An evaluation of regulatory and commercial barriers to stratified medicine development and adoption.

Today, a range of products based on genomics, proteomics and metabolomics have facilitated the development of 'stratified' medicines and companion diagnostics. This investigation profiles a series of targeted medicines and corresponding diagnostics, and their role(s) in supporting evidence-based medicine. Despite their potential benefits we found that scientific, financial and regulatory barriers impede the development and adoption of companion diagnostics. Therefore, in order to realise improvements to the risk/benefit profiles of health-care interventions-notably reducing clinical uncertainty-conferred by the use of companion diagnostics, industry representatives, health-care providers and regulators will need a coordinated response to overcome these barriers.

NMR studies on phospholipid bilayers. Some factors affecting lipid distribution.

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.

Sequence variation of the ribosomal DNA second internal transcribed spacer region in two spatially-distinct populations of Amblyomma americanum (L.) (Acari: Ixodidae).

Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

Computer-controlled olfactometer system for studying behavioral responses of ticks to carbon dioxide.

An olfactometer system that consisted of a laptop computer, a carbon dioxide analyzer, and an apparatus to control gas flows was designed to study ixodid tick behavioral responses to varying carbon dioxide (CO2) concentrations. The system provided a method to regulate and measure stimulatory CO2 concentrations (1-99 ppm) above ambient CO2 background levels. Adult Amblyomma americanum (L.) and Dermacentor variabilis (Say) demonstrated behavioral responses to CO2 concentrations as low as 9 ppm above the mean ambient background. Behavior (questing, initial movement, or activity rate) of adult ticks was observed before and during CO2 stimulation. Between the species, significantly more D. variabilis quested during stimulation, whereas significantly more A. americanum moved. Questing behavior between the sexes for either species was not significantly different. Initial movement for male A. americanum was significantly greater than for females. Activity rate was greater in A. americanum than in D. variabilis and significantly greater in male D. variabilis than in females.

Fecundity of partially engorged female Dermacentor albipictus (Acari: Ixodidae) removed by cattle grooming.

Eight Hereford heifer calves were infested 28 times over a 64-d period with approximately 49,560 Dermacentor albipictus (Packard) larvae per calf. The fecundity of partially engorged and small, fully engorged female ticks, prematurely removed by grooming behavior of the calves, were compared. A total of 3,532 females of various engorged weights was collected from all calves, resulting in a mean female tick yield of 1.78% based on the number of larvae used for all infestations. The ratio of partially to fully engorged females was 1:16. Female oviposition success was 96%. The mean (+/- SE) values for female weight, number of larvae, and percentage larval eclosion of 50 partially engorged females that ranged in weight from 54 to 397 mg was 174 +/- 99, 1,272 +/- 931 and 85 +/- 14, respectively. The correlation between preoviposition weight and number of larvae from partially engorged females was highly significant (r = 0.95, P less than 0.0001). A prediction model (Y = -354.6 + 9.07X) showed that the preoviposition weight of partially engorged females and larval fecundity from a separate study was highly correlated (r = 0.92, P less than 0.0001). There was no significant difference between mean values of larval numbers and percentage eclosion for partially engorged and small, fully engorged females of similar weight.

Occurrence of the Gulf Coast tick (Acari: Ixodidae) on wild and domestic mammals in north-central Oklahoma.

Parasitic life stages of Amblyomma maculatum Koch were collected from domestic cattle and several species of wild mammals during a 3.5-yr study (May 1998-October 2001) in north-central Oklahoma. Adult ticks were the predominant life stage collected from cattle, white-tailed deer, coyotes, and raccoons, whereas only immature ticks were collected from cotton rats and white-footed mice. The prevalence of adult A. maculatum on white-tailed deer (n = 15) examined in June, July, and August 1998 was 80, 100, 100%, respectively. The prevalence of adult A. maculatum on cattle (n = 84) ranged from 52% in February 1999 to 100% in May 1999. The prevalence of adult A. maculatum on coyotes (n = 16) was 100% in April 1998 and 43% on coyotes (n = 7) examined in January 2001. The prevalence of adult A. maculatum on raccoons (n = 23) examined during May, June, and July 1999 was 13%. No A. maculatum of any life stage were recovered from opossums (n = 7). Nine hundred forty-five rodents were trapped over 294 trap-nights; prevalence of A. maculatum larvae and nymphs on cotton rats (n = 395) was 34 and 15%, respectively, whereas on white-footed mice (n = 517), prevalence was 1.5 and 1.4%, respectively. No A. maculatum were recovered from pack rats (n = 33). There were significant differences (P = 0.0001) in larval infestation prevalence between cotton rats and white-footed mice in the spring, summer, and fall and for nymphs in the spring and summer. Results of A. maculatum parasitism and seasonal occurrence on hosts in this study are compared with previous research conducted in Oklahoma and with collection records of A. maculatum in the Entomology Museum at Oklahoma State University.

Isolation of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) from Ixodes scapularis and Dermacentor albipictus ticks (Acari: Ixodidae) in Oklahoma.

Borrelia burgdorferi was isolated from Ixodes scapularis Say and Dermacentor albipictus Packard that were removed as partially fed adults from white-tailed deer, Odocoileus virginianus Zimmermann, in Oklahoma. Isolation in media was accomplished only after homogenates of pooled field-collected ticks were inoculated into laboratory-reared Peromyscus leucopus and reisolated from the urinary bladder into BSK II media. Both isolates were confirmed by western blot analysis and reactivity with monoclonal antibody H5332. These are the first reported isolates of B. burgdorferi from Oklahoma from these two tick species and are the first isolates from ticks from the south-central United States that were infective for laboratory-reared P. leucopus.

Attempted transmission of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) (JDI strain) by Ixodes scapularis (Acari: Ixodidae), Dermacentor variabilis, and Amblyomma americanum.

Laboratory-reared Ixodes scapularis Say, Amblyomma americanum (L.), and Dermacentor variabilis (Say) were fed on New Zealand white rabbits experimentally infected with Borrelia burgdorferi (JDI strain). At repletion, spirochetes could be detected by dark-field microscopy only in I. scapularis. Acquisition rates were 18 and 21%. When previously exposed nymphs of each species were fed on susceptible rabbits, I. scapularis was the only tick of the three species that transmitted B. burgdorferi. When a single rabbit was experimentally infected with B. burgdorferi and infested at 7-d intervals with I. scapularis, A. americanum, D. variabilis, and a second time with I. scapularis, B. burgdorferi was detected again only in cultures from the two groups of I. scapularis. When molted nymphs from each tick species were allowed to feed on susceptible rabbits, spirochetes again were isolated only at necropsy from the rabbits on which the two groups of I. scapularis fed.

Failure to transmit Ehrlichia canis (Rickettsiales: Ehrlichieae) with Otobius megnini (Acari: Argasidae).

An ear tick, Otobius megnini (Dugès) recovered from a child who had serologic evidence of ehrlichiosis, was examined for Ehrlichia species microscopically and by inoculation into a susceptible dog; no evidence of infection was found in the tick. Experimental transmission of E. canis by laboratory-reared O. megnini was attempted; neither transstadial nor transovarial transmission occurred.

Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae).

Ehrlichia chaffeensis Anderson, Dawson & Wilson, causative agent of human (predominantly monocytic) ehrlichiosis, was successfully transmitted experimentally by Amblyomma americanum (L.) to white-tailed deer, Odocoileus virginianus (Zimmerman). Deer were needle-exposed intravenously to E. chaffeensis in tissue-culture canine macrophage (DH82) cells, and 11 d later were exposed to laboratory-reared A. americanum larvae, nymphs, and adults for acquisition feeding. Three months after this feeding, naive deer and dogs were exposed to recently molted nymphs and adults. Attempted reisolation of the pathogen by way of tissue culture was successful from one needle-exposed deer but not from the tick-exposed deer or dogs. Based on serologic evidence and polymerase chain reaction data, both nymphal and adult ticks transmitted E. chaffeensis to naive deer but not to dogs.

Bovine resistance to Amblyomma americanum ticks: an acquired immune response characterized by cutaneous basophil infiltrates.

Purebred Holstein calves acquired resistance to Amblyomma americanum adult ticks after 1 infestation and expressed resistance during a challenge infestation 26 days later. Tick yields from resistant animals were normal, but mean tick weight and egg-mass weight were reduced significantly (31 and 32%, respectively). Cutaneous cellular responses at tick feeding sites in primary (naive) hosts consisted almost exclusively of mononuclear cells, until 24-h post-infestation when granulocytes appeared. At 48 h, basophils were the most abundant granulocyte and comprised 19% of the total cellular response. Secondary and tertiary hosts expressed significant cutaneous basophil responses as early as 6 h post-tick infestation, indicative of an anamnestic response. Cutaneous basophil levels in tertiary hosts (16-34% of the infiltrate) were increased by 2-fold over secondary hosts (12-16% of the infiltrate). Neutrophils and eosinophils were fairly abundant in secondary hosts (22-33%, and 3-13%, respectively), but decreased significantly in tertiary hosts corresponding to increased basophil levels. Mast cells comprised less than 10% of the cells found in primary hosts, and less than 5% in secondary and tertiary hosts representing a consistent, but insignificant component of the total cells. Basophils were not observed in the blood of hosts during any tick infestation, but a peripheral blood basophilia (0.1-1.1%) marked the end of each tick feeding period. A neutropenia was observed in the blood of tertiary hosts corresponding to a decreased tissue neutrophil response. Blood eosinophil, lymphocyte and monocyte levels remained unchanged during each tick infestation compared to controls. In summary, calves acquired immunity to A. americanum ticks, resulting in significant decreases in feeding and ovipositional success associated with a local cutaneous basophil response and peripheral blood basophilia. These findings suggest that basophils are involved in bovine immunity to ticks as has been established in the guinea pig model.

Attempted transmission of human granulocytotropic Ehrlichia (HGE) by Amblyomma americanum and Amblyomma maculatum.

Transstadial transmission of human granulocytotrophic Ehrlichia (HGE) was attempted in dogs using Amblyomma americanum (L.) and A. maculatum Koch, two species that, as adults, feed readily on human beings. Larvae and nymphs were acquisition-fed on a dog that was parasitemic with HGE. Two months later, following digestion of the blood meal and subsequent molting to nymphal or adult stage, these ticks were fed to repletion on HGE-naive dogs. None of the dogs developed clinical evidence of ehrlichiosis. Parasites were not observed in blood smears by light microscopy, HGE DNA was not detected by polymerase chain reaction, and none of the dogs seroconverted. Based on this trial, we conclude that, unlike E. chaffeensis, HGE is probably not transmitted from dog to dog by either A. americanum or A. maculatum.

Experimental transmission of Ehrlichia canis (Rickettsiales: Ehrlichieae) by Dermacentor variabilis (Acari: Ixodidae).

Four trials were conducted in which laboratory-reared Dermacentor variabilis nymphs were exposed to Ehrlichia canis by feeding on experimentally infected dogs as soon as classical morulae were detected in peripheral blood monocytes. After molting 25, 50 or 90 adult tick pairs were permitted to feed on 7 Ehrlichia-naive dogs. Transmission occurred in trials 1 (1/1 dog), 3 (1/1 dog) and 4 (2/2 dogs) but not in trial 2 (0/3 dogs), with 4 of 7 dogs becoming infected. Successful transstadial transmission was demonstrated by detection of morulae in peripheral blood lymphocytes and by seroconversion to Ehrlichia canis 30 d post-exposure. Incubation periods ranged between 17 and 22 days (mean = 19). Clinical signs, typical of ehrlichiosis, included mucopurulent ocular discharge, lymphadenopathy and malaise with accompanying pyrexia, leukopenia and thrombocytopenia. Pyrexia, thrombocytopenia and erythrophagocytosis and vacuolization of the cytoplasm of monocytic cells were observed 1-4 d prior to detection of morulae. This is the first demonstration that a tick other than Rhipicephalus sanguineus is capable of transstadial transmission of this important pathogen of dogs.

Experimental transmission of a granulocytic form of the tribe Ehrlichieae by Dermacentor variabilis and Amblyomma americanum to dogs.

Transstadial transmission of granulocytic Ehrlichieae in dogs was attempted using ticks, Amblyomma americanum and Dermacentor variabilis. Ticks were exposed by feeding as nymphs on acutely infected pups; adult ticks then fed to repletion on susceptible adult dogs that were monitored daily for signs of infection. Evidence of transmission was not observed in control dogs or in those exposed to D variabilis. In contrast, dogs exposed to A americanum developed serologic or clinical evidence of infection, but organisms were not seen in blood smears until corticosteroids were administered, causing recrudescence and accompanying parasitemia. At 12 days after subinoculation of blood obtained from donor adult dogs, before corticosteroids were administered, a febrile response, thrombocytopenia, and appearance of morulae in neutrophilic granulocytes were observed in 2 susceptible recipient dogs.

Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture.

OBJECTIVES: To compare the transmissibility by the brown dog tick, Rhipicephalus sanguineus, of a recent isolate of Ehrlichia canis (Ebony) with that of another isolate (Oklahoma) that had been passaged in cell culture, and to assess the genetic similarity of the 2 isolates as reflected in the nucleotide (NT) sequence of 16S rDNA. ANIMALS: 13 healthy dogs of various ages and breeds. PROCEDURE: Larval and nymphal ticks were acquisition fed on acutely infected dogs, and, after molting, they were transmission fed as nymphs and adults, respectively, on Ehrlichia-naive dogs. All dogs were monitored daily by blood smear evaluation for evidence of parasitized leukocytes and by physical examination for clinical signs of ehrlichiosis. Serologic and hematologic values were measured weekly. Using a nested polymerase chain reaction, the 16S rDNA was amplified, and the NT sequence of the template DNA was determined. RESULTS: The Ebony isolate of E canis was successfully transmitted to dogs by nymphal and adult ticks. In contrast, no ticks that fed on dogs harboring the cell-cultured isolate (Oklahoma) transmitted it to dogs. On the basis of 16S rDNA sequence, the 2 isolates were 99.9% similar, with only 1 NT difference. CONCLUSIONS: These results reconfirm the vector potential of R sanguineus for E canis. Passage of the Oklahoma isolate of E canis in cell culture apparently adversely affected its transmissibility by ticks, raising the possibility that cell-cultured isolates of this rickettsia may lose their affinity for ticks. Determination of 16S rDNA sequence suggests minor strain variation within the species E canis.

Isolation of Borrelia burgdorferi from Peromyscus leucopus in Oklahoma.

Borrelia burgdorferi was isolated from a field-caught Peromyscus leucopus from central Oklahoma (USA). The strain was identified as B. burgdorferi by reaction with monoclonal antibody H5332 specific for the outer surface protein OspA of B. burgdorferi. This represents the first isolation of B. burgdorferi from a wild mouse outside of the normal range of the known vectors Ixodes dammini and I. pacificus.

Naturally occurring hepatozoonosis in coyotes from Oklahoma.

Nine of 16 free-ranging coyotes (Canis latrans) from central Oklahoma (USA) had naturally acquired infections of Hepatozoon americanum. Infections were confirmed by recognition of tissue stages closely resembling H. americanum in skeletal and cardiac muscle. At the time coyotes were collected they were infested with a variety of ticks, including adult Gulf Coast ticks (Amblyomma maculatum). We propose that the high prevalence of H. americanum in this small sample of free-ranging coyotes and the ability of these same animals to harbor adult populations of A. maculatum is an important component of the epizootiology of canine hepatozoonosis in North America.

Transmission of Babesia odocoilei in white-tailed deer (Odocoileus virginianus) by Ixodes scapularis (Acari: Ixodidae).

Laboratory reared Ixodes scapularis proved to be an efficient vector of Babesia odocoilei Emerson and Wright between white-tailed deer (Odocoileus virginianus). Transtadial survival of the babesia occurred between nymph and adult stages of the tick, and the adult stage transmitted the babesia.

Muscular sarcocystosis in coyotes from Oklahoma.

In a recent survey in Oklahoma (USA), 52 free-ranging coyotes were examined for the presence of sarcocysts. Two of these coyotes were found infected with sarcocysts in skeletal muscle. By light microscopy, the cyst wall was thin and smooth. Ultrastructurally, the cyst wall had minute villar protrusions. The sarcocysts were 14.4 to 50.4 microm wide and 46.8 to 99 microm long. This is the first report of Sarcocystis sp. sarcocysts in the skeletal muscle of coyotes.

Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma.

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.