Association of umbilical vein flow with abnormal fetal growth and adverse perinatal outcome in low-risk population: multicenter prospective study.

OBJECTIVES: To investigate the relationship of umbilical vein flow (UVF) measured close to term with abnormal fetal growth and adverse perinatal outcome in a cohort of pregnancies at low risk of placental insufficiency. METHODS: This was a prospective multicenter observational study conducted across two tertiary maternity units. Patients with a singleton appropriate-for-gestational-age fetus between 35 and 38 weeks' gestation were included. Pregnancies at higher risk of placental insufficiency or with fetal anomalies were excluded. At ultrasound examination, the abdominal circumference (AC), umbilical vein diameter and peak velocity of the umbilical vein were measured, and, using these variables, a new variable, UVF/AC, was calculated. The primary outcome was the occurrence of severely stunted fetal growth, defined as a greater than 40-percentile drop between estimated fetal weight at the third-trimester ultrasound and birth weight between the third-trimester ultrasound and delivery. The occurrence of adverse perinatal outcome, defined as one of the following: neonatal acidosis (umbilical artery pH < 7.15 and/or base excess > 12 mmol/L) at birth, 5-min Apgar score < 7, neonatal resuscitation or neonatal intensive care unit admission, was analyzed as a secondary outcome. RESULTS: Between April 2021 and March 2023, 365 women were included in the study. The mean UVF/AC at enrolment was 6.4 ± 2.6 mL/min/cm, and 35 (9.6%) cases were affected by severely stunted fetal growth. Severely stunted fetal growth was associated with a lower mean UVF/AC (5.4 ± 2.6 vs 6.5 ± 2.6 mL/min/cm; P = 0.02) and a higher frequency of UVF/AC < 10th percentile (8/35 (22.9%) vs 28/330 (8.5%); P = 0.01). Moreover, UVF/AC showed an area under the receiver-operating-characteristics curve (AUC) of 0.65 (95% CI, 0.55-0.75; P = 0.004) in predicting the occurrence of severely stunted fetal growth, and the optimal cut-off value of UVF/AC for discriminating between normal and severely stunted fetal growth was 7.2 mL/min/cm. This value was associated with a sensitivity and specificity of 0.77 (95% CI, 0.60-0.90) and 0.33 (95% CI, 0.28-0.39), and positive and negative predictive values of 0.11 (95% CI, 0.07-0.15) and 0.93 (95% CI, 0.87-0.97), respectively. Regarding the occurrence of adverse perinatal outcome, this was associated independently with maternal age (adjusted odds ratio (aOR), 0.93 (95% CI, 0.87-0.99); P = 0.04), UVF/AC Z-score (aOR, 0.53 (95% CI, 0.30-0.87); P = 0.01) and augmentation of labor (aOR, 2.69 (95% CI, 1.28-5.69); P = 0.009). UVF/AC showed an AUC of 0.65 (95% CI, 0.56-0.73; P = 0.005) in predicting the occurrence of adverse perinatal outcome, and the optimal cut-off value of UVF/AC for discriminating between normal and adverse perinatal outcome was 6.7 mL/min/cm. This value was associated with a sensitivity and specificity of 0.70 (95% CI, 0.54-0.83) and 0.40 (95% CI, 0.34-0.45), and positive and negative predictive values of 0.14 (95% CI, 0.09-0.19) and 0.91 (95% CI, 0.85-0.95), respectively. CONCLUSIONS: Our data demonstrate an association between reduced UVF close to term, severely stunted fetal growth and adverse perinatal outcome in a cohort of low-risk pregnant women, with a moderate ability to rule out and a poor ability to rule in either outcome. Further studies are needed to establish whether the assessment of UVF can improve the identification of fetuses at risk of subclinical placental insufficiency and adverse perinatal outcome. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Use and comparison of serologic assays to detect anti-Neospora caninum antibodies in farmed red deer (Cervus elaphus).

Neospora caninum is a protozoan parasite that cause abortion in different ruminant species, including red deer ( Cervus elaphus). There are no validated assays to be performed with sera from red deer. At the present work, we evaluated the agreement among indirect fluorescent antibody test (IFAT), competitive inhibition ELISA based on a recombinant protein (ciELISA tSAG1) and immunoblot (IB) to detect anti- N. caninum antibodies in a red deer herd that presented reproductive losses due to N. caninum. In addition, we analyzed the relationship between the serologic results and 15 hinds were analyzed by IFAT, ciELISA tSAG1 and IB to detect anti- N. caninum antibodies. In the three assays, the cut-off established for cattle was used. Besides, sera were analyzed by IFAT to detect anti- Toxoplasma gondii antibodies. The hinds were monitored by ultrasound scanning during the gestational period to detect abortions. Gwet's agreement coefficient (AC1) and the percentage of agreement were used to estimate the agreement between pairs of assays. Chi-square test and odds ratio (OR) were used for the statistical association between abortion and seropositivity to N. caninum or to T. gondii. The N. caninum seropositivity rate was 53.9% (62/115), 57.4% (66/115) and 55.7% (64/115) for IFAT, ciELISA tSAG1 and IB, respectively. The AC1 and percentage of agreement were 0.760% and 87.8% for the pair ciELISA tSAG1 /IFAT, 0.793% and 89.6% for the pair IFAT/IB, and 0.966% and 98.3% for the pair IB/ciELISA tSAG1. The T. gondii seropositivity rate was 53.0% (61/115). Seropositive hinds to N. caninum were more likely to abort than seronegative hinds by the 3 assays. The OR for the association between N. caninum seropositivity and abortion was 72.70, 22.96 and 83.24 when ciELISA tSAG1, IFAT or IB assays were used, respectively. between T. gondii seropositivity and abortion. The three serologic assays were useful to detect N. caninum infected hinds. The validation of the assays for use in red deer would be an improvement for diagnosis of neosporosis in this species.

Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

Babesia bovis biological clones and the inter-strain allelic diversity of the Bv80 gene support subpopulation selection as a mechanism involved in the attenuation of two virulent isolates.

The virulence phenotype of Babesia bovis subpopulations was evaluated using biological clones derived from the high-virulence BboS2P and the low-virulence BboR1A strain and two original virulent isolates, BboL15 and BboL17, multiplied extensively in vitro or attenuated by successive passages in splenectomized calves. The virulence phenotype was assessed both by inoculation of normal Holstein adult steers and by analyses of polymorphic fragments of the single-copy Bv80 gene as a subpopulation marker. BboS2P and its nine derived clones contained a single 750 bp fragment with identical nucleotide sequences and numbers of repeats. A single fragment of approximately 850 bp was observed in BboR1A and its derived clones (Ca3B1, Ca2B1). Ca3B1 and Ca2B1 were differentiated by a stable deletion of 15 contiguous nucleotides in the Bv80 allele of Ca3B1. Both alleles were identified in the parental strain. Original isolates BboL15 and BboL17 contained two Bv80 fragments of different sizes. Interestingly, the heavy and light fragments persisted in the in vivo-attenuated strains and the virulent in vitro-multiplied strains, respectively. Despite the inter-strain allelic diversity of the Bv80 gene, the fragments had identical nucleotide sequences and numbers of repeats compared to their respective parental Bv80 genes. The high-virulence and low-virulence phenotypes remained unchanged after they were multiplied in vitro. In conclusion, the polymorphic B. bovis Bv80 gene, was a useful marker for differentiating subpopulations with different phenotypes. The brevity of the procedure to isolate one parasite from the original isolate or strain before in vitro cloning and the fact that the continuous in vitro multiplication did not modify the virulence phenotype of B. bovis clones strongly suggest that the in vivo-attenuated subpopulations existed in the original isolates before they were selected by passages in splenectomized calves.

The novel protein BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis.

The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.

Antibody dynamics in Holstein Friesian heifers vaccinated with Brucella abortus strain 19, using seven serological tests.

The serological response induced by Brucella abortus strain 19 was evaluated in 52 Holstein females from a brucellosis-free herd using seven serological tests. Each calf was vaccinated at an age of 4 and 8 months old with 3 x 10(10) CFU B. abortus S19 and the antibody response was determined as the proportion of positive results to each test. The antibody dynamics, measured with the buffered plate antigen (BPA) test and the rapid automated presumptive (RAP) test, were similar. The proportion of positive reactions in these tests reached 100% one week after vaccination and remained at this level for seven weeks, after which the proportion of positive samples slowly declined to 8% (BPA) and 2% (RAP) at week 50. The response in the indirect enzyme immunoassay (i-ELISA) was similar, but shorter than that observed with the BPA/RAP. The antibody dynamic, measured using the seroagglutination test (SAT) in parallel with the 2-mercaptoethanol (2-Me) test and the complement fixation test (CFT) were similar to the RAP/BPA, but of slightly shorter duration. The competitive ELISA (c-ELISA) was positive in all animals for 3 weeks, followed by a rapid decline. The fluorescence polarization assay (FPA) reached a maximum of 68.5% positive animals at week 4 and then declined. Based on these data, the c-ELISA and FPA discriminated residual antibody activity due to vaccination more efficiently than the other tests.

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200. Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.

Nevus comedonicus: case report and brief review of the literature.

We report extensive nevus comedonicus in a female patient that involved half of her body entirely, with infected cystic lesions as well as typical scars, limited by the midline. The lesions worsened at the beginning of puberty. A brief review of the literature highlights the histopathologic, etiopathogenic, and therapeutic aspects.

Frequency of qh+ chromosomal variants in a) radioexposed, b) Down syndrome and c) control subjects.

"Paranormal" variants of human chromosomes, devoid of phenotypical effects (since what appears to vary is heterochromatic, non-genic DNA) are known to be heritable. Some very large variants (especially the qh+ variants on chromosomes 1 and possibly 16 and Y) were reported to be associated with increased reproductive pathology (sterility, fetal wastage, chromosomal aberrations). These variants are currently assessed by the C-band techniques; very large C-bands correspond to morphological alterations (elongation or deformation) of the chromosome. A study of qh+ morphological variants of chromosomes 1, 9 and 16 in 40 professionally radioexposed subjects, in 40 Down-syndrome patients and in 40 controls is reported, indicating that the frequency of each variant is lowest among controls, intermediate among professionally radioexposed subjects and highest among Down-syndrome patients. These findings, if confirmed, suggest a possible use of the qh+ variants as heritable indicators of chromosomal damage.

Duchenne muscular dystrophy: alterations in lymphocyte fluidity.

We have studied lymphocyte membrane from 7 patients with Duchenne Muscular Dystrophy (DMD) and 8 normal controls using fluorescence polarization of 1,6-diphenyl-hexatriene (DPH). The microviscosity of lymphocytes membrane is significatively higher in DMD patients than in normal controls. Our results support the hypothesis of a generalized membrane defect in Duchenne Muscular Dystrophy.

[Interaction of the genes of progressive muscular dystrophy (PMD) and blood groups (ABO)]. / Interazione tra i geni delle distrofie muscolari progressive (D.M.P.) e dei gruppi sanguigni (ABO).

Verification of the hypothesis that the inheritance of the Progressive Muscular Dystrophies (PMD) may involve dispersed genes, throiugh the study of a sample of 105 patients revealed that 82% of the Duchenne type belong to blood group O, while 79% of the limb-girdle type belong to group A. The fact that the Duchenne gene is sex-linked while the ABO locus is on chromosome 9 confirms the "dispersed genes" hypothesis, with important implications for further research, for genetic counselin and possibly for prevention.