Attempt to detect evidence for tick-borne encephalitis virus in ticks and mammalian wildlife in The Netherlands.

To investigate if tick-borne encephalitis virus (TBEV) is present in mammalian wildlife species or ticks in The Netherlands, serum samples and ticks were tested for TBEV antibodies and TBEV RNA, respectively. Serum samples were collected from wild boar (666), deer (13), fox (399), and rodents (90), and were tested for TBEV antibodies, using ELISA, and SN test or HI test. Over a period of 4 years, a total of 906 ticks was collected from seven regions in The Netherlands. In four different regions, this was done on a monthly basis and during four consecutive summers. All ticks were tested for TBEV RNA by RT-PCR. TBEV antibody was detected by ELISA in two (0.5%) sera of foxes and 49 (7%) sera of wild boar, but not confirmed by HI or SNT. TBEV RNA was not detected in any of 906 ticks. It was concluded that there is no real evidence for a TBEV reservoir in ticks or wildlife in The Netherlands.

Molecular characterizations of human and animal group a rotaviruses in the Netherlands.

To gain more insight into interspecies transmission of rotavirus group A, human and animal fecal samples were collected between 1997 and 2001 in The Netherlands. A total of 110 human stool samples were successfully P and G genotyped by reverse transcriptase PCR. All strains belonged to the main human rotavirus genotypes G1 to G4, G9, [P4], [P6], [P8], and [P9]. [P8]G1 was predominant, and 5.5% belonged to the G9 genotype. Eleven percent of all P[8] genotypes could be genotyped only by a recently published modified primer. Rotavirus-positive fecal samples from 28 calf herds were genotyped by DNA sequencing. Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus-positive calf herds, respectively. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found. Genotype G6 bovine rotaviruses were divided into three clusters: UK-like, VMRI-29-like, and Hun4-like. DNA sequencing of a part of the VP7 gene was shown to be useful as a quick determination of uncommon or novel strains of which the genotyping cannot be done by genotyping PCR. Of equine strains, both VP4 and VP7 genes could be used for genotyping: two [P12]G3 and four [P12]G14 equine rotaviruses were determined. We did not find indications for rotavirus interspecies transmissions, although the recently published human G6-Hun4 is genetically related to our G6 bovine isolates. All bovine, porcine, and equine rotaviruses were within genotypes previously reported for these animal species.

Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands.

Hepatitis E virus (HEV), a major cause of viral hepatitis in much of the developing world, has recently been detected in swine in North America and Asia, raising concern about potential for zoonotic transmission. To investigate if HEV is commonly present in swine in the Netherlands, pooled stool samples from 115 swine farms and nine individual pigs with diarrhea were assayed by reverse transcription-polymerase chain reaction (RT-PCR) amplification. HEV RNA was detected by RT-PCR and hybridization in 25 (22%) of the pooled specimens, but in none of the individual samples. RT-PCR amplification products of open reading frames 1 and 2 were sequenced, and the results were compared with published sequences of HEV genotypes from humans and swine. HEV strains from swine in the Netherlands were clustered in at least two groups, together with European and American isolates from swine and humans. Our data show that HEV in swine in the Netherlands are genetically closely related to HEV isolates from humans. Although zoonotic transmission has not been proven, these findings suggest that swine may be reservoir hosts of HEV.

Characterisation of a recently isolated lyssavirus in frugivorous zoo bats.

In July 1997 a lyssavirus was isolated in Denmark from a colony of Egyptian flying foxes (Rousettus aegyptiacus) originating from a Dutch zoo. Sequencing of a 400 nucleotides coding region of the nucleoprotein and of a major part of the G-protein ectodomain encoding region of the newly isolated virus, revealed a very high similarity with European Bat Lyssavirus subtype 1a (EBL-1a). For characterisation of the recently isolated lyssavirus in frugivorous zoo bats, 16 frugivorous bats (Rousettus aegyptiacus) of the same colony and 80 mice were experimentally infected with the Rousettus isolate or with a well defined EBL-1a strain isolated from a Dutch insectivorous bat (Eptesicus serotinus). Inoculation viruses were titrated in mice to determine LD50's of both isolates. Clinical signs of inoculated bats were recorded during 6 weeks. After showing neurological signs or at the end of the experimental infection all animals were euthanized. During the experimental infection sera and various tissues of inoculated bats were collected. Immunoassays, mouse inoculation tests (MIT) and polymerase chain reaction (PCR) were employed for detection of lyssavirus specific antibodies, antigen or RNA. Five bats inoculated with the Rousettus isolate and 2 bats inoculated with the Eptesicus isolate showed neurological signs. The remaining 9 bats survived and cleared the virus; at least under the detection limit of the used assays. Despite a much higher pathogenicity of the Rousettus isolate observed in mice, LD25's in bats were quite the same for the 2 isolates. The pathogenicity of both isolates suggested that like many other mammals, Rousettus aegyptiacus bats could be victims of lyssavirus infection besides reservoir hosts of infectious EBL1a. There was no significant difference in detecting the different lyssavirus isolates in Rousettus aegyptiacus bats. An employed immunoperoxidase staining (IP) method was very useful for sensitive detection and localization of lyssavirus antigen in histologic preparates.

Norwalk-like calicivirus genes in farm animals.

Viruses closely related to Norwalk-like viruses (NLVs) were recently found in stored stool samples from two calves (United Kingdom and Germany) and four pigs (Japan), sparking discussions about the potential for zoonotic transmission. To investigate if NLVs are commonly present in farm animals, pooled stool samples from 100 pig farms, 48 chicken farms, 43 dairy cow herds, and 75 veal calf farms from the Netherlands were assayed by reverse transcription- polymerase chain reaction amplification, using primers specific for the detection of NLVs from humans. NLV RNA was detected in 33 (44%) of the specimens from veal calf farms and two (2%) specimens from pig farms. Our data show that NLV infections until recently thought to be restricted to humans occur often in calves and sometimes in pigs. While zoonotic transmission has not been proven, these findings suggest that calves and pigs may be reservoir hosts of NLVs.

Molecular detection and epidemiology of Sapporo-like viruses.

Sapporo-like viruses (SLVs) are associated with acute gastroenteritis in humans. Due to a limited supply of available reagents for diagnosis, little is known about the incidence and pathogenicity of these viruses. We have developed a first-generation generic reverse transcriptase (RT) PCR assay based on a single primer pair targeting the RNA polymerase gene. With this assay, 55 (93%) of the 59 stool specimens collected in a 10-year period of time (1988 to 1998) and containing typical caliciviruses by electron microscopy tested positive and could be confirmed by Southern hybridization. By phylogenetic analysis, most SLV strains could be classified into one of the three recently described genotypes. However, three samples clustered separately, forming a potential new genotype. We sequenced the complete capsid gene of one of the strains in this cluster: Hu/SLV/Stockholm/97/SE. Alignment of the capsid sequences showed 40 to 74% amino acid identity among strains of the different clusters. Phylogenetic analysis of the aligned sequences confirmed the placing of Hu/SLV/Stockholm/97/SE into a new distinct genetic cluster. This is the first report on the development of a broadly reactive RT-PCR assay for the detection of SLVs.

Isolated systolic hypertension in Dutch middle aged and all-cause mortality: a 25-year prospective study.

In the early 1950s, the blood pressure of 3901 Dutch civil servants and their spouses aged 40-65 years was measured in a general health survey. Isolated systolic hypertension (systolic pressure greater than 160 mmHg, diastolic pressure less than 90 mmHg) was observed in 6.3% of the women and 3.0% of the men. The prevalence increased with age and it was more common in women in all age groups. Using logistic regression, with adjustment for potential confounders (age, smoking, serum cholesterol, Quetelet index, alcohol consumption, haemoglobin level, pulse rate and diastolic blood pressure) the association of 15- and 25-year total mortality with isolated systolic hypertension was determined. Compared to normotensive people (systolic pressure less than or equal to 135 mmHg, diastolic pressure less than 90 mmHg), the risk of death from all causes was significantly higher for men with isolated systolic hypertension after 15 and 25 years of follow-up (odds ratio OR = 2.4, 95% confidence interval (CI) 1.2-4.8 and OR = 3.2, 95% CI 1.3-8.0). For women 15-years mortality risk was strongly associated with isolated systolic hypertension (OR = 3.7, 95% CI 1.4-9.7). The increased risk was less pronounced after 25 years of follow-up (OR = 1.7, 95% CI 0.96-3.0). Our results support those of other studies and indicate that isolated systolic hypertension is an important independent risk factor for all-cause mortality. Since isolated systolic hypertension may be an indicator for the early onset of ageing, it is important to study its determinants and to pay more attention to its diagnosis and treatment in middle-aged populations.

Scoring of prudent dietary habits and its relation to 25-year survival.

To evaluate the effect on longevity of a diet that is concurrent with common dietary guidelines, a simple diet scoring system was developed and applied in a follow-up study of 2,820 middle-aged Dutch civil servants and their spouses. In the early 1950s those civil servants were seen for a health examination that included a dietary survey. Consumption frequency data of the quantitatively most important food items at that time were used for the diet scoring. Overall survival after 25 years was 46.8% among men and 68.6% among women. In men, a significant positive association between prudent diet score and 25-year, age-adjusted survival could be demonstrated. Of the 10 food items that constituted the diet score, a higher intake of brown bread, porridge and/or yogurt, vegetables, fish, and fruit was associated with a slightly better survival. In a separate analysis we had found a significant inverse relationship between coffee consumption and survival. A similar trend, which, however, was not significant, was observed for alcohol intake. In women, the results for the separate food items were inconsistent, and no effect of a prudent diet score on longevity was observed. The proposed diet scoring system provides a means for evaluating the effects of the individual's food choice behavior on subsequent health and longevity.

Dietary sodium, calcium, and potassium, and blood pressure.

The cross-sectional association of systolic blood pressure with dietary sodium, calcium, and potassium, as estimated from dietary histories, was investigated by multiple regression analysis of data gathered in the Netherlands in the early 1950s in a general health examination of 2,291 middle-aged civil servants and spouses of civil servants. A statistically significant negative trend with systolic blood pressure was seen for calcium intake in both males and females, even after adjustments for covariates. For sodium and potassium intake, the observed negative trends were not significant after multivariate analyses. In addition, no consistent associations were found between diastolic blood pressure and the micronutrients after multivariate analyses, except for a significant negative association with calcium intake in females. In this study population, blood pressure was a strong independent risk factor of total mortality: 15- and 25-year mortality was about twice as high for hypertensives (greater than or equal to 160 mmHg) as for normotensives (less than 160 mmHg). These findings support the conclusion in recent epidemiologic studies that higher intakes of calcium are associated with lower systolic blood pressure, and they extend the evidence to an earlier time period.

Retinopathy as an independent indicator of all-causes mortality.

The association between retinopathy and mortality in hypertensive patients is well established. At the same level of blood pressure the prognosis gets worse with increasing seriousness of retinopathy. To assess the prognostic value of retinopathy for all-causes mortality, we studied the data from a general health survey of 1583 men and 1508 women after 15 and 25 years of follow up. Among the men a clear association was found in the first 15 years. Among the women a weaker association was found only after 25 years of follow-up. This association was independent of hypertension or other cardiovascular risk indicators, such as obesity, serum cholesterol, smoking or proteinuria.

Weight, smoking, and mortality.

A controversy has existed concerning the relationship between body habitus and mortality. Original studies found that overweight persons were at risk for increased mortality, but later studies suggested that the very lean were also at high risk. The existence of a U-shaped relationship between mortality and body mass index was confirmed in a reanalysis of a 25-year follow-up of 1,503 men and 1,464 women, initially aged 40 to 65 years. Cigarette smoking did not explain the higher mortality among persons in the lowest body mass index category.

Parental survival, an independent predictor of longevity in middle-aged persons.

In a 25-year follow-up of 2370 middle-aged civil servants and spouses of civil servants, the authors studied the effect of the number of parents (none, one, or both) which a middle-aged person still has alive on this person's survival. Among men, no relationship was found with the number of parents alive after 15 years of follow-up and a weak gradient was found after 25 years. Among women, there was a clear gradient of mortality according to the number of parents alive, both after 15 and after 25 years. In a logistic regression analysis of mortality, after 25 years, the age-adjusted effect of having both parents alive relative to none was significant in men and women (for men: odds ratio = 0.63, 95% confidence interval = 0.42-0.97; for women: odds ratio = 0.36, 95% confidence interval = 0.19-0.69). These estimates did not change materially upon inclusion of other determinants for chronic disease and death into the model (smoking, blood pressure, serum cholesterol, and body mass index). The authors concluded that the parental effect was independent of these risk factors.