Fate of Escherichia coli O157:H7 during silage fermentation.

The survival characteristics of Escherichia coli O157:H7 in silage derived from contaminated grass were investigated. The survival of other enteric bacteria was also investigated to determine if E. coli O157:H7 demonstrates enhanced acid tolerance in comparison. Samples of chopped grass were treated as follows: (i) no additive (control); (ii) inoculation with E. coli O157:H7 to a final concentration of log10 4.0 CFU g(-1); (iii) addition of an 85% solution of formic acid at 3.0 ml kg(-1) grass; and (iv) addition of both E. coli O157:H7 and formic acid, at the above concentrations. Treated 6-kg grass samples were packed into laboratory silos, sealed, and stored at 15 degrees C for up to 180 days. Individual replicate silos were removed from storage periodically and subjected to microbiological and chemical analyses. Chemical analyses of the silage samples indicated that lactic acid-dominant fermentations, with a rapid drop in pH, occurred. Numbers of enteric bacteria decreased from log10 7.0 to 8.0 CFU g(-1) to undetectable levels within 19 days' storage. E. coli O157:H7 did not survive the silage fermentation process, with numbers declining from approximately log10 4.0 CFU g(-1) to undetectable levels within 19 days of ensiling. The pattern of decline in numbers of E. coli O157:H7 was the same as that for the enteric bacteria, indicating that under the conditions tested, the acid tolerance of E. coli O157:H7 was not significantly different from the acid tolerance of other enteric bacteria. This study found that E. coli O157:H7 did not survive a good silage fermentation process, indicating that properly ensiled grass that is correctly stored is unlikely to be a vector for the transmission of the pathogen among cattle.

Use of parasite antigen detection to monitor the success of drug therapy in Dirofilaria immitis-infected dogs.

Recently the authors developed a monoclonal antibody-based enzyme immunoassay for circulating Dirofilaria immitis antigen and demonstrated its utility as a diagnostic tool for canine dirofilariasis. In the present study, serum parasite antigen measurements were used to monitor the success of thiacetarsamide therapy in 2 controlled trials that involved 24 naturally infected dogs. Parasite antigen levels correlated significantly with adult worm burdens in untreated control dogs. Antigen levels fell dramatically by 8 wk after treatment in successfully treated dogs and were undetectable 12 wk after treatment in dogs that were parasitologically cured. Microfilarial counts exhibited seasonal periodicity in both treated and control dogs and were not useful in monitoring the success of adulticide therapy. Parasite antigen detection is quite useful in monitoring the efficacy of adulticide therapy for dogs infected with D. immitis. This approach may lead to improved clinical use of thiacetarsamide, and it should facilitate evaluation of new drugs for this important infection.

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs.

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.

Efficacy of ivermectin against third-stage Dirofilaria immitis larvae in ferrets and dogs.

Ivermectin prevented maturation of Dirofilaria immitis when given per os to ferrets at a dosage of 0 X 1 mg ivermectin per kg bodyweight two days after inoculation with third stage heartworm larvae. Similar results were found in dogs treated one day after infection at a dosage of 0 X 05 mg ivermectin per kg. No heartworm were found in the hearts and lungs of five treated dogs compared to an average of 11 worms per dog in five control animals.

Experimental Trichinella spiralis infection in the ferret, Mustela putorius furo.

Laboratory ferrets (Mustela putorius furo) were each inoculated with 500 larvae of a strain of Trichinella spiralis that had been passaged in mice for many generations. The recovery of adult worms from ferrets on Days 4 and 7 of infection represented a mean of 32% of the inoculum, with most of the worms being in the anterior three quarters of the small intestine. Larvae subsequently became encysted in the ferret musculature, with the diaphragm alone yielding as many as 5,750 larvae upon digestion. For comparative purposes, mice were each inoculated with 200 T. spiralis from the same batch of larvae. The recovery of adult worms from mice represented a mean of 54% of the inoculum. An abrupt decline in worm population, typical of infections in the mouse, was observed in both mouse and ferret hosts; in neither species had the decline begun on Day 7 of infection, but it had clearly begun by Day 11 and was essentially ended by Day 14.

Immunization of ferrets against Dirofilaria immitis by means of chemically abbreviated infections.

Ferrets were exposed to two successive inoculations with 30 third-stage larvae of the canine heartworm Dirofilaria immitis, the inoculations being approximately 5 months apart. Each infection was terminated by ivermectin therapy approximately 2 months after inoculation. The ferrets were challenged with 30 larvae 3 weeks after the termination of the second infection, and were necropsied approximately 6 months after challenge. Of the four ferrets that survived this protracted experimentation, two were entirely free of heartworm while the other two had only a single female worm each. In contrast, 14 control ferrets that had not been immunized (four of which had been given ivermectin doses) were all infected at necropsy, yielding a mean of 6.6 worms per ferret.

Efficacy of Ivermectin against Dirofilaria immitis larvae in dogs 31, 60, and 90 days after injection.

Ivermectin, given as a single oral dose of 0.05 mg/kg, prevented the maturation of Dirofilaria immitis in dogs if given at 31 or 60 days after infection. When treatment was delayed 90 days after injection, maturation of worms occurred in 3 of 5 dogs.

Efficacy of avermectin B1a against microfilariae of Dirofilaria immitis.

Avermectin B1a given at a dose level of 0.05 or 0.1 mg/kg of body weight caused rapid removal of Dirofilaria immitis microfilariae from the blood of dogs with heartworm infections. If the adult worms were also killed with an arsenical (melarsoprol), the removal of microfilariae was permanent.

Avermectins, new family of potent anthelmintic agents: efficacy of the B1a component.

When given to sheep as a single oral dose at 0.1 mg/kg, the B(1a) component of the avermectins caused a reduction of >95% in the numbers of Haemonchus contortus, Ostertagia circumcincta (including inhibited L(4) larvae), Trichostrongylus axei, Trichostrongylus colubriformis, Cooperia oncophora, and Oesophagostomum columbianum. When given to cattle as a single oral dose at 0.1 mg/kg, avermectin B(1a) was >95% effective in reducing the numbers of Haemonchus placei, Ostertagia ostertagi (including inhibited L(4) larvae), T. axei, T. colubriformis, C. oncophora, Cooperia punctata, Oesophagostomum radiatum, and Dictyocaulus viviparus. Avermectin B(1a) was similarly effective, with the exception of a detectable loss in activity against adult C. oncophora, when administered to cattle as a parenteral injection. Some of these ruminant parasites were fully susceptible to dosages of avermectin B(1a) at 0.025 mg/kg, e.g., D. viviparus, O. radiatum, O. ostertagi, and H. contortus. Avermectin B(1a) removed 83 to 100% of Ancylostoma caninum from dogs given a single oral dose of 0.003 to 0.005 mg/kg. The poultry nematodes Capillaria obsignata and immature Ascaridia galli were effectively removed by avermectin B(1a) at 0.05 and 0.1 mg/kg, respectively, but 0.1 mg/kg was not effective for Heterakis gallinarum. Thus, the avermectins would appear to have unprecedented potency and spectrum of biological activity.

Trial of avermectin B1a, mebendazole and melarsoprol against pre-cardiac Dirofilaria immitis in the ferret (Mustela putorius furo).

Avermectin B1a was found active against the pre-cardiac stage of Dirofilaria immitis in ferrets. The drug was given orally, at 0.2 mg/kg, on days 38--42 of infection. At necropsy, 5 1/2 months after inoculation, worms were almost totally absent from the hearts of these ferrets. The efficacy of melarsoprol against developing Dirofilaria, which has been demonstrated in dogs by other workers, was demonstrated in ferrets given oral doses, at 100 mg/kg, on days 38--42 of infection. The reported efficacy of mebendazole was not evident in the ferret host in this study, probably because the treatment (100 mg/kg, orally, on days 38--42) was shorter and later than that reported for dogs.

Dirofilaria immitis: experimental infections in the ferret (Mustela putorius furo).

The ferret, Mustela putorius furo, was found to be susceptible to Dirofilaria immitis infection when exposed to low (14) or high (280-420) numbers of infective larvae harvested from Aedes aegypti. Eight ferrets (half of them cortisonized) were inoculated subcutaneously with 14 larvae each. All of them were subsequently found to harbor D. immitis in the heart, and all but one of them had worms of both sexes. Six of these ferrets were examined for microfilaremia at 31 to 35 weeks after inoculation; 3 were positive (one observed only at postmortem examination) and there was evidence that fertilization of female worms had occurred in one other. Females up to 25.5 cm and males up to 16.0 cm were recovered. There was no evidence that the cortisonization of some ferrets had affected the infections. Both male and female ferrets became infected. Four cortisonized ferrets were inoculated with 280 or 420 larvae of D. immitis (divided equally between subcutaneous and intraperitoneal routes). All of them died 16 to 18 weeks after inoculation, yielding 102 to 125 immature D. immitis. In these lethal infections, worms were recovered from the heart and adjoining vessels, and also from vascular and extravascular sites throughout the body.

The in vivo and in vitro analysis of immunity to Trichinella spiralis in mice and rats.

Wistar-Furth strain male rats and CFW strain male mice were immunized against Trichinella spiralis using an antigenic fraction derived from a cell-free homogenate of mature muscle larvae. In rats, animals immunized with 250 mug of antigen harboured significantly fewer (135000) muscle larvae 30 days after oral challenge than controls (231000). Further 7-day-old adult worms derived from immunized rats shed 48% fewer (P less than 0.001) newborn larvae over a 24h period in vitro than adult worms from non-immunized animals. Mice were injected with either 10 or 100 mug of antigen. In comparison with non-immunized controls, mice immunized with 100 mug of antigen harboured significatnly fewer adult worms at days 7 and 9 after oral challenge infection, while female worms recovered from immune mice on days 6-10 after challenge shed fewer newborn larvae in vitro. Finally, mice immunized with 100 mjg of antigen harboured significantly fewer (10391) muscle larvae at 30 days after challenge than did controls (47750). Immunization of mice with 10 mug of antigen did not induce a statistically significant reduction in adult worms at either day 7 or 9 after challenge (P less than 0.5). However, adult female worms from mice receiving 10 mug of antigen still shed fewer larvae than did adults from control mice (P less than 0.05). Mice immunized with 10 mug of antigen harboured significantly fewer (13700) recoverable muscle larvae than did controls at 30 days after challenge (39000).