Arthropod cell lines in the isolation and propagation of tickborne spiroplasmas

The ability of several continuous tick cell culture lines to support growth of tickborne spiroplasmas (helical, wall-less prokaryotes in the classMollicutes) was assessed. Seven triturates, prepared from pools ofIxodes pacificus ticks naturally infected with theSpiroplasma sp. (group VI) organism, were retrieved from frozen (–70°C) storage and passaged in three distinct tick cell lines, in antibiotic-free tick cell culture medium alone, or in spiroplasma culture medium (SP-4 formulation). Six spiroplasma strains were recovered in the RML-19 cell line fromDermacentor variabilis, and five isolations were made in another cell line (RML-15) from this tick species. None was recovered in aRhipicephalus sanguineus cell line (RML-23), in tick cell culture medium, or in SP-4 broth medium. One of the spiroplasma isolates (Y43) was maintained through four consecutive weekly refeedings of theD. variabilis cell line and for three feedings ofR. sanguineus cells, where numbers of spiroplasmas in cell supernatants reached levels comparable to those obtained in the SP-4 medium.A laboratory-adapted strain (SMCA) ofSpiroplasma mirum, a second helical mollicute of tick origin (the suckling mouse cataract agent), grew in three tick cell lines (RML-15, RML-23, and RML-16 cells fromD. parumapertus), in three mosquito cell lines (fromAedes albopictus, Ae. aegypti, andCulex quinquefasciatus), and in both cell culture medium alone and in SP-4 medium. The organisms survived for 1–2 weeks, but failed to multiply, in cell lines fromC. tritaeniorhynchus, Antheraea eucalypti, orXenopus laevis. Some evidence of cytopathic effect ofS. mirum on tick cell lines was seen, although growth of the organism in mosquito cell cultures was not associated with cell toxicity. The use of arthropod cell lines appears to have value in the primary isolation of arthropod- or insect-derived mollicutes and for the study of cytopathogenicity of these wall-less prokaryotes.     

How Specific are Host-Produced Kairomones to Host-Seeking Ixodid Ticks?

Ixodid ticks respond to host-produced substances (kairomones) that influence the ticks’ host-finding behavior. In the laboratory adult blacklegged ticks, Ixodes scapularis Say, lone star ticks, Amblyomma americanum L., and American dog ticks, Dermacentor variabilis (Say) became akinetic on residues rubbed from their principal hosts (deer for the former two species and dogs for the latter). However, arrestment also occurred when adults of these species were tested using the same method bioassay, but with host substances reversed (i.e., I. scapularis and A. americanum against canine substances, and D. variabilis against deer gland substances). Although adult D. variabilis exhibited arrestant responses to deer substances and are often found along trails used by deer, they apparently make little use of deer as hosts. It is unclear whether responding to deer-produced kairomones may have disadvantages for D. variabilis. Until the active components of host-produced arrestment kairomones are isolated, identified and evaluated in behavioral tests, this host-finding strategy remains only partially understood. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genomic evidence for host-associated differentiation in an animal parasite,Dermacentor variabilis,the American dog tick

The process responsible for the formation of genetically distinct populations associated with different host species is known as host-associated differentiation (HAD). Many insect parasites of plants have been shown to exhibit HAD but there have been fewer studies of HAD in parasites of vertebrate animals. Previous to this study, HAD has been documented in at least three species of ticks. The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae) was chosen as the focal species for this study due to its importance as the vector of tularemia and Rocky Mountain spotted fever. Previous population genetic studies of this tick found the existence of various haplotypes but the tick’s host origins were unknown. In this study, ticks were collected from 15 vertebrate host species to test for HAD using single nuclear polymorphisms (SNPs). In total, 136 individual D. variabilis ticks were sequenced using ddRADseq. Genomic evidence was found to point to D. variabilis exhibiting HAD on eight different hosts. A STRUCTURE analysis showed that the highest posterior probability was obtained with a population size of eight and these populations correlated with host species. Pairwise F_ST values were as high as 0.622 and indicated a range of genetic distinction between host groups. In addition, ticks collected from the vegetation appeared as one homogenous distinct genotype suggesting the existence of nidicolous (nest dwelling) and non-nidicolous genotypes. The identification of host race formation occurring in this animal parasite has implications for the understanding of D. variabilis pathogen transmission and targeted control efforts because genetically distinct populations can differ in traits relevant to these applications.     

Allomonal defence secretions of the American dog tick Dermacentor variabilis (Acari: Ixodidae) promote clustering

The release of the defence secretion from the large wax glands (sensilla sagittiformia) of Dermacentor variabilis ticks modifies the behaviour of other ticks by inducing clustering. A coating of natural tick secretion onto test objects (delipidized ticks, glass beads or filter paper discs) also elicits a clustering response, but a topically applied squalene, its major component, does not have this effect. The clustering response appears to be species specific: D. variabilis cluster on conspecific ticks that secreted but they fail to cluster on Amblyomma americanum or Ixodes scapularis ticks unless coated with secretions from D. variabilis. Volatile components in the defence secretion are involved in recruiting conspecific ticks to those that secreted. When attacked by predatory fire ants, Solenopsis invicta, D. variabilis clustered on individuals that had released the defence secretion. This suggests that the secretion protects ticks from predation by functioning as an alarm pheromone. If confirmed, this is the first report of an alarm pheromone and its glandular source in ticks. The terminology applicable to the integumental glands of ticks is discussed.     

Surface lectin binding toAnabaena variabilis and to cultured and freshly isolatedAnabaena azollae

Five fluorescein isothiocyanate (FITC)-labeled lectins and Calcofluor white ST were tested for their binding abilities to vegetative cells and heterocysts of culturedAnabaena variabilis (AVA) and toA. azollae from four species ofAzolla; toAnabaena azollae freshly isolated fromAzolla pinnata (AP),A. caroliniana (AC),A. mexicana (AX), andA. filiculoides (AF); and to cultured akinetes ofAnabaena variabilis and four isolates ofA. azollae. Heterocysts of cultured cells of threeAnabaena isolates (APC, ACC, AXC) were most intensively surface-stained with soybean agglutinin fromGlycine max (SBA)-FITC; those of AX and AC were dimly stained with wheat germ agglutinin fromTriticum vulgaris (WGA); and only heterocysts of AX were dimly stained withDolichos biflorus agglutinin (DBA). Akinetes of cultured cells stained only with ConA. Vegetative cells and heterocysts of all four fresh isolates stained with Jack Beam aglutinin fromCanavalia ensiformis (ConA). None of the cell types were stained with either peanut agglutinin fromArachis hypogea (PNA) or Calcofluor.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Genetic variation in the mitochondrial 16S rRNA gene of the American dog tick,Dermacentor variabilis (Acari: Ixodidae)

Genetic variation in the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene was examined for the American dog tick, Dermacentor variabilis (Say, 1821). Nine different haplotypes were detected among 369 adult D. variabilis collected from four localities in Canada. There were eight variable nucleotide positions in the 404 bp sequence alignment. Individuals of haplotype 1 occurred at frequency of >75% at all localities. Five haplotypes were detected at only one of the four localities. High haplotype diversity and low nucleotide diversity, combined with significantly negative F_s values for ticks at three localities, suggest a recent population expansion. Genetic differences were found between populations at different localities, but a Mantel regression analysis revealed no association between genetic differences and geographical distances. There was also no association between tick haplotype and the prevalence of the bacterium, Rickettsia montanensis Weiss and Moulder, 1984, in D. variabilis among localities or on opposite sides of Blackstrap Lake (Saskatchewan). The 16S rDNA haplotypes from Canadian populations of D. variabilis formed a clade with those from the eastern and central U.S.A., to the exclusion of D. variabilis from geographically isolated populations in the western U.S.A. Although sample sizes for D. variabilis in the eastern U.S.A. are small, there may be genetic divergence between populations in Canada and those in the eastern U.S.A., which may have implications for studies on the pathogenic agents transmitted by D. variabilis to its hosts.     

Ixodid ticks associated with feral swine in Texas

Ixodid ticks were collected from feral swine in eight Texas ecoregions from 2008–2011. Sixty‐two percent of 806 feral swine were infested with one or more of the following species: Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor albipictus, D. halli, D. variabilis, and Ixodes scapularis. Juvenile and adult feral swine of both sexes were found to serve as host to ixodid ticks. Longitudinal surveys of feral swine at four geographic locations show persistent year‐round tick infestations of all gender‐age classes for tick species common to their respective geographic locations and ecoregions. Amblyomma americanum, A. cajennense, A. maculatum and D. variabilis were collected from 66% of feral swine harvested through an abatement program in seven ecoregions from March to October in 2009. These results indicate westward geographic expansion of D. variabilis. Summary results show feral swine are competent hosts for ixodid species responsible for the transmission of pathogens and diminished well‐being in livestock, wildlife, and humans.     

Ultrastructure of type-I salivary-gland acini in four species of ticks and the influence of hydration states on the type-I acini ofAmblyomma americanum

We describe the ultrastructure of type-I salivary-gland acini in two argasid and two ixodid species. The basic cell types in the agranular or type-I acini, and their associations, are very similar in argasids and ixodids; therefore, we propose an anatomical nomenclature for cells in the type-I acinus based on the adult ixodidsAmblyomma americanum andDermacentor variabilis, and the argasid adultArgas (Persicargas) arboreus and on nymphalOrnithodoros moubata. Four cell types were present in all specimens: one central lamellate cell, a variable number of peripheral lamellate cells, a variable number of peritubular cells depending on the species, and one circumlumenal cell. The lamellate cells had infolded basal plasma membranes that presented an amplified surface area to the hemolymph. These cells most likely secreted the fluid involved in water vapor uptake by ticks. ForAmblyomma americanum females, abundant K⁺-dependent, ouabain-sensitive Na⁺, K⁺-ATPase complexes were located on the infolded basal plasma membranes of the lamellate cells. Apical membranes of the lamellate cells, and plasma membranes of other cell types in the acinus had little or no evidence of Na⁺, K⁺-ATPase activity. Only the central lamellate cell extended from the hemolymph of the acinus to its lumen; peripheral cells did not contact the lumen. Except when the ticks were rehydrating, lipid inclusions were common features in the lamellate cells of the ixodids. Lipid inclusions were not seen in argasid type I acini; however, glycogen deposits were common. To determine if acinar cells respond to the changing hydration state of the tick, unfed femaleA. americanum were subjected to dehydration/rehydrating conditions. During rehydration, mitochondria in the lamellate cells changed from a matrix of medium electron-density and intermembrane space (orthodox configuration) to a matrix of greater density and larger intermembrane space (condensed configuration). The orthodox configuration was consistently observed in control and dehydrating ticks. The condensed configuration was the norm for mitochondria in lamellate cells of rehydrating ticks. Lipid inclusions were depleted in the rehydrating ticks compared to control or dehydrating ticks. Acini appeared to be reverting to the control or desiccated state when ticks were returned to low humidity, suggesting that these changes were cyclical. Nymphs ofO. moubata subjected to the same dehydration/rehydrating conditions showed no obvious ultrastructural changes.     

Hybridization in natural sympatric populations of Dermacentor ticks in northwestern North America

Hybridization in ticks has been described in a handful of species and mostly as a result of laboratory experiments. We used 148 AFLP loci to describe putative hybridization events between D. andersoni and D. variabilis in sympatric populations from northwestern North America. Recently, D. variabilis has expanded its range westward into the natural range of D. andersoni. Using a sample of 235 D. andersoni and 62 D. variabilis, we identified 31 individuals as putative hybrids: four F₂ individuals and 27 backcrosses to D. andersoni (as defined by NewHybrids ). We found no evidence of hybrids backcrossing into D. variabilis. Furthermore, all hybrids presented 16S mtDNA signatures characteristic of D. andersoni, which indicates the directionality of the hybrid crosses: female D. andersoni × male D. variabilis. We also discovered 13 species‐specific AFLP fragments for D. andersoni. These loci were found to have a decreased occurrence in the putative hybrids and were absent altogether in D. variabilis samples. AFLP profiles were also used to determine the levels of genetic population structure and gene flow among nine populations of D. andersoni and three of D. variabilis. Genetic structure exists in both species (D. andersoni, Φ_ST = 0.110; D. variabilis, Φ_ST = 0.304) as well as significant estimates of isolation by distance (D. andersoni, ρ = 0.066, P = 0.001; D. variabilis, ρ = 0.729, P = 0.001).     

Host utilization and seasonal occurrence of Dermacentor

A total of 3,235 Dermacentor variabilis (Say) specimens were collected from birds, mammals, and by dragging vegetation, and 2,683 D. albipictus (Packard) ticks were collected from deer from 1993 to 1996. Peak seasonal occurrence of adult D. variabilis was from May through July with a precipitous decrease in August. Nymphal D. variabilis populations peaked in June. Peak activity of larvae was bimodal, with one activity peak during late summer (September) and a second peak in winter or early spring. The raccoon, Procyon lotor (L.), was the principal host of adults followed by the Virginia opossum, Didelphis virginiana Kerr. Rodents and the eastern cottontail rabbit, Sylvilagus floridanus (J. A. Allen), were the primary hosts of nymphs. The marsh rice rat, Oryzomys palustris (Harlan), was the principal host of larvae followed by the pine vole, Microtus pinetorum (Le Conte), and white-footed mouse, Peromyscus leucopus (Rafinesque). All stages of D. albipictus were found only on white-tailed deer, Odocoileus virginianus (Zimmermann). Numbers of adult and nymphal D. albipictus peaked in November, whereas larvae peaked in September.     

A quantitative comparison of two sample methods for collecting <Emphasis Type="Italic">Amblyomma americanum</Emphasis> and <Emphasis Type="Italic">Dermacentor variabilis</Emphasis> (Acari: Ixodidae) in Missouri

In studies of tick communities, sampling methodology may influence observed patterns. The objective of this study was to compare two sampling methods, dragging and dry ice baiting, in two habitats to assess abundance of off-host ticks. Tick communities were monitored from March to December in a forested and an old-field habitat in northeast Missouri. In each habitat, eight drag and eight dry ice bait samples were collected every 2 weeks on a permanent grid. The most common ticks collected were all life stages of Amblyomma americanum L. and adult and larval Dermacentor variabilis Say. Capture data was analyzed to determine if there were differences due to sampling method, habitat or an interaction for each life stage of each species across the entire monitoring period. Data indicate that there were differences in the methods. Significantly more A. americanum nymphs were captured by bait sampling. Dragging captured significantly more larval A. americanum. Significantly more larval and nymphal A. americanum and larval D. variabilis were caught in the forest, whereas significantly more adult D. variabilis were collected in the field. Significant interaction between site and method was found for A. americanum adults and D. variabilis larvae. These differences are likely due to differences in behavior among species and developmental age that interact with microclimate. These data indicate that community monitoring studies should use multiple sampling methodologies to avoid bias. While comparing these methods, the first documented collection of off-host Ixodes scapularis Say in Adair County, Missouri was made.     

Ultrastructure of the preseptal part of metanephridia in Nais variabilis and Dero digitata (Annelida, Clitellata)

The composition and arrangement of cells in the preseptal region of metanephridia have been examined by ultrastructural methods in two naidid species, Nais variabilis and Dero digitata. Within this region special attention has been paid to the portion around the orifice and the region where the metanephridium penetrates the septum. In N. variabilis, the preseptal region is composed of four cells and, in D. digitata, three cells are present. In both species three cells correspond in position and ultrastructural details and, hence, are interpreted as homologous. These are the mantle cell, the flame cell, and the canal cell. The mantle cell covers the preseptal region and surrounds the opening. The margin around the orifice is endowed with cilia, which extend into the coelomic space and beat irregularly. They do not enter the orifice and, thus, are not part of the internal ciliary flame. Posteriorly, in D. digitata, the mantle cell originates from the septal wall, i.e., its extensions spread in the plane of the frontal coelothelium of the septum. In N. variabilis, the mantle cell is continued by a further cell, enwrapping the posterior region of the preseptal part. This cell, called the septal cell, is anchored in the septal wall like the mantle cell in D. digitata. Both cells are interpreted as mesodermal components of the metanephridium. The flame cell lies beneath the mantle cell. In front, on its dorsal wall, many cilia are inserted which extend posteriorly into the nephridial canal forming a flame. In D. digitata, the caudal extension of this cell was examined in more detail; it originates from an intraseptal position. The canal cell lines the anterior lumen of the nephridial duct. While the mantle cell and flame cell enclose the organ from a dorsal position, the canal cell lies opposite embracing the lumen from a ventromedial position. Behind, it extends into the postseptal region for a certain distance. It is concluded that metanephridia in the Clitellata have a coelothelial component and, probably, are not just descendants of a single cell, the nephridioblast. The results further indicate that a flame cell and a mantle cell or some corresponding coelothelial cells may be constitutive elements of the ground plan of the clitellate metanephridium. Phylogenetic consequences for non-clitellate Annelida are discussed. Accepted: 21 December 1999     

<Emphasis Type="Italic">Dermacentor marginatus</Emphasis> and <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks versus L929 and Vero cell lines in <Emphasis Type="Italic">Rickettsia slovaca</Emphasis> life cycle evaluated by quantitative real time PCR

Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment.     

Spatial and temporal factors affecting parasite genotypes encountered by hosts: Empirical data from American dog ticks (Dermacentor variabilis) parasitising raccoons (Procyon lotor)

The American dog tick (Dermacentor variabilis) is an important vector of numerous pathogens of humans and animals. In this study, we analysed population genetic patterns in D. variabilis at scales of the host individual (infrapopulation) and population (component population) to elucidate fine-scale spatial and temporal factors influencing transmission dynamics. We genotyped D. variabilis collected from raccoons (Procyon lotor) trapped in two habitat patches (located in Indiana, USA) which were spatially proximate (5.9 km) and limited in size (10.48 Ha and 25.47 Ha, respectively). Despite the fine spatial sampling scale, our analyses revealed significant genetic differentiation amongst component populations and infrapopulations (within each component population), indicating a non-random pattern of encountering tick genotypes by raccoons at both scales evaluated. We found evidence for male-biased dispersal in the ticks themselves (in one component population) and an age-bias in spatial scales at which raccoons encountered ticks in the environment. At the scale of the component population, our analyses revealed that raccoons encountered ticks from a limited number of D. variabilis family groups, likely due to high reproductive variance amongst individual ticks. Finally, we found evidence for a temporal effect with raccoons encountering ticks in the environment as “clumps” of related individuals. While the genetic structure of parasite populations are increasingly being investigated at small spatial scales (e.g. the infrapopulation), our data reveal that genetic structuring can originate at scales below that of the infrapopulation, due to the interaction between temporal and biological factors affecting the encounter of parasites by individual hosts. Ultimately, our data indicate that genetic structure in parasites must be viewed as a consequence of both spatial and temporal variance in host-parasite interactions, which in turn are driven by demographic factors related to both the host and parasite.     

Kinetics of ingested host immunoglobulin G in hemolymph and whole body homogenates during nymphal development of Dermacentor variabilis and Ixodes scapularis ticks (Acari: Ixodidae)

Patterns in the utilization of host immunoglobulin G (IgG) during nymphal development differed between Dermacentor varibilis (Say) and Ixodes scapularis Say ticks. In unfed nymphs of D. variabilis, host IgG was readily detectable in both hemolymph and whole body homogenates. In unfed nymphs of I. scapularis, host IgG was absent in hemolymph and at very low concentrations in whole body homogenates. Host IgG in unfed nymphs was undoubtedly the remnants of IgG acquired during the larval bloodmeal that persisted through metamorphosis to the nymphal stage. In both tick species, host IgG crossed the midgut into the hemocoel during the latter phases of engorgement. Concentrations of host IgG in I. scapularis declined considerably after replete nymphs molted to the adult stage. In contrast, concentrations of host IgG in D. variabilis remained elevated throughout metamorphosis to the adult stage. When larval D. variabilis were fed on a rat, then 2 months later as nymphs on a rabbit, the rat IgG (“old IgG”) present in unfed nymphs was totally replaced by rabbit IgG (“new IgG”) within 2 d of nymphs attaching to the rabbit. Presumably, the old IgG acquired from a previous bloodmeal was secreted via saliva into the new host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tick species establishment in Oklahoma County,Oklahoma, U.S.A., identified by seasonal sampling in residential and non‐residential sites

In recent years, human tick‐borne disease occurrence has risen in Oklahoma, U.S.A., but year‐round data on tick presence in frequently used recreational areas is not widely available. In this study, ticks were collected monthly for one year at residential and non‐residential sites in a suburban area of Oklahoma County, OK, U.S.A. At each trapping site, dry ice traps were used in both woodland and grassland areas and fabric tick drags were used in grassland areas. Four species were collected from each park: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. Prior to this study, A. americanum was the only species with an established population in Oklahoma County. Consistent with this, A. americanum was collected in all months of the year and accounted for over 90% of ticks collected at each site. Based on our tick survey, we report that A. maculatum, D. variabilis, and I. scapularis, which were each collected in numbers greater than six within a single sampling occasion, are now each confirmed as established populations in Oklahoma County.     

Porphyra cell cultures: isolation,growth and polysaccharide production

A range of cell lines was isolated fromPorphyra umbilicalis L. (Rhodophyta) tissue using a variety of methods, the most successful involving exposure to a limpet acetone powder enzyme extract for 24 h, homogenisation and filtration through a series of polyester meshes. All established lines grew as 0.1–5 mm diameter aggregates in liquid culture; most were stable and have been grown in shake-flask or air-lift culture for periods in excess of 1 yr without reverting to the foliose growth form. An investigation of the medium used to grow these lines indicated that it was not nitrogen-deficient and that the sodium chloride concentration was optimal. The addition of an organic buffer increased the final cell yield. None of these cell lines grew heterotrophically in medium supplemented with a range of fixed carbon sources. The infrared spectra of polysaccharides isolated fromPorphyra aggregates and from tissue grown under identical conditions indicated that the structures of the two isolates were analogous.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

Relative utilization of reptiles and rodents as hosts by immature Ixodes scapularis (Acari: Ixodidae) in the coastal plain of North Carolina,USA

The interaction of immature black-legged ticks, Ixodes scapularis, with reptiles and rodents was investigated in various woodland habitats in the coastal plain of North Carolina. Reptiles were sampled from April 1 to September 30, 1991. No ticks were found on 95 specimens representing 16 species of snakes. Ticks were found on 54 (36.7%) of 147 lizards. I. scapularis was the only tick recovered from lizards. Some lizards were collected in drift fence traps each month of the study except August. Capture rates averaged one lizard per 16 trap-days. Larvac and nymphs of I. scapularis were removed from the southeastern five-lined skink (Eumeces inexpectatus), the ground skink (Scincella lateralis), the broad-headed skink (E. laticeps) and the eastem glass lizard (Ophisaurus ventralis), but ticks were not found on three other lizard species. Tick infestation rates and loads for parasitized species are presented. Ticks were almost exlusively attached at the base or in the axils of forelimbs of skinks and in the lateral grooves of eastern glass lizards. Rodents were live-trapped at sites where lizards were sampled and at other sites from 1 July, 1990 to 30 January, 1992. Capture rates averaged one rodent per 47 trap-nights. Ticks were found on 23 (17.8%) of 129 animals inspected. Five species of rodents were examined but only four species were found to be tick-infested. In contrast to lizards, few I. scapularis were collected. Rodents, principally the white-footed mouse (Peromyscus leucopus) and cotton mouse (P. gossypinus) were most frequently infested with immature American dog ticks, Dermacentor variabilis, during winter and early spring months. Burdens of D. variabilis on these rodents averaged 0.3 ticks per rodent. Effects of the diversion of ticks from feeding on Peromyscus mice on the transmission of the Lyme disease spirochete are discussed.The use of trade names in this publication does not imply an endorsement by the North Carolina Agricultural Research Service or the College of Veterinary Medicine, North Carolina State University of the products named, nor criticism of similar ones not mentioned. Partial support for this research was provided by the U.S. Marine Corps, The Centers for Disease Control and Prevention, and the North Carolina Agricultural Research Service and College of Veterinary Medicine, North Carolina State University.