B lineage cells in cell culture. These results suggest that no new immunoglobulin rearrangements are generated during bovine adult life. Thus it is likely that the animal manages its whole life with the peripheral B cell pool produced during the fetal and neonatal period.