Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions.

BACKGROUND: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. OBJECTIVES: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 5' untranslated region (5' UTR)/exon 1 of the tat gene of EIAV. STUDY DESIGN: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RT-PCR (RT-qPCR) along with the AGID test. METHODS: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 5' UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. RESULTS: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. MAIN LIMITATIONS: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. CONCLUSIONS: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or "serologically silent" equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions.

On-site detection of equid alphaherpesvirus 3 in perineal and genital swabs of mares and stallions.

Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species. Analytical sensitivity and specificity of the iiPCR were compared with a qPCR, using a plasmid containing the target region of the EHV3 glycoprotein G gene and an Argentinian EHV3 isolate (E/9283/07 C3A). In order to evaluate the diagnostic performance of the iiPCR, nucleic acids of 85 perineal and genital swabs (PGS) of mares and stallions were extracted by tacoTM mini and tested by both techniques. EHV3 was detected in 46 and 45 of the 85 PGS by the iiPCR and qPCR, respectively. There was almost perfect agreement between the two diagnostic methods (98.82%; 95% CI: 95.03-100%; κ = 0.98). The iiPCR had a limit of detection of 95.00% at 6 genome equivalents per reaction and a detection endpoint for viral DNA comparable to that of the qPCR, and did not react with six non-targeted equine pathogens. The iiPCR represents a sensitive and specific method for the rapid on-site diagnosis of EHV3 infection. Its routinely implementation in breeding facilities, and artificial insemination and embryo transfer centers, will contribute to prevent the dissemination of this venereal, highly contagious disease in horses.

Epidemiology and molecular characterization of chicken anaemia virus from commercial and native chickens in Taiwan.

Chicken infectious anaemia (CIA) is a disease with a highly economic impact in the poultry industry. The infected chickens are characterized by aplastic anaemia and extreme immunosuppression, followed by the increased susceptibility to secondary infectious pathogens and suboptimal immune responses for vaccination. Commercially available CIA vaccines are routinely used in the breeders in Taiwan to protect their progeny with maternal-derived antibodies. However, CIA cases still occur in the field and little is known about the genetic characteristics of Taiwanese chicken anaemia viruses (CAVs). In this study, CAV DNA was detected in 72 of 137 flocks collected during 2010-2015. Among the PCR-positive samples, the coding regions of 51 CAVs were sequenced. Phylogenetic analysis of the VP1 gene revealed that, although most of Taiwanese CAVs belonged to genotypes II and III, some isolates were clustered into a novel genotype (genotype IV). Moreover, a Taiwanese isolate in this novel genotype IV appeared to be derived from a recombination event between genotypes II and III viruses. Five Taiwanese CAV isolates were highly similar to the vaccine strains, 26P4 or Del-Ros. Taken together, these results indicate that the sequences of CAVs in Taiwan are variable, and inter-genotypic recombination had occurred between viruses of different genotypes. Moreover, vaccine-like strains might induce clinical signs of CIA in chickens. Our findings could be useful for understanding the evolution of CAVs and development of a better control strategy for CIA.

Horizontal root fractures in posterior teeth without dental trauma: tooth/root distribution and clinical characteristics.

AIM: To describe the clinical characteristics and radiographic findings of horizontal root fractures (HRF) in posterior teeth without a history of dental trauma. METHODOLOGY: A total 24 patients and 31 HRF cases in 28 posterior teeth were collected from 2006 to 2015. Clinical examinations and radiographic imaging were evaluated. Value of confidence intervals of the proportions was calculated for data presentation. RESULTS: The number of males (54%) was similar to females (46%). The patients were predominantly between 50 and 70 years of age (75%). Most HRF cases were found in nonendodontically treated teeth (79%), without crown and bridge restorations (82%), and maxillary molars (54%). Many roots of maxillary molars had developed HRF, and the probability was nearly equal. Fractured teeth usually presented with periodontal and apical bone loss, and most patients (92%) were diagnosed with full mouth chronic periodontitis. Tooth wear was another common clinical feature amongst these patients. CONCLUSIONS: HRF in posterior teeth without dental trauma occurred mainly in patients aged between 50 and 70, in nonendodontically treated teeth, teeth with attrition but without crown and bridge restorations, maxillary molars and with periodontal and periapical bony destruction. Periodontal condition, occlusal wear and patients' age at diagnosis were the possible related factors. HRF in posterior teeth without dental trauma is a diagnostic challenge and even misdiagnosed. A thorough clinical examination, radiographic analysis and recognition of the clinical characteristics are helpful in the early diagnosis and treatment of HRF.

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

Prostaglandin F(2alpha) stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cells.

AIM: To study prostaglandin F(2alpha) (PGF(2alpha)) receptor expression and downstream signalling in cultured human dental pulp cells and the effect of PGF(2alpha) on the alkaline phosphatase (ALP) activity of dental pulp cells. METHODOLOGY: Human dental pulp cells were cultured and exposed to PGF(2alpha). The expression of PGF(2alpha) (FP) receptors was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The activation of extracellular regulated kinase (ERK) and cAMP responsive element binding protein/activating transcription factor-1 (CREB/ATF-1) signalling was determined by Western blotting. The expression of ALP in pulp cells after exposure to PGF(2alpha) was evaluated by ALP staining and PCR. RESULTS: Dental pulp cells expressed FP receptor mRNA and protein. Exposure to PGF(2alpha) revealed little cytotoxicity to pulp cells. PGF(2alpha) induced both ERK and CREB/ATF-1 phosphorylation in pulp cells. Exposure to PGF(2alpha) (>1 micromol L(-1)) further decreased the ALP activity and mRNA expression. However, U0126 (an inhibitor of MEK1) showed little preventive effect on the decline of ALP activity in dental pulp cells by PGF(2alpha). CONCLUSION: PGF(2alpha) may potentially activate FP receptors leading to ERK/CREB-ATF-1 activation during its production in inflamed dental pulp. PGF(2alpha) attenuated the ALP activity of pulp cells possibly via pathways not solely by MEK/ERK activation. PGF(2alpha) is a contributing factor of pulpal inflammation by regulating the activities of pulp cells.

Comparative cytotoxicity of five root canal sealers on cultured human periodontal ligament fibroblasts.

AIM: To evaluate the cytotoxicity of current root canal sealers to periodontal ligament (PDL) fibroblasts. METHODOLOGY: Five root canal sealers (Canals, Canals-N, Topseal, Sealapex, Tubliseal) were prepared and placed into transwells. After initial setting for 1 h, the transwells with sealers were placed into cultured PDL fibroblasts. They were cultured for further 3 or 18 h. Morphological changes were observed. Cell viability was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay. RESULTS: Marked retraction and death of PDL fibroblasts were observed after exposure to Canals or Topseal for 3 h. A 3-h exposure of PDL fibroblasts to Tubliseal stimulated MTT reduction. Canals-N showed little cytotoxicity even after an exposure of 18 h. CONCLUSION: Canals was the most toxic sealer, followed by Topseal. Sealapex and Tubliseal had comparable and moderate cytotoxicity to PDL fibroblasts, whereas Canals-N showed little cytotoxicity. Exposure to Tubliseal may modulate MTT reduction in PDL fibroblasts. Canals-N had good biocompatibility.

Schwannoma of the auricle.

OBJECTIVES: We report an extremely rare case of schwannoma of the auricle. METHODOLOGY: A case report and review of the world literature concerning schwannoma of the auricle are presented. RESULTS: Schwannoma is a benign, encapsulated, slow-growing neoplasm. Approximately 25-45% of all schwannomas occur in the head and neck, whereas schwannomas of the external ear are extremely uncommon, with only nine cases reported in the English literature since the first report in 1977. In this report, we describe the case of a 47-year-old male patient who presented with a 2-year history of a slow-growing mass in the left auricle, which was managed by wide surgical excision. We believe this case to be the second case of schwannoma of the auricle reported. CONCLUSIONS: To our knowledge, this is the second report in the world literature of auricle schwannoma. Schwannoma should be considered in the evaluation of any tumour of the auricle.

Is planned surgery important in sinonasal inverted papilloma?

OBJECTIVE: To evaluate which clinical characteristics of inverted papilloma (IP) can result easily in misdiagnosis by clinicians and to determine the recurrence rate of inverted papilloma depending on diagnosis confirmation pre-operatively, intra-operatively or post-operatively. PATIENTS AND METHODS: Fifty-one patients were treated for IP and 47 patients attended regular post-operative follow-up appointments for at least one year (mean: 54 months). RESULTS: The patients were assigned to 3 groups: group Ia, in which IP was not confirmed until the post-operative pathology findings; group Ib, in which IP was confirmed during the operation; and group II, in which IP was confirmed before the operation. There were nineteen (37%) patients in group Ia, nine patients (18%) in group Ib and twenty-three patients (45%) in group II. The recurrence rates for groups Ia, Ib and II were 33%, 11% and 5% respectively. CONCLUSIONS: The initial presentation of bilateral sinonasal disease can easily lead the doctor into the misdiagnosis of IP. Bony erosion, remodelling, and a widening of the natural orifice of the sinuses on a CT scan are useful signs indicating IP. The correct diagnosis prior to surgery is a determinant of outcome. The use of frozen sections should, however, improve outcome as well. In this study we highlight the high prevalence and relatively low control rate of misdiagnosed IP (pathology undetermined pre-operatively) and hope to remind clinicians that early recognition of the tumour is most beneficial to patients.

Horizontal/oblique root fractures in the palatal root of maxillary molars with associated periodontal destruction: case reports.

AIM: To report two cases of palatal root fracture in maxillary molars that were successfully managed in the short term by root canal treatment and root amputation. SUMMARY: In the first case, a 48-year-old woman with bony destruction and a deep periodontal pocket on the palatal root of tooth 26 (FDI) underwent root canal treatment. Bleeding into the palatal canal and radiolucent lines over the root suggested a fracture. Further evidence was provided by an electronic apex locator. Subsequent surgery confirmed the presence of a horizontal root fracture and the fractured root was removed. In the second case, a 75-year-old woman presented with pain from the left posterior teeth. Clinical examination revealed an oblique root fracture of tooth 27 palatal roots with abscess formation and a deep periodontal pocket. Palatal root amputation and odontoplasty were performed. This was followed by root canal treatment. Both teeth were preserved in the short term and early healing of these two cases was uneventful. KEY LEARNING POINTS: Horizontal/oblique root fracture of the palatal root in molars is rare. A combination of periodontal and root canal treatment and palatal root amputation may allow short-term preservation of functional teeth.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization.

BACKGROUND AND PURPOSE: Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. EXPERIMENTAL APPROACH: We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. KEY RESULTS: HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. CONCLUSIONS AND IMPLICATIONS: HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

Cytokine-induced prostaglandin E2 production and cyclooxygenase-2 expression in dental pulp cells: downstream calcium signalling via activation of prostaglandin EP receptor.

AIM: To determine whether (i) proinflammatory cytokines stimulate prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX) gene expression in dental pulp cells, and (ii) pulp cells that express different prostaglandin E(2) receptor (EP) isoforms and their activation by PGE(2) leads to downstream Ca(2+) signalling. METHODOLOGY: Cultured human dental pulp cells were exposed to interleukin (IL)-1beta and tumour necrotic factor-alpha (TNF-alpha). The expression of COX-1 and COX-2 was measured with reverse transcriptase-polymerase chain reaction (RT-PCR). The production of PGE(2) was measured using an enzyme-linked immunosorbent assay. Expression of prostaglandin EP receptor isoforms was studied by RT-PCR, whereas fura-2 fluorescence was used to measure calcium mobilization. The Kruskal-Wallis test and Wilcoxon sum rank test with Bonferroni correction were used for statistical analysis. RESULTS: Interleukin-1beta and TNF-alpha stimulate PGE(2) production of human dental pulp cells (P < 0.05). IL-1beta stimulated the COX-2 but not COX-1 mRNA expression. Pulp cells express mainly EP2, EP3 and EP1 receptors as analysed by RT-PCR. PGE(2) (0.25-2 micromol L(-1)) stimulated the Ca(2+) mobilization as indicated by increase in fura-2 fluorescence. CONCLUSIONS: Interleukin-1beta and TNF-alpha may stimulate PGE(2) production in dental pulp cells. Activation of prostaglandin EP receptors in dental pulp cells by PGE(2) may induce Ca(2+) signalling to regulate cellular biological activity during inflammation.

Folliculosebaceous cystic hamartoma arising within a port-wine stain.

A 34-year-old woman presented with 2-year history of a dome-shaped papule on a well-circumscribed, thickened, port-wine stain on the left side of the chin. Squeezing on the port-wine-stain plaque revealed many comedos within dilated follicular orifices. The papule was excised and submitted for histological examination. Histopathological study showed a lobular neoplasm, comprising dilated, cystic pilosebaceous structures surrounded by fibrous stroma, bearing the characteristics of folliculosebaceous cystic hamartoma. The reported case shows that, in addition to the vascular nature, both ectodermal and mesenchymal abnormalities may be involved in port-wine stains.

Cesarean scar pregnancy: issues in management.

OBJECTIVE: To evaluate our experience with the diagnosis and treatment of Cesarean scar pregnancy. METHODS: During a 6-year period, 12 cases of Cesarean scar pregnancy were diagnosed using transvaginal color Doppler sonography and treated conservatively to preserve fertility. Incidence, gestational age, sonographic findings, beta-human chorionic gonadotropin ( beta-hCG) levels, flow profiles of transvaginal color Doppler ultrasound, and methods of treatment were recorded. RESULTS: The incidence of Cesarean scar pregnancy was 1:2216 and its rate was 6.1% in women with an ectopic pregnancy and at least one previous Cesarean section. Gestational age at diagnosis ranged from 5 + 0 to 12 + 4 weeks. The time interval from the last Cesarean section to the diagnosis of Cesarean scar pregnancy ranged from 6 months to 12 years. High-velocity and low-impedance subtrophoblastic flow (resistance index, 0.38) persisted until beta-hCG declined to normal. Patients were treated as follows: transvaginal ultrasound-guided injection of methotrexate into the embryo or gestational sac (n = 3), transabdominal ultrasound-guided injection of methotrexate (n = 2), transabdominal ultrasound-guided injection of methotrexate followed by systemic methotrexate administration (n = 2), systemic methotrexate administration alone (n = 2), dilatation and curettage (n = 2), or local resection of the gestation mass (n = 1). Eleven of the 12 patients preserved their reproductive capacity; the remaining patient, treated by dilatation and curettage, underwent a hysterectomy because of profuse vaginal bleeding. The Cesarean scar mass regressed from 2 months to as long as 1 year after treatment. Uterine rupture occurred in one patient during the following pregnancy at 38 + 3 weeks' gestational age. CONCLUSION: Ultrasound-guided methotrexate injection emerges as the treatment of choice to terminate Cesarean scar pregnancy. Surgical or invasive techniques, including dilatation and curettage are not recommended for Cesarean scar pregnancy due to high morbidity and poor prognosis.

Prenatal three-dimensional ultrasound diagnosis of a camptomelic dysplasia.

We describe a case of camptomelic dysplasia identified prenatally with the assistance of three-dimensional ultrasonography. The typical skeletal dysplasia of camptomelic dysplasia - including anterior bowing of the tibia, with skin dimpling over a convex surface at the point of maximal deformity, and talipes equinovarus - was successfully identified using the techniques of surface-rendering, multiplanar displays and rotated volume data. Three-dimensional ultrasonography allows the diagnosis of camptomelic dysplasia, which has a poor prognosis as it is accompanied by respiratory insufficiency and spinal deformities, to be made without delay and provides information supplementary to that provided by two-dimensional ultrasound for early diagnosis of skeletal dysplasia.

A biomarker for the identification of swine fecal pollution in water, using the STII toxin gene from enterotoxigenic Escherichia coli.

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

Comparison of the outcomes of primary twin pregnancies and twin pregnancies following fetal reduction.

High-order multifetal pregnancy is an inevitable consequence of assisted reproduction; and is associated with an increase in perinatal morbidity and mortality. Fetal reduction appears to be a safe and efficacious method for improving obstetric outcome. We made a retrospective study of study population 54 high multiple pregnancy following assisted reproduction that were reduced to twins at Shin Kong Memorial Hospital, Taipei,Taiwan from September 1992 to March 2000. Our study compares the outcome of multifetal pregnancy reduced to twins with the outcome of primary twin pregnancy. Maternal age, birth weight, gestational age at delivery and the incidence of preterm contractions were the only statistically significant differences between the two groups.

Effects of solvent evaporation rate on the properties of protein-loaded PLLA and PDLLA microspheres fabricated by emulsion-solvent evaporation process.

This paper investigated the effects of the rate of solvent removal by varying the ambient pressure at a fixed temperature on the morphology, particle size, encapsulation efficiency and release pattern of albumin-loaded PLLA and PDLLA microspheres, prepared by the W/O/W emulsion-solvent evaporation process. For PLLA microspheres prepared either with a fast rate of solvent evaporation (FRSE) or a normal rate of solvent evaporation (NRSE) process, the difference in morphology was minor. In contrast, the different processes did affect the morphology of PDLLA microspheres. Large (surface) pores were observed for PDLLA microspheres fabricated with a FRSE process, while a smooth surface was seen in those with a NRSE process. With the FRSE process, both PLLA and PDLLA microspheres showed smaller particle sizes and lower albumin encapsulation efficiencies than those prepared in the NRSE process. PLLA microspheres prepared with the NRSE process had higher drug encapsulation efficiencies than PDLLA ones, but this was not the case for the FRSE process. An initial burst release of albumin was observed for both PLLA and PDLLA microspheres prepared with the NRSE process, while a lesser burst release was seen for those prepared with the FRSE process. In subsequent stages of drug release, PLLA microspheres prepared with the two different processes showed differences, but this was not the case for PDLLA ones.

Fetal meconium peritonitis in single and twin pregnancy. Two cases report.

We present two cases of fetal meconium peritonitis in a single and twin pregnancy, respectively. The first case diagnosis was made at 30 weeks and was confirmed after delivery of the twins by cesarean section at 37 weeks. The second case diagnosis was made at 31 week and was confirmed at 37 weeks. Meconium peritonitis is a rare prenatal complication that results from intrauterine perforation of small bowel with spillage of sterile meconium into peritoneal cavity. We now report two cases of meconium peritonitis diagnosed at 30 and 31 weeks gestation.

A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli.

This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.