A ring trial to harmonize Toxoplasma gondii microsatellite typing: comparative analysis of results and recommendations for optimization.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

First Case of Idiopathic Eosinophilic Meningoencephalitis in a Sheep.

A 1.5-year-old ewe was presented with neurological signs that had been observed from about 2 days prior to death. There had been no clinical response to anti-inflammatory and antibiotic treatment. Histopathological examination of the brain revealed a severe and widespread eosinophilic meningoencephalomyelitis of unknown aetiology. Defining histological features included diffuse angiocentric eosinophilic infiltrates in the neuroparenchyma and meninges, neuronal necrosis, astrocytosis, neuropil vacuolation and occasional glial scars. Differential diagnostics for eosinophilic meningoencephalitis were taken into account and investigated by means of special stains, immunohistochemistry, bacteriology and polymerase chain reaction. No pathological changes or ancillary tests were supportive or revealed a specific aetiology for the condition and therefore it was considered idiopathic. Idiopathic meningoencephalitis is a rare disease, mainly described in man and rarely in dogs, with no apparent aetiological cause or potential breed predisposition. To our knowledge this is the first case of idiopathic eosinophilic meningoencephalitis in a sheep and provides a histopathological guideline for prospective comparative pathology studies.

Prevalence and Genetic Diversity of Toxoplasma gondii in Free-Ranging Chickens from the Caribbean.

PURPOSE: Toxoplasma gondii is a zoonotic parasite capable of infecting a wide range of hosts. Free-range chickens are important sentinels in the epidemiology of this parasite as they feed from the ground and are likely to ingest oocysts shed in the faeces of infected cats. Atypical strains of T. gondii are known to dominate in South America where they are associated with more severe disease in humans, yet relatively little is known about the strains circulating in neighbouring Caribbean islands. METHODS: In this study, hearts and brains were collected from free-range chickens in Antigua and Barbuda (n = 45), Dominica (n = 76) and Trinidad (n = 41), and DNA was extracted for nested ITS1 PCR and PCR-RFLP. Sera were collected and screened for antibodies using the modified agglutination test (MAT). RESULTS: Antibodies to T. gondii were detected in 20.5, 38.2 and 17.1% of chickens in Antigua and Barbuda, Dominica and Trinidad, respectively. Toxoplasma gondii DNA was also detected by PCR in 24.4, 17.1 and 17.1% of chickens, respectively, giving an overall prevalence of 31.1, 42.1, and 29.3% for each of the 3 island nations. Results of PCR-RFLP revealed 2 new atypical genotypes (designated ToxoDB #281 and #282) and one Type III (ToxoDB #2) in chickens from Antigua. Partial genotyping of a further 8 isolates (7 from Antigua and one from Trinidad) revealed different allele-types at five or more markers for 7 of the isolates, suggesting atypical genotypes. CONCLUSIONS: This is the first study to report the prevalence of T. gondii in free-range chickens in Antigua and Barbuda, Dominica and Trinidad and Tobago. It is also the first to report the presence of atypical genotypes in Antigua and Barbuda and Trinidad and Tobago.

Cytokine expression in the brains of Toxocara canis-infected mice.

Toxocara canis can infect a number of hosts including mice and humans. In the murine host, larvae exhibit a predilection for the central nervous system, resulting in an increasing number of parasites migrating to the brain as infection progresses. Previous studies have shown that larval burdens vary between individual outbred mice receiving the same inocula, suggesting a role for immunity in the establishment of cerebral infection. Although the systemic immune response to T. canis has been widely reported, there has been no investigation of the cerebral immune response. The aim of the present study was to characterize the cerebral immune response in two inbred strains of T. canis-infected mice (BALB/c and NIH) at several time points post-infection (p.i.). Relative quantification of gene expression in the brains of these mice showed increased expression of IL-5, IL-10, IFN-gamma and inducible nitric oxide synthase (iNOS). This response was detected as early as 3 days p.i., persisting up to 97 days p.i., and was more pronounced in BALB/c-infected mice. These results have implications for the role of these cytokines and iNOS in the cerebral establishment of T. canis, and in the cerebral pathology reported during infection.

Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

A binary-BAC system for plant transformation with high-molecular-weight DNA.

A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E. coli and A. tumefaciens. In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A. tumefaciens virulence genes virG and virE were constructed. These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A. tumefaciens host. This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.

Multichannel plating unit for high-throughput plating of cell cultures.

High-throughput genomic approaches to gene function or target identification have led to the development and implementation of the 96-well format for many standard molecular biology manipulations. The apparatus described here, a Multichannel Plating Unit, is designed to plate out individual cultures efficientlyfrom standard 96-well culture blocks. Following transformation, aliquots of culture are loaded onto sterile beads that are rolled along individual channels of agar media. After the beads traverse the channel, they drop into the exit alley for disposal via an exit pore. The apparatus presented has 12 individual lanes, and the spacing is compatible with a standard 12-channel pipettor Thus, the unit allows for the rapid plating of 12 individual cultures at a time. For one 96-well block of transformants, this method reduces the labeling and plating effort from 96 culture dishes that are spread individually to eight multichannel plates. The savings in time, materials, and storage space is significant

Evaluation of positive controls for the in vitro unscheduled DNA synthesis assay using hepatocytes from induced (Aroclor 1254) and uninduced male cynomolgus monkey.

We have evaluated the use of four different positive control compounds for assessing UDS in monkey hepatocytes and have found three of these, methylmethanesulfonate, benzo[a]pyrene, and dimethylbenz[a]anthracene, to produce strong positive responses in vitro. Dimethylnitrosamine induced only weak responses. We also report that the strength of the response induced by procarcinogens was not enhanced in hepatocytes taken from Aroclor 1254-pretreated monkeys, even though substantial induction of cytochrome P450 enzymes was demonstrated in these cells. These studies raise the question of the utility of employing an in vivo induction system to enhance the monkey UDS assay.

Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds.

The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with 3H-thymidine following in vivo treatment by gavage. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). Controls from both sexes of both species yielded less than 0.0 NG. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. 11-Aminoundecanoic acid, benzyl acetate, bis(2-chloro-1-methylethyl)ether (BCMEE), C.I. Solvent Yellow 14, cinnamaldehyde, cinnamyl anthranilate, dichloromethane, dichlorvos, glutaraldehyde, 4,4'-methylenedianiline (MDA), 4-nitrotoluene, 4,4'-oxydianiline, a polybrominated biphenyl mixture (PBB), reserpine, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, trichloroethylene, and 2,6-xylidine all failed to induce UDS in rats and/or mice. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induce UDS in the liver; however, the significant S-phase responses induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarcinogenicity.

Automated constant-current coulometric assay system for ascorbic acid and sodium ascorbate.

The performance of an automated constant-current coulometric system for the assay of ascorbic acid and sodium ascorbate is described. After loading, it is capable of analyzing 25 samples and printing out the titer values with no operator attention for 2.5 hr. Under optimum conditions, ascertained by evaluating various electrochemical parameters, the accuracy and precision (95% ts) were found to be +/- 0.3%.

Factors that affect the sensitivity of the in vivo-in vitro hepatocyte DNA repair assay in the male rat.

Measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) in rat hepatocytes following in vivo exposure has been shown to be a useful indicator of the genotoxicity of chemicals in rat liver. We have examined some of the parameters of this assay in an attempt to increase its sensitivity and reduce cytoplasmic backgrounds. Fischer-344 rats were treated with a low dose of a known positive chemical, water, or corn oil. Livers were perfused with a collagenase solution and isolated hepatocytes were incubated with [3H]thymidine (3H-TdR) followed by overnight incubation in unlabeled TdR, then cell fixation and washing. UDS was measured by quantitative autoradiographic grain-counting as net grains/nucleus (NG). Incubation in 3H-TdR ranging in age from 1 week to more than 12 months gave highly variable background (BKG) and NG counts and a slight overall decrease in NG when the 3H-TdR used was more than 4 months old. Control BKG was 3 times higher after 19 h than after 4 h of incubation in 3H-TdR, with little change in NG. Incubation in unlabeled TdR also reduced BKG significantly. Reduction of autoradiogram exposure from two to one week cut BKG in half without significantly reducing NG. A half-hour wash in fixative (1:3 acetic acid:ethanol) followed by two water washes was as effective in reducing BKG as three 10-min washes in fixative followed by 6 water washes, and resulted in better overall cell attachment. An examination of the distribution of historical control data shows that vehicle control animals never exceed zero NG. This suggests that any NG response greater than zero should be viewed as a possible positive response.

Effects of soya bean flakes and liquorice root extract on enzyme induction and toxicity in B6C3F1 mice.

Both soya bean flakes (SBF) and liquorice root extract (LRE) have previously been reported to have anticarcinogenic properties, which have been thought to be related to an increased activity of specific enzymes responsible for the detoxification of chemical carcinogens. 30- and 90-day studies were conducted in male B6C3F1 mice to determine which, if any, of several detoxification enzymes are induced by SBF or LRE. Mice fed 8 and 25% LRE showed a variety of adverse clinical signs, poor weight gain and 30% mortality. Significant increases in liver:body weight ratios were observed in both the SBF and LRE groups. No significant treatment-related gross autopsy findings were observed in any of the SBF groups. A number of abnormalities were observed in the LRE groups, including lesions of the kidney, liver, spleen and thymus. Liver samples from the 90-day study were analysed for 7-ethoxycoumarin O-deethylase (7-ECOD), benzo[a]pyrene hydroxylase (BPH), superoxide dismutase (SOD), glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) at 90 days, and at an interim 30-day autopsy. No treatment-related increases were observed for BPH or SOD. Both SBF and LRE induced modest increases in UDPGT activity. SBF induced modest increases in GST activity, but LRE decreased this activity. 7-ECOD activity was significantly increased by LRE and decreased by SBF. Samples from a 30-day study in which both LRE and SBF were administered at various dose levels were examined for UDPGT activity; all dose groups showed decreases in UDPGT activity relative to controls. The results suggest that both SBF and LRE may alter the activities of specific enzymes involved in the detoxification of chemical carcinogens; however, the combination of these two foodstuffs may not produce an additive effect in B6C3F1 mice.

The pathogenesis of neosporosis in pregnant cattle: inoculation at mid-gestation.

To investigate the pathogenesis of bovine neosporosis, 14 pregnant cattle were each inoculated subcutaneously with either 10(7) or 5 x 10(8) Neospora caninum (strain NC1) tachyzoites at 140 days' gestation. Serial necropsies were then carried out over an 8-week period. In the placenta, Neospora DNA and histopathological changes were observed in samples taken 14 days post-inoculation (dpi), with focal necrosis of maternal caruncular septa and fetal placental villi, serum leakage, and a maternal and fetal inflammatory response. At subsequent samplings, pathological changes in the placenta showed signs of resolution. No parasitaemia was detected in the dams in the two weeks following inoculation. In the fetus, Neospora DNA was detected at 14 dpi, and histopathological changes in the fetal central nervous system at 28 and 42 dpi consisted of small foci of necrosis and inflammation. Resolution of placental lesions during the experiment indicated that the disease was being controlled, and fetal infection, although established, did not appear to be progressing to a fatal outcome. The two doses of tachyzoites produced similar results, but the higher dose elicited earlier and more extensive lesions in the placenta and fetus. Control animals remained negative for all parameters recorded. It is concluded that in bovine neosporosis the placenta plays a central role in the pathogenesis and epidemiology of the infection, and that while primary tissue destruction by the parasite may endanger the fetus, the maternal and fetal inflammatory responses may also be damaging.

The effect of Fasciola hepatica infection on respiratory vaccine responsiveness in calves.

Fasciola hepatica is a common parasite in cattle, and bovine fasciolosis causes significant production losses, as well as being a zoonotic disease of global importance. F. hepatica has been shown to have immunoregulatory effects and the aim of this research was to establish whether F. hepatica infection influences the response to vaccination against respiratory pathogens in calves. A total of 48 calves were randomly and equally allocated to two groups. The experimental group was infected with F. hepatica, while the other group was used as a control. At week 2 and 6 after infection calves from both groups were administered a vaccine containing inactivated PI-3, BRSV and Mannheimia haemolytica, pathogens commonly associated with bovine respiratory disease. Blood samples were taken weekly over 12 weeks to measure specific antibodies against all vaccine antigens and against F. hepatica, as well as IgG1 and IgG2 isotypes for PI-3 and BRSV specific antibodies. Faecal samples were examined for F. hepatica eggs and routine haematology and liver enzyme biochemistry were performed and cytokine production in vitro measured. Liver enzymes (GGT and GLDH) and eosinophils were significantly higher in the experimental group, whereas neutrophil numbers were higher in the control group. There was no significant difference between the groups in terms of vaccine-specific total responses to PI-3, BRSV and M. haemolytica. IgG1 levels were higher in comparison to IgG2 levels in both PI-3 and BRSV specific antibodies. IL-4 levels from stimulated and unstimulated PBMC were significantly higher in the control group. IFN-γ levels were significantly higher in PBMC from the control group when cultured in medium only. No significant differences were noted in the levels of other cytokines measured. In this work, no effect of early F. hepatica infection on the antibody responses to a range of respiratory vaccine antigens in calves was shown. However, differences in cytokine responsiveness of PBMC between control and infected groups were observed, indicating that further work in measuring the effect of F. hepatica infection response to challenge infection following vaccination would be warranted.

A murine model for cerebral toxocariasis: characterization of host susceptibility and behaviour.

Toxocara canis, the parasitic roundworm of dogs, can infect a number of paratenic hosts, such as mice and humans, due to the widespread dissemination of its ova in the environment. In these paratenic hosts, larvae have been shown to exhibit a predilection for the central nervous system, resulting in an increasing number of parasites migrating to the brain as infection progresses. In an initial experiment, we investigated the differential brain involvement of T. canis in 7 strains of inbred mice, and chose 2 strains, susceptible (BALB/c) and resistant (NIH) to cerebral infection. In a second experiment, both strains were investigated in terms of course of migration, larval accumulation, and behavioural response to T. canis infection. Results revealed that infected BALB/c mice took significantly longer to drink from a water source (following a period of deprivation), compared with control mice, indicating some degree of memory impairment. Cerebral larval recoveries from both strains of mice demonstrated variation between the two experiments, suggesting that larval burdens may not be a reliable indicator of susceptibility or resistance to T. canis infection. The percentage of total recovered larvae in each organ may be a better representation of larval distribution. Our model system may provide insights into the impact of chronic geohelminth infection on cognitive development.

Eating disorders in adolescence.

Primary anorexia nervosa presents with severe disorder of eating behaviour occuring predominantly in young women. The aetiology is unknown and treatment ranges from psychoanalysis to leucotomy. Management is difficult because of the patient's inability to accept traditional methods of psychiatric treatment and also because of family attitudes which result in perpetuation of the condition. Because it was noted in a study of patients suffering from anorexia nervosa that response to conventional methods of psychiatric treatment was limited, further investigations were made: (1) A comparative study of 12 patients with anorexia nervosa and 12 patients suffering from psychoneurotic illness was made. Patients were matched for age and intelligence. (2) The fact that significant differences emerged indicated that the concept of anorexia nervosa as a psychoneurotic condition was in question. (3) Group therapy, recently introduced, is mentioned.

A Strain of Rosa damascena Cultured Cells Resistant to Ultraviolet Light.

A strain of cultured cells of Rosa damascena Mill. which showed unusual resistance to damage by short wave (254 nanometers) ultraviolet radiation was isolated. The resistant cells were 2.2 to 2.8 times larger and had about twice the amount of DNA and more chromosomes than the parental, sensitive cells. The resistant cells also produced larger quantities of polyphenolic compounds, principally flavonoids, during the later phases of culture growth. At 10 days, resistant cells had 4 times more nonflavonoid polyphenolics and 14 times more flavonoids than parental cells. The resistance, which was also observed only in the later phases of culture growth, was best correlated with the production of polyphenolics, which apparently shielded ultraviolet-sensitive target molecules from damage.