Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

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.

Direct genetic characterization of Toxoplasma gondii from clinical samples from Denmark: not only genotypes II and III.

Genetic variation within Toxoplasma gondii can have both clinical and epidemiological significance, while the genotypes circulating in many parts of the world, including the Nordic country Denmark, are still unknown. We genetically characterized T. gondii strains that had been detected in human clinical samples in Denmark in 2011-2016. Samples that had tested positive for T. gondii DNA and had a quantification cycle value <33 were included in this study and subjected to direct genetic characterization of T. gondii based on length-polymorphism of 15 microsatellite markers. A total of 23 DNA samples from 22 individual patients were analyzed. The results were consistent with genotype II with 15/15 markers amplified from seven samples from the central nervous system (CNS) including two samples from one patient, four ocular samples, and one unspecified sample; with genotype III with 15/15 markers amplified from two ocular samples; with genotype Africa 1 with 15/15 markers amplified from one amniotic fluid sample and from one CNS-sample; with atypical genotype with 15/15 markers amplified from one CNS-sample and with 11/15 markers amplified from one CNS-sample; and with HG12-like genotype with 9/15 markers amplified from one CNS-sample. Genotype II, which is endemic in Europe, was predominant, but more than a third of the successfully genotyped strains were non-type-II. The possibility that clinical toxoplasmosis is caused by a strain that is not considered endemic to the region is definitely not negligible.

Hepatitis E Virus Antibodies in Finnish Veterinarians.

We investigated hepatitis E virus (HEV) infections in Finnish veterinarians engaged in different practice specialties and evaluated the effect of different background factors on HEV exposure by examining total HEV antibodies in samples collected from the participants of the 2009 National Veterinary Congress in Helsinki, Finland. Finnish veterinarians commonly have total HEV antibodies with seroprevalence of 10.2%. Of the non-veterinarians, 5.8% were seropositive. Increasing age was associated with HEV seropositivity, and, surprisingly, the highest HEV seroprevalence (17.8%) among veterinarians was detected among small animal practitioners. Although no positive correlation between swine contacts and HEV seropositivity was found, 22.7% of veterinarians who had had needle stick by a needle that had previously been injected into a pig versus 9.0% of those who had not were seropositive, even though the finding was statistically non-significant (P = 0.07). Our results suggest that, although contact with swine is a known risk factor for HEV infection, the sources of HEV infections are probably numerous, including travelling abroad and possibly also other reservoirs of HEV than pigs.

Molecular identification of Taenia spp. in the Eurasian lynx (Lynx lynx) from Finland.

Cestodes of the genus Taenia are parasites of mammals, with mainly carnivores as definitive and herbivores as intermediate hosts. Various medium-sized cats, Lynx spp., are involved in the life cycles of several species of Taenia. The aim of the present study was to identify Taenia tapeworms in the Eurasian lynx (Lynx lynx) from Finland. In total, 135 tapeworms from 72 lynx were subjected to molecular identification based on sequences of 2 mtDNA regions, the cytochrome c oxidase subunit 1 and the NADH dehydrogenase subunit 1 genes. Available morphological characters of the rostellar hooks and strobila were compared. Two species of Taenia were found: T. laticollis (127 samples) and an unknown Taenia sp. (5 samples). The latter could not be identified to species based on mtDNA, and the rostellar hooks were short relative to those described among other Taenia spp. recorded in felids from the Holarctic region. In the phylogenetic analyses of mtDNA sequences, T. laticollis was placed as a sister species of T. macrocystis, and the unknown Taenia sp. was closely related to T. hydatigena and T. regis. Our analyses suggest that these distinct taeniid tapeworms represent a putative new species of Taenia. The only currently recognized definitive host is L. lynx and the intermediate host is unknown.

Relationship of growth and blood pressure in inbred rats.

Blood pressure and body weight rise with age, and hypertension is hypothesized to result from an early disorder of growth. We used the statistical technique of spline fitting to examine patterns of growth and rising blood pressure in individual inbred rats of the Wistar-Kyoto, spontaneously hypertensive (SHR), and stroke-prone SHR strains. Our results indicate that growth is phasic and that a well-defined growth spurt peaks at approximately 50 days of age in each strain. In addition, the time of maximal weight gain corresponds to the onset of a blood pressure spurt. The timing of this blood pressure rise was similar in the three strains, but the maximal rate of blood pressure rise was significantly higher in the hypertensive strains. Our results suggest that growth and rising blood pressure are intimately linked. Blood pressure appears to rise in response to a growth spurt, suggesting that there are growth-related events that trigger the cardiovascular response.

Blood pressure and acidic renin forms in stroke-prone hypertensive rats.

Six forms of renin, distinguishable by their isoelectric focusing characteristics, are found in rat kidney and plasma (forms 1-6, form 1 having the most basic pI). To test the hypothesis that high blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP) is associated with a particular fraction of the renin forms, systolic blood pressure (SBP) and renin profile were measured in SHRSP, normotensive Wistar-Kyoto rats (WKY), and their genetic crosses (F1). Adult SHRSP showed an increase in the proportion of forms 4, 5, and 6 compared with WKY. F1 showed values between SHRSP and WKY. A strong positive correlation was noted between the fraction of the acidic forms of renin and SBP. The mean blood pressure and the fraction of the acidic renin forms in SHRSP and WKY pups also fitted the same regression line. However, no correlation existed between renal renin content (RRC) and SBP. SHRSP pups had a higher RRC than age-matched WKY or F1. RRC in normal adult SHRSP was lower than that in WKY or F1 of the same age. In old animals, SHRSP again showed the highest RRC, and microangiography of the kidney of old SHRSP revealed a loss of fine vascularity with few visible glomeruli. These results indicate that the established phase of hypertension in SHRSP is associated with an increased proportion of the acidic forms of renin, which may have a unique role in the establishment or maintenance of hypertension. It is also suggested that high RRC in the early developmental stage plays a role in the developing phase of hypertension.

Genetic relationship between a lymphocyte membrane abnormality and blood pressure in spontaneously hypertensive stroke prone and Wistar-Kyoto rats.

The fluxes of sodium and potassium across the lymphocyte membrane in spontaneously hypertensive, stroke prone rats (SHRSP) are greater than those in Wistar-Kyoto rats (WKY). In order to define the relationship of these abnormal membrane traits to arterial pressure elevation, their distributions were studied in F1, F2, and backcross progeny of the cross of SHRSP and WKY rats. In the F1 generation, arterial pressure and potassium efflux values resembled those of the WKY parents suggesting that these traits are dominant in the normotensive rat; sodium influx values resembled those of the SHRSP parents. There was poor correlation (r = 0.45) between blood pressure and sodium influx in the F2 generation. There was a high correlation between blood pressure and potassium efflux (r = 0.86), suggesting that these two traits may be under the control of a common genetic mechanism.

Genetic basis for altered vascular responses to ouabain and potassium-free solution in hypertension.

Many properties intrinsic to vascular smooth muscle are altered in hypertension. It is unknown whether these abnormalities are primary traits that may contribute to the etiology of hypertension or whether these vascular differences between the hypertensive and normotensive strains are inherited independently of genetic factors that predispose to hypertension. To determine if genetic factors responsible for the predisposition for hypertension may be the same as or linked to genetic factors determining a specific vascular response, adult stroke-prone spontaneously hypertensive rats (SHRSP), normotensive Wistar-Kyoto (WKY) rats, and progeny of genetic crosses of SHRSP and WKY rats (F1, F2, F1 X WKY, F1 X SHRSP) were studied. Rats were killed and helical aortic strips were mounted in a tissue bath for isometric force recording. Contractile responses to 10(-3) M ouabain (expressed as a percent of force generated to a maximal depolarizing stimulus) were greater in aortas from SHRSP (90 +/- 9%) compared with aortas from WKY rats (38 +/- 5%, P less than 0.05). The half time for contraction in K+-free solution was more rapid in aortas from SHRSP (21 +/- 4 min) when compared with aortas from WKY rats (48 +/- 4 min, P less than 0.05). A significant positive correlation between blood pressure (tail-cuff method) and the contractile response to 10(-3) M ouabain was observed in the segregating F2 progeny. In contrast, no correlation between blood pressure and the half time for contraction in K+-free solution was observed in the segregating F2 progeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Renin heterogeneity in stroke-prone hypertensive and normotensive rats.

Six forms of renin are found in the rat kidney. We studied their secretion in renal slices from spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar-Kyoto rats (WKY). Incubation media from renal slices were subjected to isoelectric focusing. Six peaks of renin activity with different isoelectric points were found. The renin concentration of each form was expressed as a percentage of the total recovered from the gel. We established that the forms secreted by renal slices of SHRSP differed from those of WKY: SHRSP slices released a higher proportion of forms focusing at the more acidic pH. The distribution of the six renin forms and of blood pressure (BP) among animals of the F1, F2, and backcross progenies resulting from the cross of SHRSP and WKY rats were studied. In the F1, BP, percentage of renin form 2, and a combination of the percentage of forms 4 + 5 + 6 were intermediate between the parental lines. The backcross rats showed BP and percentages of forms closer to their SHRSP or WKY parent. In the F2, the distribution of BP, percentage of forms 2 and 4 + 5 + 6 take the form of a unimodal distribution with a significantly larger variance than F1. The increase in the correlation between percentage of renin forms and BP, and between renin concentration of BP, in the segregating progenies over that observed in the parental lines and the F1, are support for the hypothesis that these traits are under the control of common genetic mechanisms.

Genetic association of hypertension and vascular changes in stroke-prone spontaneously hypertensive rats.

Isolated tail arteries from stroke-prone spontaneously hypertensive rats (SHRSP) exhibit oscillatory contractile activity in response to norepinephrine, whereas those from normotensive Wistar-Kyoto rats (WKY) do not. To determine whether the norepinephrine-induced oscillations are related to high blood pressure or to separable genetic differences between strains, the response to norepinephrine was studied in adult SHRSP, WKY, and progeny of genetic crosses of SHRSP and WKY (F1, F2, F1 X SHRSP, F1 X WKY). Helical tail artery strips were mounted in a tissue bath for isometric force recording. Rats were classified as responders if oscillatory activity in the presence of 1.8 X 10(-7) M norepinephrine exceeded 250 mg/10 min (milligrams of force amplitude during a 10-minute interval). The blood pressures (mm Hg +/- SEM; tail cuff method) and percentage of rats exhibiting norepinephrine-induced oscillations were as follows: WKY: 109 +/- 3, 0%; F1: 129 +/- 4, 0%; F2: 150 +/- 4, 38%; F1 X WKY: 137 +/- 3, 9%; F1 X SHRSP: 188 +/- 7, 71%; SHRSP: 207 +/- 7, 100%. The distribution of the frequency of animals with oscillatory activity among the progenies was consistent with the hypothesis that a single gene locus determines the observed difference in oscillatory activity between the WKY and SHRSP strains. The allele from the SHRSP that determines the activity phenotype is recessive to the allele contributed by the normotensive WKY strain. In the segregating F2 progeny, the blood pressure of the responders was higher than that of the nonresponders (161 +/- 6 vs 144 +/- 4 mm Hg; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological evidence of vestibular pathology in long-term experimental diabetes mellitus. I. Microvascular changes.

The influence of long-term experimental diabetes on the microvasculature of the saccule and utricle was investigated using quantitative light and electron microscopic techniques. Basal lamina thickening or reduplication, typically seen in diabetic microangiopathy, were not observed. However, morphometric analysis did reveal a statistically significant increase in capillary diameters along with an increased vascularization of both the saccule and utricle. Both of these microvascular abnormalities may have been caused by the hemodynamic alterations known to occur in diabetes. These alterations include decreased deformability of red blood cells and increased blood viscosity. Either of these factors can lead to a greater stress on the capillary wall and possibly to a reduced oxygen delivery to the tissues.

Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats.

Evidence was found for different outcomes to middle cerebral artery occlusion in the young genetically hypertensive stroke-prone rat (SHRSP) compared to sham operated controls and the Wistar Kyoto rat (WKY). Qualitatively and quantitatively different gross lesions marked by Evans blue-albumin, cortical atrophy, large areas of strikingly altered cortical histology, postoperative survival and motor behavioral deficits differentiate young SHRSP from sham operated controls and the normotensive WKY. We conclude that the limited focal lesion observed in normotensive and sham operated rats is primarily due to surgical trauma of exposing the vessel and passing the ligature deep to it. The grossly larger and qualitatively different lesion in the SHRSP is the result of an inadequate circulation provided by the dorsal cerebral arterial collaterals. Since the 5-6 week old SHRSP were only mildly hypertensive (systolic blood pressure 140 mm Hg), the inadequate collateral circulation appears to be related to either a genetic or acquired problem rather than being secondary to a vascular lesion of chronic hypertension.

Dorsal cerebral arterial collaterals of the rat.

This study demonstrated that distal branches of the anterior cerebral artery (ACA) are joined by interarterial anastomoses to rami of the middle cerebral artery (MCA) in the normal Wistar rat. Arteries of 36- and 56-day-old animals were dilated with papaverine and injected with Vultex. Vultex arrived at corresponding ACA and MCA collaterals simultaneously as determined by microscopy through a skull window and photography. There were about 29 ACA-MCA junctions per hemisphere. Junction density was nearly constant along the frontal-occipital axis. The anastomoses were most numerous between 2 and 3 mm lateral to the midline and were less than 120 micron in internal diameter. No significant difference was found between total numbers of junctions for right versus left hemispheres or between age groups. The most evident collateral pattern was characterized by two ACA end rami joining two MCA end branches to form a closed, diamond-shaped collateral unit. Considerations were given to alternate routes of blood flow into the MCA tissue field. We conclude abundant dorsal anastomoses exist in 36- and 56-day-old rats and are the prime potential source for ACA collateral supply to the MCA tissue field.

Lamellated osmiophilic bodies in mouse gallbladder epithelium.

Mouse gallbladder epithelium was studied in the electron microscope after glutaraldehyde and osmium tetroxide fixation and Epon embedding. Special attention was paid to dense cytoplasmic bodies. Heterogeneous dense bodies with osmiophilic and loose granular components were identified. Many of these bodies consisted of lamellated osmiophilic structures, similar to the phospholipid bodies of the large alveolar (type II) cells of the mammalian lung. The dense bodies containing the lamellar structures were membrane-limited and 0.5-1.0 mum in diameter. They were located in the supranuclear cytoplasma and were often closely associated with the Golgi apparatus. Acid phosphatase activity was demonstrated within these bodies and in some of the Golgi vacuoles. The contents were disposed in concentric lamellar osmiophilic structures, sometimes appearing as scroll-like configurations. These morphological findings were interpreted as indication of these presence of phosphlipid absorbed from the gallbladder lumen and stored in phagosomes in the central cytoplasm of the gallbladder epithelial cells.