Colorado Strain Research Articles

In vitro and in vivo encystment of the cercariae of Echinostoma caproni.

In vitro and in vivo studies were conducted on the cercariae of Echinostoma caproni. Of the 15 media tried, 2 resulted in effective in vitro encystment in petri dish cultures maintained at 23 +/- 1 C. They were a Locke's--artificial springwater (ASW) (1:1) medium (67% encystment) and a Biomphalaria glabrata embryonic cell line medium (23% encystment). To obtain large numbers of in vitro--formed cysts, finger bowl cultures containing 40 ml of the Locke's-ASW (1:1) medium were used at 23 +/- 1 C. Of 3,000 cercariae tested, 1,890 (63%) were encysted in this medium by 48 hr. Most of these cysts looked similar to those formed in vivo, although some showed abnormalities in the outer cyst wall and other malformations. A total of 200 in vitro-formed cysts treated in an alkaline trypsin-bile salts (TB) medium for 2 hr at 41 C showed 94% excystation. In vitro-formed cysts fed to mice produced ovigerous adults within 2 wk postinfection (PI). Eggs from these worms gave rise to miracidia that produced patent intramolluscan infections in B. glabrata snails. In vivo encystment was studied in lab-raised juvenile Helisoma trivolvis (Colorado strain) snails, 1-3 mm in shell diameter. From 6 to 24 hr PI, 93-100% of the cercariae were recovered as metacercarial cysts in the snail tissue. Treatment of these cysts in the TB medium resulted in 96% excystation within 2 hr at 41 C.

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli.

Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry. Experimental infections are typically less severe than those observed in field cases. Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents. Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV). In the current study, we examined the role of lentogenic NDV in the APV disease process. Two-week-old commercial turkey poults were infected with the Colorado strain of APV. Three days later, these poults received an additional inoculation of either NDV or E. coli. Dual infection of APV with either NDV or E. coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses. These lesions were not present in the single APV, NDV, or E coli groups. These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.

Lack of Antigenic Relationship between French and Recent North American Non-A/Non-B Turkey Rhinotracheitis Viruses

Twelve turkey rhinotracheitis viruses (TRTVs) including the Colorado isolate and two French non-A/non-B viruses were serologically compared. Six enzyme-linked immunosorbent assay (ELISA) antigens derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado TRTVs were used. Virus neutralization (VN) tests were performed with four Ma-104-adapted viruses derived from subgroup A, subgroup B, a French non-A/non-B, and the Colorado viruses. French strains isolated since 1995 were assigned to subgroup B in both ELISA and VN, whereas those isolated in 1985 and 1986 appeared more diverse: two strains belonged to subgroup B, one to subgroup A, and two others appeared antigenically different from both the A and B subgroups and are classified as non-A/non-B. The Colorado strain appeared different from these three groups of TRTVs. Assignment to subgroup A or B was confirmed by reverse transcription-polymerase chain reaction, but neither the French non-A/non-B strains nor the Colorado virus could be classified with the subgroup-specific G-based primers. These results suggest that at least three antigenically different viruses were present in France in 1985-86 and that the Colorado strain is different from all European TRTVs. Further serologic and phylogenic studies will be necessary to evaluate their actual prevalences and relationships.

A Multiplex Reverse Transcription-Polymerase Chain Reaction Assay for Newcastle Disease Virus and Avian Pneumovirus (Colorado Strain)

Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

The Sensitivity and Specificity of a Reverse Transcription-Polymerase Chain Reaction Assay for the Avian Pneumovirus (Colorado Strain)

A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR.

This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 x 10(-5) TCID50 (0.0323 microg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota.

A Reverse Transcription-Polymerase Chain Reaction Assay for the Detection of Avian Pneumovirus (Colorado Strain)

A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

Maintenance of Helisoma trivolvis naturally infected with Echinostoma trivolvis in spring water at 4 degrees C for 300 days.

Helisoma trivolvis (Pennsylvania strain) snails naturally infected with Echinostoma trivolvis larvae were maintained for 10 months (300 days) at 4 degrees C in artificial spring water to determine the effects of storage on parasite survival. Three of 20 snails were alive at 10 months and when isolated in spring water at 22-24 degrees C released active cercariae. The mean number of cercariae released in 2 h from each stored snail was significantly less than that from freshly collected snails. Infectivity of cercariae from stored hosts to experimentally infected, laboratory-raised Helisoma trivolvis (Colorado strain) snails was significantly less than that of cercariae from fresh hosts based on cyst recoveries in the Colorado strain of H. trivolvis within 24 h p.i. There was no significant difference in the redial number or the number of cercariae per redia in stored versus fresh snails. In conclusion, some reduction in cercarial emergence and decrease in cercarial infectivity to a second intermediate snail host occurred following storage of H. trivolvis naturally infected with E. trivolvis in ASW at 4 degrees C for 10 months.

HPTLC Determination of Amino Acids in Snail-Conditioned Water From Biomphalaria glabrata, Two Strains of Helisoma trivolvis, and Lymnaea elodes

High performance thin-layer chromatography (HPTLC) was used to analyze amino acids in water conditioned by Biomphalaria glabrata, a Pennsylvania and Colorado strain of Helisoma trivolvis, and Lymnaea elodes. The snail-conditioned water (SCW) samples were dried with air and reconstituted in 10% n-propanol and then applied to cellulose HPTLC plates and developed with n-propanol-water (7:3). Amino acids were detected with ninhydrin reagent and the resulting colored sample zones were compared to known standards. The amino acids present in SCW were identified as alanine, aspartic acid, leucine, phenylalanine, serine, tryptophan, and valine along with one unidentified ninhydrin positive zone. The valine zone was quantified by densitometric scanning at 610 nm, and the concentrations ranged from 1.3-2.6 μg mL−1 snail−1 in the SCW for all four strains of snails. There were no significant differences in the concentrations of valine in water conditioned by the four strains of snails based on Student's t-test.

In vivo and Ectopic Encystment of Echinostoma revolutum and Chemical Excystation of the Metacercariae

In vivo and ectopic encystment of the cercariae of Echinostoma revolutum from Lymnaea elodes snails in Indiana and chemical excystation of the metacercariae were studied. In vivo encystment occurred in adults of Biomphalaria glabrata and Helisoma trivolvis (Colorado strain) snails and in neonatal and juvenile L. elodes snails. These results were expected because 37-collar-spined Echinostoma species show broad specificity in their second intermediate gastropod hosts. Encysted metacercariae of E. revolutum and Echinostoma trivolvis removed from experimentally infected snails and treated in a trypsin-bile salts excystation medium at 39 C showed 30.3% excystation for the former and 55.7% for the latter at 4 hr. The ducts and openings of the paraesophageal glands of excysted metacercariae of E. revolutum from cysts formed in snails did not stain with neutral red. Abnormal ectopic cysts with distorted outer walls and granular inner walls were obtained within 48 hr of placing E. revolutum cercariae in Locke's 1:1 plus 1% dextrose. These metacercariae excysted rapidly in the excystation medium and their paraesophageal gland ducts and openings stained with neutral red. Differences in ectopic encystment and chemical excystation in vitro can be used to distinguish these closely related species in the E. revolutum complex.

Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins.

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.

Experimental infection of Helisoma trivolvis (Colorado strain) snails with cercariae of Echinostoma trivolvis

A Colorado (CO) strain of Helisoma trivolvis resistant to miracidial infection, but susceptible to Echinostoma trivolvis cercarial penetration, was used to study encystment of cercariae released from naturally infected H. trivolvis (Pennsylvania strain). All 12 snails, 8–10 mm in shell diameter, each exposed to 25 cercariae and necropsied 1–4 days post-exposure were infected with between 9 and 19 cysts per snail (average 14). In all infectivity experiments, regardless of the size (age) of the Colorado strain snails used, encysted metacercariae were recovered from the kidney-pericardial region of experimentally infected snails within 24 h post-exposure. Snails exposed individually with 500 cercariae averaged 230 cysts per snail at 24 h p.i. Cercariae encysted at 6–8 h p.i., but not at 2 and 4 h p.i. Light microscopical observations of the cysts suggested that greater than 99% were viable. Metacercariae subjected to in vitro excystation in an alkaline trypsin-bile salts medium showed excystation rates of 60–85%. In conclusion, H. trivolvis (CO strain) is an excellent experimental second intermediate host for cercarial encystment of E. trivolvis.

Intraspecific and interspecific chemoattraction inBiomphalaria glabrata andHelisoma trivolvis (Gastropoda: Planorbidae)

A Petri dish bioassay previously used to examine food preferences in planorbid snails was used to study intraspecific and interspecific chemoattraction inBiomphalaria glabrata (albino strain, M-line) andHelisoma trivolvis (Colorado strain) snails.B. glabrata snails showed significant intraspecific chemoattraction in the absence of visual cues and snail thigmotaxis.H. trivolvis snails also showed significant intraspecific chemoattraction. Interspecific chemoattraction between these species occurred in the bioassay, suggesting that the chemoattractants were not species specific. Artificial spring water conditioned by aqueous excretory-secretory products (snail-conditioned water) ofB. glabrata elicited significant intraspecific chemoattraction. However, lipophilic excretory-secretory products ofB. glabrata elicited significant chemorepulsion. Repellant factors in the lipophilic fraction were not characterized.

Fatty acid composition of two strains of Helisoma trivolvis (Gastropoda)

Gas-liquid chromatographic studies were done to determine the fatty acid composition of total lipids, triacylglycerols, and phospholipids of two strains of Helisoma trivolvis snails maintained on a lettuce-tetramin diet, Linolenic (18:3), oleic (18:1), palmitic (16:0), and linoleic (18:2) accounted for about 50% of the Tetramin diet, Linolenic (18.3), oleic (18:1), palmitic (16.0), and linoleic (18:2) accounted for about 50% of the fatty acids in the total lipids of both the Colorado (CO) and Pennsylvania (PA) strains. In the Colorado strain, about 50% of the fatty acids in the triacylglycerol fraction consisted of linolenic (18:3), palmitic (16:0), and eicosodienoic (20:2) acids; in the Pennsylvania strain, linelenic (18:3), palmitic (16:2), and oleic (18:1) acids made up about 50% of this fraction. The following fatty acids comprised about 50% of the phospholipids in the Colorado strain: linolenic (18:3), lauric (12:0),linoleic (18:2), eicosodienoic (20:2) and palmitic (16:0); linolenic (18:3), lauric (12:0) myristic (14:0), stearic (18:0), and eocosodienoic (20:2) made up about the same percentage of the phospholipid fatty acids in the Pennsylvania strain. Significant differences, both qualitatively and quantitatively, were seen in some of the fatty acids identified in this study, supporting the contention that lipid profiles may be useful in the chemotaxonomy of related strains of snails.

Patterns of Sugar Feeding in Diapausing and Nondiapausing Culex pipiens (Diptera: Culicidae) Females

In the laboratory, diapausing Culex pipiens L. females fed on a continuously available sugar source more frequently and longer after adult emergence than did non-diapausing females. They also showed an extended nocturnal pattern of sugar feeding compared with that of non-diapausing females. Differences between diapausing and non-diapausing females with respect to sugar feeding and host-seeking behavior also were related to responses to volatiles from fruit and vertebrate hosts. In this Colorado strain of Cx. pipiens, increased sugar feeding by diapausing females during the first few weeks of adult life may be an adaptation that enhances overwintering survival.

Densitometric thin-layer chromatographic studies on neutral lipids-in two strains of Helisoma trivolvis (gastropoda)

1. 1. Densitometric thin-layer chromatographic studies were used to determine differences in the neutral lipid content of Helisoma trivolvis Pennsylvania strain versus H. trivolvis Colorado strain of snails fed on a lettuce-Tetramin diet ad libitum for 2 weeks. 2. 2. The major neutral lipid fractions in the digestive gland-gonad (DGG) complex and snail bodies of both strains were triacylglycerols and free sterols, along with lesser amounts of free fatty acids and sterol esters. 3. 3. Both the DGG and the snail body of the Colorado strain showed significantly greater amounts of triacylglycerols and free sterols than those of the Pennsylvania strain. 4. 4. The major neutral lipid fraction in the hemolymph and plasma of both strains was free fatty acids. Quantitative differences in the neural lipid fractions of the blood of the Colorado versus Pennsylvania strains were not apparent.

SEROLOGICAL EVIDENCE OF BOVINE HERPESVIRUS 1-RELATED VIRUS INFECTION IN THE WHITE-TAILED DEER POPULATION ON ANTICOSTI ISLAND, QUEBEC

High white-tailed deer (Odocoileus virginianus) population densities and the occurrence of harsh environmental conditions are present on Anticosti Island, located in the Gulf of Saint-Lawrence (Quebec, Canada). This island is the northernmost region of white-tailed deer distribution in northeastern North America. The aim of this work was to determine whether a herpesvirus serologically related to bovine herpesvirus 1 (BHV1) may occur in a stressed white-tailed deer population. One hundred one deer sera were collected from apparently healthy animals during the hunting season from September to late November 1985. Fifty-three percent of tested deer were positive to the seroneutralization test using Colorado strain of BHV1 virus. Higher percentages of seropositivity were observed in animals of both sexes greater than 4-yr-old. Analysis of antibody titers in seropositive animals according to age suggests that BHV1-related viral infection is endemic in the Anticosti Island deer population. It is postulated that environmental stress may induce immunosuppression of certain infected and/or carrier animals in their population that shed virus for long periods of time.

Effects of hormone-induced superovulation on latent infectious bovine rhinotracheitis virus in cattle

Twelve nulliparous, sexually mature heifers free of antibodies to infectious bovine rhinotracheitis (IBR) virus were exposed intranasally to Colorado strain IBR virus. After 3 mos, when the postexposure antibody titers had stabilized, the heifers were divided into three groups. Individuals in each group were treated with either saline, dexamethasone or follicle stimulating hormone (FSH) for five consecutive days. Blood samples were taken at predetermined intervals for isolation of virus, and for determination of serum cortisol levels. No changes occurred in the saline-treated group, except that one heifer had a slightly elevated serum neutralizing antibody titer. Recrudescense of typical clinical lesions was observed in the dexamethasone-treated group, and the IBR virus was isolated from nasal swab samples taken from all heifers. In the FSH-treated group, no changes occurred, with the exception of slightly reduced serum cortisol levels. Results indicate that FSH-induced superovulation does not cause reactivation of IBR virus in heifers previously infected by the intranasal route, and has no effect on serum neutralizing anti-IBR virus antibody titers.

Infectivity of Echinostoma revolutum Miracidia for Laboratory-Raised Pulmonate Snails

With respect to the first intermediate host, Echinostoma revolutum is considered to be less specific than most trematodes. In his studies on that species, Beaver (1937, Illinois Biological Monographs 15: 1-96) utilized Helisoma trivolvis as that host and tabulated, from the world literature, pulmonate species representing 6 genera and reported to serve E. revolutum in that capacity. After the discovery of E. revolutum in H. trivolvis from ponds in eastern Pennsylvania, periodic examinations of snails from that area revealed the infection only in that species. More recently, laboratory-raised snails were exposed to miracidia ofE. revolutum; H. trivolvis became infected and Physa heterostropha did not (Fried, 1985, Proceedings of the Pennsylvania Academy of Science 59: 27-28). In this study, that procedure was repeated and extended to include other species of laboratory-raised pulmonate snails. Miracidia were derived from E. revolutum adults raised in hamsters (Franco et al., 1986, Journal of Parasitology 72: 142-147). The uteri of worms were teased, eggs were embryonated in tap water at 22-24 C for 2-3 wk, and miracidia were used within 4 hr of emergence from eggs. Most snails were exposed individually in multiwell chambers (Anderson and Fried, 1987, Journal of Parasitology 73: 49-54) for 4 hr to 10 miracidia per snail. In some trials with Helisoma trivolvis (Pennsylvania strain), snails were also exposed to either 1, 5, or 20 miracidia/snail (see Table I). Following exposure, snails were placed in 10-20/1,000 ml of aerated artificial spring water in glass jars and maintained at 22-24 C under diffuse overhead fluorescent light for 12 hr/day and fed ad libitum on boiled lettuce. They were necropsied 4-6 wk postexposure. Laboratory-raised, helminth-free Helisoma trivolvis (PA strain), Physa heterostropha (PA strain), and Biomphalaria glabrata (Puerto Rican strain) were raised in the laboratory from eggs (Anderson and Fried, 1987, loc. cit.), in addition to a highly inbred strain of Helisoma trivolvis (Colorado strain) maintained in laboratory culture since the mid 1960's (Kater, 1974, American Zoologist 14: 1017-1036). Helisoma trivolvis (PA strain) is a first intermediate host of E. revolutum (Fried, 1985, loc. cit.). To assure the viability ofE. revolutum miracidia, H. trivolvis (PA strain) was always exposed to miracidia as a positive control for this experiment. The shell diameters of snails used in this study were: 3-10 mm for P. heterostropha; 3-16 mm for B. glabrata and Helisoma trivolvis (PA strain); and 8-12 mm for H. trivolvis (Colorado strain). A total of 183 H. trivolvis (PA) was exposed to miracidia; another 100 H. trivolvis (PA) were left as controls and were not exposed to miracidia.

A comparison of the virulence of three strains of infectious bovine rhinotracheitis virus

The virulence of three strains of infectious bovine rhinotracheitis (IBR) virus was compared in six-month-old Ayrshire-cross calves. The strains were an isolate from a recent severe outbreak of IBR in Scotland (Strichen strain), the prototype British strain (Oxford strain) and a North American isolate (Colorado strain). The Colorado and Strichen strains produced the characteristic clinical signs and pathological lesions of severe IBR three to four days post infection (p.i.). The Strichen strain was slightly more virulent, possibly as a result of its having been passaged fewer times in tissue culture. In contrast, the Oxford strain produced a mild clinical response with minimal pathological lesions. Virus was recovered from nasal swabs for a longer period from the calves infected with the Strichen strain (up to 13 days p.i.) and Colorado strain (up to 12 days p.i.) than from the animals infected with the Oxford strain (up to 10 days p.i.). These findings support the suggestion that the recent epidemic of severe IBR in Britain had resulted from the importation of a “new” strain of virus.