Advances in laboratory diagnosis of parasitic infections of sheep.

Parasitic infections constitute an important group of diseases in sheep concerning the health status, welfare and productivity. On a global scale, there are considerable differences concerning the epidemiological situation with respect of the various parasite species. However, there are also numerous species, which occur on all continents and, potentially, in every country. Accordingly, the present review aims to providing an overview about the recent developments in methods and technologies for the laboratory diagnosis of parasite infections in sheep. Following in principle a systematic order the review encompasses publications addressing the diagnosis of helminthes (i.e., trematodes, cestodes and nematodes) and arthropod species. New approaches using conventional (e.g., microscopic), immunological and molecular techniques are being considered. The diagnosis of anthelmintic resistance is highlighted separately, due to its significant importance. The review ends with an outlook into the future by discussing most recent technological advances, which might become of use for the diagnosis of parasite infections in sheep in the future.

Evaluation of the efficacy of emodepside+praziquantel topical solution against cestode (Dipylidium caninum, Taenia taeniaeformis, and Echinococcus multilocularis) infections in cats.

Emodepside+praziquantel topical solution was developed to provide broad-spectrum anthelmintic activity against gastrointestinal parasites in cats. Eight controlled studies were conducted to evaluate the efficacy of a topical solution of emodepside (3 mg/kg) and praziquantel (12 mg/kg) (Profender, BayerAG, Leverkusen, Germany) against feline infections with three species of cestodes. Studies featured naturally acquired infections of Dipylidium caninum or Taenia taeniaeformis, or experimental infections with Echinococcus multilocularis that were placebo-controlled, randomized and blinded. Cats were euthanatized and necropsied between 2 and 11 days after treatment, depending on the target parasite. The efficacy of emodepside+praziquantel topical solution was 100% against D. caninum and T. taeniaeformis, and 98.5- 100% against E. multilocularis. No significant systemic or local adverse reactions to treatment were noted in cats that received the combination. Topical treatment of cats with emodepside+praziquantel topical solution was safe and highly effective against cestode infections.

Crossreacting IgG antibodies against fox mite antigens in human scabies.

Scabies continues to be an important parasitic disease of mammals. There remain, however, major gaps in the understanding of the human host immune response, and a simple diagnostic test is lacking. In contrast to human mites, red fox mites (Sarcoptes scabiei var. vulpis) can be collected easily and have been used, due to crossreactivity, for enzyme-linked immunosorbent assay (ELISA) studies in dogs and pigs. We wanted to investigate the possibility that crossreactivity might also exist for the human mite, and determined titers against fox mite antigens by ELISA in 41 patients with scabies. Specific IgG was significantly higher in patients with scabies than in healthy controls (P=0.01). The sensitivity was, however, only 48%, although it increased slightly during treatment (P=0.86). A positive correlation was also noted between disease duration and severity of infestation (r=0.5), with specific IgG titers increasing in parallel with severity of symptoms (P=0.01). Patients with symptomatic scabies for more than 4 weeks had furthermore significantly higher IgG titers than patients with a shorter duration of disease (P=0.007). In conclusion, these findings demonstrate IgG antibodies in human scabies that crossreact with fox mite antigens, thus encouraging the search for improved ELISAs with more specific mite antigens to produce a more sensitive detection system for scabies in humans.

Detection of antibodies in sera of weaned pigs after contact infection with Sarcoptes scabiei var. suis and after treatment with an antiparasitic agent by three different indirect ELISAs.

Three commercial enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of antibodies to Sarcoptes scabiei var. suis using experimental sera of six 8-week-old pigs after contact infection with Sarcoptes scabiei var. suis. Six non-infected pigs were monitored as a control group. Blood sera were taken once a week from all animals. After successful infection the pigs were treated with an antiparasitic agent (12 weeks post infection (p.i.)) and the antibody titres were monitored until they were negative. The antibody levels of the experimental pigs reached the cut-off level 5 weeks after introduction of an infected animal to the group and were positive by both the Sarcoptes-ELISA 2001 PIG and the Acar-Test P-ELISA. Four weeks after treatment mean results showed optical densities (% OD) below the cut-off level in the Sarcoptes-ELISA 2001 and 8 weeks after treatment in the Acar-Test P-ELISA. In the Chekit Sarcoptest pigs had elevated antibody levels in comparison to control animals, but ODs remained below the given cut-off level at all times. In a second examination with Chekit Sarcoptest (different lot) and at a lower cut-off level, the sera of most of the piglets tested positive. Eight weeks after treatment, four from six pigs still had positive OD values. Therefore this investigation showed a higher sensitivity for the Sarcoptes-ELISA 2001 and the Acar-Test P-ELISA than for the Chekit Sarcoptest. Different test sensitivities must be considered when serologic methods are used for the diagnosis of swine sarcoptic mange, especially for monitoring and controlling eradication programs.

[Liver fluke infection and sarcoptic mange in red foxes in Berlin]. / Leberegelbefall und Räude beim Rotfuchs in Berlin.

396 red foxes originating from the city of Berlin were examined for opisthorchiid liver flukes and clinical sarcoptic mange between January 1997 and March 1998. Out of 232 (= 58.6%) foxes positive for opisthorchiid flukes 221 animals harboured Metorchis bilis and 70 were infected with Opisthorchis felineus. Pseudamphistomum truncatum was found only in 8 foxes. M. bilis occurred as mono-infection in 154 animals. M. bilis in combination with O. felineus was found in 61 cases. Pure Opisthorchis infection as well as other fluke combinations were found in a small number of animals only. 85 (= 21.5%) foxes showed clinical sarcoptic mange. Liver fluke positive foxes showed a higher mange prevalence than uninfected animals. However, significant associations between flukes and manage were only found when comparing uninfected foxes with those having the highest worm burden. The association of liver flukes and mange could be established for adult female foxes by a significant Odds Ratio of 4.3.

[Eradication of Chorioptes bovis mange in a dairy herd with turn-out to graze by Eprinex Pour-On]. / Versuch der Tilgung der Chorioptes bovis-Räude in einem Milchviehbestand mit Weidehaltung mit Eprinex Pour-On.

The possibility to control or even eradicate chorioptes manage by a single herd treatment with EPRINEX Pour-On (dosage: 0.5 mg Eprinomection/kg bodyweight) during pasture season was investigated in a dairy herd of 320 dairy cows. A further aim of the study was to evaluate whether such mid-summer treatment with EPRINEX Pour-On due to its endo-ectocide action would at the same time also result in a metaphylaxis of gastro-intestinal and lung worms and in a reduction of fly infestation. Due to clinical symptoms manage prevalence in the herd prior to treatment was 11 percent. After treatment, clinical symptoms disappeared within 2 months completely and did not re-occur during the subsequent housing period. Chorioptes mites during the entire trial period no longer were detected. The final clinical and parasitological investigation shortly before turn-out the next year (April 1999) demonstrated chorioptes mange to be eradicated clinically and parasitologically. Due to the mid-summer treatment infestation with gastrointestinal helminths also was eliminated and clinical symptoms of helminth infestations during the pasture season no longer were observed. However, at housing in November, low numbers of eggs of gastrointestinal nematodes were detected in 6% of dairy cows, 32% of second-season and 63% of first-season heifers, respectively. Milk yield per cow and day on average increased by 1 litre after treatment with EPRINEX Pour-On. This increase in production is likely a result of the reduction in total parasite burden of lactating cows.

'Babesia gibsoni' of dogs from North America and Asia belong to different species.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

Detection of a new pathogenic Babesia microti-like species in dogs.

Small babesiae in dogs are generally considered to belong to Babesia gibsoni. Here we describe the genotypic characterisation of small piroplasms found in the blood of a dog which suffered from clinical babesiosis. Pairwise identities as well as distance, parsimony and maximum likelihood analyses of the 18S rDNA clearly demonstrated that this isolate was only distantly related to the other canine piroplasms characterised genetically so far, including B. gibsoni. It was more closely related to B. microti, B. rodhaini, and Theileria equi. It is concluded that the small canine piroplasms described in this study represent a hitherto unknown species and that the fauna of piroplasms occurring in dogs is more diverse than assumed so far.

Lymphocyte stimulatory capacity of Theileria annulata-infected ovine lymphoblastoid cells.

T. annulata, the causative agent of tropical theileriosis in cattle, is transmitted by ticks of the genus Hyalomma. Sporozoites of this parasite invade their target cells, where they differentiate to macroschizonts. T. annulata additionally invades and transforms ovine and caprine leukocytes. T. annulata infection in the ovine system is poorly studied, thus we used a mixed lymphocyte culture (MLC) to analyze the capacity of these cells to activate naïve uninfected ovine cells. The peak response was observed on day three or four and the response could not be induced by lysates of infected cells or their supernatants. The stimulated cells expressed IL-2 and secreted an IL-2-like growth factor.

Generation of cytotoxic T lymphocytes and cytostatic acting cells in T. annulata-immune cattle.

Cattle immunized against Theileria annulata with schizont containing autologous cell lines are immune to challenge with a homologous parasite strain. Two cell types have been detected in the peripheral blood of the immunized animals: cytotoxic T lymphocytes (CTL) and cytostatic acting cells (CAC). Killing the target cells by CTL is infection associated and is MHC class I restricted. Hence, no cytotoxicity was observed against target cells that were treated with the theilericidal drug buparvaquone or autologous Con A-blasts. The growth inhibition of CAC is MHC unrestricted, and not mediated by cytokine interferon gamma (IFN-gamma).

Is interleukin 2 necessary for the autocrine proliferation of Theileria-infected bovine cells?

Theileria-infected bovine lymphoblastoid cells are induced to proliferate permanently such that the division of the parasites and that of their host cells occur synchronously. The mechanism by which the parasites induce the transformation of their host cells is unknown. We investigated the growth-factor dependency of a number of Theileria-infected cell lines. Supernatants of the majority of the cell lines tested in our study showed no growth-enhancing activity. However, supernatants of a limited number of cell lines occasionally enhanced the growth of parasitized cells that were used as responder cells. Thus, they contained a growth factor whose biological effect was not eliminated by an anti-interleukin-2 (anti-IL-2) antibody. Moreover, neither the proliferation of T. parva-infected cells nor that of T. annulata-infected cells was impaired by this antibody. In contrast, the anti-IL-2 antibody substantially prevented bovine peripheral blood mononuclear cells from undergoing a proliferative response upon stimulation with concanavalin A (Con A). In line with these results we observed that unlike Con A-stimulated lymphocytes, the infected cell lines did not express IL-2 mRNA. Taken together, our results suggest that Theileria-infected cells do not secrete IL-2 and that IL-2 does not play an important role in the autocrine proliferation of the parasitized host cells.

[Pseudo-scabies transmitted by red fox]. / Pseudoskabies durch Rotfuchs übertragen.

Pseudoscabies, i.e. infestation of human skin with animal mites may occasionally occur and should be considered in the differential diagnosis of pruritic and papular skin disease. We report here on a 52-year-old woman with pseudoscabies or canine scabies (Sarcoptes scabiei var. canis), transmitted by indirect contact with a red fox in the urban area of Berlin. Red foxes may live in unhabited areas of metropolitan large cities, i.e. in garages, car wrecks and cellars. Full remission of the prolonged and pruritic rush was seen after topical administration of lindane together with systemic corticosteroids.

Investigation of skin samples from red foxes (Vulpes vulpes) in eastern Brandenburg (Germany) for the detection of Borrelia burgdorferi s. l.

During earlier investigations a high prevalence of Borrelia (B.) burgdorferi s. l. in unfed Ixodes (I.) ricinus ticks in the Federal State of Brandenburg has been demonstrated. In the present study skin samples were obtained from 100 red foxes (Vulpes vulpes) from the districts where the highest B. burgdorferi prevalences had previously been found (i.e. Uckermark, Barnim, Märkisch-Oderland, Oder-Spree). BSK- and MKP-medium including inhibitory substances were used for cultivation of spirochaetes. Non-motile spirochaete-like organisms were observed in 26% of the samples. Additionally, by subcultures it was not possible to obtain motile helical forms characteristic for B. burgdorferi. On tryptose agar, the bacteria which produced nonmotile forms appeared as corynebacterium-like-colonies. Investigations by electron microscopy showed that the immobile spiral forms were giant whips (flagellae) which belonged to the contaminant flora. These forms proved to be negative for B. burgdorferi s. l. by the use of a nested-PCR. In a further study, the same skin samples were investigated for the presence of B. burgdorferi s. l.-DNA using a nested-PCR. Seven out of 100 samples were positive.

Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs.

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target.

Detection and differentiation of Theileria annulata and Theileria parva using macroschizont-derived DNA probes.

A lamda gt11 expression library based on T.annulata-infected cells was screened with an antiserum raised in rabbits against partially purified schizonts of T.annulata. Two clones were detected, sequenced and designated as SA288 and SB288 (Shayan et al., submitted for publication). From the sequences of these two genes oligonucleotide primers were designed for specific amplification of parasite DNA by polymerase chain reaction (PCR). We could show that these genes are of parasitic origin and do occur in all T.annulata stocks tested in the present study. In addition, a target sequence for SA288 could also be identified in T.parva-schizonts. None of them reacted with genomic DNA of different Babesia spp. A third primer pair was designed from the DNA-sequence of a gene encoding for the T.parva-specific casein kinase II-alpha subunit. Using this primer pair, a target sequence could only be detected in T.parva. Taken together, the primers described here can be used as molecular tools in PCR for the detection of Theileria parasites and to distinguish T.annulata from T.parva.

Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998.

The horse-parasitizing species Babesia equi Laveran, 1901 was redescribed as Theileria equi Mehlhorn, Schein 1998 and, thus, transferred from one valid genus to another. This transfer was needed since it turned out that this horse parasite showed the relevant characteristics of theilerians with regard to biological data, morphological features, biochemical properties, and molecular biological relationships.

Genital myiasis (Wohlfahrtiosis) in camel herds of Mongolia.

Genital infestations caused by Wohlfahrtia magnifica (Schiner, 1862) has been reported as being a common problem encountered in female camels in the Gobi desert. Thus, a field investigation to establish the prevalence of wohlfarthiosis in camel herds in the eastern Gobi district was carried out between May and July 1994. The objectives of the study were to establish the relevance of genital myiasis, and to describe the disease patterns, clinical course and pathomorphological changes associated with it. The field study was designed to suit the specific ecological, infrastructural and logistic conditions. Two cross-sectional samples were taken from 45 selected herds in six different areas of the Chatanbulag Sumon to obtain data on herd structure, age distribution, and the breeding history of infested female animals. Post partum events and previous occurrence of genital myiasis were recorded by use of a standardised questionnaire. Clinically infested animals were first detected visually. All camels to be investigated further were subsequently immobilised using ropes and the genitals were examined for tissue lesions and the presence of larvae. Examination of 1676 Bactrian camels from 45 selected herds in six different areas of the Chatanbulag Sumon in the Eastern Gobi district, Mongolia, led to an estimate of Wohlfahrtia magnifica infestation rates between 8-10%. Most myiasis cases were found in older females (> 4 yr), younger animals were infested at a lower rate. Highest prevalence rates were discovered during June and July, highest levels of infestation occurred in the Aman Us Chudak region with infestation rates up to 15%.

Macroschizonts of Theileria annulata as vaccine and diagnostic tools.

T. annulata-infected cells present infection-associated peptides. These peptides represent target molecules of the cytotoxic acting cells. Their preparation and characterization may help to develop a sub-unit vaccine. Our studies show that macroschizont-infected bovine cells can be used as parasite antigen in serology for the detection of parasite-specific antibodies in serum of infected animals. Primers derived from the macroschizont of T. annulata can be used as molecular tools for the detection of parasite DNA in blood samples of carrier cattle.