Genotypic characterization of Staphylococcus chromogenes and Staphylococcus simulans from Swedish cases of bovine subclinical mastitis.

Staphylococcus chromogenes and Staphylococcus simulans are commonly found in intramammary infections (IMI) associated with bovine subclinical mastitis, but little is known about genotypic variation and relatedness within species. This includes knowledge about genes encoding antimicrobial resistance (AMR) and potential virulence factors (pVF). The aim of this study was therefore to investigate these aspects by whole-genome sequencing of milk isolates from Swedish dairy cows with subclinical mastitis in an observational study. We also wanted to study if specific genotypes were associated with persistent IMI and the inflammatory response at udder quarter level. In total, 105 and 118 isolates of S. chromogenes and S. simulans, respectively, were included. Isolates were characterized using a 7-locus multilocus sequence typing (7-MLST), core genome analysis and in-silico analysis of AMR and pVF genes. Forty-seven sequence types (ST) and 7 core genome clusters of S. chromogenes were identified, and the most common ST were ST-6 and ST-109, both belonging to cluster VII. A 7-locus MLST scheme for S. simulans was not available, but 3 core genome clusters and 5 subclusters were described. Overall, substantial variation in ST and clusters among cows and herds were found in both species. Some ST of S. chromogenes were found in several herds, indicating spread between herds. Moreover, within-herd spread of the same genotype was observed for both species. Only a few AMR genes [blaZ, strpS194, vga(A)] were detected in a limited number of isolates, with the exception of blaZ coding for ß-lactamase, which was identified in 22% of the isolates of S. chromogenes with ST-19, ST-102, and ST-103 more commonly carrying this gene compared with other ST. However, the blaZ gene was not identified in S. simulans. The average total number of pVF detected per isolate was similar in S. chromogenes (n = 30) and S. simulans (n = 33), but some variation in total numbers and presence of specific pVF or functional groups of pVF, was shown between ST/clusters within species. Differences in inflammatory response and potentially in persistent IMI at udder quarter level were found between S. chromogenes subtypes but not between S. simulans subtypes. In conclusion, the results from the present study generates new insight into the epidemiology of bovine S. chromogenes and S. simulans IMI, which can have implications for future prevention and antimicrobial treatment of infections related to these species.

A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus.

Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory.

Longitudinal study of vascular structure and function during normal pregnancy.

OBJECTIVE: To examine alterations in maternal vascular structure and function during normal pregnancy. METHODS: We assessed brachial and central blood pressure, pulse-wave velocity and augmentation index (by pulse-wave analysis and applanation tonometry), common carotid artery structure (by ultrasonography) and endothelial function in the brachial artery (by postischemic hyperemia-induced flow-mediated vasodilatation by glyceryl trinitrate) and in the forearm skin microcirculation (by laser Doppler perfusion imaging during iontophoretic administration of acetylcholine and sodium nitroprusside) in 52 healthy nulliparous women at 14, 24 and 34 weeks' gestation, and at 9 months postpartum. RESULTS: During pregnancy, brachial and central systolic and diastolic blood pressures initially decreased but subsequently increased (all P < 0.05). Flow-mediated vasodilatation in the brachial artery increased during early pregnancy (P < 0.05), whereas non-specific vasodilatation by glyceryl trinitrate decreased (P < 0.01), indicating improved endothelial function. Thus, endothelial function index (forearm blood flow/glyceryl trinitrate) increased during pregnancy (0.30 ± 0.18 in the non-pregnant state at 9 months postpartum and 0.51 ± 0.19, 0.61 ± 0.39 and 0.49 ± 0.30 in the first, second and third trimesters, respectively) (P < 0.001). Endothelium-dependent skin microvascular reactivity to acetylcholine also increased (P < 0.01). Carotid-femoral pulse-wave velocity decreased during pregnancy (5.88 ± 0.91 m/s in the non-pregnant state and 5.55 ± 0.67, 5.12 ± 0.66 and 5.62 ± 0.74 m/s in the first, second and third trimesters, respectively) (P < 0.001). CONCLUSION: During normal pregnancy, the blood volume expansion necessary for sufficient fetal growth is accommodated by early and marked changes in the matvascular system. This seems to be dependent on normal adaptive endothelial and vascular function. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Stereological assessment of placental morphology in intrahepatic cholestasis of pregnancy.

OBJECTIVE: To apply stereology for the detection of possibly morphological abnormalities in placentas of women with intrahepatic cholestasis of pregnancy (ICP). STUDY DESIGN: Prospective case-control study of placentas from untreated and UDCA-treated ICP, respectively, and normal pregnancies, examined for morphological differences by systematic random sampling generated by computerized stereology methodology. MAIN OUTCOME MEASURES: Volume of placenta, surface area of terminal villi and capillaries, volume fraction of collagen, number of syncytial knots, and chorangiosis. RESULTS: Surface area of terminal villi and capillaries, and number of syncytial knots were higher in placentas from all ICP, as compared to controls (p < 0.01). A reduction of collagen was found in placentas from UDCA-treated ICP, both in comparison to placentas from untreated ICP and controls (p < 0.05). CONCLUSION: ICP affects the placenta morphologically as shown by increased terminal villous and capillary surface area, and number of syncytial knots.

Placental IGF-I, estrogen receptor, and progesterone receptor expression, and maternal anthropometry in growth-restricted pregnancies in the Swedish population.

BACKGROUND/AIMS: Fetal growth restriction is a complex problem of pregnancy arising from multiple etiologies. Key regulatory elements of growth are the insulin-like growth factor (IGF) axis, and estrogen and progesterone receptors. The aims were to determine the relations of expression of IGF-I, estrogen receptors α and ß (ERα and ERß, respectively), and progesterone receptor (PR), with maternal anthropometry, focusing on birth weight outcomes. METHODS: Placental samples were obtained from 33 patients following delivery. mRNA expression was determined by a solution hybridization technique. Samples were divided into normal control (NC) and growth-restricted (GR) groups. RESULTS: IGF-I expression was lower in the GR as compared to the NC group. PR levels correlated positively with IGF-I expression, infant anthropometry, and gestational age (GR). ERα correlated positively with PR expression (NC), and maternal BMI at delivery (GR). ERß correlated positively with maternal delivery weight and gestational age (NC). CONCLUSION: The differences in placental expression of IGF-I emphasize its key role in birth weight outcomes. We further suggest the importance of PR expression in the pathogenesis of intrauterine growth restriction, as there were direct correlations of PR expression with both IGF-I expression and infant anthropometric parameters, as well as gestational age.

Detection of bluetongue virus RNA in field-collected Culicoides spp. (Diptera: Ceratopogonidae) following the discovery of bluetongue virus serotype 1 in white-tailed deer and cattle in Louisiana.

In November 2004, bluetongue virus (family Reoviridae, genus Orbivirus, BTV) serotype 1 (BTV-1) was detected for the first time in the United States from a hunter-killed deer in St. Mary Parish, LA. In 2005, sera surveys were conducted on three cattle farms near the area where the deer was found, and BTV-1-seropositive cattle were found on two of the three farms; in 2006, sera surveys from the cattle on the three farms did not detect any BTV-1-positive animals. The purpose of this study was to survey ceratopogonid populations at the three farms and test field-collected specimens for the presence of BTV and epizootic hemorrhagic disease virus (family Reoviridae, genus Orbivirus, EHDV). Miniature CDC light traps and New Jersey traps were used to capture ceratopogonids on the three farms from January 2006 through November 2007. In total, 3,319 ceratopogonids were captured, including 1,790 specimens of 10 different species of Culicoides. IR-RT-polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in 264 pools representing 2,309 specimens collected at the farms. All positive samples were sequenced for serotype determination. Five pools of 275 (1.8%) were positive for BTV. Pools of four species of Culicoides were found to be positive: Culicoides crepuscularis (Malloch), Culicoides debilipalpis Lutz (two pools), Culicoides haematopotus Malloch, and Gulicoidesfurens (Poey). The amplicons of the positive specimens were sequenced and found to be identical to both BTV-17 and BTV-13. During our study, no BTV-1 transmission was detected in cattle, and no BTV-1 was detected in specimens of ceratopogonids.

Bilateral versus unilateral cochlear implantation in young children.

OBJECTIVES: To compare the preverbal communication skills of two groups of young implanted children: those with unilateral implantation and those with bilateral implantation. MATERIAL AND METHODS: The study assessed 69 children: 42 unilaterally and 27 bilaterally implanted with age at implantation less than 3 years. The preverbal skills of these children were measured before and 1 year after implantation, using Tait Video Analysis that has been found able to predict later speech outcomes in young implanted children. RESULTS: Before implantation there was no significant difference between the unilateral group and the bilateral group. There was still no difference at 12 months following implantation where vocal autonomy is concerned, but a strongly significant difference between the groups for vocal turn-taking and non-looking vocal turns, the bilateral group outperforming the unilateral group. Regarding gestural turn-taking and gestural autonomy, there was a strongly significant difference between the two groups at the 12 month interval, and also a difference before implantation for gestural autonomy, the unilateral group having the higher scores. Multiple regression of non-looking vocal turns revealed that 1 year following implantation, bilateral implantation contributed to 51% of the variance (p<0.0001), after controlling for the influence of age at implantation and length of deafness which did not reach statistical significance. CONCLUSIONS: Profoundly deaf bilaterally implanted children are significantly more likely to use vocalisation to communicate, and to use audition when interacting vocally with an adult, compared with unilaterally implanted children. These results are independent of age at implantation and length of deafness.

Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.

We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.

Distribution of bluetongue in the United States of America, 1991-2002.

Bluetongue virus (BTV) distribution in the United States of America (USA) is limited by the range of the vector Culicoides spp. Regional differences exist with the north-eastern states being free of BTV, while the central and north-western states are seasonally free of virus. Activity of the virus can be observed throughout the year in the southern USA. Serological evidence defining the distribution of BTV in selected regions of the USA is gathered regularly through serological surveys conducted on samples from slaughter cattle. From 1991 to 2002, ten serological surveys were completed. Results from Alaska, Hawaii, Michigan, Minnesota, New York, Wisconsin and New England consistently demonstrated a seropositive rate of less than 2%, confirming BTV-free status. Antibody against BTV was sporadically detected in cattle originating from states contiguous to the BTV-free regions. Additional information on BTV distribution in the USA is obtained through identification of BTV or BTV RNA in diagnostic, surveillance and export specimens submitted to the National Veterinary Services Laboratories. Results confirm that BTV serotypes 2, 10, 11, 13 and 17 are present in the USA.

Bluetongue surveillance methods in the United States of America.

Historical surveillance for bluetongue virus (BTV) exposure in the United States of America (USA) has relied on periodical serological surveillance using samples collected from cattle at slaughter. Most of this surveillance has been focused on the north-eastern portion of the USA due to the lack of competent vectors of BTV in this region. For most of the states tested in this region, the prevalence of seropositive animals has been less than 2%. Recently, a study was conducted in north-central USA using sentinel cattle herds. Results of serological testing showed an increasing gradient of exposure from north to south. In addition, detection of Culicoides sonorensis showed a similar gradient with detection in the northern areas being relatively rare. The results of these studies indicate that cattle herds in the northern and north-eastern areas of the USA are likely to be free of BTV.

Equine West Nile encephalitis, United States.

After the 1999 outbreak of West Nile (WN) encephalitis in New York horses, a case definition was developed that specified the clinical signs, coupled with laboratory test results, required to classify cases of WN encephalitis in equines as either probable or confirmed. In 2000, 60 horses from seven states met the criteria for a confirmed case. The cumulative experience from clinical observations and diagnostic testing during the 1999 and 2000 outbreaks of WN encephalitis in horses will contribute to further refinement of diagnostic criteria.

Detection of North American West Nile virus in animal tissue by a reverse transcription-nested polymerase chain reaction assay.

A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues.

West Nile virus outbreak among horses in New York State, 1999 and 2000.

West Nile (WN) virus was identified in the Western Hemisphere in 1999. Along with human encephalitis cases, 20 equine cases of WN virus were detected in 1999 and 23 equine cases in 2000 in New York. During both years, the equine cases occurred after human cases in New York had been identified.

Foxtail-induced ulcerative stomatitis outbreak in a Missouri stable.

Twenty of 25 horses in a well-managed Missouri boarding stable were diagnosed with gingivitis/stomatitis. Gross examination of the affected horses revealed varying degrees of gingivitis ranging from mild periodontal swelling to marked swelling and erythema with ulceration and hemorrhage. Fine hair-like material was embedded within the intensely affected areas. Gingival biopsies from 4 affected horses contained pyogranulomatous inflammation with, in some cases, numerous eosinophils and several grass awns in cross and longitudinal section. Numerous foxtail seed heads were identified in hay samples. Examination of the records revealed that all of the affected horses had been fed the suspect hay, with the exception of 1 horse. Although not deliberately fed the suspect hay, this horse did have access to the hay when turned out into the exercise paddock. The lesions resolved following a change in hay source.

Presence of CD30(+) and CD30L(+) cells in human placenta and soluble CD30 levels in cord blood are independent of maternal atopy.

The intrauterine environment is characterized by a Th2 dominance during pregnancy, a milieu that also promotes atopic allergy. The aim of this study was to compare the presence of CD30, a molecule associated with Th2 related disorders such as atopic allergy, and its ligand (CD30L) in placenta in order to investigate if the placenta environment differs between atopic and non-atopic women. Serum concentrations of soluble CD30 (sCD30) from the mothers and their newborns were also elucidated. There were no differences in the immunohistochemical expression of CD30 and CD30L in placenta from atopic (n=28) compared with non-atopic (n=37) women. CD30 was expressed on the decidual stromal cells alone, while CD30L, previously not described in placenta, was detected on macrophage-like HLA-DR(+) cells throughout the mesenchymal chorionic villi. Serum sCD30 in atopic mothers was significantly elevated compared with serum sCD30 in non-atopic mothers (P< 0.05), while sCD30 levels in cord blood were similar in both groups independently of maternal atopic heredity. We suggest that sCD30 in cord blood and CD30 expression by decidual cells may reflect the Th2 environment surrounding the fetus, and both CD30 and CD30L could have immune regulatory functions in placenta.

Fibronectin is a marker for organ involvement and may reflect the severity of preeclampsia.

OBJECTIVE: We have studied whether plasma fibronectin is related to a rise in blood pressure during normal pregnancy, whether it can be used for the early prediction of preeclampsia, and whether plasma fibronectin is a marker for organ involvement in preeclampsia. STUDY DESIGN: Two hundred twenty-eight healthy pregnant nullipara women were examined prospectively during pregnancy. Analyses of fibronectin in plasma were performed in pregnancy weeks 16, 24, 28, 32, and 36. During the same period, 177 patients with suspected preeclampsia and/or intrauterine growth retardation (IUGR) were tested for plasma fibronectin, mainly in the third trimester. RESULTS: In the normal population of pregnant women (n=222/228), fibronectin levels were 0.35 +/- 0.06 g/L in pregnancy week 16 and 0.43 +/- 0.12 g/L in week 36. These levels showed a positive correlation to blood pressure elevation during pregnancy (r=0.21, p=0.006). The six patients in this group (n=6/228) who later developed preeclampsia had higher fibronectin values 0.42 +/- 0.07 g/L already in week 16 (p=0.023). In the population of women with suspected preeclampsia (preeclampsia, n=129; IUGR alone, n=17; hypertension or proteinuria during pregnancy, n=31), fibronectin values were significantly higher, 0.75 +/- 0.27 g/L than in the normal population. Patients with preeclampsia and laboratory signs of organ involvement (n=56) showed significantly higher fibronectin values (0.85 +/- 0.27 g/L) compared to preeclampsia without organ involvement (n=73) [0.76 +/- 0.22 g/L (p=0.03)]. CONCLUSION: Our data show that fibronectin is related to blood pressure in pregnancy. Fibronectin values in women who develop preeclampsia are elevated already in pregnancy week 16 and were higher in those with laboratory signs of organ involvement.

Decreased expression of thioredoxin and glutaredoxin in placentae from pregnancies with pre-eclampsia and intrauterine growth restriction.

Pre-eclampsia is one of the major contributors to perinatal morbidity. This study was performed to test a hypothesis which suggests that pre-eclampsia is associated with inadequate control by the thioredoxin system and other related reducing systems. Placental tissue from normal pregnancies (NC), severe pre-eclampsia with fetuses small for gestational age (SPE), mild pre-eclampsia with fetuses small for gestational age (MPE) and pregnancies with small fetuses for gestational age without any sign of pre-eclampsia (IUGR) was collected immediately after delivery. The mRNA levels for thioredoxin and glutaredoxin were determined using a solution hybridization method and the distribution of the proteins in a normal placenta was analysed by immunohistochemistry. Results showed that the thioredoxin mRNA level in the SPE group was decreased to one third of the level in the NC group. Also the IUGR group showed a significant decrease. The glutaredoxin mRNA level in the SPE group was one half of that seen in the NC group. There was significant correlation between the mRNA levels for thioredoxin and glutaredoxin, both in the normal and growth restricted pregnancies. We conclude that the thioredoxin and glutaredoxin reducing systems are affected in placenta from pregnancies with pre-eclampsia and/or growth restriction of fetuses, and that the decrease correlates to the severity of the condition.

Termination of second trimester pregnancy with mifepristone and gemeprost. The clinical experience of 197 consecutive cases.

BACKGROUND: Earlier controlled clinical trials have demonstrated that combined treatment with the antiprogestagen, mifepristone and a suitable prostaglandin reduce the induction to abortion time in second trimester abortion. The aim of this study was to describe the results of the 197 consecutive second trimester terminations performed in routine clinical practice at our Department from 1996 to 1998. METHODS: The report is based on 197 consecutive second trimester abortions including live pregnancies and missed abortions, carried out in 192 women. The women were treated with 600 mg mifepristone followed 24 to 48 hours later by 1 mg gemeprost administered every 6 hours four times. If abortion had not occurred, 1 mg gemeprost was administered every 3 hours for the next 12 hours. After expulsion of the fetus a surgical evacuation of the uterus was routinely performed up to 18 weeks gestation and thereafter when needed. The induction to abortion time was defined as the interval between the insertion of the first gemeprost pessary and expulsion of the fetus. RESULTS: The median abortion time was 9.0 (1.4-40.5) hours for primigravidae and 7.2 (0-152.5) hours for multigravidae. The medium number of gemeprost pessaries to induce abortion was two and all except seven women aborted within 24 hours. Significantly more abortions occurred before 6, 7 and 8 hours in multigravidae than among primigravidae. The induction to abortion interval was also significantly shorter for nulliparous than for parous women. Except for one case of heavy bleeding, no serious complications occurred. CONCLUSION: The study confirms the efficacy and safety of mifepristone, together with gemeprost, for termination of second trimester pregnancy when routinely used in the clinic.

Fetal erythropoietin and endothelin-1: relation to hypoxia and intrauterine growth retardation.

BACKGROUND: We have examined whether endothelin-1 (ET-1) and erythropoietin (EPO) in amniotic fluid, and EPO in fetal serum obtained by cordocentesis from fetuses with signs of intrauterine growth retardation (IUGR), were correlated to fetal growth and/or chronic fetal hypoxia. METHODS: Amniotic fluid and fetal serum were obtained by cordocentesis from 28 fetuses suspected to have IUGR and subsequently analyzed for EPO and ET-1 by ELISA. These data were correlated to blood gas results and fetal/maternal parameters at delivery. RESULTS: A novel finding was that ET-1 correlated to PO2 in amniotic fluid. The average level of ET-1 in amniotic fluid was 48.3+/-4.7 pmol/L. The results also showed a correlation between EPO levels in amniotic fluid and EPO in fetal serum. Furthermore, EPO correlated weakly to birth weight at delivery. Children with the lowest birth weights had the highest EPO levels. High EPO values, similarly to ET-1, correlated to low pO2 values. The level of EPO in amniotic fluid was 8.0+/-1.6 mIU/ml and in cord blood 29.5+/-9.6 mIU/ml. CONCLUSIONS: The results indicate that ET-1 levels may be a marker for short-term hypoxia, but not for fetal growth, since ET-1 in amniotic fluid was correlated to PO2 at the time of cordocentesis, but not to birth weight. The results also indicate that EPO levels in amniotic fluid and in fetal cord serum are highly correlated, and thus both can be used as markers for fetal growth and chronic hypoxia before the onset of labor.