Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse - an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.

Rat parvovirus type 1: the prototype for a new rodent parvovirus serogroup.

A newly recognized parvovirus of laboratory rats, designated rat parvovirus type 1a (RPV-1a), was found to be antigenically distinct. It was cloned, sequenced, and compared with the University of Massachusetts strain of rat virus (RV-UMass) and other autonomous parvoviruses. RPV-1a VP1 identity with these viruses never exceeded 69%, thus explaining its antigenic divergence. In addition, RPV-1a had reduced amino acid identity in NS coding regions (82%), reflecting phylogenetic divergence from other rodent parvoviruses. RPV-1a infection in rats had a predilection for endothelium and lymphoid tissues as previously reported for RV. Infectious RPV-1a was isolated 3 weeks after inoculation of infant rats, suggesting that it, like RV, may result in persistent infection. In contrast to RV, RPV-1a was enterotropic, a characteristic previously associated with parvovirus infections of mice rather than rats. RPV-1a also differed from RV in that infection was nonpathogenic for infant rats under conditions where RV infection causes high morbidity and mortality. Thus, RPV-1a is the prototype virus of an antigenically, genetically, and biologically distinct rodent parvovirus serogroup.

Monospecific nematode infections of donor calves with Cooperia punctata.

During a 25 year period, 48 calves from three states were raised helminth-free from birth and inoculated with Cooperia punctata. These calves served sequentially as donors of the parasite. The following aspects of the monospecific infection were of value in successful donor management and contributed to understanding the host-parasite relationship. Calves with no previous experience with C. punctata were the best hosts for establishing initial infections. Male Holstein calves were satisfactory donors of the parasite. Inoculation of calves 6-24 weeks of age with 10,000-11,200 infective third-stage larvae (L3) over a 2 day period produced the desired donors. Occasional over-feeding of calves appeared to reduce the patent period. Superimposed infection was more likely when calves were still patent from previous infection, whereas reinfection was less likely after patency of the earlier infection had ended. Strong immunity eventually developed following oral administration of L3.

Immunogenic effects of culture-derived exoantigens of Cooperia punctata on calves before and after challenge exposure with infective larvae.

The parasitic stages of Cooperia punctata were cultivated in vitro, and the incubation fluid from these stages containing exoantigen (ExoAg; metabolites, secretions, excretions) was diafiltered (retaining material of approximate molecular weight greater than 10,000), concentrated, and lyophilized. Using serum of calves immune to C punctata, ExoAg activity was verified by agar gel immunodiffusion. The ExoAg was combined with adjuvants and was inoculated into calves. Results of preliminary experiments indicated that the ExoAg had potent biological activity capable of inducing severe respiratory distress and death. Subsequently, however, improved inoculation procedures and combination of ExoAg with aluminum hydroxide gel or Freund complete adjuvant consistently prevented this reaction in 25 calves. In helminth-free calves, the ExoAg induced marked eosinophilia, which generally was greater during the ExoAg-exposure period than after challenge exposure with infective larvae. Of 27 calves exposed to the various ExoAg inoculation schedules, 12 had nematode reductions greater than 80%, 10 had less than 16% reduction, and 5 had reductions between 39 and 72%. This polarization of the nematode recovery data generally thwarted statistical difference in overall consideration of all ExoAg inoculation schedules. Additional manifestations of resistance, ie, prolonged prepatent period, reduced patent period, reduced number or lack of nematode eggs passed in feces, and reduced nematode length, were observed in calves of certain ExoAg inoculation schedules. In vitro blastogenic responses of blood lymphocytes was observed in calves exposed to ExoAg. Quantitatively, this cell-mediated immune response to ExoAg was as great as or greater than that after subsequent challenge exposure with infective larvae. Intradermal inoculation of ExoAg into a calf resistant to C punctata induced delayed-type hypersensitivity. However, this reaction to ExoAg was not observed in nonimmunized, helminth-free calves used as controls. Humoral antibodies against ExoAg were detected by an agar gel immunodiffusion test. Calves exposed to ExoAg always seroconverted and usually remained seropositive until necropsy. There were no false-positive results; however, ExoAg did not consistently coprecipitate (by agar gel immunodiffusion) with serum from infected calves that were not exposed to ExoAg.

Economic value and course of infection after treatment of cattle having a low level of nematode parasitism.

To determine whether it is economically advantageous to treat calves having inapparent parasitism, we conducted experiments from 1971 to 1976, involving more than 1,800 calves from 30 pens or lots, using formulations of thiabendazole, levamisole, and crufomate (ruelene). Differential egg counts, cultured larvae, and cultured parasitic stages were used to estimate the kind and degree of nematode parasitism. Differentiation of infective larvae consistently established Cooperia as the predominating (%) genus in all fecal samplings. Bunostomum, when initially present, decreased or disappeared, whereas Trichostrongylus increased; other genera fluctuated less consistently. These qualitative generic fluctuations were not primarily the result of treatment, but more likely were seasonal variation. Judged by average daily gain (ADG), anthelmintic treatment was statistically advantageous at one or more points during the observation periods in 10 of 13 treated groups. In seven treated groups, the observation periods were concluded with statistical advantage in ADG, whereas in three groups, compensatory gain by corresponding controls had neutralized earlier advantages. The comparative influence of the various anthelmintics was not consistent from year to year. When total cost/kilogram gain was calculated from feed efficiency measuremnts and other costs, economic treatment advantage was evident in seven of 11 tests (7 of 10 treatment groups) from 1973 through 1976. This financial advantage, due primarily to feed efficiency and noted after 28 to 51 days, justified anthelmintic treatment. This advantage was not likely lost by the animals in subsequent periods (to 218 days) on pasture or in lots, since ADG indicated the treated calves performed either as well as, or better than, the nontreated controls. Considering all aspects of the study, the results indicate calves coming into Kansas from southern states and weighing 184 to 267 kg may possess a level of subclinical (symptomless) nematode parasitism that when treated will result in a response justifying expense for using anthelmintics.

Utilization of propionic acid by the L4 and adult stages of Cooperia punctata (Nematoda: Trichostrongylidae) grown in vitro.

Cultures of Cooperia punctata, a nematode parasitic in cattle, were studied in Ae medium at a pH of 7.2 to 7.3 under air. In fourth and fifth stages of development, they absorbed or otherwise took into their free pool and tissues, carbon derived from 14C-labeled sodium propionate and converted, by pathways not elucidated, a portion of this carbon into protein and lipid fractions. Thirty minutes postincubation in balanced slat solution was adequate to reduce the amount of isotope in the nematode gut to a constant level. Activity from specifically-labeled 14C-propionate was recovered (as glucose pentaacetate) from worm glucose and from CO2 evolved from cultures consisting of L4 and adult stages. Use of propionate by these worms, for whatever metabolic purpose, would result in depriving the ruminant host of some of its necessary glucogenic precursors, and could account for a specific pathogenic mechanism attendant to heavy infections with this parasite.

Detection of activity for various anthelmintics against in vitro-produced Cooperia punctata.

The in vitro-grown parasitic stages of Cooperia punctata were used to evaluate 28 compounds with different kinds and degrees of in vivo activity. Using presumptive and confirmatory tests, it was possible to establish a group order of in vitro potency that compared favorably with an order based on established in vivo use of these compounds. The procedure lends itself to evaluating activity against a given parasitic growth stage and gives a quantitative estimate (range) of the concentration that produces 50% nematode kill. The system was most successful in detecting compounds with in vivo activity for C punctata, followed in order by compounds active against Cooperia spp, other gastrointestinal nematodes of ruminants, and other nematodes of non-bovine hosts. The procedure showed some differentiation between activity against nematodes versus that against cestodes, trematodes, and arthropods. The system permits considerable flexibility in experimental design, thus making possible the acquisition of the particular information desired. In addition to establishing lethal effects on the nematode, the procedure detected compounds with nematode-anesthetizing effects. The results indicate this in vitro system can be used with some expediency as a preliminary screening method in the search for new anthelmintic compounds.