Growth of singletons born after frozen embryo transfer until early adulthood: a Finnish register study.

STUDY QUESTION: Are there growth differences between singleton children born after frozen embryo transfer (FET), fresh embryo transfer (ET), and natural conception (NC)? SUMMARY ANSWER: Adolescent boys born after FET have a higher mean proportion and increased odds of overweight compared to those born after fresh ET. WHAT IS KNOWN ALREADY: Children born after FET have higher mean birthweights and an increased risk of large-for-gestational-age compared to those born after fresh ET and even NC. This raises questions about possible growth differences later in childhood. Previous studies on child growth after FET report partly conflicting results and lack long-term data until adolescence. STUDY DESIGN, SIZE, DURATION: This was a cohort study based on national population-based registers, the Finnish Medical Birth Register and the Register of Primary Health Care visits, including singletons born after FET (n = 1825), fresh ET (n = 2933), and NC (n = 31 136) in Finland between the years 1995 and 2006. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proportions of overweight (i.e. age- and sex-adjusted ISO-BMI for children ≥ 25) were compared between the groups. Odds ratios (ORs) and adjusted odds ratios (aORs) of overweight were calculated. Adjustments were made for birth year, preterm birth, maternal age, parity, and socioeconomic status. Mean heights, weights, and BMIs were compared between the groups each year between the ages of 7 and 18. MAIN RESULTS AND THE ROLE OF CHANCE: FET boys had a higher mean proportion of overweight (28%) compared to fresh ET (22%, P < 0.001) and NC (26%, P = 0.014) boys. For all ages combined, the aOR of overweight was increased (1.14, 95% CI 1.02-1.27) for FET boys compared to fresh ET boys. For girls, the mean proportions of overweight were 18%, 19%, and 22% for those born after FET, fresh ET, and NC, respectively (P = 0.169 for FET vs fresh ET, P < 0.001 for FET vs NC). For all ages combined, FET girls had a decreased aOR of overweight (0.89, 95% CI 0.80-0.99) compared to NC girls. Growth measurements were available for 6.9% to 30.6% of FET boys and for 4.7% to 29.4% of FET girls at different ages. LIMITATIONS, REASONS FOR CAUTION: Unfortunately, we were not able to adjust for parental anthropometric characteristics. The growth data were not available for the whole cohort, and the proportion of children with available measurements was limited at the start and end of the follow-up. During the study period, mainly cleavage stage embryos were transferred, and slow freezing was used for ART. WIDER IMPLICATIONS OF THE FINDINGS: The risk of overweight among FET boys warrants further research. Future studies should aim to investigate the mechanisms that explain this sex-specific finding and combine growth data with long-term health data to explore the possible risks of overweight and cardiometabolic disease in adulthood. STUDY FUNDING/COMPETING INTEREST(S): Funding was obtained from the Päivikki and Sakari Sohlberg Foundation, the Alma and K.A. Snellman Foundation (personal grants to A.M.T.), and the Finnish Government Research Funding. The funding sources were not involved in the planning or execution of the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Health of singletons born after frozen embryo transfer until early adulthood: a Finnish register study.

STUDY QUESTION: Is the health of singletons born after frozen embryo transfer (FET) comparable to that of singletons born after fresh embryo transfer (ET) until early adulthood? SUMMARY ANSWER: The health of singletons born after FET does not differ from that of singletons born after fresh ET. WHAT IS KNOWN ALREADY: The differences in perinatal outcomes of children born after FET and fresh ET are well known. FET is associated with an increased risk of large-for-gestational-age but diminished risks of preterm birth (PTB), small-for-gestational-age and decreased perinatal mortality compared to fresh ET. However, knowledge on the long-term health after FET is scarce. STUDY DESIGN, SIZE, DURATION: This retrospective register-based cohort study compares singletons born after FET (n = 1825) between the years 1995 and 2006 to those born after fresh ET (n = 2933) and natural conception (NC, n = 31 136) with a mean follow-up time of 18-20 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Singletons born after FET were compared to those born after fresh ET and NC regarding the frequencies of diagnoses in the main ICD-10 chapters (International Statistical Classification of Diseases and Related Health Problems, 10th revision), the number of outpatient visits and hospital admissions, and mortality. Adjustments were made for PTB, maternal age, parity, socioeconomic status based on mother's occupation and offspring sex. The study combines data from the Finnish Medical Birth Register, the Finnish Care Register for Health Care (CRHC) and the Cause-of-Death Register at Statistics Finland. The Student's T-test was used for continuous variables, and the Chi-square test was used for categorical variables. Cox regression was used to estimate crude and adjusted hazard ratios (HRs and aHRs, respectively). A general linear model was used to compare the means of outpatient visits, hospital admissions and lengths of hospital stays per person. MAIN RESULTS AND THE ROLE OF CHANCE: No significant differences between the FET and fresh ET groups were found in the frequency of diagnoses in any of the ICD-10 chapters or in the parameters describing the need for hospital care. However, compared to the NC group, higher proportions in the FET group had outpatient visits in the hospital (93.5% vs 92.2%, aHR 1.23, 95% CI 1.17, 1.30) or hospital admissions (48% vs 46.5%, aHR 1.28, 95% CI 1.19, 1.37). Compared to the NC group, the FET group had elevated adjusted risks of diagnoses of infectious and parasitic diseases (aHR 1.24; 95% CI 1.11, 1.38), neoplasms (aHR 1.68; 95% CI 1.48, 1.91), diseases of the eye and adnexa, the ear or mastoid process (aHR 1.11; 95% CI 1.01, 1.21), the respiratory system (aHR 1.15; 95% CI 1.06, 1.23), the digestive system (aHR 1.17; 95% CI 1.05, 1.32), the skin or subcutaneous tissue (aHR 1.28; 95% CI 1.14, 1.43) and the genitourinary system (aHR 1.27; 95% CI 1.11, 1.45), as well as congenital malformations or chromosomal abnormalities (aHR 1.31; 95% CI 1.14, 1.50) and symptoms, signs or abnormal clinical or laboratory findings (aHR 1.25, 95% CI 1.16, 1.34). LIMITATIONS, REASONS FOR CAUTION: Only hospital-based inpatient and outpatient care is covered by the CRHC register, excluding milder cases diagnosed elsewhere. We were not able to study the effect of ART treatments and subfertility separately in our setting. In addition, although our cohort is reasonably sized, even larger cohorts would be needed to reliably study rare outcomes, such as cancer. WIDER IMPLICATIONS OF THE FINDINGS: For many ICD-10 chapters, we present the first published data on the long-term outcome of singletons born after FET. The results on FET versus fresh ET are reassuring, whereas the results on FET versus NC warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S): Finnish government research funding was obtained for this study. Funding was also obtained from the Finnish Medical Society Duodecim, the Päivikki and Sakari Sohlberg Foundation, Orion Research Foundation, Finnish Society of Obstetrics and Gynaecology (research grants to A.M.T.) and Finnish government research funding. The funding sources were not involved in the planning or execution of the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

High birth weight and large-for-gestational-age in singletons born after frozen compared to fresh embryo transfer, by gestational week: a Nordic register study from the CoNARTaS group.

STUDY QUESTION: When do the differences in birth weights become apparent between singletons born after frozen embryo transfer (FET) and fresh embryo transfer (fresh ET)? SUMMARY ANSWER: Mean birth weights after FET become significantly higher starting from gestational week (GW) 33 among boys and from GW 34 among girls. WHAT IS KNOWN ALREADY: In recent years, there has been a steep rise in recorded FET treatments, enabling widespread use of elective single embryo transfer, thus reducing the risks associated with multiple gestations. However, singletons born after FET are heavier and there is a higher risk of large-for-gestational-age (LGA) (birth weight > 90 percentiles) compared to fresh ET. In contrast, risk of small-for-gestational-age (SGA, birth weight < 10 percentiles) is lower in singletons born after FET compared to fresh ET. The reasons, timing and consequences of these differences remain largely unclear. There is limited evidence about whether this difference in growth develops before the last trimester of pregnancy. STUDY DESIGN, SIZE, DURATION: This retrospective Nordic register-based cohort study compared singletons born after FET (n = 17 500) to singletons born after fresh ET (n = 69 510) and natural conception (NC, n = 3 311 588). All live born singletons born between the years 2000 and 2015 in Denmark, Norway and Sweden at gestational age ≥22 weeks were included from the population-based Committee of Nordic ART and Safety (CoNARTaS) study population. PARTICIPANTS/MATERIALS, SETTING, METHODS: Children born after FET were compared to those born after fresh ET and NC for mean birth weight and proportion of LGA and SGA for each GW at birth. Chi-square test and tests for relative proportions were used to compare categorical variables and Student's t-test was used to compare continuous variables. Adjusted odds ratios (aORs) for LGA and SGA were calculated using logistic regressions, adjusting for year of birth, maternal age, parity, BMI, chronic hypertension, diabetes, smoking and offspring sex. MAIN RESULTS AND THE ROLE OF CHANCE: Mean birth weights were significantly higher after FET compared to fresh ET starting from GW 33 (range from 75 g to 228 g by week) for boys and starting from GW 34 (range from 90 g to 236 g by week) for girls. Boys born after FET had a significantly higher proportion of LGA (11.0-15.1%) at birth between GW 36 and 42, compared to those born after fresh ET (7.1-9.4%) (range from P < 0.001 to P = 0.048 by week). For girls born after FET, the difference was seen between GW 37 and 42 (10.6-13.4%) compared to those born after fresh ET (6.6-8.0%) (range from P < 0.001 to P = 0.009 by week).The proportion of SGA was significantly lower among boys born after FET (7.6-8.7%) compared to fresh ET (11.9-13.6%) between GW 36 and 42 (range from P < 0.001 to P = 0.016 by week). For girls born after FET, the difference was seen between GW 38 and 42 (7.0-9.3%) compared to those born after fresh ET (13.0-14.6%) (P < 0.001). The proportion of LGA (12.3-15.1%) was significantly higher for boys born after FET between GW 38 and 41 (P < 0.001) and for girls born after FET (12.6-13.4%) between GW 37 and 40 (range from P < 0.001 to P = 0.018 by week), compared to naturally conceived boys (9.7-9.9%) and girls (9.0-10.0%). All singletons born after FET had a higher risk of LGA compared to singletons born after fresh ET (aOR 1.87, 95% CI 1.76-1.98) and singletons born after NC (aOR 1.28, 95% CI 1.22-1.35). LIMITATIONS, REASONS FOR CAUTION: There may be residual confounding factors that we were not able to control for, most importantly the causes of preterm birth, which may also influence foetal growth. A further limitation is that we have no knowledge on growth patterns between implantation and GW 22. Finally, the number of children born extremely preterm or post-term was limited even in this large study population. WIDER IMPLICATIONS OF THE FINDINGS: This is, to date, the largest study on birth weights among preterm and term ART singletons with a population-based design and NC control group. The results suggest that the freeze-thaw process is associated with higher birthweights and greater risk of LGA at least in the last trimester of pregnancy. This is an important aspect of the safety profile of ART. More research is needed on the long-term outcome of these children. STUDY FUNDING/COMPETING INTEREST(S): The CoNARTaS collaboration has received the following funding: the Nordic Trial Alliance: a pilot project jointly funded by the Nordic Council of Ministers and NordForsk [71450], the Central Norway Regional Health Authorities [46045000], the Norwegian Cancer Society [182356-2016], the Nordic Federation of Obstetrics and Gynaecology [NF13041, NF15058, NF16026 and NF17043], the Interreg Öresund-Kattegat-Skagerrak European Regional Development Fund (ReproUnion project) and the Research Council of Norway's Centre of Excellence funding scheme [262700]. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER: ISRCTN11780826.

Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci.

Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism, Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow-growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies.

Physical health of singleton children born after frozen embryo transfer using slow freezing: a 3-year follow-up study.

STUDY QUESTION: Are there differences in the physical health of singleton children born after frozen embryo transfer (FET) compared with children born after fresh embryo transfer (fresh ET)? SUMMARY ANSWER: Register-based health indicators were similar among FET and fresh ET singletons during a 3-year follow-up. WHAT IS KNOWN ALREADY: Large cohort studies have shown that perinatal outcomes are similar or even better in FET than fresh ET children. The early childhood morbidity among FET and fresh ET children has been shown to be quite similar, but so far these studies have been small. The short-term health outcomes of assisted reproductive technology (ART) children have been shown to be slightly worse compared with spontaneously conceived children. STUDY DESIGN, SIZE, DURATION: This register-based study includes women who had undergone ART treatments leading to singleton live births (n = 4758 children) in 1995-2006. A 10% random sample of women with spontaneous pregnancies from the Finnish Medical Birth Register (FMBR) served as the reference group (n = 31 137 children). The children were identified through the FMBR by using the mother's personal identification (ID) number. Children's ID numbers were linked with two nationwide registries; the Finnish Hospital Discharge Register and the Cause-of-Death Register at Statistics Finland. Information on all visits was received until 2009 using ICD-10 codes. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study includes 1825 children born after FET, 2933 children born after fresh ET and 31 137 children born after spontaneous pregnancies. The risk estimates for diseases were adjusted for the child's year of birth and maternal age, parity, socio-economic status and prematurity. The study focused on the differences between FET and fresh ET children. MAIN RESULTS AND THE ROLE OF CHANCE: Most health indicators were similar among FET and fresh ET children during the 3-year follow-up. The most common discharge diagnoses, including gastroenteritis and colitis, otitis, upper and lower respiratory diseases, asthma and allergies were similar between the ART groups. A large proportion of FET children (70.1%) and fresh ET children (69.9%) had visited a hospital at least once (P = 0.877). The risk of hospital admission did not differ between the two groups after adjusting for premature births [adjusted odds ratio (aOR) 1.01; 0.88-1.17]. Comparing with children born after spontaneously conceived pregnancies, the risk of hospital admission was slightly increased in the ART group, even after adjusting for premature births (aOR 1.10; 1.02-1.19). LIMITATIONS, REASONS FOR CAUTION: Due to the study design, we were not able to control for some parental background factors, such as the cause and length of infertility. Furthermore, the health registries do not include data on the growth of the children. Our findings are generalizable only to the slow-freezing method. WIDER IMPLICATIONS OF THE FINDINGS: Our study provides further evidence of the safety of embryo cryopreservation. The early physical health of FET children is similar to that of children born after fresh ET. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the University Hospital of Oulu and Helsinki, Finland. The National Institute for Health and Welfare (THL) covered the data linkages and the work of Mika Gissler. There are no competing interests to be reported.

Major congenital anomalies in children born after frozen embryo transfer: a cohort study 1995-2006.

STUDY QUESTION: Is there a different risk for major congenital anomalies (CAs) in children born after frozen-thawed embryo transfer (FET) compared with children born after fresh embryo transfer (ET)? SUMMARY ANSWER: Children born after FET have a similar risk of developing major CAs as children born after fresh ET. WHAT IS KNOWN ALREADY: The perinatal outcome in children born after FET is as good as that after fresh ET. Children born as a result of assisted reproductive technology (ART) have an increased risk for CAs when compared with spontaneously conceived children, but the knowledge on the risk for CAs in specific organ systems of children born after FET is limited. STUDY DESIGN, SIZE, DURATION: This register-based cohort study includes women who have undergone ART treatments with ET leading to singleton births (n = 4772) between the years 1995 and 2006. The women were identified from the registers of the infertility clinics, and the corresponding births were matched with data from the Finnish Medical Birth Register (FMBR). The 10% random sample of women with spontaneous pregnancies from the FMBR served as the reference group (n = 31,243). The study data were linked with the Register of Congenital Malformations using the mothers' and children's personal identification numbers to get information on CAs. Furthermore, the personal identification numbers of the ART women were linked with the Register of Induced Abortions to find their selective terminations of pregnancy for severe foetal anomalies. PARTICIPANTS, SETTING, METHODS: The study was focused on singleton births and included 1830 children born after FET, 2942 children born after fresh ET and 31 243 children born after spontaneous pregnancies. Only major CAs were analysed in keeping with European Concerted Action on Congenital Anomalies and Twins. The risk estimates for CAs were adjusted for the children's year of birth and maternal age, parity and socioeconomic status. The total prevalence of major CAs was counted, including both births and selective terminations of pregnancy for major fetal anomalies (n = 33). MAIN RESULTS AND THE ROLE OF CHANCE: Among singletons at least one major CA was reported in 77 cases (4.2%) in the FET group, 132 cases (4.5%) in the fresh ET group and 994 cases (3.2%) in the reference group. The risk for at least one major CA of the children born after FET was not increased compared with the children born after fresh ET [adjusted odd ratio (aOR) 0.95; 0.71-1.27]. Furthermore, no increased risks according to the organ system affected were found between these two ART groups. When comparing the children born after ART (FET and fresh ET) with the reference group children, the risk of having at least one major CA was moderately increased in the ART group (aOR 1.24; 1.05-1.47). LIMITATIONS, REASONS FOR CAUTION: Because of the study design we were neither able to examine the aetiology of infertility nor could we compare the data with a group of subfertile women to account for the effect of infertility per se on CAs. WIDER IMPLICATIONS OF THE FINDINGS: Perinatal outcomes of FET children, including the risks for CAs, are good and comparable with outcomes of other ART children indicating that slow freezing is a safe method to use during ART treatments. STUDY FUNDING/COMPETING INTEREST(S): University Hospital of Oulu and Helsinki, Finland. THL covered the data linkages and the work of Annukka Ritvanen and Mika Gissler. There are no competing interests to be reported.

Distribution and epidemiology of genotypes of the crayfish plague agent Aphanomyces astaci from noble crayfish Astacus astacus in Finland.

The crayfish plague agent Aphanomyces astaci was isolated from 69 noble crayfish Astacus astacus samples in Finland between 1996 and 2006. All isolates were genotyped using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Altogether, 43 isolates belonged to the genotype group of Astacus strains (As), which is assumed to represent the genotype originally introduced into Europe around 1860 and into Finland in 1893. There were 26 crayfish plague isolates belonging to the group of Pacifastacus strain I (Ps1), which appeared in Europe after the stocking of the North American species signal crayfish Pacifastacus leniusculus. The geographical distribution of the 2 genotypes in Finland corresponded with the stocking strategies of signal crayfish. The majority of Ps1-strains (83%) were associated with a classical crayfish plague episode involving acute mortality, compared with only 33% of the As-strains. As-strains were found more often by searching for reasons for population declines or permanently weak populations, or through cage experiments in connection with reintroduction programmes. In some water bodies, isolations of the As-strains were made in successive years. This study shows that persistent crayfish plague infection is not uncommon in noble crayfish populations. The described epidemiological features suggest a difference in virulence between these 2 genotypes.

Perinatal outcome of children born after frozen and fresh embryo transfer: the Finnish cohort study 1995-2006.

BACKGROUND: The number of children born after frozen embryo transfer (FET) is steadily rising. However, studies on obstetric and perinatal outcomes are limited. Our primary aim was to compare the perinatal health of children born after FET and fresh embryo transfer, and to use data from children born after spontaneous conception as a reference. METHODS: In a register-based cohort study we evaluated the obstetric and perinatal outcomes of children born after FET (n = 2293), fresh embryo transfer (n = 4151) and those born after spontaneous pregnancy (reference group; n = 31 946). Data were collected from the registers of two infertility outpatient clinics, two university hospitals and the Finnish Medical Birth Register (1995-2006). RESULTS: After adjusting for confounding factors the FET group showed decreased risks of preterm birth [adjusted odd ratio (AOR) 0.83, 95% confidence interval (CI) 0.71-0.97], low birthweight (AOR 0.74; 0.62-0.88) and being small for gestational age (AOR 0.63; 0.49-0.83) compared with the fresh embryo transfer group. Mean birthweight was 134 g higher in the FET singletons versus the fresh embryo transfer singletons (P< 0.0001). When FET singletons were compared with the reference group, increased risks of preterm birth (AOR 1.45; 1.25-1.68) and low birthweight (AOR 1.22; 1.03-1.45) and a decreased risk of being small for gestational age (AOR 0.71; 0.54-0.92) were found. No excess of perinatal and infant mortality occurred between the groups. CONCLUSIONS: Embryo freezing does not adversely affect perinatal outcome in terms of prematurity, low birthweight and being small for gestational age versus the fresh embryo transfer and the outcome is similar or even better, particularly regarding fetal growth. Our study, which is one of the largest on FET pregnancies, provides further evidence on the safety of FET.

Comparison of Finnish meat inspection records and average daily gain for cattle herds differing in Mycoplasma bovis test-status.

Detecting Mycoplasma bovis on cattle farms represents a challenge in the absence of an outbreak or cases of M. bovis mastitis, yet identification of an infection is essential to control the spread of the disease successfully. The objectives of this study were: (1) to determine whether meat inspection records can aid identification of cattle farms supporting M. bovis infection, and (2) to compare the average daily weight gain estimated from carcass weight for cattle originating from farms differing in M. bovis test-status. Meat inspection records were collected from two abattoirs in 2015; 80 677 animals in total. All the dairy and mixed breed cows and bulls used for meat production were categorized according to known M. bovis infection status of the farms from which the cattle were derived; positive, contact or control farms. The associations between animals from different M. bovis categories and lung lesions of bulls and cows (pneumonia and pleuritis), identified during meat inspection, and estimated average daily gain (ADG) of bulls, were investigated. The odds ratios for lung lesions, especially pleuritis, were higher in M. bovis test-positive or contact farms compared with control farms. Additionally, odds ratios for pleuritis were higher among animals from M. bovis test-positive farms and animals from contact slaughtering farms originating from M. bovis-free rearing farms. Bulls originating from M. bovis test-positive farms had higher estimated average daily gain than cattle from control farms. Meat inspection records can be used alongside other methods to detect M. bovis-positive farms where M. bovis causes lung lesions.

Characterisation of the course of Mycoplasma bovis infection in naturally infected dairy herds.

Mycoplasma bovis causes bovine respiratory disease, mastitis, arthritis and otitis. The importance of M. bovis has escalated because of recent outbreaks and introductions into countries previously free of M. bovis. We characterized the course of M. bovis infection on 19 recently infected dairy farms over 24 months. Our objective was to identify diagnostic tools to assess the efficacy of control measures to assess low risk infection status on M. bovis infected farms. PCR assays and culture were used to detect M. bovis, and in-house and BioX ELISAs were used to follow antibody responses. Cows and young stock were sampled on four separate occasions, and clinical cases were sampled when they arose. On 17 farms, a few cases of clinical mastitis were detected, mostly within the first eight weeks after the index case. Antibodies detected by in-house ELISA persisted in the serum of cows at least for 1.5 years on all farms, regardless of the M. bovis infection status or signs of clinical disease or subclinical mastitis on the farm. Six out of 19 farms became low risk as the infection was resolved. Our results suggest that, for biosecurity purposes, regular monitoring should be conducted on herds by screening for M. bovis in samples from cows with clinical mastitis and calves with pneumonia, in conjunction with testing young stock by screening longitudinally collected nasal swabs for M. bovis and sequential serum samples for antibody against recombinant antigen.

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds.

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.

A quantitative risk assessment of the public health impact of the Finnish Salmonella control program for broilers.

In order to study the public health effects of the Finnish Salmonella control program (FSCP), a quantitative risk assessment model of Salmonella from slaughtered broiler flocks to consumers was developed. Based on the model, approximately 0.21% of domestically produced broiler meat mass was contaminated with Salmonella (95% probability interval 0.05-0.48%). This model was combined to the model on primary production of broilers. By this way, the effect of eliminating breeder flocks from production which have tested positive for Salmonella and heat-treating the meat of detected positive broiler flocks on public health could be simulated. Based on the whole model, if detected positive breeder flocks were not removed this would result in 1.0-2.5 more reported human cases compared to the expected number of cases under current FSCP (95% predictive interval). Without heat treatment of meat the increase would be 2.9-5.4-fold and without both interventions 3.8-9.0-fold. In scenarios with one grandparent or five parent flocks infected, the combined effect of these two interventions was 9.3-25.8-fold and 4.9-11.7-fold compared to the baseline level under each scenario, respectively. The scenario analyses suggest that with a higher infection level, inclusion of both interventions will be more effective than either of the interventions alone. Replacement of half of the current retail broiler meat by meat with 20-40% contamination could result in 33-93 times more human cases compared to the expected value under current situations. On the basis of the model, the interventions applied in FSCP clearly protect the public health.

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection.

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.

Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance.

Immunoblot analysis of bacterial polysaccharides: application to the type-specific polysaccharides of Streptococcus suis and Streptococcus agalactiae.

A method for the immunoblot analysis of the type-specific capsular polysaccharides of streptococci was developed. The capsular polysaccharides were extracted by sonication and subjected to polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulphate (SDS). After electrophoresis the polysaccharides were transferred to charged PVDF-N membranes and probed with the type specific antibodies. A characteristic ladder-like pattern of polysaccharide bands specific for the serotype (1, 2, 4, 7) was observed for capsular extracts of Streptococcus suis. Human immune sera against type-specific group B streptococcal polysaccharides reacted most strongly with the immunizing polysaccharide type (Ia, II, III). The previously observed crossreactions between the group B streptococcal type-specific capsular preparations were shown to be due to binding to the isomeric polysaccharide molecules. Thus, gel electrophoresis combined with immunoblot analysis seems to provide a novel method for the molecular and immunochemical characterization of bacterial polysaccharides and for the study of the specificity and properties of antibodies to capsular polysaccharides.

Virulence factors of Escherichia coli isolated from bovine clinical mastitis.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.

Eradication of endemic Brachyspira pilosicoli infection from a farrowing herd: a case report.

Brachyspira pilosicoli and B. innocens were isolated repeatedly from a herd of 60 sows which mostly produced feeder pigs but also raised some fattening pigs. Postweaning diarrhea had been a severe problem in this herd for years. The B. pilosicoli eradication plan was based on the general guidelines for elimination of B. hyodysenteriae, with some modifications. The eradication measures were run in August 1997. In-feed medication with 200 p.p.m. tiamulin lasted for 18-30 days, depending on the age group. The piggery unit was emptied, cleaned, disinfected and dried, and all worn surfaces were repaired. The animals were removed to temporary sheds situated 0-100 m from the piggery unit. Only the sows and the boar returned to the piggery unit. All other pigs were sold from the sheds within 3 months after the eradication. Immediately after the eradication, the clinical postweaning diarrhea disappeared. The success of the program was monitored four times bacteriologically, and the last control sampling was in December 1999, 7 months after the total withdrawal of antimicrobial feed additives. The primary cultures from the last three samplings were also analysed with B. pilosicoli-specific PCR. All the samples were negative for B. pilosicoli. However, B. innocens could be isolated from each batch of samples. The analysis of B. innocens isolates by pulsed-field gel electrophoresis indicated that at least one genotype persisted in the herd. The clinical and laboratory findings suggest that the eradication of B. pilosicoli had succeeded in this herd.

Vaccination influences growth of Arctic charr.

There is limited knowledge about the effects of oil-based vaccines on the growth of Arctic charr Salvelinus alpinus, in particular at different rearing temperatures. One-year-old Arctic charr were immunized intraperitoneally at 2.9 degrees C with a metabolizable oil-adjuvanted, bivalent vaccine containing killed typical and atypical Aeromonas salmonicida bacteria. After vaccination the non-vaccinated (controls) and vaccinated individually marked fish were held for 20 d at 10.0 degrees C and then for 7 wk at 10.3, 14.1 or 18.1 degrees C. During the first 20 d at 10.0 degrees C the growth rate (G) was higher for non-vaccinated than vaccinated fish. Thereafter vaccinated charr had higher G than control fish at 10.3 and 14.1 degrees C. In contrast, at 18.1 degrees C there was no difference in G and therefore no compensation of earlier growth suppression in vaccinated fish was observed at that temperature. The study indicates that vaccination has no ultimate negative effects on the growth of Arctic charr at temperatures ranging from 10.3 to 14.1 degrees C.

Temperature effect on the immune defense functions of Arctic charr Salvelinus alpinus.

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.

Suicide among military conscripts in Finland: a psychological autopsy study.

This study describes suicides during compulsory military service and compares them with other young male suicides as part of a nationwide psychological autopsy study of all suicides in Finland during a single year. The victims' relatives and attending health care personnel were interviewed, and data from records were collected. Five of the seven conscript suicides had a depressive syndrome, often of short duration. Two victims previously had suicidal thoughts or attempted suicide. The most common suicide method was with firearms. Compared with other young male suicides, physical illness in conjunction with a psychiatric diagnosis was less common in the conscript victims. Civilian male suicides tended to have more substance abuse and more severe psychological impairment. They also committed suicide under the influence of alcohol more often than conscripts. Interpersonal separation was more often the precipitating event of the suicide among the conscripts than among the other suicides. Conscripts should be encouraged to utilize health and social services when they have psychosocial problems or even minor psychiatric symptoms.