Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs

A recombinant LipL 32 antigen-based dipstick ELISA was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95.9 per cent, 93.8 per cent and 94.8 per cent, respectively.     

Diagnosis of bovine brucellosis by skin test: conditions for the test and evaluation of its performance.

Brucellergene OCB (Rh?ne-Mérieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin test was also evaluated for its capacity to solve problems associated with false positive reactions in serological tests. The optimal reading delay for the skin test was 72 hours. The brucellosis allergic reaction was two to three times less intense than the tuberculosis allergic reaction. An increase of 1.1 mm or more in the skin thickness was therefore considered to be an adequate cut-off. The specificity calculated for 1192 brucellosis-free animals (including animals from brucellosis-free herds in which false positive serological reactions had been reported) was 99-83 per cent (95 per cent confidence interval [CI] 99-40 to 99-98 per cent). The sensitivity determined from 27 experimentally infected heifers ranged from 93 per cent (95 per cent CI 76 to 100 per cent) to 78 per cent (95 per cent CI 58 to 91 per cent) when measured respectively one and six months after the infection. Allergic reactions could be detected in vaccinated animals up to four-and-a-half years after the vaccination. On the other hand, no sensitisation was recorded in na?ve animals after up to eight monthly injections of the allergen. The skin test gave valuable information, in combination with the serological tests, in both acute and chronic brucellosis. The skin test discriminated brucellosis clearly from false positive serological reactions due to infections with Yersinia enterocolitica O9.     

Prevalence of enterotoxigenic Escherichia coli in calves in Scotland and northern England

Eighty-eight of 1529 (5.7 per cent) Escherichia coli isolates from diarrhoeic and clinically normal calves in Scotland and northern England were found to possess the K99 pilus antigen (K99+). There was complete correlation between possession of K99 antigen, heat stable enterotoxin production and ability to dilate intestinal loops. The diagnosis of calf enterotoxigenic E coli infections may therefore be based on the detection of K99 antigen alone. Enterotoxigenic E coli was isolated from 23 of 306 (7.5 per cent) diarrhoeic calves from eight of 70 (11.4 per cent) farms and was not isolated from clinically normal calves. Infected calves were between one and three days old. A survey by an enzyme-linked immunosorbent assay found 3.0 per cent and 3.9 per cent of sera from calves and cows respectively to contain antibodies to K99 antigen. The prevalence of other enteropathogenic organisms in calf faeces is also discussed.     

Microbiology of calf diarrhoea in southern Britain

Faeces samples from calves with diarrhoea in 45 outbreaks were examined for six enteropathogens. Rotavirus and coronavirus were detected by ELISA in 208 (42 per cent) and 69 (14 per cent) of 490 calves respectively; calici-like viruses were detected by electron microscopy in 14 of 132 calves (11 per cent). Cryptosporidium were detected in 106 of 465 (23 per cent), Salmonella species in 58 of 490 (12 per cent) and enterotoxigenic Escherichia coli bearing the K99 adhesin (K99+ E coli) in nine of 310 calves (3 per cent). In the faeces of 20 per cent of calves with diarrhoea more than one enteropathogen was detected; in 31 per cent no enteropathogen was found. Faces samples from 385 healthy calves in the same outbreaks were also examined. There was a significant statistical association of disease with the presence of rotavirus, coronavirus, Cryptosporidium and Salmonella species (P less than 0.001). Healthy calves were not examined for calici-like viruses and the association of K99+ E coli with disease was not analysed because there were too few positive samples. Rotavirus infections were more common in dairy herds and single suckler beef herds whereas Salmonella infections were more often found in calf rearing units. Cryptosporidium were more common in single and multiple suckler beef herds. K99+ E coli were found in one dairy herd and one multiple suckler beef herd both with unhygienic calving accommodation. Variations in coronavirus detection among different farm types were not statistically significant. In this survey rotavirus was the most commonly detected agent in calf diarrhoea and Cryptosporidium were found in approximately one quarter of affected calves. Infection with Salmonella species was widespread, but K99+ E coli infections were less common in the United Kingdom than in other countries.     

Enzyme-linked immunosorbent assay for detecting antibodies to Aujeszky's disease virus in pigs

An indirect micro enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Aujeszky's disease virus in pigs is described. A control antigen prepared from infected cells was included for each serum tested. Of 243 sera from serologically positive farms, 175 (72 per cent) and 147 (60 per cent) were positive by the ELISA test and microtitre serum neutralisation test, respectively. Failure to include a control antigen for each serum would have resulted in 14 sera (6 per cent) being differently recorded. Results for sera from experimental and field infections indicated that seroconversion was more quickly detected by the ELISA test than the microtitre serum neutralisation test. In addition to greater sensitivity the ELISA test has other advantages over the serum neutralisation test. ELISA is a rapid, cheap test which is not dependent on a continuous supply of cell cultures and which can be readily automated.     

Persistent anthelmintic activity of ivermectin in cattle

Two studies are described which demonstrate the persistent activity of ivermectin injected subcutaneously into cattle at 200 micrograms/kg in preventing the establishment of induced infections with the gastrointestinal parasites Ostertagia ostertagi and Cooperia oncophora and the lungworm Dictyocaulus viviparus. These results indicated a reduction in mean worm count compared with the control group for O ostertagi of more than 99, 45 and 94 per cent with a seven, 14 or 21 day interval between treatment with ivermectin and the administration of infective larvae, respectively, in trial 1 and more than 99, more than 99 and 99 per cent at seven, 10 or 14 days, respectively, in trial 2. Corresponding values against C oncophora were 99, 0 and 45 per cent at seven, 14 and 21 days in trial 1 and more than 99, 84 and 31 per cent at seven, 10 and 14 days in trial 2. Against D viviparus, reduction in counts were more than 99, 98 and more than 99 per cent at seven, 14 and 21 days, respectively, in trial 1 and 100, 100 and 100 per cent at seven, 10 and 14 days, respectively, in trial 2. The relevance of these results to the build-up of infective larvae on pasture and infection in cattle is discussed.     

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (ELISA) and complement fixation test

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.     

Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus)

Between 1982 and 1999 blood samples were collected from 500 polar bears (Ursus maritimus) captured in the Beaufort and Chukchi seas, to determine the seroprevalence of Brucella species, Toxoplasma gondii, and Trichinella species infections. The bears were classified into four age groups, cubs, yearlings, subadults and adults. Brucella and Toxoplasma antibodies were detected by agglutination (a buffered acidified card antigen and rapid automated presumptive test for brucellosis and a commercial latex agglutination test for toxoplasmosis); an ELISA was used to detect Trichinella antibodies. The overall seroprevalence of Brucella species was 5 per cent, and subadults and yearlings were 2-62 times (95 per cent confidence interval 1.02 to 6.82) more likely to be seropositive for Brucella species than adults and their cubs. The antibody prevalence for Toxoplasma gondii was 6 per cent, and for Trichinella species 55.6 per cent. The prevalence of antibodies to Trichinella species increased with age (P<0.001).     

Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11

To establish for how long protective antibody levels may be maintained, lambs were vaccinated with the gut membrane antigen H11 and challenged with Haemonchus contortus 14, 84, 126 or 168 days later. Compared to controls, mean faecal egg counts of vaccinated lambs were reduced by 97 per cent, 99 per cent, 92 per cent and 86 per cent respectively. Total worm burdens at postmortem five weeks after infection were reduced by 87 per cent, 94 per cent, 92 per cent and 62 per cent respectively. In vaccinated lambs, antibody levels to H11 peaked at about 60 days after the first vaccination and were maintained for the duration of the experiment. There was evidence of secondary antibody responses to H11 following challenge.     

Efficacy of ivermectin against induced gastrointestinal nematode infections in goats

The efficacy of ivermectin (0.08 per cent w/v oral solution) at different dose levels was evaluated against induced infections of adult Haemonchus contortus (21 days old) and Trichostrongylus colubriformis (21 days old) and fourth stage larvae of Oesophagostomum columbianum (17 days old), Ostertagia circumcincta (five days old) and Strongyloides papillosus (five days old). Twenty-five Boergoats (mutton goats) were randomly allocated by bodyweight within each sex to an untreated control group and four ivermectin treatment groups; ivermectin was administered at either 25, 50, 100 or 200 micrograms/kg orally, once. The goats were killed and processed for worm recovery 25 to 27 days after treatment. At 25 micrograms/kg the efficacy of ivermectin varied from 43 per cent for adult T colubriformis to more than 99 per cent for fourth larval stage O columbianum. Ivermectin at 50 micrograms/kg or higher was 99 per cent or more effective against all induced parasite infections with the exception of ivermectin at 50 micrograms/kg against S papillosus (97 per cent). For all parasites there was a statistically significant (P less than 0.05) difference between the control group and the pooled treated groups. No adverse reactions to ivermectin treatment were observed in the goats.     

The complement fixation test for the diagnosis of Brucella ovis infection in rams

Complement fixation tests using three B. ovis antigen preparations in warm fixation tests (WCFT) and cold fixation (CCFT) tests were done on 541 ram sera. Semen samples from the same rams were examined culturally to identify B. ovis excretors. The CCFT, using an antigen prepared by heat extraction of B.ovis cells, had a sensitivity of 97% in 124 rams which were shedding B.ovis. The specificity was 99% in 144 rams from non-infected flocks. Seventy-seven per cent of 156 rams which reacted to this test were shedding B. ovis in their semen. Tests with other antigens were inferior in sensitivity and/or specificity. The WCFT gave lower titres than CCFT. Vaccination caused large numbers of false positive reactions in 4 flocks.     

Persistent anthelmintic activity of topically administered ivermectin in cattle

The persistent anthelmintic effect of ivermectin as a topical treatment at 500 µg/kg was evaluated against induced infections of Ostertagia ostertagi, Trichostrongylus axei and Dictyocaulus vivparus in calves. The results showed a highly significant (P<0.001) anthelmintic activity for at least 14 days against O. oslertagi and T: axei (>99 per cent efftcacies) and for at least 28 days (98 per cent efficacy) against D. viviparus.     

A Brief Overview of Transgenic Farm Animals

A rapid recombinant antigen-based latex agglutination test (LAT) has been developed to detect specific anti-leptospiral antibodies from human and dog sera. The recombinant LipL32 antigen developed and used for detecting the antibodies is specific in detection of the pathogenic serovars of Leptospira as the expression of the LipL32 antigen is restricted only to the pathogenic leptospires. The sensitized latex beads are stable and could be stored at 4^°C for more than three months without showing loss of activity for both weakly and strongly positive samples. The test is found to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT). Moreover, the recombinant antigen-coated latex beads could detect the specific anti-leptospiral antibodies in the acute phase of the illness. The test is simple and inexpensive, and is rapid in the management of large numbers of patients.     

Efficacy of ivermectin against benzimidazole-resistant nematodes of sheep

Two trials involving a total of 36 Dorset horn lambs were conducted to assess the anthelmintic efficacy of ivermectin against experimental infections of benzimidazole-resistant strains of Haemonchus contortus and Ostertagia circumcincta. Two resistant strains of each of the two species were used and in each trial the lambs were allocated to three groups. One group was given 200 micrograms ivermectin/kg bodyweight orally, the second group was given 5 mg oxfendazole/kg bodyweight orally and the third group remained untreated as controls. Fourteen days after treatment the lambs were necropsied. Ivermectin was found to be more than 99 per cent to 100 per cent effective against all four benzimidazole-resistant strains, whereas oxfendazole was 78.6 per cent and 83.8 per cent effective against the H contortus strains, and 25.6 per cent and 39.8 per cent effective against the O circumcincta strains.     

Bile agar migration test: a novel method for testing the viability of Dictyocaulus viviparus larvae in lungworm vaccine

A bile agar migration (BAM) test was used to determine the viability of Dictyocaulus viviparus larvae in lungworm vaccines. Percentage migration values for 10 batches of vaccine varied from 8.8 to 33.2 per cent compared with 99 per cent of unirradiated larvae tested under similar conditions. On the other hand a conventional viability count (CVC), based on subjective assessment of larval shape, gave figures of 95.1 to 98.7 per cent for the 10 vaccine batches. The BAM test may therefore have potential for providing a more discriminatory assessment of vaccine quality than CVC.     

The use of spleen antigen in the tube agglutination test for diagnosis of anaplasmosis in cattle

An antigen was prepared from the spleen of an infected bovine calf and was used in the tube agglutination test. Cases having rare and very low parasitaemia as well as the carrier cases were detected by this procedure and no non-specific reaction was observed with other haemoprotozoan infections. The maximum titre obtained with the Anplasma positive sera was 1:20, about 60 per cent showed a titre of 1:10 and 90 per cent showed a titre of 1:5 or more. The test provided satisfactory results in detecting the cases even at the early phase of the infection. The test also proved useful in judging the efficacy of a drug employed to destroy the carrier status.     

Echinococcus granulosus,Taenia hydatigena,T. ovis: Evaluation of cyst fluids as antigen for serodiagnosis of larval cestodes in sheep

In order to monitor the progress of New Zealand's hydatids eradication campaign, a specific, serological, diagnostic test is required to identify infected sheep. An indirect haemagglutination test, using pyruvic aldehyde-stabilized sheep erythrocytes as the antigen carrier, was developed for the serodiagnosis of larval cestode infections of sheep. Using cyst fluid from Echinococcus granulosus, Taenia hydatigena and T. ovis as the antigens in this test, it was shown that the larval cestode species, responsible for an infection in sheep harbouring a single specific infection, could be identified by the higher titre given with the homologous antigen, in comparison to that given with the heterologous antigens. Sera of sheep infected with two or more species were also tested by this method, and the only specific infections to be diagnosed by differential titres were those due to the presence of live E. granulosus cysts. These antigens cannot be used for the diagnosis of specific larval cestode infections in the field because of the cross-reactivity between cyst fluids. However, the test did show that infection with larval cestodes could be diagnosed on a non-specific basis.     

Laboratory investigations in sheep with a new anthelmintic.

The efficacy of fenbendazole against the following gastrointestinal nematodes was tested in experimentally infected lambs: O circumcincta, H contortus, T colubriformis and N filicollis. As low a dose as 0.5 mg per kg reduced the egg output of a patent infection with H contortus and T colubriformis by 85 per cent to 100 per cent. A dose of 3.5 mg per kg has a 100 per cent effect on three or 10-day-old stages of H contortus and one of more than 99 per cent or 100 per cent T colubriformis. The same dose reduces seven-day-old stages of O circumcincta and N filicollis by more than 94 per cent or 100 per cent, while 10 mg per kg produces a 100 per cent elimination of seven-day-old stages of O circumcincta. In field trials and efficacy of more than 99 per cent was determined after oral dosage with 5.0 mg per kg against Ostertagia, Haemonchus, Trichostrongylus, Cooperia, Nematodirus and Chabertia. Fenbendazole has a wide therapeutic-toxic dose ratio. Discoloration of the wool does not occur. Fenbendazole can be administered as a drench or with the feed.     

Osmotic fragility of erythrocytes in clinically normal dogs and dogs infected with parasites

The osmotic fragility of the erythrocytes was measured in blood samples collected from randomly selected healthy and infected dogs at a dogs' rescue shelter. The dogs were classified into six groups on the basis of the final diagnoses from clinical, post mortem and laboratory findings. The minimum (less than 5 per cent) and maximum (more than 90 per cent) haemolysis of the erythrocytes of the clinically normal dogs (group 1), occurred in 0·60 per cent and 0·30 per cent solutions of sodium chloride (NaCI). For the non-anaemic hookworm-infected dogs (group 2a) the respective values were 0·8 per cent and 0·4 per cent NaCl, and for the anaemic hookworm-infected dogs (group 2b) they were 0·85 per cent and 0·5 per cent NaCl, respectively. The erythrocytes from dogs with Babesia canis (group 3), concurrent hookworm and B canis (group 4) and Ehrlichia canis infections (group 5) had minimum haemolysis in 0·75 per cent NaCl and maximum haemolysis at between 0·20 per cent and 0·35 per cent NaCI solutions. The derivative fragiligrams for groups 2a, 2b, 3 and 4 were shifted to the left, whereas the fragiligram for group 5 was similar to that for the clinically normal dogs (group 1). The left shift for the hookworm-infected dogs was due to the increased osmotic fragility of a minor sub-population of the erythrocytes, but for the dogs, infected with B canis major sub-populations of the erythrocytes had an increased osmotic fragility.     

Bovine serum albumin and cell counts in the diagnosis of subclinical udder infection

Summary

Puncture of the milk cisterns was performed in 120 bacteriologically positive quarters of forty‐seven lactating dairy cows on three farms. This method was used to determine whether the existing infection was an infection of the teat canal or one of the udder. The results were related to the concentration of bovine serum albumin (BSA) and the cell count in the milk. Of the bacteriologically negative quarters, both BSA levels (in 91 per cent of the quarters the BSA concentration was 0.20 mg. per ml. of milk or less) and cell counts (92 per cent contained less than 500,000 cells per ml. of milk) were low. In cases of udder infection with primary pathogenic bacteria there was a marked increase in cell count (90 per cent more than 500,000 cells per ml. of milk), whereas the increase in BSA was rather small (51 per cent still contained 0.20 mg. BSA per ml. of milk or less). While the difference in cell counts of milk from quarters with udder infections and teat canal infections with primary pathogenic bacteria was significant, the difference between the BSA levels of these two groups was not. Therefore, the cell count supplies more reliable information than does the BSA level of the milk. Of all infections, 23 per cent were found to be infections of the teat canal.