Väitöskirjat - Doctoral thesis


Recent Submissions

  • Viljamaa-Dirks, Satu (Evira, 2016)
    Evira Research Report 3/2016
    Crayfish plague is a severe disease of European crayfish species and has rendered the indigenous crayfish populations vulnerable, endangered or even extinct in the most of Europe. Crayfish plague is caused by an oomycete Aphanomyces astaci, a fungal-like water mould that lives its vegetative life in the cuticle of crayfish and infects other crayfish by producing zoospores. Zoospores swim around for a few days in search of crayfish, and when they find one they attach onto its surface, encyst and germinate to start a new growth cycle as new growing hyphae penetrate the crayfish tissues. Unrestricted growth of A. astaci leads to the death of the infected animal in just a few weeks. Crayfish plague induced mortalities started in Italy around 1860. Although the disease was known about since 1860 its cause remained unknown for several decades. Little was done to prevent the spread of the disease. A lively crayfish trade probably facilitated the spread of the crayfish plague, which reached Finland in 1893. The crayfish plague has remained the most important disease problem of the Finnish noble crayfish Astacus astacus, since then. The consensus was that the disease killed all infected animals in a short time, and it appeared almost impossible to restore the flourishing crayfish populations to the levels that existed before. Following the example of neighbouring Sweden, a North American crayfish species, the signal crayfish Pacifastacus leniusculus that appeared resistant to crayfish plague was introduced to Finland in 1960s. As expected, the signal crayfish slowly started to replace the lost populations of the noble crayfish to become an important part of the crayfish fisheries. The introduction of the signal crayfish significantly added to the management problems of the noble crayfish stocks left. Signal crayfish appeared to be a chronic carrier of the crayfish plague agent, and spread the disease to the dwindling vulnerable noble crayfish populations. Later research showed that the crayfish plague agent is a parasite of North American crayfish that in normal circumstances does not harm the host animal. Intriguingly, the crayfish plague agent carried by the signal crayfish, genotype Ps1, is different from the pathogen originally introduced into Europe, genotype As. The diagnosis of crayfish plague especially when based on the isolation of the pathogen is challenging and accordingly the genotype difference was mostly unrecognized until recently. In this study we determined the genotype of the causative agent from most of the detected Finnish crayfish plague cases between 1996 - 2006. It appeared that most of the epidemics in the immediate vicinity of signal crayfish populations were caused by genotype Ps1, whereas genotype As was more prevalent in the noble crayfish areas. Interestingly, a difference was seen in the outcome of the infection. The Ps1 infection was always associated with acute mortalities, while As infections were also frequently found in existing but weak populations. The persistent nature of an As infection could be verified in noble crayfish from a small lake in southern Finland. This finding explained why many of the efforts to introduce a new noble crayfish population into a water body after a crayfish plague induced mortality were futile. The main conclusion from the field study data of this research was the difference in virulence between the Ps1 and As genotype strains. This was also verified in a challenge trial with noble crayfish. While the Ps1 strains did not show much variation in their growth behaviour or virulence, there was much more variation in the As strains. The As genotype arrived in Finland more than 100 years ago, and since that date it seems to have adapted to the novel host, the noble crayfish, to some extent. In order to gain insight into a possible vector of this genotype, we studied another North American crayfish species present in Europe, the spiny-cheek crayfish Orconectes limosus from a Czech pond. This crayfish species appeared to carry a novel genotype of A. astaci, named Orconectes genotype, designated “Or”. It seems possible that many of the North American crayfish species carry their own type of crayfish plague agent, with variable features such as virulence. These differences should be further tested in the future. The results of this study alleviate the necessity to study the noble crayfish mortalities for the verification of crayfish plague, including the study for the genotype of the A. astaci strain. Crayfish fisheries and conservation management decisions should not be made without a prior control of the donating population and the receiving water body for the eventual presence of a low-virulent A. astaci.
  • Ritvanen, Tiina-kaisa (Evira, 2013)
    Evira Research Reports 4/2013
    Fat is a remarkable source of energy in diet. The majority of dietary fat consists of fatty acids, which have a great influence on health. Much attention in nutritional discussion has focused on the saturation of fatty acids in food. Another group of fatty acids with significance to health is trans fatty acids (TFAs). Trans fatty acids resemble saturated fat in a physiological sense but are shown to increase the risk of cardiovascular disease even more than saturated fat. Nevertheless, there are differences in the biological activity of different TFA isomers. The analysis of fatty acids has met an increasing demand for more and more precise identification. The correlation between saturated fat and cardiovascular diseases found in earlier decades has changed the nutritional recommendations to reducedfat dairy products. Reduced-fat dairy products are a way of reducing total energy in diet, too. Dairy products may have a modified fatty acid profile if fat sources other than milk are used. Modern methods, with a greater weight on mass spectrometric methods, for fatty acid analysis and the effects of fat on the chemical and sensory characteristics of dairy products are reviewed. Fat reduction changes the properties of cheese. The aim of the first part of the study was to characterise cheeses on the Finnish market and find out specifically the appealing characteristics of reduced-fat cheeses. The cheeses in the study were Emmental, Edam and Havarti-type cheeses. Chemical composition, sensory profile and consumer liking were studied. Cheese properties and liking were linked to identify the appealing characteristics of different cheeses. The second part of the study included fatty acid analysis. Dairy products on the market have variable fat sources of vegetable origin. It is known from the literature that hardened vegetable fats may have TFAs, but the current situation on the Finnish market was not known. The aim of the second part of the study was to survey the fatty acid profiles of milk-based dairy products (cheeses, vegetable fat ice creams, and vegetable fat cream substitutes) and spreads and shortenings on the market. Products with reduced-fat or modified fat were included. Free fatty acids are produced by lipolysis from fat. They have effect on the flavour of cheeses. Lipolysis can be enhanced by homogenisation. The aim of the third part of the study was to validate chromatographic method for free fatty acid analysis from cheese and to study the effect of homogenisation on free fatty acids in Emmental. The content of free fatty acids was joined to the sensory profile to find out if they have impact on flavour in the pilot Emmental cheeses. This study shows that it is possible to create reduced-fat cheeses with appealing characteristics. The properties influencing liking were slightly different in reduced-fat cheeses than in regular fat cheeses. Generally 4 reduced-fat cheeses were lacking flavour. The liking of reduced-fat cheeses might increase if flavour intensities could be increased. However, it is desirable that flavour intensity is not increased with salt, as this has negative health effects. The second part of the study shows that several milk-based products and spreads on the Finnish market do not contain remarkable amounts of trans fat. In addition, the fatty acid profiles of the cheeses are shown not to be affected by cheese variety or fat reduction. More interestingly, products have variable amounts of essential fatty acids and n-3 fatty acids. Accurate identification requires good separation between fatty acid isomers. In this study polar and highly polar columns with 60 m or 100 m length were sufficient for cis/trans separation for nutritional purposes. The analysis of fatty acid methyl esters by GC-MSD gave detailed information on fatty acids in dairy food. Free fatty acids of Emmental cheese were quantitatively analysed by GCMSD without derivatisation. Internal standards were used to correct for the effect of sample treatment. The method was suitable for cheese and for shortand medium-chain fatty acids. These volatile fatty acids contribute to the flavour of cheese. The homogenisation of cheese milk increased the content of free fatty acids and the intensity of taste in trial cheeses. The defects, and on the other hand, the appealing characteristics of reduced-fat cheeses were studied. These results contribute to the development of appealing cheeses with reduced-fat content. The survey on fatty acids in dairy-based products gave up-to-date information on products on the market. These results have significance to nutritionists, dieticians, legislators and consumers, as this knowledge was not available before.
  • Gadd, Tuija (Evira, 2013)
    Evira Research Report 3/2013
    Viral haemorrhagic septicaemia virus (VHSV) was diagnosed after clinical symptoms for the first time in 2000 from four rainbow trout Oncorhynchus mykiss (Walbaum) farms in Åland and Pyhtää in Finland. Phylogenetic analysis based on the full-length VHSV glycoprotein (G) and nonvirion (NV) genes of the Finnish VHSV isolates in 2000–2004 revealed that all isolates are closely related and grouped in the genotype Id, which suggests the same origin of infection. Finnish isolates were shown to be closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and located close to the presumed ancestral source. Infection with the VHSV genotype Id has spread since then, and the same genotype had been isolated from rainbow trout farms in three separate locations: Åland in the Baltic Sea, and Uusikaupunki in the Gulf of Bothnia, and Pyhtää in the Gulf of Finland. The majority of isolations have been from Åland, and since 2009 have only been from there. The VHSV genotype Id was isolated from Pyhtää only in 2000 and 2001 and from Uusikaupunki once in 2004 and 2008. The pathogenicity of rainbow trout genotype Id isolates was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately from 13% to 40% and 66 % to 90%, respectively, depending on the size of the rainbow trout fingerlings. The Finnish brackish water VHSV genotype Id isolates induce lower mortality than freshwater VHSV isolates in infection experiments but they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout. The occurrence of viral haemorrhagic septicaemia virus (VHSV) was examined in the main spawning stocks of wild European river lamprey, Lampetra fluviatilis, in the rivers of Finland from 1999 to 2008. In total, 2621 lampreys as 262 pooled samples were examined virologically during 1999–2008. VHSV was isolated from five lamprey samples from the mouth of the rivers Lestijoki and Kalajoki, which flow into the Bothnian Bay of the Baltic Sea from Finland. The full-length VHSV G gene sequence revealed that the isolates were closely related to the VHSV strains isolated earlier from herring and sprat, Sprattus sprattus (L.), in Gotland and were therefore assigned to VHSV genotype II. The virulence of the lamprey VHSV genotype II isolate was evaluated by an experimental infection trial in rainbow trout fry. No mortality was induced post-infection by either waterborne or intraperitoneal challenge. To clarify the role of wild fish, especially Baltic herring, Clupea harengus membras (L.), in the epidemiology of VHSV in brackish waters, Baltic herring with no visible signs of disease were collected from the Archipelago Sea, the Gulf of Bothnia and from the Eastern Gulf of Finland. In total 7580 herring as 758 pooled 5 samples and 3 029 wild salmonid broodfish were virologically examined during 2004–2006. VHSV was isolated from 50 pooled herring samples collected from the Archipelago Sea and one pooled sample collected from the Gulf of Bothnia. Further studies based on the full-length VHSV G gene sequence revealed that the Finnish herring isolates were VHSV genotype II, closely related to the VHSV strains isolated earlier from herring and sprat in Gotland. VHSV genotype II isolated from the lamprey and herring is thought to be independent of the VHSV Id epidemic in farmed rainbow trout in Finnish brackish waters. The most varied VHSV strains are found in seawater. This would indicate that the viruses in freshwater originate from the sea. Two fish farms situated in the lake area of Finland have experienced elevated mortalities affecting fry of grayling, Thymallus thymallus, since 2002. These farms are using surface water for the production of juveniles of several fish species. Fourteen pooled samples were positive in virus isolation. Based on full-length G gene and partial RNA polymerase (L) gene sequences and the indirect fluorescent antibody technique (IFAT), the virus was classified as a perch rhabdovirus (PRV). Pairwise comparisons of the G and L gene regions of grayling isolates revealed that all the isolates were very closely related, with almost 100% nucleotide identity, which suggests the same origin of infection for the two farms. PRV isolates were closely related to the strain isolated from perch, Perca fluviatilis and sea trout, Salmo trutta trutta, caught from the Baltic Sea. The second shortest phylogenetic distances to rhabdoviruses isolated from other countries appeared to be to perch, grayling and pikeperch isolates from France and a pike rhabdovirus isolate from Denmark. This is the first time PRV has caused disease in grayling in Finland.
  • Hautaniemi, Maria (Evira, 2012)
    Parapoxviruses (PPVs) are zoonotic viruses which cause contagious pustular skin infections of sheep, goats and cattle worldwide. In addition, they have more recently been shown to infect other animals such as red deer, seals, camels and reindeer. Cases of contagious pustular stomatitis in Finnish reindeer have been reported for many years. This economically important disease occurs typically during winter and is more common in the southern parts of the reindeer herding districts than in the north. The first severe outbreak occurred in the winter 1992-1993, and during the winter of 1999-2000 and in the late winter 2007 outbreaks of the disease were again observed. Usual symptoms include diminished appetite, drooling, fever, and later erosions and ulcerative lesions in the mouth. The aims of this study were to establish specific and rapid detection methods for the causative agent of the disease and characterize the viruses circulating in Finland. The causative agent of reindeer pustular stomatitis was originally considered to be Orf virus (ORFV) of the genus Parapoxvirus. PCR methods amplifying different regions of the PPV genomes were developed to analyse clinical samples obtained from outbreaks of the disease in reindeer and later from viruses isolated from the disease of sheep and cattle in Finland. Subsequent phylogenetic analyses of the Finnish PPVs, known members of the genus Parapoxvirus and selected members of the subfamily Chordopoxvirinae were conducted to identify the virus species isolated from reindeer. The results showed that the reindeer PPV from 1999-2000 is most closely related to the cattle PPV Pseudocowpox virus (PCPV) whereas the PPV strains from the winter of 1992-1993 outbreak grouped with sheep ORFV strains. Reindeer samples from the 2007 outbreak were identified as both PCPV and ORFV. Analysis of the similarity between genes of reindeer PCPV and ORFV isolates, Finnish sheep ORFV and cattle PCPV isolates indicated that these viruses have been circulating among Finnish reindeer, cattle and sheep at least ten years. Since the initial classification of the viruses causing pustular stomatitis in Finnish reindeer relied solely on the partial sequence analysis of virion core- and EEV envelope phospholipase protein sequences, the genome of PCPV-like reindeer isolate (F00.120R) was sequenced by shotgun sequencing of plasmid sublibraries of cosmids covering the central region of the genome, and by sequencing transposon random insertion libraries of plasmids derived from each end of the genome. The F00.120R and the genomic sequence of a reference strain of PCPV (VR634) were annotated and analyzed in this study. This first characterization of PCPV genomes revealed that F00.120R and VR634 are 135 and 145 kb in length and contain 131 and 134 putative genes, respectively. The organization of their genomes was found to be similar to that of other PPVs and both included 88 predicted genes that are conserved across all sequenced poxviruses. F00.120R was found to have four, possibly fragmented, genes at the left terminus and another near the central region of the genome that are not present in ORFV or Bovine papular stomatitis virus (BPSV; another PPV) genomes. In addition, the F00.120R genome was found to lack six genes seen near the right genome terminus of other PPVs. Comparing the PPV proteomes and whole genome phylogenetic analyses confirmed the classification of PCPV as a separate species within the PPV genus and verified that the virus causing pustular stomatitis in reindeer in 1999-2000 can be classified as PCPV. The observed six gene deletion at the right terminus of the F00.120R genome was further investigated in an attempt to use it in differentiating PCPV and ORFV causing pustular stomatitis in reindeer. The preliminary PCR analyses of wild type virus and early passages of F00.120R implied that the deletion of genes may have arisen during cell culture of the virus. The sequence around the deleted region was determined by sequencing two cloned overlapping PCR fragments from F00.120R wt virus isolated from lesion material. The same region was sequenced from an Italian PCPV field isolate (It1303). Further PCR analyses together with sequence determination showed that a 5431 bp sequence containing genes 116-121 was likely to have been deleted from the F00.120R genome prior to the 7th passage in cell culture. In addition, genes 116-121 were present in It1303 and in other isolates of reindeer and bovine PCPV isolated in Finland during the years 2005-2010. These results indicate that the genome of reindeer PCPV is about 140 kbp in length and has 137 genes instead of previously estimated length of 135 kbp and 131 genes; it contains homologues of all known ORFV genes and this analysis further reinforces the close genetic relationship between PCPV and ORFV.
  • Perko-Mäkelä, Päivikki (Evira, 2011)
    Evira Research Reports 3/2011
    Campylobacter, mainly Campylobacter jejuni and C. coli, are worldwide recognized as a major cause of bacterial food-borne gastroenteritis (World Health Organization 2010). Epidemiological studies have shown handling or eating of poultry to be significant risk factors for human infections. Campylobacter contamination can occur at all stages of a poultry meat production cycle. In summer 1999, every broiler flock from all three major Finnish poultry slaughterhouses was studied during a five month period. Caecal samples were taken in the slaughterhouses from five birds per flock. A total of 1 132 broiler flocks were tested and 33 (2.9%) of those were Campylobacter-positive. Thirty-one isolates were identified as C. jejuni and two isolates were C. coli. The isolates were serotyped for heat-stable antigens (HS) and genotyped by pulsed-field gel electrophoresis (PFGE). The most common serotypes found were HS 6,7, 12 and 4-complex. Using a combination of SmaI and KpnI patterns, 18 different PFGE types were identified. Thirty-five Finnish C. jejuni strains with five SmaI/SacII PFGE types selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and HS serotypes. The discriminatory power of PFGE, AFLP and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. An HS serotype was distributed among different genotypes, and different serotypes were identified within one genotype. From one turkey parent flock, the hatchery, six different commercial turkey farms (together 12 flocks) and from 11 stages at the slaughterhouse a total of 456 samples were collected during one and the half year. For the detection of Campylobacter both conventional culture and a PCR method were used. No Campylobacter were detected in either of the samples from the turkey parent flock or from the hatchery samples using the culture method. Instead PCR detected DNA of Campylobacter in five faecal samples from the turkey parent flock and in one fluff and an eggshell sample. Six out of 12 commercial turkey flocks were found negative at the farm level but only two of those were negative at slaughter. Campylobacter-positive samples within the flock at slaughter were detected between 0% and 94%, with evisceration and chilling water being the most critical stages for contamination. All of a total of 121 Campylobacter isolates were shown to be C. jejuni using a multiplex PCR assay. PFGE analysis of all isolates with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA-SVR typing yielded nine flaA-SVR alleles. Three Campylobacter-positive turkey flocks were colonized by a limited number of Campylobacter genotypes both at the farm and slaughter level.In conclusion, in our first study in 1999 a low prevalence of Campylobacter in Finnish broiler flocks was detected and it has remained at a low level during the study period until the present. In the turkey meat production, we found that flocks which were negative at the farm became contaminated with Campylobacter at the slaughter process. These results suggest that proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination. Prevention of colonization at the farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of Campylobacter-positive poultry meat in Finland. In Finland, with a persistent low level of Campylobacter-positive flocks, it could be speculated that the use of logistic slaughtering, according to Campylobacter status at farm, might have be advantageous in reducing Campylobacter contamination of retail poultry products. However, the significance of the domestic poultry meat for human campylobacteriosis in Finland should be evaluated.
  • Venäläinen, Eija-Riitta (Evira, 2007)
    Lead and cadmium are toxic elements, which are a natural part of earth crust. They are taken up from the soil and transferred upwards in the plant and animal food chain. Cadmium is widely distributed throughout the natural environment with human activities having an important role in its dispersion into the biosphere. The natural sources of cadmium are volcanic eruptions and old granite rocks, which are an important geochemical source. Anthropogenic sources of cadmium are related mainly to mining, fertilizers and atmospheric deposition. Leaded gasoline has been most important source of atmospheric lead. However, most countries have now prohibited the use of leaded gasoline. This action has greatly reduced emissions of lead into the atmosphere. Anthropogenic sources of lead other than traffic are typically fossil fuel combustion, non-ferrous metal production and iron and steel production. Zinc and copper are important constituents in a number of different enzyme functions in man and animals. Zinc and copper reach the environment via industrial releases involving production and refining of metals. In 1997 emissions of heavy metal particulates were only a third of their values in the early 1990s due to the installation of sulphur removal systems. In this study lead, cadmium, copper and zinc levels in Finnish game animals (moose, hares) and farmed game animal (reindeer) were studied during twenty years period. About 500 moose were collected between 1980 and 1999 from south western, southern, central and south eastern Finland. About the same number of Mountain and European hare samples were collected during the period 1980 and 1993 from south western, southern, south eastern and northern Finland. About 300 reindeer were collected in 1990–1992 from southern, eastern, western and northern Lapland. After these initial projects, it was decided to collect moose and reindeer samples every year as part of national residue control program; fifteen moose and ten reindeer per year. In this residue control program, also cattle samples have been collected regularly; muscle, liver and kidney from 30 animals in every year. The lead and cadmium levels in muscle tissue has decreased in all studied animals during the monitoring years being now near to the limit of quantification; 0.01 mg/kg w.w. for lead and 0.001 mg/kg w.w for cadmium. Also the lead levels in liver and kidney samples have decreased during the monitoring period but the moose cadmium levels have increased. The zinc levels had also increased in moose samples but there were no significant correlation with increasing cadmium and zinc levels. The lead levels in liver and kidney in moose varied in 1999 from 0.04 to 0.07 mg/kg w.w. and 0.05-0.07 mg/kg w.w. The corresponding values for cadmium are 0.71-1.28 mg/kg w.w. and 4.95- 6.18 mg/kg w.w. The cadmium and lead levels in Mountain hares are higher than in European hares and the kidney cadmium levels in Mountain hares are statistically significantly higher than the kidney cadmium levels in moose. The lead levels in liver and kidney samples in both adult and calves of reindeer have been invariably below the recommended maximum level in the EU (0.5 mg/kg). However, the kidney cadmium level exceeds the maximum level (1.0 mg/kg) in almost all adult reindeer samples and also in some calves. The meat of Finnish moose, reindeer and hares does not contain residues of cadmium and lead and therefore consumers can be assured that consumption of the meat is not a health risk. The consumption of the organs of the animals studied may represent a health risk for human. However, the levels are far from the toxic levels to the animals themselves.
  • Tuomisto, Pirkko (Evira, 2009)
    The drive for risk-based food safety management, systems and control has spread world-wide in recent decades. Since the term is still internationally undefined, its use and implementation vary, producing different realizations. In this Ph.D. thesis, microbiological risk assessment (MRA) was investigated as a basis for risk-based food safety management, which was defined as ‘food safety management based on risk assessment in order to achieve an appropriate level of protection (ALOP)’. Governments are responsible for commissioning MRAs and also for setting food safety targets up to a certain point, but the practical management measures that need to be in place in order to achieve the targets are to be addressed by the operators. On the plant level, food safety is usually managed through regulation, quality assurance systems and a hazard analysis and critical control point (HACCP) programme with its prerequisites. In Finland, food safety management on the food plant level is implemented through an HACCP-like regulated system termed an ownchecking (OC) programme. A quantitative microbiological risk assessment (QMRA) was conducted on salmonella in the beef production chain according to the official standards of the Codex Alimentarius Commission (Codex Alimentarius), and utilized in determining the food safety metrics for beef production. The Finnish Salmonella Control Programme (FSCP) and the main official interventions due to it were examined in the light of risk-based food safety management. The targets set for beef processing plants by the government were converted into quantitative limits, and the results of salmonella monitoring included in the FSCP were examined by the QMRA. The goal of the FSCP was declared in 1994 to ‘maintain the present salmonella situation’, which was considered to refer to the salmonella incidence in humans at that time, and also the de facto ALOP. The requirement for a maximum salmonella prevalence of 1% at defined stages of the beef production chain was embodied in the FSCP. This statement was considered to convey performance objectives (PO) for the aforementioned stages. According to the QMRA, the de facto ALOP was achieved in the referred year 1999, and even the true prevalence levels in the FSCP were estimated to be clearly under the set PO limits with 95% credibility. However, the PO limits were set too high for the de facto ALOP to be maintained in practice. If the salmonella prevalence reached the PO limit of 1% or values near it, the public health risk would increase and overrun the de facto ALOP. The QMRA produced in this work has for the first time provided the possibility to quantitatively asses the relationships between targets set in the FSCP and their impact on public health. At present, imports of beef and beef-derived foods may impose on Finnish consumers a significantly greater exposure than domestic products. If their salmonella prevalence or their share of the foods consumed in Finland increase, the number of human cases could rapidly rise. The models for the QMRA were mainly Bayesian hierarchical models using Markov chain Monte Carlo (MCMC) techniques, which was found to be a flexible and appropriate method for this type of complex modelling. The resulting distributions were also regarded as an advantage compared to the results from models developed with the deterministic approach, because the presentation of results included the extent of the uncertainty, and also in this manner better illustrated the actual operational environment. Based on an inquiry, the personnel in food processing plants had a positive attitude towards food safety management systems, but the knowledge, training and involvement of those employees directly operating on the site with these systems were discovered to be deficient. Therefore, a generic semi-quantitative hygiene risk assessment model, Hygram®, was developed for small and medium-sized food enterprises to offer assistance in understanding, training, and, first of all, detecting the critical steps of the processes, and thereby to contribute to the development of their own-checking systems towards risk-based food safety management. Hygram® was not considered a risk-based tool as such, but whenever the critical limits of the process have been defined as equal to a risk assessment, Hygram® can be used as a risk-based management tool. It can also serve as a tool for systematic hazard analysis and CCP detection when establishing a food safety management system. To conclude, the development of risk-based food safety management is a process in which risk assessment is an essential tool. Scientific, technical, psychological and resource-bound barriers need to be overcome in order to put risk-based management systems into practice. This study showed that QMRA can be valuable in national risk management decision making, although few QMRAs are currently available. Appropriate tools for practical risk management decision making on the industrial level, such as Hygram®, need to be further developed.
  • Rikula, Ulla (Evira, 2008)
    Canine distemper (CD) is one of the longest-known infectious diseases of dogs and is still prevalent in many parts of the world. Vaccination combined with biosecurity measures is the most productive way to prevent and control infectious diseases. The beneficial effects of vaccination are realized not only on the individual but also on the population level, the latter in the form of herd immunity (HI). Control of CD among dogs relies heavily on vaccination, while in fur farms and zoos with several species or large numbers of CD-susceptible animals in close contact, biosecurity measures in some cases offer the only available means for CD control. Modified live CD virus vaccines have been successfully used to control CD among farmed mink, and since no licensed vaccines for other species kept for fur exist, mink CD vaccines have also been used for foxes and raccoon dogs in CD emergency situations. CD vaccines for dogs (Canis familiaris) and mink (Mustela vison) were studied in experimental settings for their ability to induce virus-neutralising (VN) antibodies in target species. Mink vaccines were also assessed in silver foxes (Vulpes vulpes), blue foxes (Alopex lagopus) and raccoon dogs (Nyctereutes procyonoides). Purpose-bred beagle dogs were vaccinated twice with one of three CD vaccines: Candur® SHP, Canlan®-3 or Dohyvac® DA2P, and the levels of VN antibodies were determined at the time of vaccination and one month after the second vaccination. Fur animals were vaccinated once with Distemink®, Distem®-R-TC or vaccine 3 (which was not licensed in Finland) and the levels of VN antibodies were determined at vaccination and 2-4 times 1-4 months afterwards. Significant differences among vaccine groups were found both in the proportion of animals with measurable levels of VN antibodies and in the mean titres of antibodies. The levels of VN antibodies were also determined from a large field sample (n = 4 627) of vaccinated dogs. In addition to the three CD vaccines in the seroconversion study above, additional two vaccines, Duramune®-4 and Nobivac® DHP, had been used in the field. Each dog with a known vaccination history, date of birth, sex and breed was sampled once. Based on the overall geometric mean titre of the dogs vaccinated with a single vaccine brand, vaccines were divided into high-take (Candur®, Nobivac® and Duramune®) and low-take (Dohyvac® and Canlan®) groups. The vaccine groups differed significantly among dogs less than two years of age both in the proportion of dogs with detectable VN antibodies and in the mean titres. Both the number of vaccinations and age were associated with the titre and vaccine usage. To control for possible confounding factors, the comparison of titres among vaccine usage groups was adjusted by classifying them according to the number of vaccinations (one to four) and the age group (less than one, one to two, or over two years old). The same division into low- and high-take vaccines was observed, irrespective of the number of vaccinations the dogs had received. The observations of this seroprevalence study regarding Candur® , Canlan® and Dohyvac® were consistent with the results of the seroconversion study. CD was reintroduced into Finland in 1990 after 16 years of absence. The disease remained at a low endemic level in 1990-1994, reached epidemic proportions in 1994-1995 and disappeared during 1995. The epidemic also involved vaccinated dogs. Among the virologically-confirmed cases the proportion of Dohyvac®-vaccinated dogs was higher than expected from the market shares on the assumption that all the vaccines had an equal take. As a result of this observation, Dohyvac® was withdrawn from and Nobivac® and Duramune® introduced to the market during 1995. A drastic redistribution of the market shares between the low-take and high-take vaccines took place, and this coincided with the decline and dying out of the outbreak. The observed occurrence pattern of CD from 1990-1996 was largely attributed to the changes in the level of HI, although the possible contribution of other factors, such as developments in the dog demographics, was also recognized. It was concluded that an HI above 75% is needed to keep CD in check, i.e., only sporadic cases of CD, at most, can occur. With the currently used vaccines an HI of 80% corresponds to a vaccine coverage of some 94%. It was concluded that the development of vaccine-induced immunity is a multifactorial process depending on the properties of the vaccine, on the individual variation, age, species and other factors influencing the immunocompetence of the host. On the individual level the prevention of clinical signs is sufficient, but on the population level, halting the circulation of the virus is crucial for the definitive control of CD. The ultimate test and criterion for a vaccine is its contribution to herd immunity. Heterogeneity in the dog population contributes to the occurrence of CD.
  • Nuotio, Lasse (2006)
    The monitoring and control of infectious animal diseases, limiting or prevention of their spread and efforts towards their eventual eradication are central tasks of the veterinary civil service. In addition to the cost-effectiveness of prophylaxis over disease and treatment, the animal welfare aspect is also involved. The purpose of this work is to review, describe and assess the available control measures against selected viral infections or diseases of domestic ruminants. The selected infections or diseases are bovine viral diarrhoea / mucosal disease (BVD), infectious bovine rhinotracheitis / infectious pustular vulvovaginitis (IBR), enzootic bovine leukosis (EBL) and maedi–visna (MV) of sheep. Each is recognized as a signifi cant disease of domestic animals. Decisive control and eradication measures are necessarily based on the biological, veterinary and diagnostic characteristics of the affl ictions, as well as on their epidemiology in terms of the intrinsic determinants of the hosts, host–agent relationships and sources and transmission of the infection, and occurrence of these infections or diseases. This information is compiled and presented in the fi rst part of the thesis with special reference to available or possible control and eradication measures. These measures and programmes against the four affl ictions employed in major cattle and sheep producing countries in individuals and herds and on national and international levels are outlined and assessed briefl y. In the descriptive part of the thesis the domestic and EU legislation that forms the offi cial framework for disease control and eradication are outlined. The development in the situation concerning these four infections or diseases is described from the early records to date. The fi rst recorded entries of the occurrence of BVD and EBL in Finland date back to the 1960s, those of IBR to the beginning of the 1970s and of MV to the beginning of the 1980s. Large-scale surveillance and health monitoring among dairy, suckler-cow and beef herds and sheep fl ocks, starting during the fi rst half of the 1990s, enabled the estimation of actual prevalences of these infections and diseases. A common feature of the occurrence of these infections or diseases is that none has had a prevalence of more than an estimated few percent before 1990, and a maximum of 1% since then. This has formed a very favourable starting point for the nation-wide control and eradication measures. The voluntary control programmes or schemes, as well as the offi cial control and eradication measures are described. The successful eradication of IBR and EBL in 1994 and 1996, respectively, and the signifi cant reduction in the occurrences of BVD and MV from 1990 to date, are reported in detail. The effi cacies of the offi cial control and eradication measures and of the actions of the voluntary control programmes or schemes are analyzed further, making use of a heuristic formulation for the infection reproduction number (R), i.e. the number of secondary cases produced by one infective animal. The infl uence of the measures is resolved into the three components of R: the probability of transmission, frequency of infectious contacts and length of the infectious period, and the impact of the measures on each component is graded on a three-step scale. The conclusion is drawn that the offi cial measures complemented by voluntary actions for control and eradication have for the most part been adequate. The signifi cance of fi nancial compensation from the state for the costs incurred in the control of notifi able diseases is noted. In the case of BVD the decisive measures for fi nal eradication have only been available since 2004 and their impact will be seen in the next few years. The role of continued surveillance and health moni toring for both overseeing the situation with BVD and MV, and maintaining an IBR and EBLfree status is emphasized.
  • Metlin, Artem (Evira, 2008)
    Rabies is a fatal disease that affects the central nervous system of all warmblooded mammals. The rabies virus belongs to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. This virus has a negative single-stranded RNA genome and the virions are bullet-shaped. Rabies is reported in many countries throughout the world and has been registered in all continents except Australia, where only the bat Lyssaviruses have been found, and in Antarctica where the main vectors of rabies are absent. Russia and most of the bordering countries are affected by rabies. Finland was a rabies-free country from 1959 to 1988, when a sylvatic rabies epidemic appeared with raccoon dogs as the main host and vector of infection. That epidemic was eradicated by the oral vaccination of wild carnivores and the parenteral immunization of dogs and cats; and Finland has been rabies-free since 1991. However, this status is constantly under threat because rabies is endemic in Russia and Estonia. In June 2003, a horse imported to Finland from Estonia was clinically and laboratory diagnosed as rabies positive. The close relationship of the isolated equine virus strain with the current Estonian strains was verified during subsequent molecular epidemiological studies. Because the case was imported, it did not affect Finland’s rabies-free status. Also in 2007 another 2 imported cases of rabies were recorded: one in a human being from Philippines and the other in a dog from India. Five different antigenic variants of the rabies virus were identified among rabies positive field samples from Russia, Finland, and Estonia by using antinucleocapsid monoclonal antibodies. Two rabies virus field isolates showed a different reaction pattern that was similar to that of the vaccine strains of the SAD group, which might suggest a new antigen variant or reverted vaccine strain. Nevertheless, the sequence analysis showed that the vaccine strains RV-97 and SAD B19 included in the oral anti-rabies vaccine “Sinrab” (Russia) and “Fuchsoral” (Germany), respectively, differ considerably from all the field strains. Field rabies viruses collected in recent years from different regions of the Russian Federation were chosen on the basis of mAb studies and geographical origin for molecular epidemiological studies to characterize their genetic heterogeneity and to study their molecular epidemiology. In addition to the Russian viruses, archival samples from Estonia and Finland and Russian vaccine strains were also included in this study. Among the field viruses studied, two main phylogenetic groups were found, and designated as the Pan-Eurasian and Caucasian based on their geographical origin. The Pan-Eurasian group including some reference viruses from Europe was further divided into four subgroups. All the vaccine strains were clearly different from the field strains. No recombination between the field and vaccine virus strains was observed. The critical roles of geographical isolation, the limitation of the genetic clustering, and the evolution of the rabies virus were shown during this study. The rabies virus vaccine strain RV-97 is widely used in Russia as a component of the oral anti-rabies vaccine “Sinrab”. To characterize the molecular properties of this strain, entire genome sequencing was conducted. A simple technique was developed to obtain this sequence, including the 3’- and 5’- ends. The entire genome sequence and deduced amino-acid sequences of the major viral proteins were compared with the sequences of other known fixed rabies viruses. The strain RV-97 formed a separate phylogenetic branch and seems to be more related to the group of Japanese strains. The field strains from the Caucasian group seem to be phylogenetically the nearest group to the RV-97 strain. The data shown herein makes it possible to develop molecular methods for distinguishing between the field rabies viruses from the vaccine strains for the rapid recognition of the vaccine strains that are unstable or have reverted back to their pathogenic form. The wide genetic heterogeneity verified in this study indicates that it is important to remain on permanent alert for the appearance of rabies.
  • Lindqvist, Nanna (Evira, 2008)
    Salmonellosis is one of the most significant zoonoses worldwide and also in Finland. The major serovars causing infections in humans are Salmonella Enteritidis and Salmonella Typhimurium. Salmonella Typhimurium definitive phage type (DT) 1 and Salmonella Infantis are considered endemic in Finland. These serovars have frequently caused outbreaks among humans, the source of which is often detected. For the sporadic cases they usually remain unknown. Salmonella Agona was not frequently encountered in Finland until a small outbreak among cattle farms occurred in 1994-1995. S. Agona became the third most common Salmonella serovar in cattle in Finland in 1995. The two more common serovars were S. Infantis and S. Typhimurium DT1. Bacterial typing methods are used for outbreak investigations and for surveillance, where the data can be used for risk assessment calculations in addition to the future prevention of outbreaks. In particular the identification of factors that contribute to the persistence and spread of infection in endemic situations, estimations of the effect of animal reservoirs on human cases, and the identification of other risk factors for human infections are among the important reasons for typing. Salmonella Infantis became more common in cattle in the 1980s, after it spread in the broiler chicken production in Finland in 1971. Subsequently it caused outbreaks among broilers and humans in the 1970s and 1980s. In the 1990s, S. Infantis became the predominant serovar among cattle in Finland. In 1995, a feedborne outbreak of S. Infantis in cattle occurred. We were able to identify the feedstuff-related genotype by XbaI-PFGE methodology. It belonged to the major endemic type, pf1, but differed from it by having a plasmid visible as an intensive band of 60 kb in XbaI-PFGE (plasmid subtype pf1/39). Farms infected with the feedstuff-related genotype pf1/39 or the related genotypes pf1/43, pf1/44, pf1/45, or pf1/46 containing the same 60 kb plasmid were also identified. The stability of the feedstuff-related genotype was followed on selected farms. The plasmid was stable on the farms during the follow-up period. The feedstuff-related genotype did not persist in the cattle population. Moreover, there was a general decline in bovine salmonella infections from 1997 onwards. The genetic diversity of the S. Infantis isolates taken from Finnish cattle was also assessed: the S. Infantis infection in cattle was highly clonal as 99 per cent of the isolates had XbaI-PFGE profiles clonally related to each other. The major genotype pf1 was predominant both at the starting year of our analysis in 1985 and as the infection seemed to fade out in 2003. Traditionally, only one isolate per farm is stored in the national collection. However, an infection may have existed subclinically for a long time in a herd until the first Salmonella isolates were obtained. In our analysis of successive isolates from the same herds, we frequently detected minor changes in banding patterns during long-lasting infections in individual herds. The sampling and testing of several isolates from a herd in outbreak investigations is therefore advisable. There is a trend towards less genetic diversity of the S. Infantis infection among domestic isolates from humans and poultry. Up to eight different ribo/IS200-types were detected in the 1980s isolates, whereas in the 1990s only two different ribo/IS200-types (1A, 1B) were seen. In cattle, 89 per cent of the analysed isolates possessed the ribo/IS200-type 1A, although four different ribo/IS200-types were recorded in the 1980s isolates, and two ribo/IS200-types in the 2000s isolates. The ribo/IS200-types and the most common XbaI-PFGE profiles determined amongst the analysed cattle isolates could also be detected among domestic isolates from poultry and humans. After the outbreak in cattle in the years 1994-1995, isolates of S. Agona taken from the 1984 to 1999 period were characterized by PFGE using XbaI, BlnI, SpeI, and NotI enzymes. Two outbreakrelated genotypes, which were not detected in the earlier isolates of S. Agona, were identified. Another small possibly genetically related outbreak among cattle farms occurred in 1997. In 1999, a large outbreak of S. Agona of domestic origin involving more than 50 human cases occurred. Despite epidemiological investigations carried out by the local authorities, the source of the outbreak remained unknown. Based on our typing data, this outbreak was unrelated to the cattle farm outbreaks, though it did occur in the same region of Finland. The outbreak profile for the 1999 outbreak could not be found in any of the other isolates. As no recent foreign isolates were available, a foreign source of the human infection cannot be disregarded. Salmonella Typhimurium DT1 has become the most common S. Typhimurium phage type among cattle farms in Finland, and has been detected annually since 1980. Domestic and foreign isolates of S. Typhimurium DT1 from 1981 to 1999 were characterised. Furthermore, two clusters formed by the effects of the combination of the XbaI-, BlnI-, and SpeI-PFGE profiles, IS200-profiles and possession of the serovar-specific virulence plasmid were analysed. The major cluster had no virulence plasmid and included the most common XbaI-PFGE profile 10 and IS200-profile D, typical of our endemic infection. The results of XbaI-, BlnI-, and SpeI-PFGE gave 54 different combination profiles, which can be applied in the analysis of outbreaks. In contrast, molecular subtyping by XbaI-PFGE alone is not discriminatory enough in analysing our endemic infection. The source of sporadic human infections is unknown in most cases, and molecular typing did not reveal any clear infection source. The most common XbaI-PFGE profile 10 was also seen in hedgehogs and wild birds. They might act as important reservoirs, maintaining a minimum baseline level of S. Typhimurium DT1 in the environment and consequently be possible sources of human infections
  • Jakava-Viljanen, Miia (Evira, 2007)
    The intestinal commensal microbiota of pigs comprises hundreds of different types of microorganisms (Stewart, 1997; Inoue et al., 2005). The members of the genus Lactobacillus are important residents of the gastrointestinal (GI) microbiota and have been subjects of increasing interest due to their possible role in the maintenance of GI health. Because of these putative health promoting properties, Lactobacillus species are widely used as probiotics (Ouwehand et al., 2002). One important criterion proposed for a probiotic bacterium is its ability to adhere and colonize host tissues, which enables multiplication and survival of the bacterium in the host and may prevent the colonization of pathogenic bacteria via competitive exlusion (CE). The F18 fimbriae-expressing (F18+) E. coli cause post-weaning diarrhoea (PWD) and edema disease (ED) in newly weaned piglets. The key virulence factors in diarrhoea are enterotoxins and fimbrial adhesins (Berberov et al., 2004; Zhang et al., 2006). Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. The FedF protein of F18 fimbriae was recognised as the adhesin and antibodies against FedF were found to inhibit the adhesion of F18+ E. coli to porcine enterocytes (Smeds et al., 2001; Smeds et al., 2003). However, oral immunisation with purified F18 fimbriae did not result in protection against ED by a challenge infection with F18+ verotoxigenic E. coli (VTEC) (Verdonck et al., 2007) and there are no commercial vaccines available against infections caused by F18 fimbriae-carrying E. coli strains. Like many other bacteria, several species of Lactobacillus have a surface (S-) layer as the outermost component of the cell (reviewed in Åvall-Jääskeläinen and Palva, 2005). The function of Lactobacillus S-layers characterized so far is involved in mediating adhesion to different host tissues. In addition to surface layer proteins (Slps) adhesive properties, the very large number of S-layer subunits present on the cell surface has prompted research aiming at the use of S-layers as a vehicle for the delivery of biologically active compounds, such as drug molecules, antibodies, enzymes and vaccine antigens (Sleytr et al., 2007). 13 The accurate species identification of bacteria is of fundamental importance in the development of new mucosal vaccine vectors or probiotics. Species of Lactobacillus form the most numerous genus in the heterogeneous group of lactic acid bacteria (LAB). The genus contains about one hundred described species, which are subdivided by 16S rRNA analysis, DNA-DNA hybridization and other phylogenetic methods, into eight major groups. The 16S rRNA gene is a reliable phylogenetic marker but is not an effective identification tool as it does not allow discrimination among closely related species. A multilocus sequence analysis (MLSA), on the contrary, effectively offers a high resolution and yields a robust identification system. MLSA compares the primary DNA sequences from multiple conserved protein coding loci for assessing the diversity and relation of different isolates across related taxa. The combined use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) gene sequences offers a reliable identification system for nearly all Lactobacillus species (Naser, 2006).