Medical Foods: Science, Regulation, and Practical Aspects. Summary of a Workshop.

On August 13-14, 2019, the Healthcare Nutrition Council and the ASN held the Medical Foods Workshop: Science, Regulation, and Practical Aspects. Medical food products help patients manage their disease and improve their quality of life. Yet many hurdles exist to getting patients new products. In this workshop, participants addressed some of these hurdles, with specific emphasis on topics like the statutory term distinctive nutritional requirements, the regulatory term modification of the diet alone, the role of clinical guidelines, the requirement that medical foods be used under medical supervision, and differentiation of foods for special dietary use from medical foods, as well as product innovation and future research. Real-world examples were discussed for intractable epilepsy, diabetes, end-stage renal disease, and inflammatory bowel disease.

Therapeutic challenges in the treatment of systemic inflammatory disease in pregnancy.

BACKGROUND: Granulomatosis with polyangiitis and Behçet's disease can occur during pregnancy and may be treated by ophthalmologists, rheumatologists, and obstetricians. We hypothesized that specialty training would affect the way physicians selected therapy. METHODS: Using an online questionnaire, 209 uveitis specialists, 853 rheumatologists, and 2500 obstetricians were surveyed. Respondents were given clinical vignettes containing a female patient who was contemplating pregnancy or in the first trimester and was diagnosed with granulomatosis with polyangiitis or Behçet's disease. RESULTS: In the patient with granulomatosis with polyangiitis, therapy choice between specialties for biologic versus non-biologic systemic immunosuppressive medications was significantly different for both the non-pregnant and pregnant patient (p < 0.00001, p < 0.00003). In the non-pregnant patient diagnosed with Behçet's disease, the therapy choice between biologic versus non-biologic medications was also significantly different (p < 0.0003). CONCLUSIONS: Specialty training affects how physicians manage granulomatosis with polyangiitis and Behçet's disease. Development of inter-specialty guidelines and treatment plans may improve outcomes, communication, and patient care.

Evaluation of the effect of host immune status on short-term Yersinia pestis infection in fleas with implications for the enzootic host model for maintenance of Y. pestis during interepizootic periods.

Plague, a primarily flea-borne disease caused by Yersinia pestis, is characterized by rapidly spreading epizootics separated by periods of quiescence. Little is known about how and where Y. pestis persists between epizootics. It is commonly proposed, however, that Y pestis is maintained during interepizootic periods in enzootic cycles involving flea vectors and relatively resistant host populations. According to this model, while susceptible individuals serve as infectious sources for feeding fleas and subsequently die of infection, resistant hosts survive infection, develop antibodies to the plague bacterium, and continue to provide bloodmeals to infected fleas. For Y. pestis to persist under this scenario, fleas must remain infected after feeding on hosts carrying antibodies to Y. pestis. Studies of other vector-borne pathogens suggest that host immunity may negatively impact pathogen survival in the vector. Here, we report infection rates and bacterial loads for fleas (both Xenopsylla cheopis (Rothschild) and Oropsylla montana (Baker)) that consumed an infectious bloodmeal and subsequently fed on an immunized or age-matched naive mouse. We demonstrate that neither the proportion of infected fleas nor the bacterial loads in infected fleas were significantly lower within 3 d of feeding on immunized versus naive mice. Our findings thus provide support for one assumption underlying the enzootic host model of interepizootic maintenance of Y. pestis.

Effect of hydration on spontaneous labor outcomes in nulliparous pregnant women: a multicenter randomized controlled trial comparing three methods.

OBJECTIVE: To evaluate the effect of mode and amount of fluid hydration during labor. STUDY DESIGN: The authors conducted a randomized controlled trial of uncomplicated nulliparous women in spontaneous labor at 36 weeks or more gestational age. Women were randomized to receive lactated Ringer solution with 5% dextrose at (1) 125 mL/h intravenously with limited oral intake, (2) 250 mL/h intravenously with limited oral intake, or (3) 25 mL/h intravenously with ad libitum oral intake of clear liquids. Results were analyzed by intent-to-treat analysis. RESULTS: A total of 311 out of 324 women were available for analysis. Groups 1 (n = 105), 2 (n = 105), and 3 (n = 101) above did not differ significantly for mean labor duration (11.6 ± 5.9, 11.4 ± 5.5, and 11.5 ± 5.9 hours, respectively; p = 0.998), proportion of women in labor > 12 hours (all groups 41%; p = 0.998), proportion receiving oxytocin augmentation (59, 60, and 57%, respectively; p = 0.923), or proportion delivered by cesarean (22, 17, and 17%, respectively; p = 0.309). Indications for cesarean were similar between groups. No cases of pulmonary edema, maternal aspiration, or perinatal mortality occurred. CONCLUSION: Although apparently safe, neither increased intravenous hydration nor oral hydration during labor improves labor performance.

Effects of low-temperature flea maintenance on the transmission of Yersinia pestis by Oropsylla montana.

Yersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis maintained in sylvatic foci throughout western North America. Transmission to humans is mediated most commonly by the flea vector Oropsylla montana and occurs predominantly in the southwestern United States. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at ambient temperatures, particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at subambient temperatures affected the transmissibility of Y. pestis by this vector. An infectious blood meal containing a virulent Y. pestis strain (CO96-3188) was given to colony-reared O. montana fleas. Potentially infected fleas were maintained at different temperatures (6°C, 10°C, 15°C, or 23°C). Transmission efficiencies were tested by allowing up to 15 infectious fleas to feed on each of 7 naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21 postinfection (p.i.). Mice were monitored for signs of infection for 21 days after exposure to infectious fleas. Fleas held at 6°C, 10°C, and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures (46.0% at 6°C, 71.4% at 10°C, 66.7% at 15°C) was higher than for fleas maintained at 23°C (25.4%) and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, pooled percent per flea transmission efficiencies for flea cohorts maintained at temperatures of 10°C and 15°C (8.67% and 7.87%, respectively) showed a statistically significant difference in the pooled percent per flea transmission efficiency from fleas maintained at 23°C (1.94%). This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6°C. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions.

Combining real-time polymerase chain reaction using SYBR Green I detection and sequencing to identify vertebrate bloodmeals in fleas.

Programs that aim to control vector-borne zoonotic diseases require information on zoonotic hosts and on the feeding behavior of bridging vectors that are capable of transmitting pathogens from those hosts to humans. Here we describe an assay developed to identify bloodmeals in field-collected cat fleas (Ctenocephalides felis Bouché) to assess this species' potential role as a Yersinia pestis bridging vector in a plague-endemic region of Uganda. Our assay uses a single primer set and SYBR Green I-based real-time polymerase chain reaction to amplify a segment of the 12S mitochondrial ribosomal RNA gene for identification by sequencing. The assay capitalizes on the sensitivity of real-time polymerase chain reaction and the specificity of sequencing and can be used to differentiate vertebrate bloodmeals to the genus or species level without a priori knowledge of the host community. Because real-time assays that detect vertebrate DNA are highly sensitive to human DNA contamination, we analyzed detection in artificially fed and unfed fleas to establish a Ct cutoff that optimized specificity without completely sacrificing sensitivity. Using the established cutoff, our assay detected human, rat, and goat DNA in artificially fed C. felis up to 72 h postfeeding.

Evaluation of the infectiousness to mice of soil contaminated with Yersinia pestis-infected blood.

Plague, an often-fatal zoonotic disease caused by Yersinia pestis, is characterized by epizootic and quiescent periods. How Y. pestis is maintained during inter-epizootic periods is poorly understood, but soil has been implicated as a potential reservoir. Although previous studies have suggested that Y. pestis is able to survive in soil for weeks or months, it is unclear whether or not it is infectious to susceptible hosts. Here we investigate the potential for Y. pestis to infect mice through close contact with contaminated soil under laboratory conditions. In an attempt to approximate the natural conditions under which animals would be exposed to Y. pestis-contaminated soil, mouse cages filled with soil from a plague-endemic region were held at temperature and humidity ranges observed in ground squirrel burrows. These laboratory "burrows" were contaminated with highly bacteremic blood (>10(8) cfu/mL) to simulate the introduction of infectious material from a dying animal during an epizootic. Outbred Swiss-Webster mice with scarified skin patches were held on contaminated soil for 10 days and monitored for signs of illness. Following exposure to contaminated soil, one animal of 104 became infected with Y. pestis. None of the remaining animals seroconverted following a 21-day holding period. Under our experimental conditions, which maximized the likelihood of contact between susceptible mice and contaminated soil, transmission efficiency from soil to mice was 0.96% (95% CI 0.17, 5.25%). This suggests that although transmission of Y. pestis from contaminated soils is possible, it is not likely a major transmission route under natural conditions.

Evaluation and modification of off-host flea collection techniques used in northwest Uganda: laboratory and field studies.

Quantifying the abundance of host-seeking fleas is critical for assessing risk of human exposure to flea-borne disease agents, including Yersinia pestis, the etiological agent of plague. Yet, reliable measures of the efficacy of existing host-seeking flea collection methods are lacking. In this study, we compare the efficacy of passive and active methods for the collection of host-seeking fleas in both the laboratory and human habitations in a plague-endemic region of northwest Uganda. In the laboratory, lighted "Kilonzo" flea traps modified with either blinking lights, the creation of shadows or the generation of carbon dioxide were less efficient at collecting Xenopsylla cheopis Rothchild and Ctenocephalides felis Bouché fleas than an active collection method using white cotton socks or cotton flannel. Passive collection using Kilonzo light traps in the laboratory collected significantly more X. cheopis than C. felis and active collection, using white socks and flannel, collected significantly more C. felis than X. cheopis. In field studies conducted in Uganda, Kilonzo traps using a flashlight were similar in their collection efficacy to Kilonzo traps using kerosene lamps. However, in contrast to laboratory studies, Kilonzo flea traps using flashlights collected a greater number of fleas than swabbing. Within human habitations in Uganda, Kilonzo traps were especially useful for collecting C. felis, the dominant species found in human habitations in this area.

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period.

BACKGROUND: Traditionally, efficient flea-borne transmission of Yersinia pestis, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of Xenopsylla cheopis with a wild type strain of Y. pestis and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using Y. pestis-specific antigen or antibody detection assays on mouse tissues. RESULTS: Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted Y. pestis infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures. CONCLUSIONS: Our results suggest that temperature does not significantly effect the per flea efficiency of Y. pestis transmission by X. cheopis, but that temperature is likely to influence the dynamics of Y. pestis flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether Y. pestis biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.

Biofilm formation is not required for early-phase transmission of Yersinia pestis.

Early-phase transmission (EPT) is a recently described model of plague transmission that explains the rapid spread of disease from flea to mammal host during an epizootic. Unlike the traditional blockage-dependent model of plague transmission, EPT can occur when a flea takes its first blood meal after initially becoming infected by feeding on a bacteraemic host. Blockage of the flea gut results from biofilm formation in the proventriculus, mediated by the gene products found in the haemin storage (hms) locus of the Yersinia pestis chromosome. Although biofilms are required for blockage-dependent transmission, the role of biofilms in EPT has yet to be determined. An artificial feeding system was used to feed Xenopsylla cheopis and Oropsylla montana rat blood spiked with the parental Y. pestis strain KIM5(pCD1)+, two different biofilm-deficient mutants (Delta hmsT, Delta hmsR), or a biofilm-overproducer mutant (Delta hmsP). Infected fleas were then allowed to feed on naïve Swiss Webster mice for 1-4 days after infection, and the mice were monitored for signs of infection. We also determined the bacterial loads of each flea that fed upon naïve mice. Biofilm-defective mutants transmitted from X. cheopis and O. montana as efficiently as the parent strain, whereas the EPT efficiency of fleas fed the biofilm-overproducing strain was significantly less than that of fleas fed either the parent or a biofilm-deficient strain. Fleas infected with a biofilm-deficient strain harboured lower bacterial loads 4 days post-infection than fleas infected with the parent strain. Thus, defects in biofilm formation did not prevent flea-borne transmission of Y. pestis in our EPT model, although biofilm overproduction inhibited efficient EPT. Our results also indicate, however, that biofilms may play a role in infection persistence in the flea.

Climatic predictors of the intra- and inter-annual distributions of plague cases in New Mexico based on 29 years of animal-based surveillance data.

Within the United States, the majority of human plague cases are reported from New Mexico. We describe climatic factors involved in intra- and inter-annual plague dynamics using animal-based surveillance data from that state. Unlike the clear seasonal pattern observed at lower elevations, cases occur randomly throughout the year at higher elevations. Increasing elevation corresponded with delayed mean time in case presentation. Using local meteorological data (previous year mean annual precipitation, total degrees over 27 degrees C 3 years before and maximum winter temperatures 4 years before) we built a time-series model predicting annual case load that explained 75% of the variance in pet cases between years. Moreover, we found a significant correlation with observed annual human cases and predicted pet cases. Because covariates were time-lagged by at least 1 year, intensity of case loads can be predicted in advance of a plague season. Understanding associations between environmental and meteorological factors can be useful for anticipating future disease trends.

Demonstration of early-phase transmission of Yersinia pestis by the mouse flea, Aetheca wagneri (Siphonaptera: Ceratophylidae), and implications for the role of deer mice as enzootic reservoirs.

The role of deer mice and other species of Peromyscus as enzootic reservoirs for plague remains controversial. In this study, we evaluated early-phase vector efficiency of Aetheca wagneri Baker, a common flea species infesting deer mice, to determine the likelihood that Y. pestis could be spread mouse to mouse by this species. We showed that A. wagneri could transmit plague bacteria to laboratory mice as early as 3 d postinfection (p.i.), but transmission efficiency was quite low (1.03%; 95% CI: 0.19-3.34%) 1-4 d p.i. compared with that for the established plague vector Oropsylla montana Baker (10.63%; 95% CI: 4.18-25.91). Using this early-phase transmission efficiency estimate, we determined through parameterization of a simple predictive model that at least 68 A. wagneri per deer mouse would be required to support levels of transmission adequate for enzootic maintenance. Because deer mice typically harbor fewer than three A. wagneri per host, our data do not support the notion of an independent deer mouse--A. wagneri transmission cycle.

Source of host blood affects prevalence of infection and bacterial loads of Yersinia pestis in fleas.

Yersinia pestis, the etiological agent of plague, is transmitted by multiple flea species. Previous studies have reported wide variability in transmission efficiency among competent vectors. However, it is unclear to what extent such variation is explained by methodological differences among studies. To optimize an artificial feeding system where fleas are infected with controlled numbers of Y. pestis under standardized laboratory conditions that could be used to systematically compare vector efficiency, we sought to test the effect of host bloodmeal source on (1) the flea's ability to remain infected with Y. pestis and (2) bacterial loads in fleas. Here, we demonstrate that both prevalence of infection with a virulent strain of Y. pestis (CO96-3188) and bacterial loads in rock squirrel fleas (Oropsylla montana) are affected by host-associated blood factors. The generality of this observation was confirmed by repeating the study using the rat flea (Xenopsylla cheopis) and a commonly used avirulent laboratory strain of Y. pestis (A1122). Implications of the results for rate of spread of Y. pestis in naturally infected host populations are discussed.

Early-phase transmission of Yersinia pestis by cat fleas (Ctenocephalides felis) and their potential role as vectors in a plague-endemic region of Uganda.

In recent decades, the majority of human plague cases (caused by Yersinia pestis) have been reported from Africa. In northwest Uganda, which has had recent plague outbreaks, cat fleas (Ctenocephalides felis) have been reported as the most common fleas in the home environment, which is suspected to be a major exposure site for human plague in this country. In the past, C. felis has been viewed as only a nuisance-biting insect because limited laboratory studies suggested it is incapable of transmitting Y. pestis or is an inefficient vector. Our laboratory study shows that C. felis is a competent vector of plague bacteria, but that efficiency is low compared with another flea species collected in the same area: the oriental rat flea, Xenopsylla cheopis. On the other hand, despite its low vector efficiency, C. felis is the most common flea in human habitations in a plague-endemic region of Uganda (Arua and Nebbi Districts), and occasionally infests potential rodent reservoirs of Y. pestis such as the roof rat (Rattus rattus) or the Nile rat (Arvicanthis niloticus). Plague control programs in this region should remain focused on reducing rat flea populations, although our findings imply that cat fleas should not be ignored by these programs as they could play a significant role as secondary vectors.

Claudin profiling in the mouse during postnatal intestinal development and along the gastrointestinal tract reveals complex expression patterns.

Members of the claudin protein family are key regulators of tight junction selectivity and are implicated in influencing development and cellular differentiation in the intestine and other tissues. The goal of the present study was to profile claudin gene expression and protein location during postnatal development of the mouse jejunum and in the adult mouse gut from duodenum to distal colon as a first step in understanding both normal claudin function and the pathologic implications of altered expression patterns. The relative expression of claudins 1-19 and other tight and adherens junction genes was determined by quantitative RT-PCR from six regions of normal mouse intestine and colon. Immunofluorescent localization was performed for claudins 1-5, 7, 8, 10, 12, 15, and 18. Transcripts for claudins 1-5, 7-13, 17, and 18 were all detected in adult intestine, although their relative abundance differed up to 1000-fold within individual segments. In contrast to the unchanging expression and localization of ZO-1, occludin, and JAM, most claudins were expressed in decreasing or increasing gradients or in more complex patterns along the longitudinal axis of the intestine and the crypt to villus/surface differentiation axis. During neonatal development at days 1, 14, 28, and 90 several claudins showed striking increases or decreases in transcript expression as well as changes in tissue localization along the crypt-villus axis. Claudin-19 was only detected at days 1 and 14. This database provides a resource for investigating regional and developmental differences in permselectivity, crypt to villus/surface differentiation and neoplastic changes along the gut and during postnatal development.

Mineral content changes in bone associated with damage induced by the electron beam.

Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200x embedded, 200x unembedded, and 1000x embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200x, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200x, 1000x data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.

Cement lines of secondary osteons in human bone are not mineral-deficient: new data in a historical perspective.

Using qualitative backscattered electron (BSE) imaging and quantitative energy dispersive X-ray (EDX) spectroscopy, some investigators have concluded that cement (reversal) lines located at the periphery of secondary osteons are poorly mineralized viscous interfaces with respect to surrounding bone. This conclusion contradicts historical observations of apparent highly mineralized (or collagen-deficient) cement lines in microradiographs. Such conclusions, however, may stem from unrecognized artifacts that can occur during scanning electron microscopy. These include specimen degradation due to high-energy beams and the sampling of electron interaction volumes that extend beyond target locations during EDX analysis. This study used quantitative BSE imaging and EDX analysis, each with relatively lower-energy beams, to test the hypothesis that cement lines are poorly mineralized. Undemineralized adult human femoral diaphyses (n = 8) and radial diaphyses (n = 5) were sectioned transversely, embedded in polymethyl methacrylate, and imaged in a scanning electron microscope for BSE and EDX analyses. Unembedded samples were also evaluated. Additional thin embedded samples were stained and evaluated with light microscopy and correlated BSE imaging. BSE analyses showed the consistent presence of a bright line (higher atomic number) coincident with the classical location and description of the cement line. This may represent relative hypermineralization or, alternatively, collagen deficiency with respect to surrounding bone. EDX analyses of cement lines showed either higher Ca content or equivalent Ca content when compared to distant osteonal and interstitial bone. These data reject the hypothesis that cement lines of secondary osteons are poorly mineralized.

Genetic variability among serotype G6 human rotaviruses: identification of a novel lineage isolated in Hungary.

Rotavirus serotype G6 has been demonstrated to be a rare cause of gastroenteritis in man. To date, only a few well characterized strains have been described from Italy, Australia, and the United States. Nucleotide sequencing of G6 VP7 genes shows that these strains belong to two distinct G6 lineages, one for strains of serotype P11[14],G6 (PA169-like strains) and one for strains of serotype P3[9],G6 (PA151-like strains). In this study, we sequenced the VP7 genes and VP8* gene fragments of human rotavirus G6 strains detected in Hungary. Phylogenetic analysis demonstrated that the VP7 genes of Hungarian G6 strains fell into three lineages, represented by a single PA169-like strain, three PA151-like strains, and two novel G6 strains, respectively. The amino acid sequence identity of VP7 was 97.2-100% within each lineage and 92-93.9% between any two lineages. The sequence analysis of VP8* revealed that the single PA169-like Hungarian G6 strain belonged to genotype P[14] and was phylogenetically closely related to P11[14],G6 strains characterized previously. In contrast, the VP8* of PA151-like Hungarian G6 strains clustered in accordance with their VP7 genes representing genetically distinguishable variants of genotype P[9]. This finding raises the possibility that Hungarian genotype P[9],G6 strains might have been generated through independent reassortment events. Serotype G6-specific primers for each human G6 lineage were also developed. The use of these primers in reverse-transcription polymerase chain reaction genotyping may help determine the epidemiological role of G6 strains in humans.

Quantitative trait loci conditioning transovarial transmission of La Crosse virus in the eastern treehole mosquito, Ochlerotatus triseriatus.

Quantitative trait loci (QTL) affecting the ability of the Eastern treehole mosquito, Ochlerotatus triseriatus, to transovarially transmit (TOT) La Crosse virus (LAC) were mapped in an F1 intercross. The Holmen strain of O. triseriatus, previously selected for TOT refractoriness, was crossed to the AIDL strain that had been selected for TOT permissiveness. In P1 and F1 parents and 49 F2 individuals, regions of 10 cDNA loci were analysed with single strand conformation polymorphism (SSCP) analysis to identify and orientate linkage groups. Genotypes were also scored at fifty-six random amplified polymorphic DNA (RAPD)-SSCP loci. Twenty-eight F2 offspring were individually analysed for TOT. Three QTL for TOT were detected with standard interval mapping on chromosomes II and III. Alleles at the three loci contributed additively towards determining the overall TOT rate and cumulatively accounted for approximately 53% of the phenotypic variance in TOT.