Novel insights into the pathogenic importance,diagnosis and treatment of the rumen fluke (Calicophoron daubneyi) in cattle

Recently, sharp increases in the prevalence of rumen fluke infections have been recorded throughout Western Europe. However, scarce information is available on the diagnosis, pathogenic importance and control of this parasite. We undertook 3 pilot studies to gain more insights into these aspects of rumen fluke biology in cattle. First, we evaluated the diagnostic performance of mini-FLOTAC to detect adult rumen fluke infections based on faecal egg count in an abattoir survey and found high sensitivity (0.94) and specificity (0.98). Moreover, there was an association between ruminal fluke burden (assessed by visual scoring) and faecal egg count and a cut-off of 200 eggs per gram is proposed to detect highly infected animals (>200 flukes present in the rumen and/or reticulum). There was also a significant association between ruminal fluke burden and faecal consistency. However, in a second study, we performed a case-control field survey to investigate the association between rumen fluke infection and herd-level problems with diarrhoea and no association was found. Finally, we evaluated the use of closantel (Flukiver^®, Elanco Animal Health, subcutaneous administration at 10 mg/kg) to treat rumen fluke infection on 3 herds, but no significant reduction in egg output post-treatment was found. Because this result is in contrast with a previous study using an oral dose of closantel, more research is required into the effect of administration route on the efficacy of closantel on rumen fluke.     

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

The use of cysteine proteinases from Fasciola hepatica adult flukes for the serodiagnosis of caprine fasciolosis by means of an indirect ELISA test was studied. Two proteolytic fractions from adult fluke homogenates, with apparent molecular weights of 28 and 34 kDa (P28 and P34 respectively), were characterised as cysteine proteinases using azocasein assays and gelatin gel analysis. Both P28 and P34 fractions were electroluted and used as antigens in two different indirect ELISA tests. Serum IgG levels against P28 and P34 in goats given an experimental primary infection with 200 metacercariae or in goats given two experimental infections with 200 metacercariae were determined and compared with those observed in an uninfected control group. ELISA tests using both cysteine proteases showed a rapid and consistent detection of specific IgG in all experimentally infected goats. The IgG response to P28 was the first to be detected as early as 2-3 weeks post-infection and remained elevated throughout the experiment. The response to P34 was detected later (4-6 wpi) and disappeared in some animals at 18 wpi, while flukes were still present in the bile ducts. No significant differences were observed between the anti-P28 and anti-P34 IgG responses between animals receiving a primary or a challenge infection. The results of our study, although preliminary, are promising since the P28 ELISA described here may be a reliable method for the immunodiagnosis of F. hepatica infection in goats.     

Efficacy of fenbendazole against adult Dictyocaulus viviparus in experimentally infected calves.

Fenbendazole, methyl-5(phenylthio)-2-benzimidazolecarbamate, a new broad-spectrum anthelmintic, was evaluated against the adult Dictyocaulus viviparus, lungworm of cattle, in artificially infected calves. At a dosage of 5 mg/kg of body weight, fenbendazole removed 100% of the worms if given as an oral suspension, and 99.7% of the worms if given as a feed additive.     

Detection of toxoplasmosis in experimentally infected goats by PCR

PCR was used to diagnose toxoplasmosis in two pairs of Barbari goats infected by oral administration of doses of either 10(4) or 10(5) oocysts of Toxoplasma gondii. Blood and lymph node aspirates were collected from the infected goats and control goat at intervals, and tissues were also collected from a fetus that was aborted and a doe that died during the trial. Both processed and unprocessed samples were used for the PCR, using primers directed to the multicopy B1 gene. None of the blood samples was positive, but a specific signal was obtained from the lymph node aspirates after partial DNA extraction. Direct PCR of the lung, muscle and mesenteric lymph node of the doe and lung tissue of the aborted fetus yielded the target fragment. The simplified PCR protocols, including partial DNA extraction and direct assay of lung tissue, were effective for the diagnosis of toxoplasmosis.     

A macro- and light microscopical study of the pathology of Calicophoron microbothrium infection in experimentally infected cattle

Twelve Tuli weaner steers aged 1 year were randomly subdivided into three groups of four animals and infected with different doses of Calicophoron microbothrium metacercariae. Each animal in Group I received a low dose (LD) of 5 000 metacercariae, Group II a medium dose (MD) of 15 000 metacercariae, Group III a high dose (HD) of 25 000 metacercariae and one additional animal was kept as an uninfected control (C). After infection, one animal from each group was slaughtered on Day 28, 42, 56 and 84 post infection (pi) and samples from the ileum, jejunum, duodenum, abomasum and the rumen were collected for histopathological and cytological examination. On Day 28 pi, the gross pathological lesions observed in the duodenum of the LD and the MD animals were similar and comprised duodenal thickening, corrugation, hyperaemia, petechiation and ulceration. In the HD animal the duodenal lesions were similar but more severe. The abomasal folds were severely oedematous in the MD group and nearly occluded the abomasal lumen. Moderate oedema of the abomasal folds was also present in the LD and HD animals. The gross pathological lesions regressed in all the infected groups with increasing age of infection and had disappeared completely by Day 56 pi. On Day 28 pi the histopathological lesions in the duodenum and jejunum of the LD and MD groups were similar, comprising subtotal villous atrophy, hyperplasia of Brunner's glands and Peyer's patches and moderate infiltration of eosinophils, mast cells and a few globule leukocytes, basophils and lymphocytes in the lamina propria. The HD group had total villous atrophy, severe hyperplasia and cystic dilatation of Brunner's glands, which had expanded to cover the entire submucosa. On Day 42 pi the histopathological lesions were still present in the MD and the HD groups comprising subtotal villous atrophy and hyperplasia of Brunner's glands. Heavy infiltrations of eosinophils, moderate amounts of mast cells and a few basophils, globule leukocytes and lymphocytes were still present in the lamina propria of all three groups. On Day 56 pi, a few glands were still cystic in the MD and the HD groups. Moderate cell infiltrations were still present in the lamina propria of all the three groups and by Day 84 pi complete regeneration had occurred in all animals.     

Antibody response in goats experimentally infected with Toxoplasma gondii

Serum samples of five goats inoculated with Toxoplasma gondii were analyzed using the enzyme linked immunosorbent assay (ELISA), indirect hemagglutination (IHA) and western blotting (WB). Antibodies detected by ELISA peaked between 19 and 62 days after inoculation and persisted throughout the experiment with no association to parasitaemia. Using WB, the main antigens detected had molecular weights of approximately 68, 62, 50, 48, 42, 34, 28, 26, 22 and 19 kDa. Antibody titers of between 1:256 and 1:32000 were observed using IHA, with a significant drop in activity after treatment with 2-mercapto-ethanol between days 12 and 48. This coincided with the parasitaemic period that occurs between 5 and 64 days after inoculation.     

Serologic diagnosis of toxoplasmosis in experimentally infected pregnant goats and transplacentally infected kids

Eight pregnant goats were inoculated orally with 10 to 1,000 oocysts of Toxoplasma gondii at 83 to 102 days of gestation. Serum samples from the goats and from the kids born to them were analyzed, using the Sabin-Feldman dye test (DT), a commercially available modified agglutination test (MAT), and a latex agglutination test. Six of the does were observed for greater than 1 year; during this time, they delivered twice. All does developed DT and MAT antibody titers of greater than or equal to 1:2,048 within 29 days after inoculation, and the high titers persisted through the 2nd pregnancy; therefore, serologic results alone should not be relied on for the diagnosis of T gondii-induced abortion in does. On the other hand, all transplacentally infected kids had DT or MAT antibody titers of 1:2,048 before ingesting colostrum, indicating the usefulness of serologic evaluation of the fetus or stillborn kid in the diagnosis of abortion. Antibody was not found in the sera of noninfected kids born to Toxoplasma-infected does. The passively acquired colostral antibody declined by 5 months. Therefore, specific antibody found in adult goats is probably actively acquired. The commercially available MAT was simple, sensitive, and reliable for the diagnosis of caprine toxoplasmosis. The latex agglutination test needs further improvement, as titers rarely exceeded 1:256.     

Observations on mass production of Calicophoron microbothrium metacercariae from experimentally and naturally infected Bulinus tropicus

In an attempt to establish an ideal method for mass production of Calicophoron microbothrium metacercariae, a study was carried out to compare the shedding capacities of Bulinus tropicus naturally and experimentally infected with C. microbothrium. A total of 906 F1 B. tropicus between 4 and 5 weeks old were each experimentally infected with two C. microbothrium miracidia and monitored for 12 weeks. The infected snails were fed on dried lettuce and fish flakes and were kept in 1 l plastic aquaria housed in a snail room where temperature, light and humidity were controlled. Seventy-four percent of the experimentally infected snails died during the prepatent period and of the remaining, only 13.2% developed patent infection, while 12.5% were refractory. Snail growth rate was poor and the average shedding rate was 20 cercariae per snail per day. Compared to the experimentally infected snails, 2200 adult B. tropicus, collected from the field and naturally infected with C. microbothrium, yielded high numbers of metacercariae. Eighty-four percent of the snails died within 7 weeks of the study with peak mortality occurring from the 2nd to the 4th week of infection and coinciding with an overall decrease in the number of cercariae shed.     

Ocular lesions associated with Trypanosoma evansi in experimentally infected goats

Ocular lesions associated with Trypanosoma spp. infection have been described in man and many animal species. However, loss of vision has not been demonstrated in humans presenting Chagas disease or in animals affected by different trypanosome species. In order to assess the possible ocular disorders caused by Trypanosoma evansi infection, six goats were inoculated with 1 x 10(5) T. evansi and maintained for 12 months and four goats were used as control. The inoculated animals became positive at serological and parasitological tests at 1-month post-inoculation and showed a subclinical course of the disease. Unilateral superficial corneal ulceration and retinochoroiditis were observed in two inoculated animals. Data from ocular neurologic examination and electroretinography showed no significant differences between inoculated and non-inoculated goats. It could be concluded that Trypanosoma evansi can produce ocular lesion but without apparent loss of vision in goats.     

Histochemical alterations in the duodenum of goats experimentally infected with Paramphistomum cervi

Certain histochemical alterations in the different tunics of duodenum in kids were studied 20, 40, 60 and 80 days post-infection (DPI) with Paramphistomum cervi and the results compared with those of uninfected kids. There was a general reduction of polysaccharide complex substances and glycogen at 20 DPI. A marked decrease in polysaccharide complex substances and glycogen at 20 DPI. A marked decrease in polysaccharide complex substances and glycogen was especially discernible in the Brunner's gland and muscularis mucosa 20 DPI. Thereafter, these substances gradually increased and at 80 DPI this decrease was fully replenished. A slight reduction in mucin and protein content of the infected duodenal goblet cells was noticed at 20 DPI. It is suggested that juvenile P. cervi utilize host-tissue polysaccharide complex substances and glycogen for their growth and development during duodenal migration.     

Development and distribution of rinderpest virus antigen in experimentally infected goats

Three goats, experimentally infected with rinderpest virus were examined for the development and distribution of precipitating antigens in various tissues and secretions using the agar gel immunodiffusion test. Virus antigens were detected in ocular secretions and lymph node biopsies from the second to the fourth and fifth days of pyrexia, respectively, but were not detected in nasal secretions. Precipitating antigens were demonstrated in various lymphoid organs, the lung and abomasum of a goat killed on the fourth day of pyrexia. These findings are discussed in relation to the epidemiology of rinderpest in goats in Africa.     

Eprinomectin 'pour-on' and the shedding of Haemonchus contortus eggs in experimentally infected goats

The effect of eprinomectin on goats experimentally infected with the nematode Haemonchus contortus was investigated. Faecal analysis showed that a single dose of eprinomectin 'pour-on' delayed the onset of shedding of eggs for 10 to 17 days.     

Efficacy of milbemycin oxime against fourth-stage larvae and adults of Ancylostoma tubaeforme in experimentally infected cats

The efficacy of milbemycin oxime against fourth-stage (L4) larvae and adults of Ancylostoma tubaeforme was investigated in a trial involving 24 young domestic shorthair cats. The animals were inoculated with approximately 300 infective stage three (L3) larvae and divided into three groups. After 12 days, eight cats (group 1) were treated with medicated tablets containing 4 mg milbemycin and 10 mg praziquantel to test the efficacy against L4 larvae; eight cats in group 2 were treated with the same tablets after 33 days to test the efficacy against adult worms; and eight cats in group 3 were treated with a placebo tablet. Faecal egg counts were determined periodically in each cat and after 40 or 41 days the number of worms in each animal was determined postmortem. The egg count reduction was determined by comparing the geometric mean numbers of eggs per gram of faeces in the placebo and medicated groups, and the worm reduction by comparing the geometric mean numbers of worms. The egg count reduction was more than 99 per cent in both treated groups, while the number of worms in groups 1 and 2 were reduced by 94.7 per cent and 99.2 per cent, respectively.     

Effects of condensed tannins on goats experimentally infected with Haemonchus contortus

Although the use of tanniferous plants or condensed tannins as an alternative to anthelmintics to control gastrointestinal nematodes has been largely documented in sheep, studies remain scarce in goats. The objective of this study was therefore to assess the possible impact of condensed tannins in goats infected with adult Haemonchus contortus. Two groups of cull goats were experimentally infected with 10.000 L3 of H. contortus. After 4 weeks, quebracho extracts, representing 5% of the diet DM, were administered for 8 days to one of the two groups. Goats of the second group remained as controls. One week after the end of quebracho administration, the goats were euthanised. Individual egg excretion and pathophysiological parameters were measured weekly during the study. At the end of the study, worm counts were assessed and histological samples from the abomasa were taken to count the numbers of mucosal mast cells, globule leukocytes and eosinophils. The administration of tannins was associated with a significant decrease in egg excretion, which persisted until the end of experiment. This reduction was not associated with any difference in worm number but with a significant decrease in female fecundity. No significant changes in the mucosal density of the three inflammatory cell types were detected between the two groups. These results indicate that the major consequence of tannin consumption in goats is a reduction in worm fecundity and egg output, which does not seem related to significant changes in the local mucosal response.     

Caprine herpesvirus-1-specific IgG subclasses in naturally and experimentally infected goats

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect caprine herpervirus-1 (CpHV-1)-specific IgG1 and IgG2 in sera from 43 naturally infected goats. The analysis of the IgG subclasses showed a dual pattern of distribution in seropositive goats with a major group of animals (36 out of 43) exhibiting significantly higher levels of IgG2 over IgG1 and a minor group (7 out of 43) possessing equal levels of IgG1 and IgG2. Four goats were experimentally infected with a virulent CpHV-1 Ba.1 strain by the intranasal or the intravaginal route and the kinetics of appearance of CpHV-1-specific IgG, IgG1 and IgG2 in the serum were studied. Two weeks following infection, both IgG1 and IgG2 levels increased although convalescent sera (i.e., collected 5–8 weeks post-infection) showed a clear prevalence of the IgG2 subclass. To determine the contribution of the different IgG subclasses to herpesvirus immunity, serum neutralization (SN) assays were performed in both naturally and experimentally infected goats. The kinetics of SN showed that neutralization activity was mainly associated to the IgG1 subclass and this was also confirmed in naturally infected goats. The results are discussed from the standpoint that the profile of the IgG subclasses is instrumental to study immune responses to CpHV-1 and that vaccination strategies may benefit from this information.     

Changes in levels of transaminases in goats experimentally infected with Trypanosoma congolense.

Goats were experimentally infected with Trypanosoma congolense and then treated with Berenil after 9 days of infection. The infection produced increases in glutamate oxalacetate transaminase (GOT) and glutamate pyruvic transaminase (GPT) values. Mean GOT values in infected West African dwarf goats were generally lower than in infected Red Sokoto goats. Treatment with Berenil did not produce any significant effect on their levels probably because of the relapse infection recorded in this study.     

Clinical and pathological observations on goats experimentally infected with Pseudomonas pseudomallei

The effects in goats of the subcutaneous injection of varying doses of Pseudomonas pseudomallei (90 to 500,000 bacilli) suspended in normal saline are described. High doses (greater than or equal to 500 bacilli) caused acute, fatal infections. Lower doses (90 to 225 bacilli) caused acute or chronic disease when infection became established. However, 11 of 18 goats injected with the lower doses of bacilli showed no sign of infection on clinical or bacteriological examination. Response to antibiotic therapy with long acting tetracycline and chloramphenicol was minimal. Goats surviving the initial phase of infection tended to overcome the disease with a corresponding increase in the number of abscesses that were sterile at necropsy. In infected goats, clinical signs included undulating fever, wasting, anorexia, paresis of the hind legs, severe mastitis and abortion. At necropsy, abscesses were found predominantly in the spleen, lungs, subcutaneous injection site and its draining lymph node.