Development of short-target primers for species identification in biological studies of Carnivora.

Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling-based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high-performance short-target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short-target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short-target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.

Intra-amniotic infection and sterile intra-amniotic inflammation in women with preterm labor with intact membranes are associated with a higher rate of Ureaplasma species DNA presence in the cervical fluid.

OBJECTIVE: To determine the prevalence of Ureaplasma spp. DNA and its load in the cervical fluid in women with preterm labor with intact membranes (PTL) complicated by intra-amniotic infection (the presence of both microbial invasion of the amniotic cavity and intra-amniotic inflammation) or sterile intra-amniotic inflammation (the presence of intra-amniotic inflammation alone). METHODS: Overall, 115 women with singleton pregnancies complicated by PTL between gestational ages of 22 + 0 and 34 + 6 weeks were included in this study. Paired amniotic and cervical fluid samples were collected at the time of admission via transabdominal amniocentesis using a Dacron polyester swab. Microbial invasion of the amniotic cavity was diagnosed based on a combination of culture and molecular biology methods. Intra-amniotic inflammation was determined based on the concentration of interleukin-6 in the amniotic fluid. Bacterial and Ureaplasma spp. DNA loads were assessed in the cervical fluid using PCR. RESULTS: Intra-amniotic infection and sterile inflammation were identified in 14% (16/115) and 25% (29/115) of the women, respectively. Ureaplasma spp. DNA in the cervical fluid was identified in 51% (59/115) of women. The presence of Ureaplasma spp. DNA in the cervical fluid was higher in women with intra-amniotic infection (75% (12/16)) and sterile intra-amniotic inflammation (76% (22/29)) than in women without intra-amniotic inflammation (36% (25/70); p = .0002). Concurrent presence of Ureaplasma spp. and Mycoplasma hominis DNA was higher in women with intra-amniotic infection (42% (5/12)) than women with sterile intra-amniotic inflammation (7% (2/29)) and women without intra-amniotic inflammation (7% (5/70); p = .001). There were no differences in the load of Ureaplasma spp. DNA in the cervical fluid among women with intra-amniotic infection, sterile intra-amniotic inflammation, and those without intra-amniotic inflammation (median values; infection: 1.2 × 104 copies DNA/mL; sterile: 5.0 × 105 copies DNA/mL; without: 8.4 × 104 copies DNA/mL; p = .18). CONCLUSIONS: In PTL , both forms of intra-amniotic inflammation were associated with a higher prevalence of Ureaplasma spp. DNA in the cervical fluid. The presence of intra-amniotic infection was related to a higher rate of concurrent Ureaplasma spp. and M. hominis DNA in the cervical fluid.

Intra-amniotic infection and sterile intra-amniotic inflammation are associated with elevated concentrations of cervical fluid interleukin-6 in women with spontaneous preterm labor with intact membranes.

OBJECTIVE: To determine the concentration of interleukin-6 (IL-6) in the cervical fluid in women with spontaneous preterm labor with intact fetal membranes (PTL) complicated by intra-amniotic infection (the presence of both microbial invasion of the amniotic cavity and intra-amniotic inflammation), or sterile intra-amniotic inflammation (the presence of intra-amniotic inflammation alone). METHODS: Eighty women with singleton pregnancies complicated by PTL between gestational ages 22 + 0 and 34 + 6 weeks were included in this retrospective cohort study. Samples of amniotic and cervical fluids were collected at the time of admission. Amniotic fluid samples were obtained via transabdominal amniocentesis, and cervical fluid was obtained using a Dacron polyester swab. Microbial invasion of the amniotic cavity was diagnosed based on the combination of culture and molecular biology methods. The concentration of IL-6 in the amniotic and cervical fluids were measured using an automated electrochemiluminescence immunoassay method. Intra-amniotic inflammation was defined as an amniotic fluid IL-6 concentration ≥3000 pg/mL. RESULTS: The presence of intra-amniotic infection and sterile inflammation was identified in 15% (12/80) and 26% (21/80) of the women, respectively. Women with intra-amniotic infection (median: 587 pg/mL; p = .01) and with sterile intra-amniotic inflammation (median: 590 pg/mL; p = .005) had higher concentrations of IL-6 in the cervical fluid than those without intra-amniotic inflammation (intra-amniotic infection: median 587 pg/mL vs. without inflammation, median: 136 pg/mL; p = .01; sterile intra-amniotic inflammation, median: 590 pg/mL vs. without inflammation, p = .005). No differences were found in the concentrations of IL-6 in the cervical fluid between women with intra-amniotic infection and sterile intra-amniotic inflammation (p = .81). CONCLUSION: In pregnancies with PTL, both forms of intra-amniotic inflammation are associated with elevated concentrations of IL-6 in the cervical fluid.

Are shed hair genomes the most effective noninvasive resource for estimating relationships in the wild?

Knowledge of relationships in wild populations is critical for better understanding mating systems and inbreeding scenarios to inform conservation strategies for endangered species. To delineate pedigrees in wild populations, study genetic connectivity, study genotype-phenotype associations, trace individuals, or track wildlife trade, many identified individuals need to be genotyped at thousands of loci, mostly from noninvasive samples. This requires us to (a) identify the most common noninvasive sample available from identified individuals, (b) assess the ability to acquire genome-wide data from such samples, and (c) evaluate the quality of such genome-wide data, and its ability to reconstruct relationships between animals within a population.We followed identified individuals from a wild endangered tiger population and found that shed hair samples were the most common compared to scat samples, opportunistically found carcasses, and opportunistic invasive samples. We extracted DNA from these samples, prepared whole genome sequencing libraries, and sequenced genomes from these.Whole genome sequencing methods resulted in between 25%-98% of the genome sequenced for five such samples. Exploratory population genetic analyses revealed that these data were free of holistic biases and could recover expected population structure and relatedness. Mitochondrial genomes recovered matrilineages in accordance with long-term monitoring data. Even with just five samples, we were able to uncover the matrilineage for three individuals with unknown ancestry.In summary, we demonstrated that noninvasive shed hair samples yield adequate quality and quantity of DNA in conjunction with sensitive library preparation methods, and provide reliable data from hundreds of thousands of SNPs across the genome. This makes shed hair an ideal noninvasive resource for studying individual-based genetics of elusive endangered species in the wild.

Molecular detection, isolation and characterization of Peste-des-petits ruminants virus from goat milk from outbreaks in Bangladesh and its implication for eradication strategy.

Peste-des-petits ruminants (PPR) is a highly contagious transboundary viral disease of small ruminants, which is endemic in much of Africa, the Middle East and Asia. In South Asia, PPR is of significant concern to the Indian subcontinent including Bangladesh as more than 30% of the world's sheep and goats are farmed in this region, predominantly by small, poor and marginal farmers. PPR virus was detected and isolated from goat milk from field samples from PPR outbreaks (2012-2015) in Bangladesh and its full-length sequences obtained. Sequence analysis of the partial N gene of Bangladesh isolates showed 99.3%-100% identity whereas 98.2%-99.6% identity was observed when compared with neighbouring Indian viruses. Further analysis of the full-length genomes indicated that the Bangladesh isolates were 99.3%-99.99% identical among themselves and 98.3%-98.4% identical to neighbouring Indian viruses. These findings further support the transboundary transmission of PPR virus across the Indian and Bangladesh border. In additional, the establishment of a cross-border strategy between India and Bangladesh will be of paramount importance for the eradication of PPR in this region. Molecular detection and isolation of PPR virus from milk is of significant potential concern for spread of the disease to free areas as the major producers of goat milk globally are PPR endemic countries in particular India and Bangladesh, as well as Sudan. Milk is a noninvasive sample type and bulk goat milk sampling for the detection of PPRV would be of practical significance for regional surveillance of PPRV as progress is made towards the targeted 2030 eradication.

Cervical fluid interleukin 6 and intra-amniotic complications of preterm prelabor rupture of membranes.

OBJECTIVE: To determine if cervical fluid interleukin (IL)-6 concentrations in women with preterm prelabor rupture of membranes (PPROM) allows identification of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). METHODS: One hundred forty-four women with singleton pregnancies complicated by PPROM were included in this prospective cohort study. Cervical and amniotic fluids were collected at the time of admission and concentrations of IL-6 were measured using an ELISA and point-of-care test, respectively. Cervical fluid was obtained using a Dacron polyester swab and amniotic fluid was obtained by transabdominal amniocentesis. MIAC was diagnosed based on a positive PCR result for Ureaplasma species, M. hominis, and/or C. trachomatis and/or by positivity for the 16 S rRNA gene. IAI was defined as amniotic fluid point-of-care IL-6 concentrations ≥745 pg/mL. The women were assigned to four subgroups based on the presence of MIAC and/or IAI: microbial-associated IAI (both MIAC and IAI), sterile IAI (IAI alone), MIAC alone, and without either MIAC or IAI. RESULTS: (1) Women with microbial-associated IAI had higher cervical fluid IL-6 concentrations (median 560 pg/mL) than did women with sterile IAI (median 303 pg/mL; p = .001), women with MIAC alone (median 135 pg/mL; p = .0004), and women without MIAC and IAI (median 180 pg/mL; p = .0001). (2) No differences were found in cervical fluid IL-6 concentrations among women with sterile IAI, with MIAC alone, and without MIAC and IAI. (3) A positive correlation was observed between cervical fluid IL-6 concentrations and the amount of Ureaplasma species in amniotic fluid (copies DNA/mL; rho = 0.57, p < .0001). (4) A weak positive correlation was detected between cervical and amniotic fluid IL-6 concentrations (rho = 0.33, p < .0001). CONCLUSIONS: The presence of microbial-associated IAI is associated with the highest cervical fluid IL-6 concentrations. Cervical IL-6 can be helpful in the identification of microbial-associated IAI.

Infecciones de transmisión sexual silentes: la muestra de orina permite una adecuada detección / Non symptomatic sexually transmitted infections: urine sample allows an adequate detection

Detection of non-symptomatic sexually transmitted infections (NSSTD) has taken great relevance, primarily due to global increase. This has led to implement various laboratory techniques with the aim of early detection of these silent infections to decrease the incidence. Techniques usually used for the detection and identification of NSSTD require invasive samples (blood, citobrush, etc.), so the urine could be a simpler option and noninvasive sample when the patient be subjected to test for some of these infections.

La detección de infecciones de transmisión sexual silentes (ITSS) ha tomado gran relevancia, debido principalmente a su incremento en el mundo. Esto ha llevado a implementar diversas técnicas de laboratorio con la finalidad de la detección precoz de estas infecciones silentes para disminuir su incidencia. Las técnicas que habitualmente se utilizan para la detección e identificación de ITSS requieren de una muestra invasora (sangre, citobrush, entre otras), por lo que la orina podria ser una opción de muestra más simple y no invasora al momento que el paciente se deba someter a un examen para detectar alguna de estas infecciones.