Faculty of Veterinary Medicine


Recent Submissions

  • Haen, Silke (Helsingin yliopisto, 2020)
    The hypothalamic-pituitary-gonadal axis is the endocrinological pathway of reproduction, where fertility is adjusted and fine-tuned with respect to intrinsic and extrinsic information. The pituitary gland receives the information that determines secretion of luteinizing hormone (LH) from the hypothalamus in the form of gonadotropin releasing hormone (GnRH), it is the frequency and amplitude of the GnRH pulses that regulate LH release. LH transports the information from the brain to the gonads, which feeds back to the hypothalamus and the pituitary gland in the form of steroids. This thesis work focused on the orchestration of GnRH, LH and progesterone during the early pregnancy of the pig. Therefore, gonads refer to the ovary with their functional bodies, the corpora lutea (CLs) and their principal steroid, progesterone. CLs form from the follicles after ovulation and they are either maintained, and pregnancy continues, or they regress, enabling follicle growth and new ovulations and a new opportunity for reproduction. The corpus luteum is independent of pituitary LH support for the first eleven days after ovulation. Subsequently the maternal organism is informed of the presence of embryos and the need to maintain the CLs. Thus, dependency of the CLs on LH begins at the same time as the sow receives the positive signals that indicate pregnancy. Progesterone is the main hormone of pregnancy and its concentration is highest around the time of maternal recognition of pregnancy. LH supports the CLs, but it remains unclear for exactly how long during pregnancy the LH stimulus directly triggers secretion of progesterone. The objective of this thesis work was to study the relationship between the two hormones, LH and progesterone, during early pregnancy in pigs. Firstly, we investigated the physiological release of LH and ovarian progesterone in inseminated gilts on Day 11 after oestrus, before the CLs are thought to be dependent on LH. Secondly, we looked at the pattern of progesterone release after the CLs had become dependent on LH. We studied the pattern of progesterone release from the ovary and were interested to ascertain if that pattern is related to the LH pattern. To explore a possible causal relationship between LH and progesterone further, a GnRH agonist model was used to nullify LH pulsatility around implantation of embryos. Thirdly, we were interested to know if parity, sow age and maturity affect the relationship between the two hormones. In our studies, a catheter was inserted through the vena saphena lateralis, enabling blood sampling in the vena cava caudalis next to the venous drainage of the ovary and uterus. LH and progesterone concentration were assessed after frequent blood sampling for 8–12 hours on Day 11 after oestrus in inseminated gilts, and on Days 16 and 21 in pregnant gilts. A slow-release GnRH agonist was inserted into pregnant gilts on Day 11 and LH and progesterone were studied on Day 16 and Day 21. Additionally, a vena jugularis catheter was inserted into pregnant primiparous sows and progesterone concentrations in local (vena cava caudalis) and peripheral (vena jugularis) blood samples were compared on Day 14. In gilts that were inseminated and classified post mortem whether they were pregnant or not, the progesterone release pattern measured in the vena cava caudalis had similar basal and mean progesterone concentrations, and frequency and amplitude of pulses on Day 11. On Day 11 and Day 21 of pregnancy, there was no relation between LH pulsatility and progesterone pulsatility. In gilts that were treated with a slow-release GnRH agonist on Day 11, LH pulsatility ceased but progesterone release remained pulsatile on Day 16 and Day 21 of pregnancy. On Day 21 we found that LH pulsatile release was active when progesterone release was basal. In primiparous sows, a progesterone pulse followed 60.8% of LH pulses within one hour. Mean progesterone concentration was approximately twice as high in the vena cava caudalis than in the vena jugularis in primiparous sows and differed significantly before and after feeding in the vena jugularis. GnRH-agonist-treated gilts had an elevated progesterone concentration on Day 21 compared with the control gilts. Progesterone concentration declined from Day 11 via Day 16 to Day 21 in gilts. LH pulse amplitude declined during those days, but no decline in mean or basal LH concentration or LH pulse frequency was recorded. LH pulsatility ceased on Day 16 and Day 21 in gilts treated with the GnRH agonist. LH release was synchronized in such a way that gilts under the same management conditions exhibited LH pulses around the same time during twelve hours, indicating a synchronized rhythm of secretion. We observed that progesterone release of CLs is similar in non-pregnant gilts and pregnant gilts on Day 11, before maternal recognition of pregnancy. During early pregnancy in gilts the pulsatile progesterone release of CLs on Days 11 and 16 is not responsive to – and on Days 16 and 21 it is not dependent on – LH pulsatile secretion. On Day 21 there is an alternate pattern of activity in pulsatile release of LH and progesterone in individual gilts. In primiparous sows, we found a temporal relationship between LH pulses and progesterone pulses already on Day 14 of pregnancy. We conclude that the progesterone release of the CLs functions without LH pulsatile stimulus from Day 11 to Day 21, but associations of these hormones at an individual level are evident.
  • Argano, Martina (Helsingin yliopisto, 2020)
    The impact of perioperative factors on metastatic spread subsequent to surgery for primary cancer removal is of rising interest. Several studies show that anaesthesia for primary cancer surgery might impact cancer recurrence by influencing cell proliferation rate and regulating the expression of specific oncogenes like neuroepithelial transforming gene 1 (NET1), which has been associated with malignant behaviours and represents a novel prognostic marker in human epithelial cancers. This project investigates the effects of different concentrations of clinically available propofol and sevoflurane formulations on canine mammary tumour cell proliferation and on the expression of the NET1 gene in three prospective controlled in vitro trials. Primary (CIPp) and metastatic (CIPm) canine tubular adenocarcinoma cell lines were incubated with sevoflurane (1, 2.5 or 4 mM), propofol (1, 5 or 10 μg mL-1) or cell culture medium (control). Proliferation was assessed after 6 hours of sevoflurane and 6 - 12 hours of propofol exposures, while NET1 expression was evaluated after 6 hours of sevoflurane and 6, 12, 24 and 48 hours of propofol exposures. After 6 hours of propofol exposure, cell proliferation significantly increased in CIPp, while after 12 hours cell proliferation significantly increased in CIPp and decreased in CIPm. After 6 hours of exposure, propofol significantly augmented NET1 expression in CIPp, whereas in CIPm the highest propofol concentration significantly reduced gene expression. Propofol significantly decreased gene expression after 12 and 24 hours of exposure. No significant differences were found in CIPm after 48 hours, while the highest concentration of propofol significantly increased gene expression in CIPp after 48 hours. Sevoflurane significantly increased cell proliferation in CIPp, while it significantly decreased cell proliferation in CIPm. The NET1 gene expression was significantly increased in CIPm after exposure to the highest sevoflurane concentration. In conclusion, exposure to both propofol and sevoflurane mainly augmented proliferation in primary cells and diminished proliferation in metastatic ones. Conversely, propofol mostly decreased NET1 expression, while sevoflurane increased NET1 expression in metastatic cells exposed to high concentrations. A profound comprehension of the molecular mechanisms underlying cancer spread is necessary, however, the much more variable environment in the clinical setting makes direct translation from in vitro to in vivo conditions difficult. Even though the lack of similar trials in veterinary medicine limits the chances for comprehensive comparisons, some promising results were observed in our study. Future projects assessing the influence of anaesthetic techniques on cancer recurrence are warranted.
  • Savolainen, Saila (Helsingin yliopisto, 2020)
    Cats are increasingly popular companion animals, and their owners are willing to provide good-quality health care for their pets. However, while cats are considered to be difficult to medicate with orally administered pharmaceuticals at home, studies on medication compliance in cats have been scarce. Palatable pharmaceuticals would most likely increase compliance in feline medication, and the pharmaceutical industry would prefer synthetic flavours over organic ones. Cats’ taste receptors are sensitive to amino acids, and certain amino acids are considered to be palatable to cats. The methods of testing the palatability of pharmaceuticals in cats have varied. The objective of the present thesis was, firstly, to investigate the difficulties encountered by owners when medicating their cats with orally administered pharmaceuticals at home. Secondly, the aim was to find solutions for making feline oral medication easier by focusing on the development of methods for testing the palatability of pharmaceutical products in cats, in addition to screening synthetic flavours for future taste masking of feline pharmaceuticals. Furthermore, behavioural indicators were investigated to distinguish between a palatable food item, an unpalatable food item, and a palatable food item containing a pharmaceutically inactive placebo mini-tablet in pet cats. As a result of the included online survey, the compliance in feline medication was 76%, and most of the difficulties seemed to be related to the aversive taste of the pharmaceutical. Off-label use of pharmaceuticals was common, as approximately half of the medications were registered for use in cats. Pharmaceuticals registered for feline medication were significantly easier to administer to the cats than other pharmaceuticals. However, the voluntary acceptance rate was low. Most of the owners preferred a solid formulation for medicating their cat, and a mini-tablet was therefore selected to be used in the palatability trials. The acceptability of flavoured, pharmaceutically inactive mini-tablets was tested in pet cats with a rapid three-portal acceptance test. If the mini-tablets were not accepted voluntarily from a table or a hand, they were offered to the cat hidden inside a palatable food item. The acceptance of a placebo mini-tablet was also tested as administered inside a palatable food item. The synthetic flavours tested included amino acids, which were chosen based on the literature and because they are meat precursors, as well as synthetic meat aromas and d-(+)-maltose monohydrate, which is the predominant sugar in Cat Malt -products, in addition to thiamine hydrochloride, which is found in large amounts in yeast. The synthetically flavoured mini-tablets were not palatable to the cats, as the cats refused to consume the mini-tablets if the tablets were not hidden inside a palatable food item. Five behavioural patterns differentiated palatable food from unpalatable food: 'flick ears backwards', 'lick nose, not eaten', 'flick tail' and 'groom body' were more frequent with unpalatable food, whereas 'lick lips' was more frequent with palatable food. One indicator, 'drop item', was more frequent with palatable food containing the mini-tablet than with palatable food alone, indicating that the mini-tablet was not fully acceptable to the cats; or it is possible that the mini-tablet fell out more easily from some types of food. The feasibility of the palatability tests carried out with pet cats in their home environments was verified by the high inter-observer agreement. In conclusion, palatable and easily administered oral pharmaceuticals are needed for feline medication. Unfortunately, no promising synthetic flavours were identified for the taste masking of feline pharmaceuticals, and more investigations are required to discover synthetic flavours that are palatable to cats. The results of the palatability trials differed from the earlier literature as regards the taste preferences of cats concerning amino acids. However, the mini-tablets were acceptable by most of the cats when hidden inside a palatable food item. The investigated behavioural indicators and methods for testing palatability could be used in future palatability studies carried out in pet cats.
  • Kontturi, Miia (Helsingin yliopisto, 2020)
    Interdigital phlegmon (IP) is an infectious hoof disease of cattle. Typically, it causes severe clinical signs, such as lameness, and hence impacts cattle welfare. IP has been a well-known disease of both dairy and beef cattle all over the world for decades. In general, IP occurs as sporadic infections. Lately however, outbreaks of IP have been detected in dairy herds in Finland. Most of these outbreaks have occurred in recently built or renovated free stall barns. In these outbreaks morbidity in IP has been substantial, which has led to the extensive use of antimicrobials and heavy financial losses associated with affected herds. The aim of this thesis was to investigate the characteristics of the IP outbreaks in Finnish dairy herds and to explore the morbidity, clinical manifestation and degree of inflammation of the affected animals in these outbreaks. Moreover, we aimed to investigate the bacteriology in IP, i.e. to investigate several bacteria detected earlier from hoof diseases. An additional objective was to identify herd level risk factors behind these outbreaks. This thesis is based on three studies. The first two were observational, cross-sectional studies, which were performed on commercial free stall dairy herds. The majority of the herds suffered from an outbreak of IP and three herds were unaffected control herds. Altogether 100 cows with IP were clinically checked, diagnosed, and sampled for the bacteriological culture and PCR, and analysis of acute phase proteins; serum amyloid A, haptoglobin and albumin. Cows with other hoof diseases and control cows were sampled similarly for comparison. The third study was a survey of free stall dairy herds of ≥50 cows. We sought general herd data, barn characteristics, herd management details, and asked questions about leg and claw health of the herd. Based on the replies, the risk factors for an outbreak of IP to occur were investigated among farms that had experienced an outbreak and farms that had not. Fusobacterium necrophorum ssp. necrophorum is the main IP pathogen. The most common finding in IP samples in the early, acute stage was a combination of F. necrophorum and Dichelobacter nodosus. Trueperella pyogenes was frequently associated with IP at the later, healing stage. However, various bacterial combinations existed in IP samples. In outbreak herds, the morbidity was either high (≥50%), or moderate (9 – 33%), and no herd had intermediate morbidity. Strong acute phase response was detected among IP cows in the early stage of the disease; the values for serum amyloid A and haptoglobin were clearly elevated, and albumin decreased in comparison with the case for other hoof diseases or control cows in our study. The acute phase response was even greater in herds of high morbidity and with a bacterial combination of F. necrophorum and D. nodosus. The possible herd level risk factors for an outbreak of IP to occur were animal movement between herds, i.e. animal purchase or contract heifer rearing, enlargement of the barn within three years, and fields under organic farming. Mechanical ventilation in the barn seemed to lower the risk. Moreover, herds that had experienced an IP outbreak more often had other infectious hoof diseases. Based on our study results, the same cow may have several hoof diseases and a thorough clinical inspection is essential in diagnosing IP, also during an IP outbreak. Furthermore, IP causes severe clinical signs and a very strong APR. Thus, an anti-inflammatory should be included in the treatment of affected animals. Even though F. necrophorum is the key pathogen in IP, in the disease process several other bacteria play a role, such as D. nodosus, which may affect the severity of IP. To lower the risk of an IP outbreak, new cattle should be purchased very cautiously, if at all, and enlargement of the barn should be constructed without undue restrictions being placed on time and labour inputs.
  • Jaakkonen, Anniina (Helsingin yliopisto, 2020)
    Cattle are commonly asymptomatic carriers of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, which cause gastroenteritis in humans. Especially STEC infections may lead to severe or fatal consequences. Both STEC and C. jejuni are intermittently shed in cattle feces and can contaminate bulk tank milk via fecal contamination during milking. These bacteria are effectively eliminated from milk by pasteurization, but the consumption of unpasteurized milk, or raw milk, poses a risk of infection. In recent years, the consumption of raw milk has become more popular, with public demand to relax legislation that restricts sales of raw milk. However, on-farm epidemiology of these pathogens have warranted further investigation to support the development of on-farm risk management practices and pathogen monitoring of dairy farms that sell raw milk to consumers. These studies investigated a milkborne outbreak caused by STEC (Study I) and obtained longitudinal data on the contamination of bulk tank milk by STEC and C. jejuni, and explored on-farm contamination routes of these pathogens (Study II). Furthermore, the studies revealed strain characteristics of C. jejuni that may affect survival and persistence of this pathogen in milk (Study III). The occurrence of STEC and C. jejuni, or C. jejuni alone, was determined in bulk tank milk, in-line milk filters of the milking machine, cattle feces, and the farm environment on four dairy farms (STEC and C. jejuni in Studies I and II) or one dairy farm (C. jejuni in Study III). STEC and C. jejuni isolates from the dairy farms were further subjected to phenotypic characterization and whole-genome sequencing, followed by comparative genomic analyses to explore gene contents and phylogenetic relationships between the isolates. Furthermore, questionnaire data were collected to trace back the outbreak source (Study I) and to determine on-farm risk factors associated with milk contamination using a logistic regression model (Study II). Ultimately, the results contributed to the revision of the Finnish legislation that restrict the sales of raw milk in 2017 (Study II). Study I elucidated the reservoirs and transmission routes of atypical, sorbitol-fermenting (SF) STEC O157, which have largely been unknown. The study presents microbiological and epidemiologic evidence that an outbreak of SF STEC O157 with 11 cases originated from a recreational farm housing dairy cattle and was transmitted via the consumption of raw milk. Thus, these results strongly support bovine origin of SF STEC O157. In longitudinal monitoring (Study II), one clone of STEC O157:H7, which represented a bovine-associated lineage, was simultaneously isolated on each of the three dairy farms. STEC O157:H7 persisted in two herds for up to 12 months, and a similar but distinct clone was reintroduced in one herd 2.5 years after the previous detection. These results support evidence that few STEC O157:H7 clones persist on-farm simultaneously. Unlike STEC, both persistent and numerous sporadic C. jejuni strains appeared simultaneously on dairy farms (Studies II and III). Persistence for 11 months or longer was associated with a few C. jejuni genotypes, especially the host generalist sequence type (ST) ST-883. C. jejuni of ST-883 outperformed other STs in environmental fitness, representing the only ST that could be isolated from bulk tank milk and milk filters and the dominant ST found from environmental samples. Therefore, ST-883 imposes a higher contamination pressure on milk than other STs among the farm isolates and represents a candidate for on-farm risk-based monitoring. In the longitudinal monitoring, STEC was rarely isolated from bulk tank milk and milk filters and only simultaneously with fecal isolation. Higher detection rates were obtained from milk filters than milk by both culture methods and real-time PCR. Therefore, milk filters are more reliable sampling targets for monitoring of STEC than milk. Isolation of C. jejuni from milk and milk filters was associated with C. jejuni clone rather than sample material, but the isolation rates of C. jejuni appeared generally poor. To enhance the isolation rates for monitoring purposes, the sampling regime also warrants further consideration. Reduced milk contamination by STEC was associated with on-farm practices: pasturing and culling of dairy cows and rigorous cleansing in the barn. Higher outdoor temperatures were associated with increased milk contamination. In Study III, C. jejuni of ST-883 persistently contaminated bulk tank milk of a dairy farm for seven months or longer after having caused a milkborne outbreak. Although ST-883 survived in refrigerated raw milk longer than other STs from the same farm, the persistence of ST-883 in bulk tank milk was likely affected by other phenotypic traits such as biofilm formation. Outbreak strain of ST-883 reversibly adapted to survival in bulk tank milk, showing biofilm formation in an on/off manner among replicate cultures and cellular heterogeneity by phase variation in genes related to capsule and oxidative stress response. Furthermore, the outbreak strain harbored a pTet-like genomic element, which may have contributed to higher biofilm quantities. This study identified candidate phenotypic and genotypic mechanisms affecting survival and persistence of C. jejuni in milk. Taken together, STEC and C. jejuni can persist on dairy farms for months or longer and contaminate bulk tank milk despite stringent on-farm hygiene measures. Although these measures cannot totally prevent milk contamination, they likely reduce the contamination pressure on milk. Therefore, cost-effective hygiene measures should be applied on all farms that sell raw drinking milk to consumers. Detection of pathogens from milk may be challenging, and milk may also be contaminated by highly virulent STEC and C. jejuni strains that show atypical phenotype, increasing their environmental endurance or hampering their detection. Therefore, only heat treatment of raw milk before consumption can adequately assure its food safety.
  • Turunen, Heta (Helsingin yliopisto, 2020)
    α₂-Adrenoceptor agonists, such as medetomidine and dexmedetomidine are considered to produce reliable sedation, muscle relaxation, and antinociception when used as sedatives or preanaesthetics. One important advantage is that their effects can be reversed with an α₂-adrenoceptor antagonist, atipamezole. However, notable cardiovascular depression associated with α₂-adrenoceptor agonists limit their use to healthy animals. A peripheral α₂-adrenoceptor antagonist vatinoxan (also known as MK-467 and L 659´066) has been shown to reduce these cardiovascular disturbances, without markedly affecting sedation after concomitant use with dexmedetomidine or medetomidine. Vatinoxan is therefore proposed to improve the clinical utility of α₂-adrenoceptor agonists in dogs. The main aim of this series of experiments was to investigate the reduction of medetomidine-induced cardiovascular changes with vatinoxan, atipamezole and ketamine, not only in laboratory dogs, but also in healthy client-owned dogs of various breeds in a clinical environment. The secondary aim was to assess the quality of sedation, hypnosis and recovery when these drugs interacted together. Two of the studies were performed under laboratory conditions with eight purpose-bred beagle dogs that were instrumented prior to each experiment. Heart rate, mean arterial pressure, central venous pressure, cardiac output and arterial blood gas partial pressures were measured and sedation was assessed. Blood samples were collected for plasma drug concentration analyses. Study Ⅰ aimed to investigate the ability of atipamezole to reverse the cardiovascular and sedative effects of medetomidine in the presence or absence of vatinoxan. Study Ⅱ aimed to explore the influence of vatinoxan on the cardiovascular function and quality of anaesthesia and recovery, when the dogs were premedicated with medetomidine and butorphanol, followed by ketamine, and subsequent reversal with atipamezole. In study Ⅲ the clinical utility of vatinoxan was then evaluated in client-owned dogs, sedated with medetomidine and butorphanol combination for non-invasive diagnostic imaging. The need for atipamezole reversal was also monitored. Plasma concentrations of the studied drugs were determined in each study to detect the influence of vatinoxan. In study Ⅰ, atipamezole failed to permanently increase heart rate or cardiac index without the presence of vatinoxan when administered intramuscularly 30 minutes after medetomidine. Momentary decrease in mean arterial pressure was observed shortly after atipamezole administration with and without vatinoxan. However, no clinically relevant hypotension was detected. Atipamezole reversed the sedative effect of medetomidine more efficiently with than without vatinoxan, probably due to increased plasma clearance of medetomidine. Relapse into sedation after intial arousal was observed during recovery when vatinoxan was not included in the treatment. The medetomidine-induced increase in systemic vascular resistance index was attenuated by vatinoxan, with significantly lower values observed throughout the studies Ⅰ and Ⅱ in its presence. Medetomidine-evoked bradycardia and decrease in cardiac index were similarly mitigated by vatinoxan in all of the present studies. In study Ⅱ, heart rate and cardiac index increased temporarily after ketamine administration and the effect was potentiated by vatinoxan. Ketamine decreased mean arterial pressure and mild hypotension was detected in two out of eight laboratory dogs that were given vatinoxan premedication. Hypotension was abolished by atipamezole administration at 60 minutes following ketamine. Vatinoxan reduced the exposure to ketamine. Therefore, the duration of anaesthesia was shorter in the presence of vatinoxan. The quality of recovery after atipamezole was impaired by nausea and increased need to defaecate with and without vatinoxan. In study Ⅲ, intramuscular medetomidine-butorphanol combination with and without vatinoxan provided reliable sedation for diagnostic imaging procedure. Vatinoxan hastened and increased the peak plasma concentrations of intramuscularly co-administered medetomidine and butorphanol. The clinically observed benefit was a faster onset of deeper sedation. Mean heart rate in the treatment group with vatinoxan during the study was approximately 50% higher than in the group without vatinoxan. Atipamezole was required less frequently to reverse medetomidine-evoked sedation when vatinoxan was included in the treatment because of shorter duration of sedation. To conclude, the two α₂-adrenoceptor antagonists, vatinoxan and atipamezole, interacted favourably in medetomidine-sedated dogs. Together they provided more complete reversal of medetomidine’s effects. While medetomidine-induced cardiovascular changes were transiently attenuated by ketamine and atipamezole, more complete improvement in haemodynamics was observed only in the presence of vatinoxan. Although vatinoxan premedicated dogs were more prone to hypotension during ketamine anaesthesia, heart rate and cardiac index were well maintained. The clinical utility of vatinoxan with medetomidine and butorphanol was demonstrated in a clinical trial. Vatinoxan alleviated the medetomidine-induced bradycardia and the drug combination including vatinoxan provided adequate sedation to complete short, non-invasive procedures. Faster decline in the plasma concentrations of co-administered drugs in the presence of vatinoxan was clinically manifested as shorter duration of sedation and anaesthesia when compared to treatment without vatinoxan.
  • Telkänranta, Helena (Helsingin yliopisto, 2020)
    Tail biting is one of the most important animal welfare problems in commercial pig farming. Its severity varies from scratched skin to partially or entirely eaten tails. A similar though less studied problem is ear biting. Tail and ear biting are abnormal behaviours with multifactorial origins. One of the main factors is a scarcity of materials to fulfil pigs’ innate need to chew and root, which causes some pigs to redirect these needs at other pigs. Of the materials readily available in large enough quantities for farmers, a substantial layer of straw on the floor is considered best to fulfil these needs and to reduce or prevent tail biting. However, such use of straw is rare in intensive farming because the low consumer price of pork has led commercial farms minimising production costs, and one of the consequences is that most farms use slatted or partly slatted pen floors and manage the manure as slurry, which reduces labour costs but precludes the use of straw bedding. Various objects are used on farms as potential outlets for pigs’ needs for oral-nasal manipulation, but their ability to reduce tail biting and to sustain pigs’ interest and to reduce tail and ear biting vary widely. The main aim of the four studies presented in this thesis was to investigate the efficacy of selected materials and object designs in reducing tail and ear biting damage in intensively farmed pigs kept on slatted floors. The studies were carried out on four commercial farms in Finland, and data were collected from a total of 2064 pigs. In each of the studies, experimental treatments represented materials and object designs selected or developed by the experimenters, and control treatments represented the materials and object designs normally used on the farm in question. In Study I, the main research question was whether pre-weaning exposure to sisal ropes and paper would reduce post-weaning tail biting. In the control treatment, 29 farrowing pens were furnished with a suspended plastic ball, and a small amount of wood shavings were provided twice a day. In the experimental treatment, 30 farrowing pens were furnished with the above and in addition ten pieces of suspended sisal rope, and sheets of non-glossy newspaper were distributed twice a day. After weaning, piglets from both treatments were housed in identical pens with three pieces of suspended rope, a plastic chewing stick and wood shavings twice a day. The main findings were that pre-weaning exposure to the supplementary materials reduced the severity of post-weaning tail biting, and that redirection of oral-nasal behaviours at pen-mates was reduced by current, but not past, exposure to the materials. In Study II, the main research questions were whether providing growing-finishing pigs with objects made of recently harvested wood would reduce tail biting and whether the same effect could be attained with metal and plastic objects with moderately added complexity. In the control treatment, 17 grower-finisher pens were furnished with a straw rack and a metal chain. There were four experimental treatments, each furnished with the above and additionally with the following: 14 pens with horizontal wooden beams (W), 13 pens with horizontal plastic crosses (P), 15 pens with hanging metal chains in a branching form (B) and 14 pens with all the above objects (WPB). The main findings were that only the wooden objects reduced the prevalence of tail and ear biting as compared to the control group, and that wooden and plastic objects sustained the interest of the pigs better than metal objects. In Study III, the main research question was whether a small amount of recently harvested wood (approximately 20cm of horizontal wooden beam per pig) would reduce tail biting in breeder gilts that also were provided with minimal amounts of straw. In the control treatment, 12 breeder gilt pens were furnished with a horizontal piece of commercially sourced dry wood and a metal chain with a moderately complex structure (feeder chain), and long straw was distributed once a day, approximately 20g per pig per day. In the experimental treatment, the above solid objects were replaced with objects of recently harvested wood. The main findings were that there was no significant difference between treatments regarding tail biting; and that the frequency of oral-nasal behaviours targeted at other pigs did not differ between treatments before the provision of the minimal amount of straw, but was lower in the experimental treatment after pigs in both treatments had consumed the straw. In Study IV, the main research questions were whether a minimal amount of recently harvested wood (approximately 10cm length of wood per pig) would reduce tail biting in growing-finishing pigs that were also provided with a straw rack and whether the same quantity of medium-density polythene objects would have the same effect. In the control treatment, 16 growing-finishing pens were furnished with a straw rack. There were two experimental treatments, each furnished with the above and additionally with the following: 16 pens with horizontal wooden objects (W) and 12 pens with horizontal plastic objects (P). The main findings were that there was no significant difference between treatments in their effects on tail biting; and that the wooden but not plastic objects reduced ear biting and the latency to approach an unfamiliar person in a human approach test, as compared to the control group. The main conclusions were that it is possible to reduce post-weaning tail biting by adding pre-weaning material for manipulation, but further studies are required to determine the necessary minimum quantity and quality of the materials. Horizontally placed lengths of recently harvested wood can reduce tail and ear biting and potentially also stress in growing-finishing pigs, but further studies are needed on optimal object design and the necessary minimum quantity. The presence of recently harvested wood can slightly add to the beneficial effects of minimal amounts of straw. There also was a finding relevant to general research methodology in this field: the frequency of object-directed behaviours is not the sole reliable predictor of a material’s efficacy in reducing tail biting as the plastic objects elicited object-directed behaviours to the same extent as the wooden objects, but plastic objects did not reduce tail biting. .
  • Mikkola, Lea (Helsingin yliopisto, 2020)
    This thesis addresses the genetic background of hip dysplasia, a complex hereditary disorder common in many species, including dogs and humans. The highly polygenic nature of hip dysplasia has become evident after many years of arduous research conducted in numerous studies that have explained only a small proportion of the disease heritability in dogs. The studies included in this thesis revealed multiple new loci, a novel regulatory variant for a gene that is imperative to normal joint development and validated numerous loci that have been associated with the disorder. In Study I, genome-wide association analyses uncovered four loci that associated with the disorder with either protective or risk effects. A subsequent targeted resequencing and variant analysis found disease associated variants for multiple genes. Deletion variants in the putative regulatory region of the gene NOG were found to protect against moderate-to-severe hip dysplasia. These variants were shown to downregulate reporter gene expression in vitro. In Study II, different hip dysplasia and osteoarthritis phenotypes were investigated. The genome-wide association analyses revealed three novel loci associated with hip joint incongruity and osteoarthritis. New candidate genes were identified: NOG and NANOS1 for hip joint incongruity and NOX3 and ARID1B for osteoarthritis. In Study III, a total of 21 loci on 14 different chromosomes were validated in across- and within-breed association analyses in a large cohort of dogs comprised of 10 breeds. One locus was specific to the across-breed data. Neddylation-pathway was found to be significantly enriched in a candidate gene set that included 254 genes from the 21 validated loci. These studies have generated new, long-awaited knowledge about the complex genetic background of hip dysplasia and related osteoarthritis in dogs. They also highlighted that mild hip dysplasia and the more severe disease forms may be induced by partially different genetic factors. Validation of associated loci for hip dysplasia has not been conducted to this extent before these studies, so an important step forward was taken in our latest study. Finally, finding causal variants has been uncommon. The regulatory NOG variants were an exciting finding, although their causality in hip dysplasia is yet to be revealed. Overall, our studies emphasise the complexity of the genetic background of hip dysplasia.
  • Aalto-Araneda, Mariella (Helsingin yliopisto, 2020)
    The severe foodborne disease listeriosis is caused by the bacterium Listeria monocytogenes, known as a problematic contaminant of the food chain. This facultative anerobe tolerates many conditions used for controlling harmful bacteria, including high salinity and temperature. Some L. monocytogenes strains tolerate external stressors better than others, which may complicate the control of the bacterium in food-related environments. Strains exposed to one stress condition may also develop tolerance towards another; such cross-adaptation occurs, for instance, between osmotic and heat stress. In food production, L. monocytogenes may encounter these stresses via salting and heat treatments or hot water used in sanitation. Vacuum-packaged ready-to-eat fish products frequently contain L. monocytogenes and have caused several listeriosis outbreaks. They often do not undergo listericidal processes before consumption, and thus, their processing requires stringent preventive measures. The aims of this dissertation were to investigate the strain variability and determinants of L. monocytogenes stress tolerance and to examine the framework of fish-processing plants and their official food control for managing L. monocytogenes contamination. Using optical density measurements of microbial growth, differences in growth ability under osmotic (NaCl) stress were determined for 388 wild-type L. monocytogenes strains. Notable strain variability as well as serotype- and lineage-dependent patterns of L. monocytogenes salt stress tolerance were discovered. Lineage-I-affiliated L. monocytogenes serotype 1/2b and 4b strains grew significantly better at NaCl 9.0% than lineage-II-affiliated serotypes 1/2a, 1/2c, and 3a. By enabling this comprehensive identification of NaCl-tolerant strains, our data assembly and analysis protocol elucidated the biologically relevant intra-species variability of L. monocytogenes salt stress tolerance phenotypes. A comparative whole-genome sequencing approach was implemented to identify underlying determinants of L. monocytogenes stress tolerance phenotypes. Accessory genetic mechanisms of stress resistance were investigated by comparison of heat survival phenotypes and whole-genome sequences of a heat-resistant and a heat-sensitive L. monocytogenes strain. The comparison identified a novel plasmid, pLM58, including an open reading frame annotated as an adenosine triphosphate (ATP) -dependent ClpL-protease-encoding gene, which was present in the heat-resistant strain but absent in the heat-sensitive strain. The curing of pLM58 resulted in a reduction of heat resistance. The conjugation of clpL increased the heat survival of a natively heat-sensitive L. monocytogenes strain. This study described, for the first time, plasmid-borne heat resistance of L. monocytogenes and identified the protease ClpL as a novel mechanism of L. monocytogenes heat resistance. To examine the framework for managing L. monocytogenes contamination in the fish industry, operational practices and efficacy of official control were studied in 21 Finnish fish-processing plants producing vacuum-packaged gravad (cold-salted) and cold-smoked fish products. Product samples were investigated for the presence and quantity of L. monocytogenes in 2014–2015. Additionally, the results of official food control sampling of products and facilities were assessed to retrospectively gain information on L. monocytogenes contamination in the participating fish-processing plant facilities in 2011–2013. The production and hygiene practices of the processing plants were surveyed with an in-depth inspection questionnaire, and the occurrence, control measures, and correction of non-compliances were drawn from their official inspection records. Associations of L. monocytogenes occurrence with fish-processing plant operational practices, compliance, and aspects of official control during the respective years were investigated with statistical modeling. L. monocytogenes product contamination was associated with number of processing machines, deficiencies in the processing environment and machinery sanitation, and staff movement from areas of low hygiene to high hygiene. Performing frequent periodic thorough sanitation was associated with a decreased risk of product contamination. The increased occurrence of L. monocytogenes in the facilities and products of the fish-processing plants was associated with hygiene deficiencies in processing machinery, a lack of demanding control measures for non-compliances, and recurrence of non-compliances. These results identified areas for improvement in the preventive measures of fish-processing plants and official food control, providing ways to reduce L. monocytogenes contamination in the fish industry.
  • Rossi, Heini (Helsingin yliopisto, 2020)
    Non-infectious inflammation of the lower airways, the main theme of this thesis, is commonly observed regardless of the breed, age, or use of the horse. Due to similarities with human asthma regarding clinical presentation and several pathophysiological aspects, non- infectious chronic lower airway inflammation in horses is currently named equine asthma (previously inflammatory airway disease or recurrent airway obstruction, depending on the severity). Long-term exposure to organic dust and moulds in stable air is thought to be the most important predisposing factor for the disease. Equine asthma is characterized by airway hyperresponsiveness and mucus accumulation, severity-related airway remodelling, impaired lung gas exchange, and increased quantity of airway inflammatory cells. These changes lead to clinical signs, including chronic cough and nasal discharge, poor performance, and in a severe form of equine asthma, increased respiratory effort at rest corresponding to an “asthma attack” in humans. Despite active research over the years, the aetiology and pathophysiology of equine asthma remain incompletely defined. In this thesis, the role of a transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN), a potential new biomarker in equine asthma, and its relationship to matrix metalloproteinases (MMPs) were investigated. In Study I, the respiratory secretions of 29 horses with asthma and 15 asymptomatic controls were examined. Expression of EMMPRIN protein was identified in all horses in bronchoalveolar lavage fluid (BALF) cells, and the expression was highest for the most substantially affected horses with severe asthma and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. This is the first study to describe the relationship of EMMPRIN and MMPs in patients of any species clinically affected with asthma, and the results suggest that EMMPRIN is involved in the pathophysiologic processes of asthma in horses. EMMPRIN as a marker of inflammation provides interesting possibilities for further research of horses and other species. The second part of this thesis concentrated on two common diagnostic tools used to assess lower airway diseases in horses. The diagnosis of lower airway inflammation, including asthma, is often aided by the analysis of cytological samples obtained from the trachea (tracheal wash; TW) or from the lungs at alveolar level (bronchoalveolar lavage; BAL). There is considerable variance in the diagnostic methods of equine lower airway diseases between facilities and clinicians, and a true gold standard for diagnosing airway inflammation is lacking. In most previous studies, the cytology of TW and BAL samples has been considered to correlate poorly and BAL has been deemed a superior diagnostic tool in most cases of equine lower airway disease, but sensitivity and specificity have not been evaluated. Moreover, the conventionally used cut-off values for cytology have been largely based on observational studies and expert opinion, and recently, higher values have been proposed. The first aim of Study II of this thesis was to compare the results of these two airway sampling methods in a population of 121 horses with signs related to respiratory disease and in 33 horses with no clinical respiratory signs. A secondary aim was to evaluate the cut-off values of TW and BALF cytology for neutrophils in order to justify their use in clinical practice. TW and BAL showed substantial agreement regarding neutrophils, and only 17.5% of the horses were classified differently (healthy vs. diseased), in contrast to some previous studies with higher disagreement. The neutrophil percentage was found to correlate between TW and BAL results. The sensitivity and specificity of TW were generally higher for TW than for BAL when estimated with conventional as well as with Bayesian statistical methods. The results of this study encourage the use of the current neutrophil cut-off values in clinical diagnosis of airway inflammation instead of elevating the cut-offs. In Study III, equine lower airway inflammation was investigated with regard to general anaesthesia. The complication rate in equine general anaesthesia is high relative to anaesthesia in several other species, postanaesthetic pneumonia being one of the potential complications. However, there is limited knowledge about the effects of general anaesthesia on inflammatory responses in the lungs and on systemic inflammatory markers such as serum amyloid A (SAA) in horses. For example, the level and duration of lower-airway neutrophilia that can be expected after anaesthesia in dorsal recumbency are unknown. Thus, our primary aim was to determine when lung inflammation reaches its maximum and how rapidly BALF cytology returns to baseline after anaesthesia in dorsal recumbency. A secondary aim was to investigate the possible effect of vatinoxan, a novel a2-adrenoceptor antagonist drug, on the BALF cytology results. Anaesthesia in dorsal recumbency resulted in no clinically relevant changes in airway cytology that could be differentiated from the effect of repeated BAL sampling. A marked increase in serum amyloid A was detected in some animals, while others showed no changes from baseline. Vatinoxan as premedication did not consistently affect lung cytology or blood inflammatory markers after anaesthesia. These combined studies on equine lower airway inflammation increase our understanding of mechanisms and factors related to pathophysiology and diagnostics of the condition in the horse. Lower airway inflammation is an important cause of impaired welfare and performance of horses. Broader understanding of the aspects related to the condition will aid in developing new improved clinical and diagnostic procedures and management strategies for equine inflammatory respiratory diseases.
  • Tapio, Heidi (Helsingin yliopisto, 2020)
    Detomidine and medetomidine are α2-adrenoceptor agonists that are used in equine medicine for sedation and analgesia. These drugs, however, also influence the cardiopulmonary and gastrointestinal system, mainly by increasing systemic vascular resistance and decreasing cardiac performance and intestinal motility. Vatinoxan, in turn, is primarily a peripherally acting α2-adrenoceptor antagonist that has been demonstrated to attenuate some of the undesired effects of α2-adrenoceptor agonists with no major influence on the level of sedation in many species. One aim of the present studies was to evaluate the effects of vatinoxan with a constant rate infusion (CRI) of medetomidine in standing and anesthetized healthy horses. Another aim was to evaluate the effects of vatinoxan when it is administered after detomidine in standing healthy horses. We hypothesized that vatinoxan would alleviate the cardiopulmonary and gastrointestinal effects of these α2-adrenoceptor agonists, while preserving their sedative effects under these experimental conditions. The horses received, in a cross-over design in three separate studies, an α2-adrenoceptor agonist either alone or with vatinoxan for sedation or for premedication before general anesthesia. To evaluate the cardiopulmonary function, the heart rate, respiratory rate, cardiac output, arterial blood pressures, central venous pressure, and pulmonary arterial pressures (this last one in two of the studies) were recorded. Additionally, arterial and venous blood gases were analyzed. Systemic vascular resistance, oxygen delivery, and other selected cardiopulmonary parameters were calculated afterwards. The effects of the drugs on intestinal motility were evaluated by auscultation of the borborygmi or by determining the amount of fecal output. The level of sedation was scored, and the recovery times were recorded in anesthetized horses. Finally, the drug concentrations in plasma were determined. In standing horses receiving a CRI of medetomidine, vatinoxan attenuated the early, although minor, cardiopulmonary changes induced by medetomidine. Furthermore, the intestinal motility was markedly improved by vatinoxan. In anesthetized horses receiving an adjunctive CRI of medetomidine, premedication with vatinoxan induced significant hypotension. Despite this hypotension, which was successfully treated with dobutamine, the cardiac performance and tissue oxygen delivery were better maintained in these horses than in those receiving the adjunctive medetomidine CRI without vatinoxan. The fecal output decreased after general anesthesia, and this decrease was not influenced by vatinoxan. Finally, vatinoxan, administered after detomidine, partially reversed the cardiopulmonary effects and intestinal hypomotility induced by detomidine. Vatinoxan decreased the level of sedation at the beginning of the CRI of medetomidine in standing horses, but not when administered with a single dose of medetomidine before general anesthesia or after detomidine in standing horses. The recovery times with or without vatinoxan also did not differ from each other. The plasma concentration of both enantiomers of medetomidine mostly correlated with the level of sedation, being lower in the presence of vatinoxan when the level of sedation was reduced. To conclude, vatinoxan attenuated the cardiopulmonary changes and intestinal hypomotility induced by detomidine or a CRI of medetomidine in standing horses. The effects of vatinoxan on the level of sedation could be considered relatively minor. With the dosing used in this study, vatinoxan induced marked hypotension in anesthetized horses and did not influence the intestinal hypomotility associated with general anesthesia. Thus, vatinoxan represents a potential drug for alleviating or reversing the undesired effects of medetomidine and detomidine in standing horses. Its hypotensive effects, however, warrant more research before it could be usable in anesthetized horse.
  • Niemelä, Tytti (Helsingin yliopisto, 2019)
    Intra-articular (IA) inflammation resulting in lameness is a common health problem in horses. Exogenous IA hyaluronic acid has been shown to provide an analgesic effect in equine and human osteoarthritis. High molecular weight non-animal stabilized hyaluronic acid (NASHA) has gained popularity in the treatment of human arthritic conditions due to its long-acting pain-relieving effects. In animal models, hyaluronan has been demonstrated to reduce pain by protecting nociceptive nerve endings and blocking pain receptor channels. Inflammatory and degenerative activity inside the joint can be studied in vivo by analysis of synovial fluid (SF) biomarkers. In addition to pro-inflammatory mediators, several anabolic and anti-inflammatory substances are produced during the disease process. The response of SF biomarkers after IA hyaluronan injection, alone or in combination with other substances, has been examined only in a few equine studies. Aim of the studies was to examine the efficacy of IA NASHA in relieving lameness and related signs of IA inflammation in clinical and experimental studies. We also investigated the effect of NASHA on various SF biomarkers. In addition, we explored possible adverse effects of NASHA on clinical signs of IA inflammation and SF. In the clinical study, we compared the response to treatment of lameness localized in the equine metacarpophalangeal joint injected with NASHA or placebo (saline). We also investigated the response of SF prostaglandin E2 (PGE2), substance P, aggrecan chondroitin sulphate 846 epitope (CS846), and carboxypeptide of type II collagen (CPII) concentrations to the NASHA and placebo treatments. After collection of baseline SF samples followed by IA diagnostic anaesthesia, horses in the treatment group received 3 ml of a NASHA product (20 mg/ml) intra-articularly, and those in the placebo group received an equivalent volume of sterile 0.9% saline solution. The horses were re-evaluated and a second SF sample was obtained after a two-week period. In the experimental study, the aim was to measure the SF interleukin-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB), transforming growth factor beta 1 (TGF-β1), and tumour necrosis factor alpha (TNF-α) concentrations before and after surgically induced cartilage defect and sham operation as a control in horses. We also investigated whether the concentrations of selected biomarkers in SF changed following NASHA injection. Results of the clinical study indicate that a single IA NASHA injection is not better than a single saline injection for reducing lameness in horses with synovitis or mild osteoarthritis. However, IA NASHA may have some beneficial effects in modifying mild clinical signs. The decrease in the SF concentration of the cartilage-derived biomarker CS846 only in the NASHA group suggests that less damage and hence less repair to the cartilage has occurred post-injection. Creation of the cartilage defect and sham operation lead to an increase of synovial fluid IL-1ra and TNF-α concentrations in the experimental study, but NASHA failed to produce changes in SF biomarkers.The significant increase in SF white blood cell count after IA NASHA may indicate a mild inflammatory response. However, as no clinical adverse effects were observed, IA NASHA appeared to be well tolerated.
  • Bergman, Paula Susanna (Helsingin yliopisto, 2019)
    In sow farms, animals are actively removed and replaced to maintain target efficiency levels, herd health status and a static herd size. Sow removal has a critical effect on pig farm functionality and profitability. Excessive removal decreases lifetime production and especially during periods of small profit margins, becomes burdensome. It is also recognized as a welfare issue. The overall aim of this thesis was to investigate the current reality of piglet production in the rapidly changing Finnish production conditions with special emphasis on removal. The thesis is based on three individual studies which utilize diverse data from real commercial piglet producing farms. The types of data collected for each are different and the methods used reflect the differences. We showed systematic and temporal differences in removal between individuals, parities, farms and replacement circumstances in Finland. The results also demonstrated the economic value of improved animal health and removal. We benchmarked national culling and mortality rates retrieved from mandatory registrations. Especially, the average on-farm mortality may be considered relatively high although the rates accorded with published literature. However, the large differences between farms and several farms succeeding in obtaining low levels of removal imply that it is conceivable to strive for and reach certain special targets. Unfortunately, we demonstrated that optimal lifetime of a sow is not a fixed number and as such, no generally applicable policy for replacing sows can be determined. Neither can excessive removal be improved by single improvements only, because of limited resource reserves and other shortages within the individual farm. However, a few factors were found to be linked with an increased risk for removal and higher removal levels in these studies: e.g. the smallest litter sizes and the number of stillborn piglets at the sow level, and features indicative of semi-intensive or intensive farming compared with a combination of environmental animal welfare indicators (mortality) and a non-intensified farming style (culling) at the farm level. Traditional production approach of maximizing the net monetary income or quantitative measures in piglet production is likely to be changed in response to consumer’s increasing concerns around animal well-being, environmental sustainability and one health. We introduced an empirical base that could be used to motivate debate on future development of piglet production systems and delivered useful evidence relevant for stakeholders to engage in and promote research into identification, monitoring and management of sow removal and health. It may be more motivating for Finnish piglet producers, herd advisers and the industry to have specified removal levels to work towards. Our study can also be considered valid for emphasizing awareness of multidisciplinary approaches in integrating accurate epidemiological livestock data into larger frameworks as well as identifying current bottlenecks in available data and modelling methodology.
  • Hasan, Shah (Helsingin yliopisto, 2019)
    In modern pig production there has been a steady increase in the litter size during recent decades. Large litters represent a major challenge for the sow, increase the farrowing duration and compromise sow welfare by prolonging this stressful process. Moreover, the continuous increase in litter size is a major cause of pre-weaning piglet mortality by increasing the proportion of low birth weight piglets, which are less vital and have reduced colostrum intake. Approximately 30% of hyper-prolific sows produce insufficient colostrum for their piglets. Colostrum plays an essential role in the health, survival and growth of piglets by providing energy, immunoglobulins, growth factors and many other bioactive components. Both colostrum yield and its composition are highly variable among sows, yet mechanisms and factors that regulate colostrogenesis are not fully understood. The aim of this study was to evaluate sow colostrum IgG concentration on farm using a Brix refractometer, to thus improve the management of neonatal piglets immediately after initiation of farrowing. We established colostrum evaluation criteria, to be used at herd level, by comparing the Brix results with those from ELISA. We aimed to investigate sow physiology around farrowing, acute phase protein (APP) response and their association with colostrum yield (CY), colostrum composition and piglet colostrum intake (CI). The impact of sow CY, colostrum composition and quality on piglet performance were also investigated. We also aimed to determine the effects of yeast hydrolysate (YD) and resin acid-enriched composition (RAC) inclusion in sows’ gestation and lactation diets on CY, colostrum immunoglobulins, nutritional composition and subsequent litter performance. We also sought to determine the influence of these two feed additives on the taxonomic profile of the hindgut microbiota of sows. Additionally we aimed to determine whether changing the gut microbiota of sows influences the microbial colonization of piglets after birth. There was a correlation between Brix refractometer measurement of colostrum and the corresponding IgG concentration measurements of ELISA (r = 0.63, P < 0.001). A classification of colostrum quality was suggested; low levels of IgG (14.5 ± 1.8 mg/ml) were recorded for colostrum samples with Brix readings below 20%. Borderline colostrum IgG content (43.8 ± 2.3 mg/ml) had Brix readings of 20% to 24%, adequate colostrum IgG content (50.7 ± 2.1 mg/ml) had Brix readings of 25% to 29% and very good IgG colostrum content (78.6 ± 8.4 mg/ml) had Brix readings >30%. Sow CY was positively correlated with plasma haptoglobin (Hp) (P = 0.029), number of live-born piglets (P < 0.01) and negatively correlated with farrowing duration (P = 0.01). Piglet CI was positively associated with piglet weight at birth (P < 0.001) and negatively associated with the number of live-born piglets in the litter (P < 0.001). Both piglet CI and birth weight were positively associated with piglet average daily gain (ADG) (P < 0.001). Risk for piglet death, or a piglet being treated with an antibiotic before weaning, increase with a decrease in sow back fat thickness at farrowing (P = 0.04). Similarly, we found that piglets of litters with low BWB and low CI had a higher risk of death before weaning (P < 0.001). Piglets born from sows having lower levels of colostrum IgA and serum amyloid A (SAA) and high plasma progesterone at the end of farrowing had higher risk of neonatal diarrhea (P = 0.04; P = 0.05; P = 0.04). Piglets born from sows having higher back fat thickness at weaning had higher risk of developing weaning diarrhea (P = 0.02). There was a significant increase in CY in sows fed with YD during gestation (P = 0.04) and a higher level of fat in their colostrum (P = 0.01). In RAC fed sows there was a significant increase in colostrum IgG content. Piglets weaning weights of RAC fed sows were higher in two trial herds. Inclusion of YD and RAC in gestation and lactation diet increased the abundance of beneficial and fermentative fecal bacteria (Roseburia, Paraprevotella, Eubacterium, Romboutsia and Clostridium sensu stricto) significantly (P < 0.01) while, opportunistic pathogens, especially Proteobacteria, were suppressed. In addition, piglets from the sows fed YD had more beneficial microbial populations with higher diversity and fewer opportunistic pathogens at one week of age. However, feeding YD, higher CY, colostrum composition and piglet birth weight were all correlated with specific sow gut microbiota. In conclusion, Brix measurement of a sow’s fresh colostrum is an inexpensive, rapid and sufficiently accurate method of estimating IgG concentration, providing indication of differentiation between good and poor IgG content of colostrum. Although colostrum is vital for piglet survival, both CY and composition are highly variable among sows. Sow body condition and physiological status around farrowing affect CY and colostrum immunoglobulin contents. Piglet survival and risk of death before weaning also depend on CI. Therefore, to support CY and CI, measures should be taken to ease the process of farrowing, increase piglet vitality and improve colostrum availability for piglets. Functional feed ingredients such as YD and RAC may increase colostrum availability, improve colostrum IgG and its energy content for neonate piglets, and also promote beneficial maternal microbial sources for neonates.
  • Summa, Maija (Helsingin yliopisto, 2019)
    Human noroviruses (HuNoVs) are yearly responsible for a large number of acute human gastroenteritis cases globally in all age groups. Typically, the virus transmits via the fecal-oral route from person to person, causing strong symptoms such as nausea, vomiting, and diarrhea, which usually disappear in a few days. However, HuNoVs cause also numerous food-related illnesses in developed countries, including Finland, inducing gastroenteritis outbreaks through contaminated water and foodstuffs. According to the reports of the European Commission, both in Europe and in Finland the most common foods causing HuNoV outbreaks are shellfish, berries (especially frozen raspberries), vegetables, and mixed foods, which most likely became contaminated by a sick food handler. Noroviruses belong to the Caliciviridae family and are classified into seven genogroups. HuNoVs belong to genogroups I (GI), II (GII), and IV (GIV). Other genogroups contain only animal noroviruses. Noroviruses are generally regarded as host-species-specific, but the possibility of zoonotic transmission and infections has been discussed for over a decade for several genotypes. The purpose of this study was to develop a simple and rapid method for detection of HuNoVs in food. The potential zoonotic nature of HuNoVs, particularly whether animals can serve as transmitters for these viruses, was also investigated. In the past two decades, numerous methods for detecting HuNoVs in food have been developed. However, many of these are time-consuming and the sensitivity of the methods has been highly variable. In this work, four published extraction methods for detection of HuNoV in food (lettuce, ham, and frozen berries) were compared. The method based on alkaline elution and polyethylene glycol (PEG) precipitation was found to be the most reliable detection method for all three food matrices tested. The recovery efficiency of the method with frozen raspberries was on average 28%. Two rapid methods for detection of HuNoV in frozen raspberries were also presented. The rapid method based on direct RNA extraction yielded the same recovery levels (32%) as the PEG precipitation method. The method proved to be sensitive because it detected HuNoV also with a virus level of 100 genome copies in a 25 g sample. Moreover, the method detected HuNoV in naturally contaminated berry samples that were linked to outbreaks of disease. A treatment with either a chloroform-butanol mixture or dilution of the food samples for the RT-PCR reaction was efficient in reducing the effect of PCR inhibitors. The same effect was achieved with PEG as a supplement in the food samples. Thirty-nine frozen berry samples purchased from local stores in 2010, 2014, and 2017 were screened. All berries tested negative for HuNoVs GI and GII. HuNoV genome was detected in the feces of 31 birds, two rats, and four pet dogs. The genotypes found in six bird samples and all dog samples were the same as those commonly found in human samples at the time of sampling. HuNoVs can be detected in food samples also in small numbers using the rapid method presented in this study. The use of PEG as a supplement was found to reduce inhibition of the RT-PCR reaction in the two rapid methods, and therefore, the commonly used chloroform-butanol treatment, which easily loses viruses during processing, could be omitted. The results of animal samples strongly indicate that wild birds, pet dogs, and possibly also rats may be involved in the transmission of HuNoVs to food, water, and surfaces
  • Adam, Magdy (Helsingin yliopisto, 2019)
    The impact of the peripherally selective α2-adrenoceptor antagonist, vatinoxan, on selected pharmacodynamic and pharmacokinetic properties of two selective α2-adrenoceptor agonists, medetomidine and dexmedetomidine, were investigated in sheep. Moreover, certain interactions between vatinoxan and atipamezole, a specific α2-adrenoceptor antagonist, were evaluated. The initial objective of this study was to identify a dose of vatinoxan that would best mitigate the undesirable cardiopulmonary changes produced by intramuscular (IM) medetomidine-ketamine in sheep. Specifically, three doses of vatinoxan (150, 300 and 600 µg/kg) or saline were combined in the same syringe with medetomidine (30 µg/kg) and ketamine (1 mg/kg) and given IM. Systemic hemodynamics, arterial blood gas tensions, clinical sedation and plasma drug concentrations were compared, both before and after reversal with IM atipamezole (150 µg/kg). The middle dose of vatinoxan (300 µg/kg), which appeared to be optimal among the other doses, was then added to medetomidine (30 µg/kg) and co-administered IM, followed by atipamezole for reversal. Last, the influence of intravenous pre-treatment with vatinoxan on dexmedetomidine-induced cardiopulmonary alterations was investigated in sevoflurane-anesthetized sheep. Following concomitant IM administration, vatinoxan dose-dependently attenuated some of medetomidine’s cardiopulmonary side effects. Vatinoxan did not significantly affect the level of sedation or the plasma concentrations of drugs when ketamine was included in the same syringe. Conversely, vatinoxan significantly increased the plasma concentrations of medetomidine, and accelerated the onset and intensified the degree of sedation when compared with the agonist alone. Moreover, recoveries after atipamezole-reversal were more complete in the presence of vatinoxan. No deleterious effects were noted between vatinoxan and atipamezole. Pre-treatment with vatinoxan prevented all dexmedetomidine-induced pulmonary alterations in sheep anesthetized with sevoflurane. In conclusion, vatinoxan alleviated or prevented the unwanted cardiopulmonary effects of (dex-) medetomidine by blocking the peripheral α2-adrenoceptors. Presumably, when co-administered IM in the same syringe, vatinoxan accelerated the absorption of medetomidine and increased its concentration in blood, which resulted in a faster and more intense sedation than when the agonist was used alone. Vatinoxan also decreased later exposure to dexmedetomidine, which appeared to improve atipamezole’s efficacy to reverse both the central and peripheral effect of the agonist.
  • Pöntinen, Anna (Helsingin yliopisto, 2019)
    Listeria monocytogenes is a remarkable bacterium, as it is able to shift from a capable environmental saprophyte into a severe intracellular pathogen. As a strictly foodborne pathogen, L. monocytogenes poses a notable risk, particularly to those consumers among the risk groups for whom invasive listeriosis is potentially fatal. Furthermore, modern consumption habits and increasingly favoured ready-to-eat foods, often consumed without proper heating, increase the risk of acquiring the foodborne disease. The aim of this study was to investigate the genetic mechanisms conferring wide-ranging stress tolerance in L. monocytogenes. Two-component systems, comprising a sensor histidine kinase and a cognate response regulator, aid bacteria in sensing and adapting to changes in both surrounding environmental as well as intracellular conditions. The histidine kinases, in particular, have lacked comprehensive studies on their roles in the stress tolerance of L. monocytogenes. Thus, histidine kinases were studied by expressional analyses under cold conditions and by mutationally disrupting each histidine kinase-encoding gene in a parental model strain, L. monocytogenes EGD-e. The modified strains were individually challenged at high (42.5 degrees C) and low (3.0 degrees C) temperatures, high (9.4) and low (5.6) pH levels, and high salt (6% NaCl), ethanol (3.5 vol%) and hydrogen peroxide (5 mM) concentrations. Expressional studies and growth experiments on genetically modified strains proved lisK and yycG to respectively play central roles in the acclimation and immediate growth of L. monocytogenes at low temperatures. The most substantial increase in gene expression under cold conditions was that of the chemotaxis gene cheY with 236-fold upregulation at 3 degrees C. The disrupted ΔliaS strain displayed impaired growth in response to all the other stresses, particularly at a high temperature and under osmotic stress. These studies demonstrated the prominent importance of the histidine kinase-encoding genes yycG and lisK to cold tolerance and liaS, with roles in the growth of L. monocytogenes under multiple stresses. To shed light on the accessory genetic mechanisms that cause large strain variation in L. monocytogenes in withstanding heat treatments, heat resistance-conferring traits were further investigated by means of whole-genome sequencing. Comparing the complete genomes of heat-resistant L. monocytogenes AT3E and -sensitive AL4E strains revealed the heat-resistant strain to harbour a novel 58-kb plasmid, pLM58, which was absent in the sensitive strain. Furthermore, curing of the plasmid in AT3E produced a marked decrease in heat resistance from virtually no reduction to a 1.1 cfu/ml log10 reduction at 55.0 degrees C. In pLM58, a 2,155-bp open reading frame annotated as an ATP-dependent ClpL protease-encoding gene was identified. Conjugation of the coding sequence and the putative promoter of the clpL gene into a natively heat-sensitive L. monocytogenes 10403S strain, in turn, enhanced the survival of the strain from a 1.2 cfu/ml log10 reduction to a 0.4 cfu/ml log10 reduction in heat challenge at 55.0 degrees C. In this study, we presented the first evidence of plasmid-mediated heat resistance in L. monocytogenes and identified the protease ClpL to be a novel plasmid-borne heat-resistance mediator. The emerging resistance of L. monocytogenes to benzalkonium chloride, a quaternary ammonium compound widely used as a detergent in food-processing facilities, is a significant concern for food safety and public health. The resistance of 392 L. monocytogenes isolates from Finland (n = 197) and Switzerland (n = 195) to benzalkonium chloride was assessed. A minimal inhibitory concentration of 20 µg/ml was defined. Altogether, 11.5% of the strains proved to be resistant to benzalkonium chloride. Serotype 1/2c harboured the highest prevalence, 32.4% (11/34), of benzalkonium chloride-resistant strains, while in total, most of the resistant strains belonged to serotype 1/2a. Altogether, 68.9% of the resistant strains harboured at least one of the efflux pump system-encoding genes, bcrABC, emrE or qacH, known to confer benzalkonium chloride resistance in L. monocytogenes. We found resistant strains with partially or completely efflux pump-dependent benzalkonium chloride resistance, with the exception of the known resistance-mediating efflux pumps, suggesting the existence of other resistance-contributing efflux pump systems. The lacking of known efflux pump system-encoding genes in addition to efflux pump-independent benzalkonium chloride resistance, in turn, indicates the contribution of completely novel benzalkonium chloride resistance mechanisms. The aim of these studies was to shed light on the genes contributing to the versatile stress tolerance abilities and strain variation of the severe foodborne pathogen, L. monocytogenes. Knowledge of such traits may aid in developing targeted strategies and measures to identify and control the contamination and risks caused, in particular, by stress-tolerant L. monocytogenes strains.
  • Lopez Valladares, Gloria (Helsingin yliopisto, 2019)
    Isolates of Listeria monocytogenes (N=932) collected from human cases of invasive listeriosis in Sweden between 1958 and 2010 were serotyped and characterised with pulsed-field gelelectrophoresis (PFGE) and AscI restriction enzyme. The genotype diversity of L. monocytogenes isolates was investigated and related to genotypic results from epidemiological information on human infection, in order to detect possible clustering of L. monocytogenes genotypes over time, season, location, age, or gender (Paper I). From 1972 to 1995, serovar 4b was the predominant serovar; however, in 1996, serovar 1/2a became the major serovar among human listeriosis cases in Sweden. Based on the number and distribution of all bands in the profile, 63 PFGE types belonging to serovars 1/2b, 3b and 4b and 119 PFGE types belonging to serovars 1/2a and 1/2c were identified (Paper I). The PFGE types were further assembled into PFGE groups, based on the number and distribution of small bands below 145.5 kb (Papers II and III). As the genomic region of small bands is genetically more conservative than in large bands, the distribution of small bands establishes relatedness of strains and defines genetic markers for both lineages. Cold-smoked salmon (Salmo salar) and gravad salmon packed under modified atmosphere or vacuum from three manufacturers were purchased in Sweden and Germany in 2005 and the occurrence and levels of L. monocytogenes were analysed (Paper IV): 56 products were analysed and eleven harboured L. monocytogenes. From the positive samples, 56 isolates were analysed with AscI, and 11 isolates were further analysed with ApaI: five AscI PFGE types were identified, four belonging to serovar 1/2a and one to 4b. Forty-three (n=43: 76.8%) isolates shared serovar 1/2a and 13 (23.2%) shared serovar 4b and all AscI types were identified among human clinical strains in Sweden. Moreover, three gravad salmon samples harboured two PFGE types each from different lineages, serovar 1/2a and serovar 4b. Although, in most of the products, the level of L. monocytogenes was less than 100 cfu/g, the highest level was 1500 cfu/g. The occurrence of L. monocytogenes was 12.9% in gravad salmon, encountered in three manufacturers (A, B, C) and 28% in cold-smoked salmon only from manufacturer A. Although the level of L. monocytogenes in RTE fish products is generally low, these products, should be considered possible sources of listeriosis in Sweden. A patient may harbour more than one L. monocytogenes PFGE type that can be determined through PFGE and AscI restriction enzyme. However, to avoid misleading conclusions, several L. monocytogenes colonies should be isolated and characterised from different sites from the same patient or mother-baby pairs (Paper V).
  • Felin, Elina (Helsingin yliopisto, 2019)
    Salmonella spp., Yersinia enterocolitica, Toxoplasma gondii and Trichinella spp. are the most relevant biological hazards in the context of meat inspection of pigs in the European Union (EU). These zoonotic pathogens show no clinical symptoms or gross pathological lesions in pigs, and thus are not detectable with current meat inspection procedures, except Trichinella spp. by laboratory analysis. In this study, we analysed the serological prevalence of these pathogens in Finnish fattening pigs and evaluated serological monitoring as a control method. In total, we studied 1353 meat juice samples and 1793 serum samples of fattening pigs using commercial ELISA kits. The seroprevalence of pathogenic Yersinia spp. was the highest of the studied pathogens, and Yersinia antibodies were detected in 57% and 66% of the meat juice and serum samples at the end of the fattening period, respectively. The seroprevalences of Salmonella spp. and T. gondii were low. Salmonella antibodies were detected in 3% of the meat juice samples and in 18% of the serum samples at the end of fattening. T. gondii antibodies were detected in 3% of meat juice samples and 1% of serum samples. Trichinella spp. antibodies were not detected. The seroprevalences at the end of the fattening or at slaughter were not associated with post-mortem findings of the current batch, which was expected. This indicates that we need new tools to control these public health hazards in pork. Meat juice serology at slaughter was feasible and easy to perform. We observed huge differences between farms considering Salmonella spp., Yersinia spp. and T. gondii seroprevalences. This shows that farm-level serological data could be used as part of the food chain information (FCI) for risk-based decisions to improve food safety. Risk-based decisions include slaughtering arrangements, additional carcass processing, targeted sampling at the slaughterhouse and improved biosecurity measures at the farm. However, risk mitigation targets and procedures must be carefully adjusted for each pathogen. With targeted serological monitoring of T. gondii we could effectively target control measures and diminish the pathogen in pork. Serological monitoring of pathogenic Yersinia spp. could be the first step in the huge challenge of Y. enterocolitica in pigs, allowing for the possibility of slaughtering pigs from high-risk farms at the end of the day. Serological monitoring of Salmonella spp. would be beneficial, but would only have a limited positive impact on food safety, because the current situation is already excellent. Serosurveillance of Trichinella spp. would become meaningful, if current testing is to be diminished. In addition to food safety issues, FCI could be useful for visual-only inspection, which should be the most common inspection method. We analysed authentic FCIs and meat inspection findings of 85 slaughter batches of fattening pigs. In addition, we analysed on-farm health status indicators, assessed by a veterinarian, and the meat inspection findings of another 57 slaughter batches. The partial carcass condemnation rate of the current batch was best predicted by the partial carcass condemnation rate of the pigs from the same farm within one year. Constant coughing and tail biting at a farm were associated with partial carcass condemnations. On-farm health indicators (such as the healed tail biting rate at the end of fattening and constant coughing during fattening) together with previous meat inspection results could be used as part of the FCI to make decisions regarding the meat inspection procedure: visual-only or additional inspections. However, farmers must be properly advised to carefully report this information.
  • Pakkanen, Soile (Helsingin yliopisto, 2019)
    Alpha2-adrenoceptor agonists detomidine and romifidine are used in equine medicine for sedation and analgesia. Vatinoxan is an α2-adrenoceptor antagonist that has poor ability to cross the blood-brain barrier, limiting its actions to peripheral α2-adrenoceptors. The main objective of these studies was to evaluate the effect of vatinoxan in preventing the adverse effects, such as cardiovascular compromise, decreased intestinal motility and changes in plasma glucose concentrations, of detomidine and romifidine, while sparing the sedative action in horses. Plasma drug concentrations were investigated to elucidate the pharmacokinetic agonist-antagonist interactions. To assess cardiovascular effects of the drugs, heart rate and blood pressures were measured in horses treated with α2-adrenoceptor agonists and vatinoxan. In horses under general anaesthesia, premedicated with either detomidine alone or with vatinoxan, cardiac index and systemic vascular resistance and tissue oxygen delivery were investigated. Sedation and intestinal borborygmi were scored after treatment with detomidine, romifidine and vatinoxan. Glucose, insulin, ACTH, lactate, FFA, cortisol, triglyceride, and potassium and sodium were analysed from blood samples. Vatinoxan attenuated the bradycardia, hypertension and intestinal hypomotility induced by detomidine and romifidine, while the effect on sedation was minor. Vatinoxan alone increased heart rate, but had no effect on mean arterial blood pressure. Premedication with vatinoxan and detomidine resulted in severe hypotension in anaesthetized horses, and high doses of dobutamine were required for blood pressure support. Combined premedication also resulted in decreased systemic vascular resistance and increased cardiac index and tissue oxygen delivery. The latter two might, at least partly, be related to high doses of dobutamine. Vatinoxan increased volume of distribution and decreased plasma concentration of α2-adrenoceptor agonists. Romifidine induced hyperglycaemia in horses, which vatinoxan alleviated. Vatinoxan alone had no effect on plasma glucose concentration. Serum FFA was significantly higher after vatinoxan alone than after romifidine alone or combined with vatinoxan. In conclusion, vatinoxan alleviated the cardiovascular and intestinal effects of detomidine and romifidine in horses, while no clinically relevant effect occurred on sedation. When vatinoxan was administered before general anaesthesia, marked hypotension followed. Romifidine-induced hyperglycaemia was attenuated by vatinoxan. Vatinoxan increased the volume of distribution and decreased plasma concentrations of detomidine and romifidine. Romifidine had no significant impact on plasma concentration of vatinoxan.

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