Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.     

Outcomes in mice with abdominal angiostrongyliasis treated with enoxaparin

Abdominal angiostrongyliasis (AA) is caused by the nematode Angiostrongylus costaricensis. Parasite-associated thrombosis of mesenteric vessels may lead to intestinal infarction, which might be prevented with anti-thrombotic agents. This study assessed the effect of enoxaparin on survival and pathological findings in Swiss mice with AA. In this experiment, 24 mice were infected with A. costaricensis (10 L3 per animal) followed by treatment with subcutaneous enoxaparin (40 mg/kg/day) or water (sham), starting from 15 days post-infection (dpi) and continued until animal death. Animals were monitored until death or sacrifice at the 50th dpi. Ten mice (42%) were dead after 36 ± 8 dpi. Of these, five (50%) were treated with enoxaparin. Animals treated with enoxaparin and sham did not differ in terms of weight loss (median, 1.3 vs. 4.2 g; P = 0.303) and macroscopical findings. Microscopically, no difference was found in regard to vascular granuloma (median grade, 2 vs. 3; P = 0.293) and presence of either vasculitis (75% vs. 100%; P = 0.217), mesenteric thrombosis (33% vs. 50%; P = 0.680), or bowel necrosis (25% vs. 50%; P = 0.400). Mice dead before the 50th dpi showed more pneumonia (90% vs. 21%; P = 0.002), bowel infarction (40% vs. 0%; P = 0.02), and purulent peritonitis (60% vs. 7%; P = 0.008) compared to survivors. Prophylactic enoxaparin in mice did not prevent tissue damage and mortality related with AA. The lower prevalence of mesenteric thrombosis and bowel infarction regardless of treatment were notorious. Frequent septic complications suggest the need of studies addressing the effect of antibiotics in AA.     

The relationship between Pepper mottle virus source leaf and spread of infection through the stem of <Emphasis Type="BoldItalic">Capsicum</Emphasis> sp.

Summary.  Pepper mottle virus (PepMoV) systemically infects Capsicum sp. in a typical source-to-sink manner with movement through the stem occurring in a predictable pattern. This study was carried out to determine the relationship between the inoculated leaf as a source of inoculum and the spread of PepMoV infection through the stem. C. annuum‘Early Calwonder’ plants were mechanically inoculated onto the first leaf with PepMoV and sets of 30 plants had their inoculated leaves removed each day from 1 through 7 days post-inoculation (dpi) with the inoculated leaves tested for infection by ELISA at the time of excision. Beginning at 2 dpi, PepMoV infection in the stem of plants with the inoculated leaf excised and plants of a nonexcision control treatment was determined using immuno-tissue blot analysis. PepMoV was detected in inoculated leaves beginning at 3 dpi with the percentage of infected leaves increasing each day through 7 dpi. PepMoV was first detected in the stem of inoculated plants of the 3 dpi excision treatment. The accumulation and extent of spread of infection in the stem was similar for plants that had their inoculated leaf removed at a time preceding detection by ELISA to plants in the nonexcision control treatment. These findings suggest that once virus is allowed to enter the stem from the inoculated leaf, subsequent spread of infection through the stem is a process independent from the source leaf. Received March 25, 2002; accepted May 17, 2002 Published online July 22, 2002     

Comparison of faecal techniques including FLOTAC for copromicroscopic detection of first stage larvae of Angiostrongylus vasorum

Angiostrongylus vasorum is a metastrongylid nematode that resides in the pulmonary arteries and the right heart chambers. In dogs, infection results in respiratory, bleeding and neurological disorders and further clinical signs. In the present study, FLOTAC was evaluated for the detection of first-stage larvae (L1) of A. vasorum in canine faecal samples. This technique is based on the counting of parasitic stages (eggs, larvae, oocysts and cysts) in chambers after spinning of faecal samples onto a surface. In a first step, nine flotation solutions were evaluated using faeces of two experimentally infected dogs. Zinc sulphate (specific gravity (s.g.) 1.2) and zinc sulphate plus potassium iodomercurate (s.g. 1.45) gave good results. However, with the latter technique, the larvae were slightly deformed. Subsequently, FLOTAC, using zinc sulphate, was compared through a randomisation technique with McMaster, flotation in tube and Baermann–Wetzel technique. The mean larvae per gramme (LPG) obtained by the FLOTAC for both dogs was significantly higher (P < 0.05) than those obtained by the other three techniques (the means of the other techniques all lie below the 95% CI of the mean LPG of the FLOTAC technique). In addition, the FLOTAC results were consistent across replicates with only Poisson (or random) variation between individual replicates. The other techniques appear to be less consistent with evidence of extra-Poisson variation in at least one of the two dogs across the replicates within each technique. The FLOTAC technique may contribute to an improvement of the ability to diagnose canine lungworm infections and represent a valuable alternative for larval counting of A. vasorum in faecal samples, especially following transport or storage where there may be limited larvae viability, and larval migration techniques cannot be used.     

A comparative study of the long-term effects of piroxicam and ketoprofen on the gastric mucosa, kidney, liver, and hematopoietic system of dogs

The use of non-steroidal anti-inflammatory drugs (NSAIDs) can be associated with severe adverse effects. The aim of this experimental study was to investigate the effect of piroxicam and ketoprofen, two NSAIDs with different excretion pathways, on the gastric mucosa, kidney, liver, and hematopoietic system of dogs. Fifteen mixed-breed healthy dogs of both sexes aged between 1 to 8 years were randomly divided in three groups and treated with piroxicam (0.15 mg/kg intramuscularly (IM)), ketoprofen (1 mg/kg IM) or placebo daily for 21 days. Although not statistically significant (p > 0.05), the dogs receiving ketoprofen showed fewer and less severe lesions than the dogs in the piroxicam group. None of the dogs showed any clinical signs related to the gastric lesions. Serum biochemical and complete blood count parameters did not change significantly after NSAlD administration (p > 0.05). However, by day 14, a decreased number of platelets and prolonged bleeding time were detected in treatment groups compared with the control group (p < 0.05). The clinical significance of this prolongation is unclear. This study suggests that ketoprofen and piroxicam produce mild lesions when administered to healthy dogs for 21 days, and there is no difference between the two groups in the number and severity of lesions. There may be an indication that longer duration of drug administration may result in a greater number of gastric lesions. However, after long-term NSAID exposure (21 days in our study), gastric tolerance to the damage caused by NSAIDs will be developed.     

Dose dependency of prednisolone tertiary-butylacetate (PTBA) treatment on the establishment and site predilection of Echinococcus multilocularis in an alternative definitive host model using Mongolian gerbil (Meriones unguiculatus)

Mongolian gerbils (Meriones unguiculatus) were orally inoculated with 10,000 protoscoleces of Echinococcus multilocularis per head after being divided into five groups (A–E). Each group was dosed with prednisolone tertiary-butylacetate (PTBA) as follows: A, 0 mg; B, 0.5 mg; C, 2 mg; D, 5 mg; and E, 10 mg/head. All animals were injected subcutaneously with control solvent or PTBA every other day from 6 days pre- to 6 days post-infection. Autopsy was performed at 7 days post-infection. Doses of PTBA and the number of worms recovered showed a positive correlation (r=0.929, P < 0.0001). In groups A, B and C, the predilection site of the worms in the small intestine could not be determined, while in group D the worms were found more in the anterior part. In group E, the predilection site was the anterior part, followed by the middle and the posterior parts of the small intestine (Fisher's test: P < 0.01). The number of worms recovered from the anterior and the middle part of the small intestine also correlated positively with PTBA dose (anterior part: r=0.930, P < 0.0001, middle part: r=0.917, P < 0.0001). All groups of the PTBA-treated animals showed significant loss of weight compared to the non-treated animals (P < 0.01). Received: 26 September 1999 / Accepted: 24 November 1999     

Persistent efficacy of topical eprinomectin against nematode parasites in cattle

Six studies were conducted to evaluate the persistent efficacy of eprinomectin pour-on against experimental challenges with infective nematode larvae in calves. In each study, calves were randomly assigned to one untreated group and up to four test groups, which were treated with eprinomectin at 500 μg/kg body weight at weekly intervals before single bolus challenge. The calves were necropsied approximately 4 weeks after challenge infection for nematode recovery. Eprinomectin pour-on provided ≥90% efficacy against challenge with Haemonchus placei, Trichostrongylus axei and T. colubriformis at 21 days after treatment and against Cooperia oncophora, C. punctata, C. surnabada, Dictyocaulus viviparus, Nematodirus helvetianus, Oesophagostomum radiatum and Ostertagia ostertagi at 28 days after treatment. Received: 14 April 2000 / Accepted: 18 May 2000     

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice

CD4⁺ T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 μm vs. 294.4 ± 72.2 μm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.     

In vivo validation of Aloe ferox (Mill). Elephantorrhiza elephantina Bruch. Skeels. and Leonotis leonurus (L) R. BR as potential anthelminthics and antiprotozoals against mixed infections of gastrointestinal nematodes in goats

Aloe ferox (Mill)., Elephantorrhiza elephantina Bruch. Skeels. and Leonotis leonurus (L) R. BR. are some of the plants used by farmers in the Eastern Cape Province to control worms in goats, but information on their efficacy is lacking. The study was conducted to determine efficacy of these plants on gastrointestinal nematodes in natural mixed infections in goats. Forty-eight male goats aged 8–12 months were divided into eight groups (Treatments A–H) of six animals each, balanced in terms of liveweight and worm egg count. Treatments A to F received plant extracts, three animals in each group receiving doses of 250 mg/kg and the other three receiving 500 mg/kg at concentration of 100 mg/ml, while those in G and H received Valbazen? (11.36% albendazole) at 10 mg/kg, and 0.5 ml/kg distilled water, respectively per os. Faecal samples were collected on days 0, 3, 6 and 9 for faecal egg counts (FEC), and body weights recorded on days 1 and 9. Results showed significant reductions (P < 0.05) in strongyle eggs by A. ferox extract at dose levels of 500 mg/kg on days 3, 6 and 9, while reductions in Eimeria spp. oocysts were observed on days 3, 6 and 9 for animals that received 500 mg/kg doses. E. elephantina caused significant reduction (P < 0.05) of Trichuris spp. eggs on days 3 and 6, respectively at 250 mg/kg dose level, whereas L. leonurus also caused significant reduction (P < 0.05) in FEC of Trichuris spp. and Eimeria spp. oocysts at 250 mg/kg dose level on day 9. Albendazole caused reductions (P < 0.05) in strongyle eggs on days 3 and 6, Trichuris spp. on days 3, 6 and 9, and on coccidia, it caused a reduction (P > 0.05) on day 1, whereas on days 6 and 9, there was an increase. On total mixed infections, highest FECR% were observed with the extract of A. ferox on days 3 (53%), 6 (54%) and 9 (58%) at 500 mg/kg,whereas albendazole had efficacy levels of 39%, 44% and 29% on days 3, 6 and 9, respectively. Body weight of goats from days 1 to 9 were not significant different from the control. The study revealed efficacy of A. ferox, E. elephantina and L. leonurus against gastrointestinal parasites at high doses (500 mg/kg), showing that the plants have the potential to be used as anthelminthics.     

Toltrazuril treatment of cystoisosporosis in dogs under experimental and field conditions

Coccidia of the genus Cystoisospora cause mild to severe diarrhoea in dogs. The effects of toltrazuril treatment on cystoisosporosis were studied under experimental and field conditions. Twenty-four puppies were experimentally infected each with 4 × 10⁴ oocysts of the Cystoisospora ohioensis group. Three groups of six puppies were treated 3 dpi with 10, 20 or 30 mg/kg body weight of toltrazuril suspension (5%); the remaining six puppies served as non-treated controls. Toltrazuril suspension or microgranulate were given once in a dose of 10 or 20 mg/kg body weight, respectively, to naturally infected puppies in conventional dog breeding facilities, depending on the coproscopical evidence of infection. Oocyst excretion and clinical data were recorded. Under experimental conditions, the non-treated puppies excreted oocysts beginning at 6 dpi and suffered from catarrhalic to haemorrhagic diarrhoea. On 12 dpi, four of six non-treated puppies died. Irrespective of the dose, toltrazuril treatment totally suppressed oocyst excretion and no diarrhoea or other signs of disease were observed in the treated groups. Natural Cystoisospora infections were regularly found during the 3rd or 4th week of age in dog breeding facilities although not always associated with diarrhoea. A single oral application of toltrazuril abrogated oocyst shedding and the treated puppies remained generally coproscopically negative during the following 2–4 weeks. Cystoisospora is pathogenic for puppies and can induce severe disease. Natural infections are common in conventional dog breeding facilities. Toltrazuril treatment is suitable for controlling cystoisosporosis under experimental and field conditions. A single oral treatment for puppies in the 3rd or 4th week of age is recommended. Received: 2 February 2000 / Accepted: 4 February 2000     

Trypanosoma cruzi infection: do distinct populations cause intestinal motility alteration?

Chagas disease, caused by Trypanosoma cruzi, is an important public health problem in Latin America. Disturbances in gastrointestinal motility are observed in 15–20% of patients at the chronic phase. We previously observed a decrease in intestinal motility in mice infected with Y strain from T. cruzi. Thus, we decided to test if infection with other T. cruzi strains also caused the intestinal disturbance. Male adult Swiss mice were infected intraperitoneally with CL–Brener clone (CL-B), Brazil strain (Br), or Dm28 clone (Dm) of T. cruzi. All infected mice presented a low cumulative mortality (CL-B, 17%; Br, 8%; Dm, 25%) at 35 days post infection (dpi) and their typical parasitemia curves. Br and Dm groups exhibited a maximal reduction of intestinal motility at 35 dpi (176.8 ± 51.3 and 198.3 ± 52.6 min, respectively), when compared with non-infected mice (90.2 ± 19.5 min). However, CL mice presented the peak of delayed intestinal transit at 12 dpi (191.0 ± 33.3 min), when compared with non-infected mice (105.6 ± 26.4 min), very close to the 15 dpi for the intense alteration (310.2 ± 67.4 min) observed with the Y strain. We clearly demonstrate a reduction in intestinal motility in mice infected with different groups of T. cruzi during the acute phase of the infection. Since Br, Dm, and CL strains presented low mortality rates in adult Swiss mice, a prospective study concerning the chronic intestinal alteration is encouraged, particularly for studies of alternative therapies.     

The intraperitoneal inoculation of Lactobacillus casei in mice induces total protection against Trichinella spiralis infection at low challenge doses

The following effects of Lactobacillus casei in NIH mice were evaluated: the establishment of Trichinella spiralis adult worms in the intestine (AWI), larvae per gram of muscle tissue (LPG), levels of IgG and IgA, and levels of IL-4 and IFN-γ. One hundred and eight mice were allocated at random into 18 groups of six mice each. Each mouse in treated or non-treated groups was inoculated intraperitoneally once a week during 6 weeks with L. casei or phosphate-buffered solution. Later each mouse was challenged either with 200, 50, or 25 T. spiralis infective larvae. When the infection dose was 200 T. spiralis infective larvae, the reductions in AWI were 78.6% at 4 days after infection (dai) and 76.7% at 10 dai; while the reduction of LPG was 80.9% with respect to control groups. When the infection dose was 50 or 25 T. spiralis infective larvae, the reductions of AWI were 100% both at 4 and 10 dai; while the reduction of LPG at 30 dai was also 100% with respect to control groups. The levels of IgG and IgA anti-T. spiralis and IL-4 were significantly higher (P < 0.01) at 4 and 10 dai in mice from groups treated with L. casei than in animals in control groups; while at 10 dai, the levels of IFN-γ were higher in control mice (P < 0.01) than in L. casei-treated animals. The results suggest that frequent treatment of mice with L. casei induces a total protection against infection with low doses of T. spiralis.     

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel

The agar-gel migration technique has previously been described, however, aspects regarding the effect of timing on worm migration needed further scrutiny. In the first experiment, pigs inoculated with Oesophagostomum dentatum were slaughtered simultaneously and their intestines stored at 21–23 °C until processed pairwise 2, 4, 6, 8, 12 and 18 h after slaughter. More than 95% of the worms migrated out of the agar if processed within 6 h. In the second experiment, intestines were treated immediately after slaughter and the migratory speed of adult worms or 4th-stage larvae of O. dentatum or O. quadrispinulatum, or adult Hyostrongylus rubidus were studied. For both Oesophagostomum species, more than 90% of the worms were recovered within 1 h. H. rubidus was significantly slower; however, approximately 98% of the worms had migrated out of the agar-gel by 20 h. This information is essential in planning experiments where recovery of live worms is of value. Received: 11 July 1997 / Accepted: 15 September 1997     

Host-parasite relationships between Echinostoma caproni and RAG-2-deficient mice

The RAG-2-deficient mouse, a strain of genetically altered mice lacking B- and T-lymphocytes, was used as a host for Echinostoma caproni. In all, 12 male RAG mice were exposed to 25 cysts each, and 12 served as uninfected controls. Mice were necropsied at 2 and 3 weeks postinfection (p.i.). The mean number  ±SE (9.7 ± 2.4) of worms recovered from infected mice at 2 weeks p.i. was not significantly different from that recovered at 3 weeks p.i. (6.5 ± 2.2). The intestinal circumference of infected RAG mice was significantly greater than that of the controls at 2 and 3 weeks p.i. A significant goblet cell hyperplasia occurred at 2 weeks p.i., but the response was not effective in eliminating worms from the RAG mice. The effect of a high cyst burden was examined by exposure of 8 RAG and 8 ICR mice to 100 cysts each. The body length and area and the oral sucker area of worms grown in RAG mice were significantly greater than those of worms grown in ICR mice. Worm recovery at up to 3 months p.i. was examined in RAG mice exposed to 25 cysts and necropsied every 2 weeks p.i. The mean worm recovery recorded at 2 weeks p.i. was significantly greater than that noted at 12 weeks p.i., at which time worm rejection from the RAG mouse host first occurred. The RAG mouse is a useful host for studies on E. caproni in a murine host that lacks B- and T-lymphocytes. Received: 7 August 1998 / Accepted: 1 October 1998     

<Emphasis Type="Italic">Angiostrongylus vasorum</Emphasis> (Baillet, 1866) Kamensky, 1905: emergence of third-stage larvae from infected<Emphasis Type="Italic"> Biomphalaria glabrata</Emphasis> snails

Biomphalaria glabrata snails were experimentally infected with Angiostrongylus vasorum first-stage larvae and divided into four groups of 30 snails. To assess the shedding of third-stage larvae (L3), the snails were maintained under different stimuli: group 1 60 W light bulb for 24 h, group 2 37 °C water bath for 24 h, group 3 room temperature (23–25 °C) for 24 h, Group 4 room temperature (23–25 °C) for up to 15 days. After 24 h, a total of 512 A. vasorum L3, alive and active, were released by snails from group 1, while 2,446 L3 were released from group 2 and five L3 from group 3. After 15 days, snails from group 4 released a total of 44 L3. To evaluate the infectivity of A. vasorum L3, two mongrel dogs were successfully infected with L3 released by snails from groups 1 and 2, confirming that the infection of dogs with A. vasorum L3 was possible, independently of ingestion of the mollusk intermediate host. The results shown in these experiments suggest that angiostrongylosis could be directly transmitted to the definitive hosts, with implications for the parasites life cycle.     

Evaluation of Helicobacter pylori eradication by triple therapy plus Lactobacillus acidophilus compared to triple therapy alone

The purpose of this study was to evaluate the influence of adding Lactobacillus acidophilus to a triple regimen for Helicobacter pylori eradication in untreated patients with peptic ulcers or ulcer-scars. This was a pre-randomized, single-blind, interventional, treatment-efficacy study with active controls and parallel-assignment, set in Coimbra, Portugal, on 62 consecutive H. pylori-positive untreated adults with peptic ulcers or ulcer-scars, diagnosed by gastroduodenoscopy, with pre-treatment direct Gram-staining and culture of gastric biopsies. The first 31 patients received esomeprazole 20 mg, amoxicillin 1000 mg and clarithromycin 500 mg (EAC), all b.i.d., for 8 days. The remaining 31 added L. acidophilus, 5 × 10⁹ organisms per capsule, 3 + 2 i.d. for 8 days (EACL). The main outcome measure was ¹³C urea breath test (UBT), ≥6 weeks after completion of therapy. Successful eradication (UBT-negativity after treatment), was similar in both groups (EAC = 80.6%; EACL = 83.9%, p = 0.740) by both intention-to-treat and per-protocol analysis. The non-eradicated strains were susceptible in vitro to both antibiotics. Adding L. acidophilus to EAC triple therapy did not increase H. pylori eradication rates. Considering the cost and the burden of ingesting five extra capsules daily, supplementing the EAC therapy with L. acidophilus, at this dose, shows no benefit. Further studies with different dosages and duration of treatment, and other probiotics or probiotic combinations are required to improve eradication.     

Efficacy of various anticoccidials against experimental porcine neonatal isosporosis

The efficacies of 20 mg/kg body weight (BW) of toltrazuril (Toltra) 2 days post-infection (dpi), 2 mg/kg BW of diclazuril 2 and 3 dpi and 200 mg/kg BW of sulphadimidine 2, 3 and 4 dpi were compared in a model for piglet isosporosis. Weight gain (first 4 weeks of life) and diarrhoea and oocyst excretion from 4 to 11 dpi were evaluated (10–12 piglets/group). Additionally, animals were killed and examined for pathohistological changes of the small intestines 5, 7, 11 and 14 dpi (n = 3 per group and time point) and lengths of the intestinal villi. Diarrhoea (semi-liquid or liquid faeces) was seen from 5 dpi in all groups except Toltra, and the differences in prevalence and intensity of diarrhoea were statistically significant (p < 0.05) between Toltra and the other groups, which were similar (trial 1). Oocyst excretion was greatly reduced in the Toltra group, which was also statistically significant for the mean and median excretion rates and the percentage of excreting piglets between Toltra and the other groups (p < 0.05). Weight gain was highest in Toltra (p < 0.05). Histopathology revealed mostly villous necrosis and atrophy in the small intestines except the duodenum, which peaked at 7 dpi, in all groups except Toltra. Between 5 and 11 dpi, the Toltra group had significantly longer villi than the other groups. Reduced weight gain and diarrhoea caused by Isospora suis was controlled by a single application of Toltra in the pre-patent period, while neither diclazuril nor sulphadimidine improved the clinical picture of isosporosis.     

Effectiveness of mefloquine against Clonorchis sinensis in rats and Paragonimus westermani in dogs

The aim of the study is to explore the effect of mefloquine against Clonorchis sinensis and Paragonimus westermani. For anti-C. sinensis study, a total of 71 rats were divided into four batches for oral infection of each rat with 50 C. sinensis metacercariae. Five to 7 weeks post-infection, groups of rats were treated orally with mefloquine at single doses or multiple daily doses while infected, but untreated rats served as control. All treated rats were euthanized 2 weeks post-treatment for assessment of efficacy. For anti-P. westermani study, two batches of eight and ten dogs were each infected intraperitoneally with 100 P. westermani metacercariae. Eighty-five to 96 days post-infection, groups of two or three dogs were treated orally with mefloquine and groups of two dogs were treated with praziquantel at a single dose or multiple doses. In each batch of test, three untreated but infected dogs served as control. All treated dogs were euthanized 26–30 days post-treatment for evaluation of efficacy. In rats infected with C. sinensis and treated orally with mefloquine at a single dose of 75 and 150 mg/kg, no effect against C. sinensis was observed. When the dose of mefloquine was increased to 250 mg/kg, one third (five out of 15) rats died 3–5 days post-treatment. Although the mean worm burden was lower than that of the control, the difference between the treated and control groups was not statistically significant (P > 0.05) with worm burden reduction of 22.4%. Whereas, the group of infected rats received mefloquine at a daily dose of 100 mg/kg for 3 days, one out of five rats died after the last administration. The mean worm burden was significantly lower than that of the control with worm burden reduction of 67.6% (P < 0.01). In the first test of mefloquine against P. westermani, three infected dogs received two oral doses of the drug, 50 mg/kg, given at a 4-h interval, the mean worm burden were similar to that of the control. While other two dogs were treated with praziquantel at the same dose schedule, the worm burden reduction of 78% was observed. In the second test, three and two dogs were treated with mefloquine 50 mg/kg daily for 5 days or 100 mg/kg daily for 2 days; the mean worm burdens of the two groups were lower than that of the control with worm burden reduction of 65.6% and 51.9%, respectively. However, only the difference of mean worm burdens between mefloquine 50 mg/kg given daily for 5 days and the control was statistically significant (P < 0.05). Other two dogs treated with praziquantel at a single dose of 100 mg/kg were cured. The results indicate that under the appropriate dose schedule mefloquine exhibits less effect against C. sinensis in rats and P. westermani in dogs.     

Toxocara canis in experimentally infected silver and arctic foxes

In two experiments, thirty-six farm foxes of two species were inoculated with various doses of infective Toxocara canis eggs or tissue larvae isolated from mice. In experiment I, six adult arctic foxes (Alopex lagopus; 11-month old) were each inoculated with 20,000 eggs and sacrificed 100, 220, or 300 days post infection (dpi), while ten silver fox cubs (Vulpes vulpes; 6–9-week old) were infected with varying doses of eggs (30–3000) and necropsied 120 dpi. In experiment II, two groups of five cubs and two groups of five adult silver foxes received both a primary inoculation and either one or two challenge inoculations: primary inoculation (day 0) with 400 embryonated eggs were administered to five cubs and five adults and another five cubs and five adults received 400 larvae. At 50 dpi, the first challenge inoculation (400 eggs) was inoculated in all animals. At 100 dpi, three animals from each group were necropsied. The remaining two animals in each group were received a second challenge inoculation of 400 tissue larvae on 100 dpi and were subsequently necropsied at 150 dpi. In both experiments, the highest numbers of larvae per gram (lpg) of tissue was found in the kidneys (100–300 dpi). In adult foxes receiving a high dose (20,000 eggs), increasing larval burdens were found in the kidneys over the course of the experiment (up to 300 dpi). The larval migration from the lungs to other tissues appeared to be dose-dependent with the highest larval burdens found in adult foxes. The faecal egg excretion, larval burden and intestinal worm burdens decreased from the first to the second challenge infection.     

Improved efficacy of tetracycline by acaciasides on <Emphasis Type="Italic">Dirofilaria immitis</Emphasis>

The discovery of Wolbachia, a bacterial endosymbiont that occurs in the filarial parasite and its sensitivity to tetracycline, has fostered a new initiative in the development of suitable antifilarial drugs. The present study is an attempt to investigate whether adding acaciasides (saponins from Acacia auriculiformis) to the standard dose of tetracycline would further improve the efficacy of tetracycline treatment against Dirofilaria immitis microfilariae in vivo. Treatment of microfilaremic adult dogs (body weight range 8–12 kg) with tetracycline at 10 mg/kg/day for 40 days resulted in 72% and 83% reduction in mf count on days 15 and 30, respectively, and the maximum reduction in mf count (91%) was achieved on day 75 post-treatment. However, treatment with tetracycline (10 mg/kg/day for 40 days) followed by acaciasides (10 mg/kg/day for 7 days) resulted in almost 100% clearance of mf at a faster rate on day 45 post-treatment and ensured long-term (until 4 months post-treatment) protection against microfilaremia. Data from polymerase chain reaction analysis reveals that compared to untreated dogs, in treated dogs, there was marked reduction in Wolbachia specific wsp markers in fast depleting mf population. The present data indicate that prior tetracycline treatment enhances microfilaricidal activity of saponins. This effect may be additive or synergistic as the worms are weakened by Wolbachia depletion, and these weakened microfilariae are possibly killed by the saponins. S. Datta and S. Maitra have contributed equally in this paper.