Coccidian oöcysts as type-specimens: long-term storage in aqueous potassium dichromate solution preserves DNA.

Preservation of the exogenous oöcyst stage of coccidian parasites (phylum Apicomplexa N.D. Levine, 1970) as type-specimens of newly described species has long been problematical. Conventional fixatives have proved unsatisfactory, and compromises such as embedding oöcysts in resin or photographing them are not entirely appropriate for various reasons. As an alternative, chilled potassium dichromate solution (normally used in the laboratory to prevent putrefaction of temporary preparations of live oöcysts) has been tested as a long-term preservative of sporulated oöcysts of Eimeria brunetti P.P. Levine, 1942, E. maxima Tyzzer, 1929, E. mitis Tyzzer, 1929, E. necatrix Johnson, 1930, E. praecox Johnson, 1930 and E. tenella (Railliet & Lucet, 1891) (suborder Eimeriorina Léger, 1911; family Eimeriidae Minchin, 1903). Oöcysts from faeces of chickens Gallus gallus (Linnaeus) were placed in 2.5% w/v aqueous potassium dichromate solution (PDS) and stored in the dark at 4 +/- 2 degrees C. After 23 years in storage, oöcysts of each species were administered orally to chickens and failed to initiate infections, indicating that the oöcysts were dead. Nevertheless, after about 24 years, DNA was still recoverable from the oöcysts, and the original species identifications made by classic parasitological methods were confirmed by polymerase chain reaction assays. Furthermore, after almost 25 years, microscopical examination revealed that the walls and internal structures remained well preserved in 83-98% of the oöcysts of the six species investigated. Hence, PDS is potentially suitable for the long-term preservation of sporulated coccidian oöcysts as type-specimens for taxonomic purposes. The samples used in this study are now in the care of the Natural History Museum, London, UK. It is recommended that they be monitored in like manner, by suitably qualified scientists, at intervals of about 5 years to assess their state of preservation and the recoverability of DNA. Enough material is available to monitor it until it is at least 100 years old.

Pathogenesis of Eimeria praecox in chickens: virulence of field strains compared with laboratory strains of E. praecox and Eimeria acervulina.

The pathogenesis in chickens of the apicomplexan Eimeria praecox was compared with that of Eimeria acervulina, using intestinal lesions, mucosal integrity, body weight gain (BWG) and the feed conversion ratio (FCR) as criteria. Characteristics of each species were described by combinations of polymerase chain reaction assays and classic parasitological signs. There were considerable overlaps in lengths, breadths, shape indices and volumes of the oocysts of each species. Both species caused statistically significant reductions in BWG at the lowest inocula tested (500,000 sporulated oocysts per bird of E. praecox and 250,000 of E. acervulina). E. praecox was observed for the first time to cause actual body weight loss and marked increases in FCR, as did E. acervulina. E. acervulina caused gross, white pathognomonic lesions, but E. praecox caused micro-lesions, visible in fresh tissue only with a dissecting microscope. Occasionally, lesions of the Houghton strain of E. acervulina were observed to be rounded, rather than typically "ladder-like". Both species caused villous erosion and atrophy. No mortality occurred in birds receiving up to 1 million sporulated oocysts of either species. Using BWG and FCR as criteria, the virulence of recent field strains of E. praecox from Wales (Tynygongl) and the USA (Raleigh) was compared with English laboratory strains of E. praecox (Houghton) and E. acervulina (Houghton). E. praecox (Tynygongl) was markedly more virulent than E. acervulina (Houghton), which was more virulent than E. praecox (Raleigh) and E. praecox (Houghton).

Cryptosporidium parvum infection in orphan lambs on a farm open to the public.

A longitudinal survey was undertaken on an open farm to investigate the occurrence of Cryptosporidium species infection in orphan lambs obtained from three local flocks. During an initial pilot study, Cryptosporidium oocysts were detected by a fluorescent antibody test (fat) in the faeces of two of 21 lambs aged between one and three weeks derived from one flock (flock A). Pooled pen samples of faeces were collected weekly from lambs derived from each flock; oocysts were detected by fat in 24 (49.0 per cent) of 49 samples from lambs from flock A, 18 (30.5 per cent) of 59 samples from lambs from flock B and 14 (29.8 per cent) of 47 samples from lambs from flock C. Oocyst counts of 1 x 10(3) to more than 2 x 10(6) per gram of faeces were detected in lambs up to 12 weeks old, with the peak counts occurring at six weeks of age in the lambs from flocks A and B and at four weeks of age in those from flock C. The oocysts were confirmed by molecular analysis as Cryptosporidium parvum. Virtually all the infections were subclinical.

Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157 : H7 in young lambs.

Escherichia coli O157 : H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E. coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E. coli O157 : H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E. coli O157 : H7, (B) colostrum-deprived and inoculated with C. parvum and then E. coli O157 : H7, (C) conventionally reared and inoculated with E. coli O157 : H7, (D) conventionally reared and inoculated with C. parvum and then E. coli O157 : H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E. coli O157 : H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E. coli O157 : H7 per gram of faeces. E. coli O157 : H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E. coli O157 : H7 was observed (P = 0.038), with the lambs inoculated with E. coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E. coli O157 : H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total, four animals were euthanized, two at 24 h and two at 96 h post inoculation with E. coli O157 : H7 (two conventionally reared and two colostrum-deprived). All animals euthanized were from groups pre-inoculated with C. parvum prior to challenge with E. coli O157 : H7. On examination of tissues, in three of the four animals examined, multifocal attaching and effacing lesions were observed in the caecum, colon, rectum and at the recto-anal junction, and were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model.

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a beta-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.

The production of a truncated form of baculovirus expressed EHV-1 glycoprotein C and its role in protection of C3H (H-2Kk) mice against virus challenge.

A truncated form of the equine herpesvirus 1 (EHV-1) glycoprotein C (gC) gene was expressed in baculovirus. The gC signal sequence was substituted with the honeybee melittin signal sequence and the transmembrane region was replaced with a histidine tag. The recombinant virus produced high levels of gC in both the cells and supernatants of infected cells. The protein was present by 24 h and maximal secretion occurred at 96 h post-infection. The recombinant protein was antigenically authentic as shown by its reaction with each of a panel of individual monoclonal antibodies specific for the five distinct antigenic sites on EHV-1 gC. Recombinant gC was purified from the supernatant of infected cells by immuno-affinity chromatography and used to immunize C3H (H-2Kk haplotype) mice. This incurred a gC specific antibody response against both the recombinant protein and EHV-1 gC. 'Pepscan' analysis showed that the gC specific antibodies in serum from these mice reacted with the same epitopes on gC as those recognized by antibodies in convalescent equine sera (i.e. antibodies were specific to antigenic sites one and five). A third previously unrecognized antibody binding site at the carboxyl terminus was also detected (Antibody binding domain I). A T-cell proliferative response against EHV-1 was detected in splenocyte populations taken from vaccinated mice. Further, the recovery of virus from the lungs and turbinates following challenge of mice with EHV-1 was significantly reduced. These findings indicate that baculovirus expressed gC may contribute significantly to a subunit vaccine preparation aimed at protecting horses from EHV-1 infection.

Growth hormone therapy in achondroplasia.

A pilot study was carried out to examine the safety and efficacy of recombinant human growth hormone for growth-promoting therapy of achondroplasia. The data suggest that the agent in doses used to treat non-GH-deficient forms of short stature (0.3 mg/kg/wk) modestly increases overall height velocity in some children with achondroplasia. The effect was seen mainly in children with the lowest growth velocities prior to treatment. No untoward effects were noted. Several questions were raised that require further study.

Factors affecting the insulin autoantibody ELISA.

IgG antibodies to insulin are present in insulin-treated patients and are detected in the prodrome of untreated type I diabetes. Sporadic reports of autoantibodies to insulin suggest that they are also present in other disorders. To establish the incidence of insulin autoantibodies in other endocrine and autoimmune diseases an ELISA was used to examine sera from 529 subjects with no prior insulin therapy. These untreated patients included: normal controls (adults and children), newly-diagnosed type I diabetes, first-degree relatives of diabetics, type II diabetes, Graves' hyperthyroidism, and systemic lupus erythematosus. As a positive control group, 280 insulin-treated patients were studied. Measurement of IgG antibodies by direct binding to insulin coated plates was complicated by differences between adult and pediatric populations and by overlap of binding between treated and untreated subjects. Competitive inhibition with excess soluble human insulin overcame these problems and permitted identification of insulin specific binding. Using this approach insulin antibodies were most frequent in insulin-treated diabetics (98%) and in type I diabetics (37%) prior to treatment. The absolute numbers of subjects with insulin autoantibody in the other groups differed depending upon whether a cut-off for binding (mean + 2SD of controls) or for insulin inhibition of binding (45%) was used. Regardless of the criteria used there were subjects (2-24%) in all groups tested with circulating insulin-specific IgG autoantibody detected by ELISA. These low level antibodies detected in solid phase assays may be part of the normal immune repertoire.

Entrainment of respiratory frequency to exercise rhythm during hypoxia.

Breathing frequency (f) is often reported as having an integer-multiple relationship to limb movement (entrainment) during rhythmic exercise. To investigate the strength of this coupling while running under hypoxic conditions, two male Caucasians and four male Nepalese porters were tested in the Annapurna region of the Himalayas at altitudes of 915, 2,135, 3,200, 4,420, and 5,030 m. In an additional study in a laboratory at sea level, three male and four female subjects inspired various O2-N2 mixtures [fraction of inspired O2 (FIO2) = 20.93, 17.39, 14.40, 11.81%] that were administered in a single-blind randomized fashion during a treadmill run (40% FIO2 maximum O2 consumption). Breathing and gait signals were stored on FM tape and later processed on a PDP 11/73 computer. The subharmonic relationships between these signals were determined from Fourier analysis (power spectrum), and the coincidence of coupling occurrence was statistically modeled. Entrainment decreased linearly during increasing hypoxia (P less than 0.01). Moreover, a significant linear increase in f occurred during hypoxia (P less than 0.05), whereas stride frequency and metabolic rate remained constant, suggesting that hypoxic-induced increases in f decreased the degree of entrainment.

Evaluation of lower extremity overuse injury potential in runners.

INTRODUCTION: The purpose of this study was to identify biomechanical and anthropometric variables that contribute to overuse injuries in runners. METHODS: Comparisons were made between a group of runners who had sustained at least one overuse running injury and a group of runners who had been injury free throughout their running careers. Groups were well matched in important training variables. Synchronized kinetic and rearfoot kinematic variables of both feet were collected by filming subjects running over a force platform at a speed of 4 m x s(-1). RESULTS: The injury-free group demonstrated significantly greater posterior thigh (hamstring) flexibility, as measured by a standard sit and reach test. This was the only anthropometric variable in which the groups differed. Within each group, there were no significant differences between left and right foot landing for any biomechanical variable. Biomechanical variables that demonstrated significantly lower values for the injury free group were the vertical force impact peak and the maximal vertical loading rate, with the maximal rate of rearfoot pronation and the touchdown supination angle showing a trend toward being greater in the injury free group. CONCLUSION: These results suggest that runners who have developed stride patterns that incorporate relatively low levels of impact forces, and a moderately rapid rate of pronation are at a reduced risk of incurring overuse running injuries.

The role of maximal strength and load on initial power production.

PURPOSE: The influence of maximal strength, as measured by the maximal load lifted for one repetition (1RM), on power production in the initial 200 ms of the concentric phase for both rebound and nonrebound movements was investigated. We also investigated the effect of external load upon this relationship. METHODS: Twenty-seven male subjects (21.9 +/- 3.1 yr, 89.0 +/- 12.5 kg) were separated by previously determined bench press IRM into high (100.88 +/- 7.24 kg) and low (72 +/- 6.61 kg) RM groups. Concentric only bench presses and rebound bench presses were compared between and within groups to note the effect of RM across external loads of 40%, 60%, and 80% 1RM, on instantaneous, mean, and peak power output. RESULTS: The results of this study clearly indicated the enhancement of concentric motion by prior eccentric muscle action (336-1332% enhancement in the first 20 ms). Possessing a high RM augmented power production in the initial 200 ms of stretch-shorten cycle activity, across all the external resistances tested (P < 0.05). The temporal characteristics of this enhancement, however, differed across loads. That is, 80% IRM loading showed a later time to peak enhancement (80 ms vs 20 ms). Interestingly, the influence of RM on concentric only motion in the initial 200 ms across the external resistances tested was found to be nonsignificant. CONCLUSIONS: The results suggest that the role of maximal strength during initial power production between concentric and stretch-shorten cycle activity differs, which has important implications for the training of athletes.

The accuracy of DLT extrapolation in three-dimensional film analysis.

An analysis of errors arising from the Direct Linear Transformation (DLT) approach to three-dimensional reconstructions from two-dimensional images has been undertaken, the principal factor studied being the number and distribution of control points used in the calibration procedure. Significantly increased error was found to be associated with extrapolation to unknown points outside the control point distribution space. Differences in accuracy between two camera position set-ups and 11 vs 12 DLT parameter solutions were also examined.

Algorithms to determine event timing during normal walking using kinematic data.

Algorithms to predict heelstrike and toeoff times during normal walking using only kinematic data are presented. The accuracy of these methods was compared with the results obtained using synchronized force platform recordings of two subjects walking at a variety of speeds for a total of 12 trials. Using a 60Hz data collection system, the absolute value errors (AVE) in predicting heelstrike averaged 4.7ms, while the AVE in predicting toeoff times averaged 5.6ms. True average errors (negative for an early prediction) were +1.2ms for both heelstrike and toeoff, indicating that no systematic errors occurred. It was concluded that the proposed algorithms provide an easy and reliable method of determining event times during walking when kinematic data are collected, with a considerable improvement in resolution over visual inspection of video records, and could be utilized in conjunction with any 2-D or 3-D kinematic data collection system.

A general Newtonian simulation of an n-segment open chain model.

This paper presents a set of general Newtonian equations which govern the simulation of movement of a body represented by n open chain links. The input for the simulation consisted of the joint moment of force histories, lengths, masses and moments of inertia, the initial absolute angular displacements and velocities and, for the fixed or constrained axis of the nth segment, the acceleration history. Angular accelerations were then determined by solving n linear equations simultaneously, and angular velocities and displacements determined by integrating forwards. The final output was in the form of a graphical display of the linked figure. Applications of the simulation were demonstrated using three-segment representations of movements of the upper and lower extremities and a five-segment representation of a jump. Good agreement was achieved between the displayed angular displacements for the original and simulated movements. The potential for varying the input data has been examined and the implications of anticipating the effects of changed torques, inertial characteristics including attached prosthetic or sports implements and/or the initial conditions for a movement are discussed.

EMG signal amplitude normalization technique in stretch-shortening cycle movements.

Analysis of functional movements using surface electromyography (EMG) often involves recording both eccentric and concentric muscle activity during a stretch-shorten cycle (SSC). The techniques used for amplitude normalization are varied and are independent of the type of muscle activity involved. The purpose of this study was: (i) to determine the effect of 11 amplitude normalization techniques on the coefficient of variation (CV) during the eccentric and concentric phases of the SSC; and (ii) to establish the effect of the normalization techniques on the EMG signal under variable load and velocity. The EMG signal of the biceps brachii of eight normal subjects was recorded under four SSC conditions and three levels of isometric contraction. The 11 derived normalization values were total rms, mean rms and peak rms (100 ms time constant) for the isometric contractions and the mean rms and peak rms values of the ensemble values for each set of isotonic contractions. Normalization using maximal voluntary isometric contractions (MVIC), irrespective of rms processing (total, mean or peak), demonstrated greater CV above the raw data for both muscle actions. Mean ensemble values and submaximal isometric recordings reduced the CV of concentric data. No amplitude normalization technique reduced the CV for eccentric data under loaded conditions. An ANOVA demonstrated significant (P < 0.01) main effects for load and velocity on concentric raw data and an interaction (P < 0.05) for raw eccentric data. No significant effects were demonstrated for changes in velocity when the data were normalized using mean rms values. The reduction of the CV should not be at the expense of true biological variance and current normalization techniques poorly serve the analysis of eccentric muscle activity during the SSC.

The pathogenesis of experimental infections of Cryptosporidium muris (strain RN 66) in outbred nude mice.

Three groups of six-week-old nude outbred mice were orally infected with 400, 20,000 and 1,000,000 oocysts of Cryptosporidium muris (strain RN 66) per mouse, respectively. Oocysts were detected in the faeces from 10-18 days post-infection (p.i.) and continued to be shed in large numbers in all groups until the termination of the trial on day 89 p.i. Clinical signs were not observed in any of the infected mice and there was no significant effect on weight gain compared to uninfected controls. Histological examination revealed the presence of parasites confined to the glandular stomach. Parasitised gastric glands were dilated, hypertrophied and filled with numerous parasites. The glands had lost their normal cellular architecture and were lined with many undifferentiated cells. In some mice receiving the largest innoculum, the glandular mucosa was congested and the lamina propria infiltrated with eosinophils, polymorphs and lymphocytes.

Histopathological observations on the activity of diclazuril (Vecoxan) against the endogenous stages of Eimeria crandallis in sheep.

Doses of sporulated oocysts of Eimeria crandallis were administered to 60 housed lambs aged 3-4 weeks that had been raised coccidia-free. Thirty of the lambs were medicated with diclazuril at intervals over a 20-day period post-infection with the remaining lambs serving as untreated controls. Lambs were euthanased between 5 and 22 days post-infection (dpi) and sections of the small intestine and caecum examined histologically. Untreated lambs showed loss of surface epithelial cells and villous atrophy associated with first-generation meronts, crypt destruction and crypt hyperplasia associated with pro-gamont stages. Diclazuril appeared to have a direct effect on several stages of the parasite life cycle, in particular, the large first-generation meront. Indications were that the drug also had an effect on second-generation meronts and gamont stages. Therapeutic benefits of diclazuril treatment appeared greatest when given early in the infection before damage to the intestine occurs although removal of coccidial stages did appear to reduce the pathology of the disease.

Knee effusion and quadriceps muscle strength.

Ten healthy subjects with no musculoskeletal problems were tested using a computerized exercise dynamometer. Quadriceps femoris concentric performance was examined during maximal effort isokinetic knee extension at joint angular velocities of 2.09 and 4.18 rad s(-1). A saline and dextrose solution was injected into the knee joint cavity in 30-ml increments, and peak torque and power tests were undertaken with knee effusions of 0, 30, 60, and 90 ml. A protocol which included and excluded a local skin anaesthetic and submaximal exercise prior to the maximal effort test was also undertaken. The results showed that quadriceps peak torque and power were not significantly affected by the amount of knee effusion at either joint angular velocity when submaximal exercise was performed prior to the maximal effort test. However, peak torques were significantly decreased during isokinetic muscle action without prior submaximal muscle work. It was suggested that these findings may be related to fluid movement in the knee joint compartments, the compliance of the joint capsule, and the location that fluid was injected into the joint cavity. Joint swelling is a common symptom of individuals with osteoarthritis and rheumatoid arthritis, as well as those suffering acute joint trauma. This study has extended previous work on joint swelling by examining quadriceps function during joint movement, thus moreeffectivelysimulating joint and muscle activity which occursduring functional activities. Patients with knee joint swelling are thought to experience greater difficulty undertaking quadriceps exercises, and will not perform these exercises effectively until swelling in the joint is reduced. The current study provides some evidence to suggest that a moderate amount of swelling does not impair performance. Assuming that patients do not have other symptoms that contraindicate exercise, they can be encouraged to do more active muscle work.

Quadriceps strength deficit associated with rectus fernoris rupture: a case report.

We report a case of rectus femoris rupture of the quadriceps muscles. Isokinetic muscle testing was undertaken at joint angular velocities of 60, 120, 180, 240, and 300 deg s(-1) to establish the knee extensor muscle strength deficits associated with this condition. The mean strength deficit over these joint angular velocities was 16.6%.

Evaluation of the efficacy of chlortetracycline for the control of chronic respiratory disease caused by Escherichia coli and Mycoplasma gallisepticum.

Using different variations of challenge, three trials were conducted to evaluate the efficacy of chlortetracycline in the control of chronic respiratory disease (CRD) caused by Escherichia coli and Mycoplasma gallisepticum. Experimentally infected birds were offered either food containing chlortetracycline at 300 ppm or water containing the drug at 120 mg litre-1. In each trial, medicated food and water were effective in the control of CRD as assessed by reduction in clinical signs, lower mortality and reduced severity of air sacculitis and other post mortem lesions. Weight gain was also improved by both forms of medication. M gallisepticum antibodies were demonstrated in surviving birds.