Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy.

BACKGROUND: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. METHODS: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. RESULTS: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. CONCLUSION: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

Presence and Size of Placental Lakes on 20-Week Fetal Anatomy Ultrasound and Obstetrical Outcomes.

Our objective was to determine if placental lake presence or size is associated with adverse pregnancy outcomes. This was a retrospective cohort of patients who had fetal anatomy ultrasounds at 18-22 weeks and delivered between 2018 and 2022. Placental lakes were classified as small (>2.0 to 3.9 cm) or large (≥4 cm). Multiple gestations, placenta previas, and placenta accretas were excluded. Outcomes included low birthweight, cesarean delivery, primary cesarean for non-reassuring fetal heart tracing, fetal growth restriction, preterm birth, and severe preeclampsia. A total of 1052 patients were included; 294 had placental lakes (204 small, 90 large). No differences in pregnancy outcomes were observed.

Comparative analysis of ultrasonographic fetal lung texture in twin and singleton fetuses.

OBJECTIVES: Increased fetal lung heterogeneity has been associated with term fetal lungs in singleton gestations. The objective of this study was to determine if fetal lung heterogeneity index (HI) differs between twin and singleton fetuses in the late second and third trimesters. METHODS: Prospective cohort study of women with singleton and twin gestations with medically-indicated ultrasound examinations at 24 weeks of gestation onward. Grayscale transverse fetal lung images were obtained at the level of the four-chamber heart. A region of interest was selected in each fetal lung image. Fetal lung HI was determined with MATLAB software using a dithering technique with ultrasound image pixels transformed into a binary map form from which a dynamic range value was determined. HI averages and standard deviations were generated for twin and singleton fetuses from 24 weeks gestation onward. Two sample t-tests were used to compare the mean HI at each gestational week between singleton and twin fetuses. RESULTS: In total, 388 singleton and 478 twin images were analyzed. From 35 through 38 weeks of gestation a statistically significant divergence in mean HI was observed with higher means in singleton compared to twin fetuses. At 24 weeks of gestation there was a significantly higher HI in twin fetuses compared to singletons. No differences in fetal lung HI were observed between 25 and 34 weeks gestational age. CONCLUSIONS: Differences in fetal lung HI were observed when comparing twin and singleton fetuses. Further investigation is required to determine the potential clinical significance of these findings.

Preterm prelabor rupture of membranes in singletons: maternal and neonatal outcomes.

OBJECTIVES: To determine the effect of gestational age at delivery on maternal and neonatal outcomes in preterm prelabor rupture of membranes (PPROM) and assess various predictors of neonatal and infant mortality in these pregnancies. METHODS: United States birth data from CDC-National Center for Health Statistics natality database for years 2004-2008 was used to identify singleton pregnancies with PPROM and delivery from 32 0/7 to 36 6/7 weeks. Controls were singletons at 37-40 weeks, without PPROM. Maternal and neonatal complications reported by all states were analyzed along with neonatal outcomes such as chorioamnionitis and hyaline membrane disease, reported by a subgroup of states. OR (95% CI) were calculated after adjusting for preeclampsia, diabetes, chronic hypertension, maternal race, and infant sex. RESULTS: There were 134,502 PPROM cases and similar number of controls. There was a significant decrease in need for prolonged ventilation, hyaline membrane disease, 5 min Apgar score <7, and NICU admission with advancing gestational age. Placental abruption decreased and chorioamnionitis and cord prolapse were not different between 34 and 37 weeks. We found reductions in early death, neonatal death, and infant mortality with advancing gestational age (p<0.001 for each). Gestational age at delivery was the strongest predictor for early death, neonatal death, and infant mortality in PPROM. These differences persisted after adjusting for antenatal steroid use. CONCLUSIONS: We provide population-based evidence showing a decrease in neonatal complications and death with advancing gestational age in PPROM. Gestational age at delivery in pregnancies with PPROM is the strongest predictor of mortality risk.

Cherenkov Radiation in Optical Fibres as a Versatile Machine Protection System in Particle Accelerators.

Machine protection systems in high power particle accelerators are crucial. They can detect, prevent, and respond to events which would otherwise cause damage and significant downtime to accelerator infrastructure. Current systems are often resource heavy and operationally expensive, reacting after an event has begun to cause damage; this leads to facilities only covering certain operational modes and setting lower limits on machine performance. Presented here is a new type of machine protection system based upon optical fibres, which would be complementary to existing systems, elevating existing performance. These fibres are laid along an accelerator beam line in lengths of ∼100 m, providing continuous coverage over this distance. When relativistic particles pass through these fibres, they generate Cherenkov radiation in the optical spectrum. This radiation propagates in both directions along the fibre and can be detected at both ends. A calibration based technique allows the location of the Cherenkov radiation source to be pinpointed to within 0.5 m with a resolution of 1 m. This measurement mechanism, from a single device, has multiple applications within an accelerator facility. These include beam loss location monitoring, RF breakdown prediction, and quench prevention. Detailed here are the application processes and results from measurements, which provide proof of concept for this device for both beam loss monitoring and RF breakdown detection. Furthermore, highlighted are the current challenges for future innovation.

Disparity in Medicare payments by gender and training track in female pelvic medicine and reconstructive surgery.

BACKGROUND: Gender disparities in medicine have been demonstrated in the past, including differences in the attainment of roles in administration and in physician income. OBJECTIVE: Our objective was to determine the differences in Medicare payments based on the provider gender and training track among female pelvic medicine and reconstructive surgeons. STUDY DESIGN: Medicare payments from the Provider Utilization Aggregate Files were used to determine the payments made by Medicare to urogynecologists. This database was merged with the National Provider Identifier registry with information on subspecialty training, years since graduation, and the geographic pricing cost index used for Medicare payment adjustments. Physicians with <90% female patients and those who graduated medical school <7 years ago in obstetrics and gynecology or <8 years ago in urology were excluded. The effects of gender, specialty of training, number of services provided, years of practice, and geographic pricing cost index on physician reimbursement were evaluated using linear mixed modeling. RESULTS: A total of 578 surgeons with female pelvic medicine and reconstructive surgery subspecialty training met the inclusion criteria. Of those, 517 (89%) were trained as gynecologists, whereas 61 (11%) were trained as urologists. Furthermore, 265 (51%) of the gynecology-trained surgeons and 39 (80%) of the urology-trained surgeons were women. Among the urology-trained surgeons, the median female surgeon was paid $85,962 and their male counterparts were paid $121,531 (41% payment difference). In addition, urology-trained female pelvic medicine and reconstructive surgery surgeons performed a median of 1135 services and their male counterparts performed a median of 1793 services (57% volume difference). Similarly, among gynecology-trained surgeons, the median female payment was $59,277 with 880 services performed, whereas male gynecology-trained surgeons received a median of $66,880 with 791 services performed, representing a difference of 12% in payments and 11% in services. With linear mixed modeling, male physicians were paid more than female physicians while controlling for specialty training, number of services performed, years of practice, and geographic pricing cost index (P<.001). CONCLUSION: Although Medicare payments are based on an equation, differences in reimbursement by physician gender exist in female pelvic medicine and reconstructive surgery with female surgeons receiving lower payments from Medicare. The differences in reimbursement could not be solely explained by differences in patient volume, area of practice, or years of experience alone, suggesting that, similar to other fields in medicine, female surgeons in female pelvic medicine and reconstructive surgery are not paid as much as their male counterparts.

Introgression of ivermectin resistance genes into a susceptible Haemonchus contortus strain by multiple backcrossing.

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a "proof-of-concept" of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.

Severe skeletal dysplasia caused by a novel FLNB gene mutation.

A late adolescent primigravida was found to have a fetus with a cystic hygroma and significant shortening of the limbs on first-trimester ultrasound. She underwent chorionic villus sampling with normal microarray result. In the early second trimester, the fetus was found to have the absence of all four limbs and a thorough skeletal dysplasia workup was pursued, identifying a variant in the FLNB gene (c.62C>G). The patient underwent termination of pregnancy. The care of this patient was expedited by first-trimester sonographic evidence of limb abnormalities enabling timely clinical management.

Association of in vitro fertilization with severe maternal morbidity in low-risk patients without comorbidities.

OBJECTIVE: To determine whether an association exists between in vitro fertilization (IVF) and severe maternal morbidity among low-risk pregnant patients. DESIGN: Retrospective cohort study. SETTING: Academic healthcare system. PATIENT(S): Low-risk pregnant patients who delivered between January 2019 and December 2022. Low-risk was defined as having an obstetric comorbidity index score of 0. INTERVENTION(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): The primary outcome (dependent variable) was any severe maternal morbidity. The secondary outcome was the need for a cesarean delivery. A modified Poisson regression with robust error variance was used to model the probability of severe maternal morbidity as a function of IVF. Risk ratios and their associated 95% confidence intervals (CIs) were computed. An α value of 0.05 was considered statistically significant. RESULT(S): A total of 39,668 pregnancies were included for analysis, and 454 (1.1%) were conceived by IVF. The overall severe maternal morbidity rate was 2.4% (n = 949), with the most common indicator being blood transfusion. The overall cesarean delivery rate was 18.8% (n = 7,459). On modified Poisson regression, IVF-conceived pregnancies were associated with 2.56 times the risk of severe maternal morbidity (95% CI, 1.73-3.79) and 1.54 times the risk of having a cesarean delivery (95% CI, 1.37-1.74) compared with non-IVF pregnancies. CONCLUSION(S): In vitro fertilization is associated with higher rates of severe maternal morbidity, primarily the need for a blood transfusion, and cesarean delivery in low-risk pregnancies without major comorbidities. Recognizing this association allows healthcare providers to implement proactive measures for better monitoring and tailored postpartum care.

Immune markers in goats selected for reduced gastrointestinal nematode egg count under artificial infection conditions.

This study aimed to further investigate the mechanisms responsible for inhibition of gastrointestinal nematode egg production in a line of Scottish Cashmere Goat selected for low faecal egg count. Animals were chosen as the lowest egg producers from a line selected for low egg output (selected group) with a second group from the wider herd based on high faecal egg count as controls. All animals were artificially infected with Teladorsagia circumcincta and Trichostrongylus vitrinus, then treated with anthelmintic and challenge infected prior to euthanasia and post mortem sampling. There was no effect on body weight between groups at any point in the study. Mean faecal egg count was reduced by 68 % in the selected group when compared with the controls. Circulating eosinophil counts were consistently elevated in the selected group, but this was only marginally significant (P = 0.045). Most of the circulating and tissue antibodies (IgG, IgA and IgE) measured were slightly elevated in the selected group but not significantly. Mucosal mast cells, eosinophils and globule leukocyte levels were higher in the abomasal and intestinal tissues in selected animals. Following challenge infection there was no difference in numbers of parasites, however there were more early stage parasite larvae and fewer late stage larvae in the both the abomasum and duodenum of the selected group compared with the unselected group, indicating some inhibition of parasite development. Overall, the study further demonstrated that selection based on low egg count has resulted in a line of goats producing significantly fewer parasites under identical infection with no effect on bodyweight. This appears to be associated with elevation in antibody and effector cells. Some evidence of host inhibition of parasite development was observed in the selected animals.

Ethnic Disparities in Coronavirus Disease 2019 after the Implementation of Universal Screening in Hartford, Connecticut.

Objective The aim of this article was to estimate the prevalence of coronavirus disease 2019 (COVID-19) in Connecticut, examine racial/ethnic disparities, and assess pregnancy outcomes in pregnant women following the implementation of universal screening for the virus. Materials and methods This is a retrospective cohort study of all obstetric patients admitted to our labor and delivery unit during the first 4 weeks of implementation of universal screening of COVID-19. Viral studies were performed in all neonates born to mothers with severe acute respiratory syndrome coronavirus 2. We calculated the prevalence of COVID-19, compared the baseline characteristics and pregnancy outcomes between those who tested positive and negative for the virus, and determined the factors associated with COVID-19. Results A total of 10 (4.6%) of 220 women screened positive for the virus. All were asymptomatic. Week 1 had the highest prevalence of infection, nearing 8%. No neonates were infected. Hispanics were more likely to test positive (odds ratio: 10.23; confidence interval: [2.71-49.1], p = 0.001). Obstetric and neonatal outcomes were similar between the groups ( p > 0.05). Conclusion Although the rate of asymptomatic COVID-19 was low, ethnic disparities were present with Hispanics being more likely to have the infection. Key Points 4.6% of pregnant women in labor and delivery tested positive for COVID-19 while being asymptomatic.Hispanic women were more likely to test positive for severe acute respiratory syndrome coronavirus 2.Pregnancy outcomes were similar between COVID-19 positive and negative women.No vertical transmission was detected.

Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus.

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.

Nutritional sensitivity of resistance to Trichostrongylus colubriformis in lactating ewes.

It has been suggested that the periparturient breakdown of immunity to parasites has a nutritional basis. Our overall hypothesis is that it results from a prioritized scarce metabolizable protein (MP) allocation to reproductive functions (e.g. milk production) rather than to immune functions. We have earlier shown that the relationship between MP supply, milk production and resistance to the abomasal nematode Teladorsagia circumcincta provides support for this hypothesis. Because nutritional sensitivity of resistance to parasites may differ between parasite species, we used the same nutritional protocol to test the overall hypothesis with a different parasite. Thus, five levels of dietary MP, ranging from 0.60 to 1.2 times assumed requirements, were offered for 4 weeks post-parturition to twin-rearing Greyface ewes, experimentally infected with the small intestinal nematode Trichostrongylus colubriformis. We hypothesized that the initial increments of MP supply would increase milk production without affecting the degree of breakdown of immunity whilst later increments would reduce the degree of breakdown of immunity. Indeed, the data suggest that the first two increments of MP supply increased milk production, whilst final worm burdens were reduced from the second increment onwards. MP supply did not affect mucosal mast cell concentration but increased globule leukocyte concentration. These results support the overall hypothesis that scarce MP allocation is prioritized to milk production over immune functions. In addition, the contrast between effects of MP supply on resistance to T. colubriformis and to T. circumcincta supports the view that periparturient immunity to small intestinal nematodes may be less sensitive to nutrient scarcity than periparturient immunity to abomasal nematodes.

Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.

Mating barriers between genetically divergent strains of the parasitic nematode Haemonchus contortus suggest incipient speciation.

Haemonchus contortus, in common with many nematode species, has extremely high levels of genetic variation within and between field populations derived from distant geographical locations. MHco10(CAVR), MHco3(ISE) and MHco4(WRS) are genetically divergent H. contortus strains, originally derived from Australia, Kenya and South Africa, respectively, that have been maintained by numerous rounds of in vivo experimental infection of sheep. In order to explore potential pre-zygotic competition or post-zygotic incompatibility between the strains, we have investigated the ability of MHco10(CAVR) to interbreed with either MHco3(ISE) or MHco4(WRS) during dual strain co-infections. Sheep were experimentally co-infected with 4000 infective larvae (L3) per os of the MHco10(CAVR) strain and an equal number of either the MHco3(ISE) or the MHco4(WRS) strain L3. The adult worm establishement rates and the proportions of F1 progeny resulting from intra- and inter-strain mating events were determined by admixture analysis of microsatellite multi-locus genotypes. Although there was no difference in adult worm establishment rates, the proportions of F1 progeny of both the MHco10(CAVR) × MHco3(ISE) and MHco10(CAVR) × MHco4(WRS) dual strain co-infections departed from Mendelian expectations. The proportions of inter-strain hybrid F1 progeny were lower than the expected 50%, suggesting either pre-zygotic competition or post-zygotic incompatibility between the co-infecting strains. To investigate this further, both eggs and hatched L1 of broods from single adult female worms recovered from each dual co-infection were genotyped. Unhatched eggs from the broods revealed no inter-strain hybrid genotype deficit, suggesting there is no pre-zygotic competition between the strains. In contrast, there was a deficit in L1 inter-strain hybrid genotypes in the broods derived from MHco3(ISE) or MHco4(WRS) maternal parents, but not from MHco10(CAVR) maternal parents. This suggests that hybrid progeny of MHco10(CAVR) paternal parents have reduced post-zygotic development and/or viability consistent with incipient speciation of the MHco10(CAVR) strain. The presence of mating barriers between allopatric H. contortus strains has important implications for parasite ecology, including the ability of newly introduced anthelmintic-resistant parasite populations to compete and interbreed with populations already established in a region.

Resources That Improve Medical Board Licensing Examination Performance.

Purpose Examine the factors improving performance on national medical licensing board examinations. Rationale Accreditation Council for Graduate Medical Education (ACGME) accredited residency programs report the United States Medical Licensing Examination (USMLE) Step 1 and Comprehensive Osteopathic Licensing Examination-USA (COMLEX-USA) Level 1 scores as the most important criteria in selecting candidates to interview. Hypotheses (1) Certain resources are superior for exam preparation. (2) Certain practice tests better assess exam preparedness. (3) USMLE performance will correlate with the COMLEX-USA. Methods One-hundred and two (102) medical students were surveyed regarding preparation for and performance on COMLEX-USA Level 1 and USMLE Step 1. Results USMLE-specific question banks were positively correlated with performance on COMLEX-USA Level 1 and USMLE Step 1 while COMLEX-specific question banks showed no correlation. National Board of Medical Examiners (NBME) Comprehensive Basic Science Self Assessment (CBSSA) and National Board of Osteopathic Medical Examiners (NBOME) Comprehensive Osteopathic Medical Self-Assessment Examination (COMSAE) practice examinations were positively correlated with performance on the USMLE Step 1 and the COMLEX-USA Level 1. Scores on USMLE Step 1 and COMLEX-USA Level 1 were highly correlated. Students who took USMLE Step 1 performed better on COMLEX-USA Level 1 than those who did not. Conclusion COMLEX-specific resources may not adequately prepare students for COMLEX-USA Level 1. Students studying for COMLEX-USA Level 1 may benefit by preparing for USMLE Step 1.

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting.

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.

A method for single pair mating in an obligate parasitic nematode.

Parasitic nematode species have extremely high levels of genetic diversity, presenting a number of experimental challenges for genomic and genetic work. Consequently, there is a need to develop inbred laboratory strains with reduced levels of polymorphism. The most efficient approach to inbred line development is single pair mating, but this is challenging for obligate parasites where the adult sexual reproductive stages are inside the host, and thus difficult to experimentally manipulate. This paper describes a successful approach to single pair mating of a parasitic nematode, Haemonchus contortus. The method allows for polyandrous mating behaviour and involves the surgical transplantation of a single adult male worm with multiple immature adult females directly into the sheep abomasum. We used a panel of microsatellite markers to monitor and validate the single pair mating crosses and to ensure that the genotypes of progeny and subsequent filial generations were consistent with those expected from a mating between a single female parent of known genotype and a single male parent of unknown genotype. We have established two inbred lines that both show a significant overall reduction in genetic diversity based on microsatellite genotyping and genome-wide single nucleotide polymorphism. There was an approximately 50% reduction in heterozygous SNP sites across the genome in the MHco3.N1 line compared with the MoHco3(ISE) parental strain. The MHco3.N1 inbred line has subsequently been used to provide DNA template for whole genome sequencing of H. contortus. This work provides proof of concept and methodologies for forward genetic analysis of obligate parasitic nematodes.