Noninvasive prenatal diagnosis of genetic diseases induced by triplet repeat expansion by linked read haplotyping and Bayesian approach.

The field of noninvasive prenatal diagnosis (NIPD) has undergone significant progress over the last decade. Direct haplotyping has been successfully applied for NIPD of few single-gene disorders. However, technical issues remain for triplet-repeat expansions. The objective of this study was to develop an NIPD approach for couples at risk of transmitting dynamic mutations. This method includes targeted enrichment for linked-read libraries and targeted maternal plasma DNA sequencing. We also developed an innovative Bayesian procedure to integrate the Hoobari fetal genotyping model for inferring the fetal haplotype and the targeted gene variant status. Our method of directly resolving parental haplotypes through targeted linked-read sequencing was smoothly performed using blood samples from families with Huntington's disease or myotonic dystrophy type 1. The Bayesian analysis of transmission of parental haplotypes allowed defining the genotype of five fetuses. The predicted variant status of four of these fetuses was in agreement with the invasive prenatal diagnosis findings. Conversely, no conclusive result was obtained for the NIPD of fragile X syndrome. Although improvements should be made to achieve clinically acceptable accuracy, our study shows that linked-read sequencing and parental haplotype phasing can be successfully used for NIPD of triplet-repeat expansion diseases.Trial registration: NCT04698551_date of first registration: 07/01/2021.

[Fetoscopic laser coagulation in 100 consecutive monochorionic pregnancies with severe twin-to-twin transfusion syndrome]. / Coagulation laser en fœtoscopie des anastomoses vasculaires placentaires: étude observationnelle de 100 grossesses gémellaires monochoriales consécutives compliquées de syndrome transfuseur-transfusé sévère.

OBJECTIVES: To report pre- and post-surgical datas of large series of severe twin-to-twin transfusion syndrome (TTTS) managed with laser ablation surgery in our centre, to evaluate the incidence of complications, perinatal outcome and to compare with other cohorts. PATIENTS AND METHODS: Observational study of 100 cases of TTTS consecutively treated with fetoscopic laser coagulation between January 2004 and April 2010 in CMCO-SIHCUS of Schiltigheim. RESULTS: There are nine stage I, 49 stage II, 38 stage III and four stage 4. Median gestation at time of laser is 20.6 weeks (14-29) whereas median gestation at delivery is 32.6 weeks (16.3-39). Overall perinatal survival rate is 68.5% (137 children over 200). Eighty-five percent have one or more surviving twins. The survival rate is the same for donors and for recipients. Preterm premature rupture of the membranes are observed in 17% of cases and the median gestational age for this complication is 30 weeks (20-34). Cerebral abnormalities are present in 7% of newborns. CONCLUSION: Our results for the management of severe TTTS are comparable to the other reported series. There are still many questions remaining concerning the optimal management of TTTS.

Transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection.

Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

Bluetongue virus serotype 8-associated congenital hydranencephaly in calves.

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

Non-infectious factors associated with macroscopic and microscopic lung lesions in slaughter pigs from farrow-to-finish herds.

A cross-sectional epidemiological study was conducted in 150 randomly selected farrow-to-finish herds to investigate which non-infectious factors might act as risk indicators for the prevalence and severity of macroscopic and microscopic lung lesions in slaughter pigs. Data were collected during herd visits through inspections of the pigs and through interviews with the farmers. Macroscopic lung lesions of pneumonia and pleuritis were recorded at slaughter from 25 pigs per herd, and microscopic lung lesions of lymphohistiocytic infiltration were recorded from 10 pigs per herd. The median herd level prevalences were 24 per cent for pneumonia, 16 per cent for pleuritis and 60 per cent for lymphohistiocytic infiltration. Pneumonia lesions were negatively associated with pleuritis lesions and positively associated with lymphohistiocytic infiltration. Pleuritis lesions were negatively associated with lymphohistiocytic infiltration. The prevalence and the severity of pneumonia lesions were increased by a high frequency of purchasing gilts and by a slaughter date in January to February. The presence of a growing unit also increased the severity of pneumonia. The prevalence and the severity of pleuritis lesions were higher when there were more pig herds in the municipality, and when there were poor biosecurity measures, and their prevalence was increased by a slaughter date in January to February, and their severity by a slaughter date in March to April. An increase in the airspace stocking density in the finishing unit also increased the prevalence of pleuritis. The prevalence and the severity of lymphohistiocytic infiltration in the lung tissue were higher in herds purchasing gilts. Pigs raised in pens with slatted floors were also at higher risk of more severe lesions of lymphohistiocytic infiltration.

Herd factors associated with the seroprevalences of four major respiratory pathogens in slaughter pigs from farrow-to-finish pig herds.

The objective of this study was to investigate sero-epidemiological aspects of Mycoplasma hyopneumoniae (Mh), influenza H1N1 and H3N2 viruses and Aujeszky disease virus (ADV) in fattening pigs from 150 randomly selected farrow-to-finish pig herds. Different herd factors were examined as potential risk indicators for the percentage of pigs with antibodies against the 4 pathogens. The median within-herd seroprevalences of the pathogens were: Mh 76%, H1N1 100%, H3N2 40% and ADV 53%. There was a positive association between the seroprevalences of both influenza viruses, and a negative association between the seroprevalences of ADV and H1N1. The percentage of pigs seropositive for Mh increased with the purchase of gilts and with the season (slaughter date in March-April). The within-herd seroprevalences of both influenza viruses were higher in the case of a higher density of pig herds in the municipality. A higher number of fattening pigs per pen additionally increased the risk of being seropositive for H3N2. The percentage of pigs with anti-gE-antibodies against the wild type ADV increased with higher airspace stocking density in the finishing unit, increasing herd size, increasing number of pig herds in the municipality and slaughter date in March-April. Increased seroprevalences for these 4 respiratory pathogens were mostly associated with pig density in the herd and its vicinity, the winter period, and with the purchase of gilts. Purchase of gilts, number of fattening pigs per pen and airspace stocking density are risk factors that can be managed directly by farmers striving to attain a high respiratory health status of pigs.

Prevalence of herds with young sows seropositive to pseudorabies (Aujeszky's disease) in northern Belgium.

In Belgium, pseudorabies in swine has been the subject of a mandatory eradication programme since 1993. From December 1995 to February 1996, a survey was conducted in the five provinces of northern Belgium to estimate the provincial pseudorabies virus (PRV) herd seroprevalence. Seven hundred and twenty randomly selected herds were included in this survey. To detect recently infected animals, only young sows were sampled. The results show that 44% of these herds had an important number of PRV-seropositive young sows. The highest herd seroprevalence was observed in West Flanders (68%), followed by Antwerp (60%), East Flanders (43%), Limburg (18%), and Flemish Brabant (8%). Assuming a diagnostic test sensitivity and specificity of 95% and 99%, respectively, and a true PRV within-herd prevalence of 43%, the overall true PRV herd prevalence was estimated to be 35%. A logistic multiple-regression revealed that the presence of finishing pigs was associated with a two-fold increase in odds of a herd being seropositive (odds ratio (OR)=2.07, 95% confidence interval (CI) = 1.31-3.26); a breeding herd size > or =70 sows was associated with a four-fold increase in odds of a herd being seropositive (OR = 4.09, 95% CI = 2.18-7.67); a pig density in the municipality of >455 pigs/km2 was associated with a 10-fold increase in odds of a herd being seropositive (OR = 9.68, 95% CI = 5.17-18.12). No association was detected between the PRV herd seroprevalence and purchase policy of breeding pigs (purchased gilts, or use of homebred gilts only).

Risk indicators for the seroprevalence of Mycoplasma hyopneumoniae, porcine influenza viruses and Aujeszky's disease virus in slaughter pigs from fattening pig herds.

Epidemiological aspects of Mycoplasma hyopneumoniae (Mh), influenza H1N1 and H3N2 viruses, and Aujeszky's disease virus (ADV) were investigated in slaughter pigs from 50 fattening pig herds. Herd factors as potential risk indicators for respiratory disease were obtained by means of a questionnaire. At slaughter, blood samples were collected from each herd, and the proportion of seropositive pigs per herd was assessed for each of these pathogens. The median herd-level seroprevalence of the agents were: Mh 88%, H1N1 100%, H3N2 60% and ADV 90%. The percentage of herds in which all investigated fattening pigs were seronegative for these agents was: Mh 0%, H1N1 0%, H3N2 12% and ADV 18%. The percentage of herds in which all investigated fattening pigs were seropositive for these agents was: Mh 8%, H1N1 71%, H3N2 22% and ADV 40%. A positive association was found between influenza H1N1 and H3N2 viruses, and a negative association between influenza H3N2 virus and ADV. There were no risk indicators for the seroprevalence of Mh. Three risk indicators were associated with the seroprevalence of influenza H1N1 virus: a fully slatted floor, an increasing number of pigs in the municipality and dry feeding. Three risk indicators were found for the seroprevalence of influenza H3N2 virus: purchase of pigs from > or = two herds, an increasing number of pigs in the municipality and natural ventilation. The seroprevalence of ADV was influenced by two risk indicators: an increasing number of pig herds in the municipality and an increasing number of pigs per pen.

Epidemiologic, pathogenic and molecular analysis of recent encephalomyocarditis outbreaks in Belgium.

In 1991 EMCV was isolated for the first time in Belgium from the offspring of a sow with reproductive failure. From August 1995 until December 1996, EMCV was diagnosed in 154 Belgian pig holdings in association with myocardial failure and sudden death in fatteners and suckling piglets or with reproductive failure in sows. To clarify some epidemiological aspects 3 EMCV isolates characteristic for the different clinical pictures and outbreaks were studied. Field observations and animal experiments indicated that the pathogenicity induced by each isolate is specific for one age category and that the spread of the virus is limited. The presented data also suggest that rodents may play a role in the transmission of EMCV but that pig-to-pig transmission is at least as important. Molecular analysis of two separate regions on the genomes of the respective EMCV isolates showed that the 1995-96 EMCV epizootic in Belgium was due to a new virus introduction. Furthermore, the VP1 coding gene is proposed as a marker of virulence.

Effect of vaccination against Mycoplasma hyopneumoniae in pig herds with an all-in/all-out production system.

A multi-site field study was conducted to evaluate an inactivated Mycoplasma hyopneumoniae (Mh) vaccine in 14 pig herds infected by Mh and practising an all-in/all-out production system. In each herd, a vaccinated and control group of 250 pigs each were compared during the growing/finishing period with respect to performance parameters (major variables) and by means of clinical, serological and pathological parameters (ancillary variables). Mh vaccination significantly (P < 0.05) improved daily weight gain (+22 g), feed conversion ratio (-0.07), medication costs (-0.476 ECU/pig) (1 ECU = US$1.0269542), prevalence of pneumonia lesions (-14%) and severity of pneumonia lesions (-3%). Mortality rate, severity of coughing and carcass quality were not significantly influenced by Mh vaccination. Serological results of Mh and other respiratory pathogens are presented and discussed. A cost-benefit analysis based on significantly improved performance parameters demonstrated that Mh vaccination was economically attractive as it resulted in an increase of the net return to labour with 1.300 ECU per finishing pig sold. The sensitivity of the economic benefit was illustrated towards fluctuations in pig finishing prices.

The effect of vaccination against Mycoplasma hypopneumoniae in pig herds with a continuous production system.

An inactivated Mycoplasma hyopneumoniae vaccine was evaluated in five pig herds clinically infected with enzootic pneumonia and practising a continuous production system in the growing/finishing unit. In each herd, a vaccinated and control group of approximately 47 pigs each were individually monitored from birth until slaughter. Vaccinated pigs received the first dose at about 1 week of age and the second approximately 3 weeks later. During all production stages, an equal number of vaccinated and control pigs was present in the same pen. Both groups were compared with respect to zootechnical parameters (major variables) and by means of serological, pathological, and bacteriological parameters (ancillary variables). Daily weight gain was improved by 14 gr/day during the period from 8 days of age until slaughter (P = 0.0486) and by 25 gr/day during the growing/finishing period (P = 0.0067). Mortality rate, and the costs for curative medication were not significantly improved by vaccination. The results of the ancillary variables are presented and discussed.

Characterization and identification of Vagococcus fluvialis strains isolated from domestic animals.

Strains of Vagococcus fluvialis, a species of Gram-positive catalase-negative cocci, related to the genera Enterococcus and Carnobacterium, were isolated from various lesions of pigs, from lesions and tonsils of cattle and cats and from tonsils of a horse. Most lesion strains were isolated in mixed culture from animals with disease conditions unrelated to coccal infection. Certain differences with the species description of Vagococcus fluvialis were found: only a proportion of the strains was motile; many strains gave positive reactions to Voges-Proskauer, alkaline phosphatase and leucine arylamidase tests or produced acid from galactose and D-tagatose. SDS-PAGE of whole-cell protein patterns, however, confirmed the phenotypic identification. Guidelines for identification of Vagococcus fluvialis are given and an emended description of the species is proposed.

Characterization of 2 groups of Actinomyces-like bacteria isolated from purulent lesions in pigs.

Two groups of Actinomyces-like bacteria isolated from purulent lesions in pigs, were characterized and compared with Actinomyces pyogenes of porcine origin. One group showed characteristics which differed in many respects from A. pyogenes but which corresponded more closely to those of the non-official species 'Actinomyces suis'. Certain carbohydrate reactions differed from those given in earlier descriptions. This group was isolated only from purulent vaginal discharge and aborted foeti. The other group resembled the 'A. suis'-like bacteria but differed in its colony morphology, hippurate and to some extent also in its cellobiose and gluconate reactions and in its habitat. It was concluded that 'A. suis' may occur in lesions other than mammary actinomycosis in areas where this disease is extremely rare, and that its natural habitat is the genital tract of pigs. Furthermore a probably related group exists, which is found in purulent lesions located mainly outside the porcine genital tract. These organisms appear to be as frequent in these types of lesions as A. pyogenes, with whom they can easily be confused in routine diagnostic bacteriology.

Bèta-hemolytic streptococci from pigs: bacteriological diagnosis.

Bèta-hemolytic streptococci from lesions in pigs were identified as S. dysgalactiae biotype "equisimilis" and S. dysgalactiae serovar L, S. porcinus, S. agalactiae, E. faecalis and CO2 dependent and broad bèta-hemolytic S. suis. Data are provided which can be used in the interpretation of commercial identification systems. Tests results and physiological characteristics which complete identification procedures using coagglutination tests are proposed and discussed.

Sites of virus replication in the genital organs of boars inoculated in the cavum vaginale with pseudorabies virus.

To determine the sites of replication and the evolution of pseudorabies virus infection in boar genital organs, 5 Belgian Landrace boars were inoculated with pseudorabies virus unilaterally in the cavum vaginale of the testis. Virus replication took place only in cells of the tunica vaginalis of both cava vaginalia. Infection of the serosa led to exudative periorchitis and increased scrotal fluid, resulting in a severely swollen scrotal region. These experimental findings were similar to findings in boars with naturally acquired pseudorabies virus infection. Scrotal fluid contained large amounts of virus, making it a useful specimen for diagnosis of the disease in affected boars.

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars.

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.