Objective data on DNA success rates can aid the selection process of crime samples for analysis by rapid mobile DNA technologies.

Mobile Rapid-DNA devices have recently become available on the market. These devices can perform DNA analyses within 90min with an easy 'sample in-answer out' system, with the option of performing comparisons with a DNA database or reference profile. However, these fast mobile systems cannot yet compete with the sensitivity of the standard laboratory analysis. For the future this implies that Scene of Crime Officers (SoCOs) need to decide on whether to analyse a crime sample with a Rapid-DNA device and to get results within 2h or to secure and analyse the sample at the laboratory with a much longer throughput time but with higher sensitivity. This study provides SoCOs with evidence-based information on DNA success rates, which can improve their decisions at the crime scene on whether or not to use a Rapid-DNA device. Crime samples with a high success rate in the laboratory will also have the highest potential for Rapid-DNA analysis. These include samples from e.g. headwear, cigarette ends, articles of clothing, bloodstains, and drinking items.

Escherichia coli-nitroreductase suicide gene control of human telomerase reverse transcriptase-transduced minor histocompatibility antigen-specific cytotoxic T cells.

Adoptive immunotherapy with ex vivo generated cytotoxic T lymphocytes (CTLs) is applied for the treatment of leukemia relapses or viral infections after allogeneic stem cell transplantation. A common problem of adoptive immunotherapy strategies is the ex vivo expansion of the generated T cells to sufficient numbers. CTLs can be efficiently expanded by ectopic expression of the human telomerase gene (hTert). However, hTert transduction may also increase the chance for malignant transformation. Therefore, we explored the feasibility of suicide gene control of ex vivo generated CTLs expanded through the ectopic expression of hTert. To this end, we compared the efficacy of the new Escherichia coli-nitroreductase (E. coli-Ntr) suicide gene with the well-known herpes simplex virus-thymidine kinase (HSV-Tk). Introduction of hTert provided the transduced CTLs with a distinct growth advantage over the nontransduced CTLs. The hTert-E. coli-Ntr double-transduced CTLs retained their antigen-specific functions. Treatment of hTert-E. coli-Ntr double-transduced CTLs with metronidazole significantly inhibited the proliferation to a similar extent to the treatment of hTert-HSV-Tk double-transduced CTLs with ganciclovir. This is the first application of the E. coli-nitroreductase gene for the elimination of human T cells with metronidazole.

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories. Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male). All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data. As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland. Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.

Results of collaborative study regarding the standardization of the Y-linked STR system DYS385 by the European DNA Profiling (EDNAP) group.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control sample. Furthermore, population data from eight different European countries with samples sizes between 91 and 150 male individuals were collected. The results confirm previous observations that DYS385 is one of the most informative Y-linked STR loci. It could also be demonstrated that reproducible results can be obtained independently from the electrophoretic separation and detection methods used. Thus DYS385 may serve as a useful complementation to the routinely used autosomal STR systems in special cases.

On the consequences of DNA profile mismatches for close relatives of an excluded suspect.

If the DNA profiles of a crime stain and the reference sample from the suspect do not match, the suspect is excluded as the donor of the crime stain. However, in some situations the DNA evidence can suggest that a close relative of the suspect might match the stain, in particular when the reference sample from the suspect and the crime stain share rare alleles. This finding can be important for the authorities. The forensic scientist has to decide whether or not to notify the authorities in these circumstances. To the best of our knowledge there is not yet an objective rule for making this decision. We propose such a decision rule for brothers of the suspect, investigate its performance and address some ethical, legal, and practical aspects. Our calculations can be simply adjusted for other relatives of the suspect.

Report of the European DNA Profiling Group (EDNAP)--an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

Low molecular weight Cooperia oncophora antigens. Potential to discriminate between susceptible and resistant calves after infection.

The recognition of low molecular weight proteins by sera obtained during a single oral (primary) infection with 100,000 3rd-stage Cooperia oncophora larvae was studied in calves. Three groups of 6 or 7 calves were selected based on different egg excretion patterns. SDS-gel electrophoresis of adult Cooperia antigen under reducing conditions, followed by Western blotting, revealed that resistance of individual calves to C. oncophora might be related with antibody responses (42 days post infection) against at least 2 protein fragments (14-16 kDa and 27 kDa). The 14-16-kDa protein complex was bound, to some extent, by individual sera from all calves. The intensity of staining was negatively correlated with egg excretion on Day 42 p.i. Calves with high egg counts on Day 21 p.i. either did not or only weakly recognized the 27-kDa band. It has to be established whether the 14-16 kDa (or recombinant 14.2 kDa) provides a tool for immunodiagnostics and whether the 27-kDa fragment can help further unravel immune-mediated resistance to Cooperia.

Report of the European DNA profiling group (EDNAP): an investigation of the complex STR loci D21S11 and HUMFIBRA (FGA).

This paper describes a collaborative exercise which was intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using two complex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosen because they are commonly used by different European laboratories. D21S11 has approximately 14 common alleles (f > 0.001), whereas HUMFIBRA has 19 common alleles. Laboratories were asked to test seven blood stains, one of which was a known control, and to report the results to the coordinating laboratory. The exercise demonstrated that complex STRs were amenable to standardisation.

A Dutch population study of the STR loci D21S11 and HUMFIBRA.

To introduce a duplex PCR system consisting of the STR loci D21S11 and HUMFIBRA in forensic identity testing we analysed a Dutch Caucasian database of 205 individuals. The combined power of discrimination of the two loci is 0.9978 and there was no evidence for linkage equilibrium between the two loci (p = 0.91). However, we noticed departure from Hardy-Weinberg equilibrium for the D21S11-locus in our database (p = 0.03), but the differences between observed and expected D21S11 allele pair frequencies were of negligible practical significance in forensic calculations.

Production of dairy replacement stock in relation to level of exposure to gastrointestinal nematode infection in the first grazing season: second-year calves and heifers.

In two experiments, groups of calves which had been exposed to different levels and patterns of infection with Ostertagia and Cooperia spp. in a simulated first grazing season (FGS), were followed throughout a natural second grazing season (SGS). Milk yields in the subsequent first lactation period were also recorded. The results suggest that although there had been differences in immune status among groups, which had been infected in the FGS, prior to the SGS, weight gain among these groups was not significantly different during the SGS. Apparently, resistance to the pathogenic effects of reinfection had developed more strongly and at lower levels of exposure to infection than resistance against establishment of larvae as shown after an experimental challenge. The groups of calves not infected during the FGS did gain less than all other groups during the SGS. Further, infection-induced differences in weight gain among the infected groups in the FGS appeared to be permanent, at least up to the end of the SGS. Finally, first lactation yield was positively correlated with body weight at calving. On average, approximately 10 kg less milk was produced for each kg of lower body weight at calving. With respect to the implications for preventive control strategies in the FGS, it is suggested that parasite control should not be applied beyond a level at which weight gain reduction is prevented.

The value of bulk milk ELISA Ostertagia antibody titres as indicators of milk production response to anthelmintic treatment in the dry period.

An experiment was done to study whether estimation of the herd infection level, by assessing Ostertagia antibodies in bulk milk samples can serve to predict the effect of anthelmintic treatment on milk production. Bulk milk samples were collected from 134 farms at monthly intervals on three occasions prior to the start of the study. The ELISA titres to Ostertagia found in October at the end of the grazing season served as a basis to select 16 and 18 farms as having high and low levels of parasitism respectively. Heifers and cows within each herd were ranked by expected calving date, paired and randomly allocated to be treated with ivermectin or a placebo. Records of milk production and composition were collected for all the trial animals. The response to treatment expressed as the 305 day corrected milk yield of anthelmintic treated animals minus that of placebo treated animals was not statistically significant and amounted to 78 kg for multiparous cows and 124 kg for heifers. For cows as well as heifers the response to treatment was larger in the high antibody level herds than in low antibody level herds, but these differences also lacked statistical significance.

Report on the third EDNAP collaborative STR exercise. European DNA Profiling Group.

This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designated to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, the laboratories were also requested to report the results obtained from any other loci which would normally be used in crime case investigations. A comparison of these results showed some inter-laboratory variation.

Residual effect of injectable moxidectin against lungworm and gastrointestinal nematodes in calves exposed to high pasture infectivity levels in the Netherlands.

The residual effect of a 0.2 mg kg-1 injectable formulation of moxidectin against lungworm and gastrointestinal nematodes in cattle was studied in a grazing experiment in the Netherlands. Five groups of four calves were grazed between May and October 1991 and one similar group was used as permanently housed control group for the evaluation of the development of immunity against lungworm by challenge infections with 5000 larvae of all six groups. The main parameter used to determine the residual effect for lungworm was faecal larval counts. Additional information was derived from pasture larval counts, enzyme-linked immunosorbent assay (ELISA), respiration frequency, coughing score and, particularly for evaluating development of immunity, worm counts. For gastrointestinal nematode infections faecal egg counts and larval differentiation of faecal cultures were the main parameters used. Pasture larval counts and an ELISA for Ostertagia and Cooperia were used as additional parameters. In three treated groups lungworm larvae (re)appeared in the faeces after 67, 95 and 119 days, respectively. This implies that a 100% residual effect did not last longer than 67-21 = 46 days. The treated group with patency starting on Day 95 was exposed to extremely high infection pressure and the ELISA indicated some host-parasite interactions from 2-4 weeks after treatment. Thus some interaction between moxidectin treatment and high infection pressure delayed the onset of patency in comparison to another treated group under much lower infection pressure. In all treated groups, including the one under high infection pressure, lungworm disease was prevented and the worm counts demonstrated development of immunity. In contrast, severe lungworm disease occurred in two control groups grazing together with the 'high infection pressure' treated group. The faecal egg counts and differentiation of larvae from faecal cultures demonstrated a 100% residual effect of at least 3 weeks and indicated a high residual effect of approximately 5 weeks against Ostertagia. Moxidectin suppressed Cooperia faecal egg counts for over 98% and the results indicated a more than 95% residual effect on faecal egg output during 2-3 weeks. The ELISA results were indicative for a delay of 2 weeks in the acquisition of gastrointestinal nematode infections following moxidectin treatment.

Low molecular weight Cooperia oncophora antigens: characterization and humoral immune responses in calves mono-infected with 100,000 infective larvae.

Characteristics of the humoral immune response of Cooperia oncophora-infected calves to low molecular weight antigens of C. oncophora were studied. Immunoblotting with sera obtained from calves 6 weeks after a single oral infection with 100,000 third-stage (L3) C. oncophora larvae revealed several corresponding antigenic fragments between adult worms and the fourth-stage (L4) larvae. No reactivity in the immune sera was found against the L3 stage. A previously defined complex of low molecular weight proteins (12-15 kDa) was found on both L4 and adult Cooperia stages, but not on the L3 stage. C. oncophora adults differed from the L4 larvae at the 31/32 and 37 kDa level. Several adult and L4 proteins were bound by biotinylated Concanavalin A, as was also true for L3 proteins. A 31/32 kDa glycoprotein of adult worms was recognised by a monoclonal antibody with specificity for phosphorylcholine. Using monoclonal antibodies in ELISA and Western blotting, the serum antibody response of C. oncophora-infected calves to adult worm antigen was almost entirely IgG1. Binding of the IgG1 antibodies was restricted to a complex of reduced 12-15 kDa protein(s) and a 27 kDa fragment of adult worms. The data suggest that the systemic humoral immune response of calves during a primary infection with C. oncophora consists mainly of an IgG1 response, and is directed to a non-glycosylated Cooperia protein (molecular weight estimated at 12-15 kDa under reducing conditions and 18 kDa under nonreducing conditions). This protein is probably present in both L4 larvae and adults. Since it was not bound by immune sera from calves mono-infected with several other nematodes, the 12-15 kDa protein complex may represent a Cooperia-specific component that can be used for serodiagnosis.

Systemic antibody responses of calves to low molecular weight Cooperia oncophora antigens.

The systemic antibody responses to adult Cooperia oncophora antigen were studied using sera obtained from calves during a 6-week period following a single oral infection with either 20,000 or 100,000 third-stage C. oncophora larvae. Dose dependent increasing titres of IgG binding complete adult Cooperia antigen were found in the sera of Cooperia-infected calves. SDS-gel electrophoresis under reducing conditions, followed by Western blotting, revealed that the increase of IgG binding Cooperia antigens could be attributed mainly to specific binding of IgG to a complex of 12-15 kDa protein fragments of Cooperia adult antigen. This protein may represent a Cooperia oncophora-specific component that can be used for serodiagnosis.

Acquired immunity against Cooperia spp. and Ostertagia spp. in calves: effect of level of exposure and timing of the midsummer increase.

In two experiments, groups of calves were exposed to different levels and patterns of infection with Ostertagia spp. and Cooperia spp. The experimental design simulated the stereotypic pattern of herbage infestation, including both as normal and a delayed midsummer increase, under conditions of set-stocking. After this simulated 'first grazing season', calves were followed during the subsequent winter housing. At the end of that housing period some calves were challenged with 100,000 L3 Cooperia spp. and 40,000 L3 Ostertagia spp. and slaughtered 23 days later. All previously infected calves were protected against the establishment of the challenge infection with Cooperia spp., but not against Ostertagia spp. For the latter a significant negative correlation was found between worm count and previous level of exposure to infection. During the simulated first grazing season, changes in the ratio of Cooperia to Ostertagia eggs in the faecal egg output and the genus-specific egg count were influenced by both the level of exposure and the timing of the midsummer increase. It is concluded that acquired immunity against both parasite genera develops depending on the level of exposure to infection during a first grazing season, and that delaying the midsummer increase results in a delay of the acquisition of an effective immunity as measured by faecal egg counts and the ratio of Cooperia to Ostertagia egg output.

Weight gain and the course of some estimators of gastrointestinal nematode infection in calves during winter housing in relation to the level of exposure during the previous grazing season.

In two experiments groups of calves were exposed to different levels and patterns of infection with Ostertagia spp. and Cooperia spp. The experimental design simulated the stereotypic pattern of herbage infestation, including a normal or a delayed midsummer increase, under conditions of set-stocking. After this simulated 'first grazing season', calves were monitored throughout the subsequent winter housing period. No continuing negative effects of previous infection on growth performance were observed. Calves in all groups gained on average over 0.7 kg day-1, irrespective of previous level of exposure. Differences between the experiments with respect to either level or pattern of infection during the preceding 'first grazing season' were all, to a greater or lesser extent, reflected in faecal egg counts, pepsinogen values, gastrin values and antibody titres against Cooperia spp. or Ostertagia spp. Depending on the time of sampling, pepsinogen values and antibody titres against Ostertagia spp. particularly were useful variables for assessing differences in levels of infection to which groups of calves had been exposed.

Report on the second EDNAP collaborative STR exercise. European DNA Profiling Group.

The European DNA Profiling Group (EDNAP) has previously carried out collaborative exercises to determine which STR systems will produce results that can be reproduced by different laboratories. The first EDNAP exercise involving STR systems focused on different types of loci: a simple locus with six common alleles (HUMTH01) and a complex locus with > 35 alleles (ACTBP2). Generally the simpler STR system was found to be readily amenable for use across a wide range of different technologies, whereas a more complex locus presented difficulties. The second EDNAP STR exercise was intended to take the process of investigation a stage further. Some laboratories are developing automation, coupled with fluorescent methods of detection and multiplex applications, whereas others use manual methods involving visual detection techniques such as silver staining. The purpose of this exercise was to determine whether loci amenable to multiplexing with automation (as a quadruplex reaction) could also be successfully used with manual methods, either by multiplexing in duplex reactions or alternatively by using just a single pair of PCR primers.

A Dutch population study of the STR Loci HUMTHO1, HUMFES/FPS, HUMVWA31/1 and HUMF13A1, conducted for forensic purposes.

We report on a Dutch population study of the STR loci HUMTHO1, HUMFES/FPS, HUMVWA31/1, and HUMF13A1, in which we used multiplex amplification and automated fragment detection. Genotype and allele frequencies showed no deviation from Hardy-Weinberg and linkage equilibrium. The improved Bonferroni procedure was used to combine the results of several tests. The power of discrimination of a complete profile exceeded 0.9998. We compared the allele frequencies in the Dutch sample to the frequencies in other populations using a bipilot to visualize alleles and populations simultaneously. The Dutch sample was similar to most other Caucasian samples. The data demonstrate that the genetic systems in this report are a valuable tool for forensic identity testing in The Netherlands.