Laboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs.

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.

Detection of schistosomes polymerase chain reaction amplified DNA by oligochromatographic dipstick.

The applications of highly specific and sensitive molecular techniques based on polymerase chain reaction (PCR) have constituted a valuable tool for the diagnosis of schistosomiasis and also for the detection of schistosome infections in the snail intermediate hosts. The common method of detecting PCR amplicons is gel electrophoresis in the presence of ethidium bromide, a carcinogen, which is followed by UV transillumination. Other methods, which are available for detecting PCR products, are real-time PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELIZA) and mass spectrometry but they are cumbersome while they are sometimes complex and expensive. Therefore, a simple method of PCR product detection would be a welcome idea and a most valuable tool particularly in disease endemic countries with limited research facilities and resources. In this study, we applied a simple and rapid method for the detection of Schistosoma haematobium and Schistosoma mansoni PCR amplified DNA products using oligochromatographic (OC) dipstick. The amplicons are visualized by hybridization with a gold conjugated probe, while a control for the chromatographic migration is incorporated in the assay. The lower detection limit observed was 10fg of genomic DNA from each of the two species, while the dipstick was also specific for each of the species used in this study.

Validation of a PCR-oligochromatography test for detection of Trypanozoon parasites in a multicenter collaborative trial.

A PCR-oligochromatography test for diagnosis of human and animal trypanosomiasis was evaluated through a multicenter ring trial with six laboratories testing a set of 21 blinded samples, resulting in qualitative data (positive or negative). Results showed an intralaboratory repeatability (accordance) of 88.7% (credible interval [CI], 84.4 to 92.5%) and an interlaboratory repeatability (concordance) of 88.1% (CI, 84.3 to 92.3%).

Molecular dipstick test for diagnosis of sleeping sickness.

Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 microl of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

In vitro tests for evaluation of the hatchability of the eggs of Psoroptes mites following exposure to acaricidal compounds.

Three in vitro assays for the evaluation of the hatchability of the eggs of the mite Psoroptes ovis (Hering) (Acari: Psoroptidae) are described. Preliminary trials showed that hatching occurs at very high rates when eggs are incubated under conditions of high humidity, on a liquid medium and in agarose dishes. These three protocols were compared, taking into account the ease of preparation, follow-up and accuracy of counting. The best protocol was found to be the use of agarose dishes. It was accurate, easy to carry out and reproducible for further evaluation of existing or potentially new compounds against both adults and eggs of Psoroptes spp. The acaricidal properties of phoxim and amitraz were then evaluated against eggs and adults using the three protocols. Results showed that for both drugs, in vitro adulticidal activity was complete, whereas the in vitro ovicidal activity was only partial. Nevertheless, efficacy of amitraz against both adults and eggs was shown to be higher than that of phoxim.

Fetal infection with Neospora caninum in dairy and beef cattle in Belgium.

Neospora caninum is a protozoan parasite, which causes fetal and neonatal mortality in livestock and companion animals. In 224 abortions in Belgian cattle, different diagnostic methods were used to demonstrate infection, and the presence of N. caninum. An indirect fluorescent antibody test (IFAT) was used to analyze fetal and maternal sera and immunohistochemistry (IHC) was performed when lesions consistent with neosporosis were observed in the brain, heart or liver. Twenty dairy cattle sera out of 70 (29%) and 13 beef cattle sera out of 93 (14%) were positive by IFAT. A positive titer to N. caninum was found in seven and three fetuses born to beef and dairy cows, respectively. Lesions consistent with N. caninum infection were observed in 17 fetuses. Of nine positive beef fetuses, five were confirmed by IHC while, all but one dairy fetus were confirmed using the same technique. Age had no influence on the serological status of the mother (P = 0.486) whereas husbandry system had a borderline influence (P = 0.082). However, a strong association (P = 0.004) between the level of antibodies in the dam and the occurrence of lesions in the fetus was observed and lesions were more prominent in dairy than in beef fetuses. Additionally, the distribution of intra-cerebral lesions was more extensive in dairy than in beef fetuses (P < 0.0001). Age and serological status of the fetus were found to influence the occurrence of lesions in beef fetuses (both P < 0.001) but no such significant relationships could be demonstrated in dairy fetuses. The study indicated that N. caninum must be considered as an important cause of bovine abortion in Belgium.

Humoral immune response in calves to single-dose, trickle and challenge infections with Fasciola hepatica.

In cattle experimentally infected with Fasciola hepatica, parasite specific IgG1 and IgG2 responses were studied. Additionally parasite specific IgE production was assessed by the Passive Cutaneous Anaphylaxis reaction. The primary infection was administered either as a single-dose or as a trickle infection over a 4-week period. Animals were challenged 4 months later. Titres of IgG1 and IgG2 against excretory-secretory parasite products (FhESAg), and against a whole-worm extract (FhSomAg) were measured by enzyme-linked immunosorbent assay (ELISA) in relation to weight gain, serum hepatic enzyme levels, and fluke infection rate. At necropsy, the mean number of flukes recovered was similar in both infected groups. The two ELISAs specific for bovine IgG1 showed analogous sensitivity and specificity (92% and 94%). Cross-reactivity was observed towards Echinococcus granulosus, Cysticercus tenuicollis, and C. ovis but not towards C. bovis, Cooperia spp., and Ostertagia spp. FhESAg gave rise to apparently more stable specific IgG1 titres as compared to FhSomAg. Mean IgG1 titres were significantly higher in the single-dose-infected group than in the trickle-infected group during the early migratory phase of the infection (week 2 to week 4 (FhSomAg) or week 6 (FhESAg)). IgG2 values were consistently lower than IgG1 levels. The kinetic response of both isotypes yielded a similar pattern. Specific IgE antibodies were detected in cattle of both infected groups from week 2 post-primary infection (PPI) onwards. The mean serum glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gammaGT) activities were significantly higher in the single-dose-infected group for 3 weeks around peak levels (12-14 weeks PPI and 14-16 weeks PPI for GLDH and gammaGT respectively). Western blotting revealed a major antigenic fraction in FhESAg (26-30 kDa) recognized specifically by sera from F. hepatica infected calves as early as 6-8 weeks PPI. Experimental challenge caused no statistically significant modification of any parameter (IgG1 and IgG2 titres, enzymatic activities, immunoblotting) used to monitor the course of the infection. No correlation was found between fluke size and number, and antibody titres, suggesting that IgG1 production has little protective effect against F. hepatica infection.

Humoral and cellular immune response to a crude exo-antigen and purified keratinase of Microsporum canis in experimentally infected guinea pigs.

In order to understand better the host-parasite relationship and to compare with previous observations in Microsporum canis naturally infected cats, the humoral and cellular immune responses to both a crude exo-antigen and a 31.5 kDa purified keratinase were evaluated in 12 M. canis experimentally infected guinea pigs. Humoral and cellular responses were assessed by ELISA from days 0 to 56 postinfection (PI) and by measurement of delayed-type hypersensitivity (DTH) responses on days 14 and 57 PI, respectively. Additionally, immunohistochemical staining was performed and demonstrated that the keratinase was produced in infected guinea pig skin, as previously reported in cats. Despite a marked interindividual variation, all the guinea pigs produced specific IgG to the crude exo-antigen from day 21 PI onwards, but no anti-keratinase IgG was detected. Strongly positive DTH responses to the exo-antigen were observed on both dates, whereas the keratinase elicited no and weak DTH on days 14 and 57 PI, respectively. These results are in agreement with those previously described for naturally infected cats, and indicate that the 31.5 kDa keratinase is not a major antigen in M. canis infection.

Histopathological pattern and humoral immune response to a crude exo-antigen and purified keratinase of Microsporum canis in symptomatic and asymptomatic infected cats.

In order to understand better the mechanisms involved in the diverse clinical patterns in Microsporum canis-infected cats, the histopathological features were compared in symptomatic and asymptomatic infected cats. Additionally, the IgG immune response to a crude exo-antigen and purified keratinase of M. canis was studied by ELISA in cats of various clinical and mycological status. Acute and subacute perifolliculitis and folliculitis occurred more frequently in symptomatic than asymptomatic cats. The latter usually displayed signs of chronic inflammation and a marked infiltration of superficial dermis by mast cells, which would suggest that these animals present similarities to chronically dermatophytic humans or animals. When using a crude M. canis antigen, all infected cats were shown to have significantly higher levels of specific IgG when compared to culture negative and mechanical carrier-cats. In these non-infected animals, specific IgG was more frequently detected in adults than in young animals. No difference in anti-crude antigen specific IgG was observed between symptomatic and asymptomatic infected cats, indicating that the presence of IgG is probably unrelated to the clinical status of cats. Anti-keratinase specific IgG was only detected in one of the infected cats.

Use of excretory/secretory antigens in a competition test to follow the kinetics of infection by Fasciola hepatica in cattle.

Eight 16-18-month-old Charolais heifers were experimentally infected with Fasciola hepatica. An antigen competition assay was used to follow the kinetics of the infection and was compared to antibody tires and serum liver enzymes. The antigen competition assay was able to detect the presence of infection as soon as 6 days after the start of the experimental infection which is considerably sooner than other methods. Consequently, this assay would be useful in diagnosing fasciolosis early in the prepatent period. The animals were slaughtered at the end of the experiment, the livers recovered and post-mortem fluke burdens determined. However, only serum liver enzyme levels gave any indication of the intensity of infection in the different animals.

Field efficacy of injectable doramectin against Chorioptes bovis in naturally infected cattle.

A single subcutaneous injection of doramectin at a dose rate of 200 micrograms/kg bodyweight was effective in controlling an infection of Chorioptes bovis mites in naturally infected cattle. From 14 days after treatment, the geometric mean number of live mites was significantly lower (P < 0.001) in the doramectin-treated cattle than in the control group at each sampling until day 35. The percentage efficacy (treated versus controls) of doramectin against C bovis at day 35 was 99.9 per cent and the percentage reduction (day 35 versus day 0) in the treated animals was 99.3 per cent. At day 35, all seven controls were still positive for C bovis whereas five of the eight doramectin-treated animals were free of live mites.

Disappearance of paternal histocompatibility antigens from hybrid mouse blastocyst at the time of implantation.

Products of the major histocompatibility complex (H-2) are important in allograph rejection. In view of the close relationship between mother and foetus, we can consider the latter as an allograph which is however not rejected by an immunological reaction. We studied the presence of H-2 antigens on embryo membranes at the time of implantation, by immunochemical labeling using gold particles coupled with protein A. Results showed that the expression of H-2 antigens is different before and after implantation. It seems that after implantation, H-2 antigens disappear from trophoblastic membranes. This could explain the absence of immunological reaction of the mother against the foetus.