Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 10⁹ colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10⁶ toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.     

Fasciola hepatica alters coagulation parameters in sheep plasma in vivo and in vitro

The blood-sucking activities of the liver fluke, Fasciola hepatica, are likely to cause alterations in coagulation during the course of infection; and the effect of F. hepatica on various coagulation parameters was studied during the course of acute and chronic fasciolosis of sheep over a period of 17 weeks. Whole blood and plasma samples from infected sheep (with 800 metacercariae each) and uninfected controls were collected weekly until 17 weeks post-infection (w.p.i.) and the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were determined. Additionally, adult F. hepatica were recovered from bile ducts, incubated for the production of excretory/secretory products (ESP) or homogenised and the effect of fluke products on APTT, PT and TT was determined. Anaemia was evident in infected sheep from 8 w.p.i. until 17 w.p.i. Plasma APTT was accelerated during 8, 9, 12, 14, 16 and 17 w.p.i., while PT was prolonged at 8-11 w.p.i. and TT at 10, 14 and 17 w.p.i. Addition of worm ESP or homogenate to plasma resulted in an enhancement of the intrinsic pathway (APTT) together with a prolongation of the extrinsic and common pathways (PT, TT) of coagulation. It was concluded that F. hepatica contains and releases substances that may contribute to coagulation changes in vivo. Further characterisation of the active substance(s) in vitro revealed heat inactivation, a size >30 kDa and inhibition by the proteinase inhibitors Complete and EDTA for the APTT-accelerating substance(s). The TT-deceleration, in contrast, was increased after heating.     

Autofluorescence microscopy for the detection of nematode eggs and protozoa, in particular Isospora suis, in swine faeces

Parasites from swine faeces were examined for autofluorescence. Oocysts of Eimeria polita, E. scabra and Isospora suis, cysts of Balantidium coli and eggs of Oesophagostomum dentatum, Strongyloides ransomi and Trichuris suis (but not those of Ascaris suum) emitted light after excitation with UV light. I. suis oocyst counts in McMaster chambers utilising autofluorescence were compared to those from conventional bright field microscopy. Similarly, faecal smears containing I. suis were examined using the same techniques. Autofluorescence was superior to bright field microscopy in detecting oocysts after flotation and was highly significantly more sensitive when direct smears were examined.     

Effects of curcumin (diferuloylmethane) on Eimeria tenella sporozoites in vitro

The negative effects of coccidiosis on poultry health and productivity and increasing problems related to drug resistance have stimulated the search for novel and alternative methods of control. The present study evaluates the anticoccidial activity of curcumin (diferuloylmethane), a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric (Curcuma longa) which is a spice and food colourant commonly used in curries and also used as medicinal herb. Its effects were evaluated on Eimeria tenella sporozoites, including morphological alterations, sporozoite viability and infectivity to Madin–Darby bovine kidney (MDBK) cells. Morphological alterations of the sporozoites were recorded as deformation due to swelling and cell membrane corrugations. Curcumin at concentrations of 25, 50, 100, 200 and 400 μM showed considerable effects on sporozoite morphology and viability in a dose-dependent manner after incubation over 3, 6, 18 and 24 h while lower curcumin concentrations (6.25 and 12.5 μM) were not effective. In comparison to the untreated control, sporozoite infectivity was reduced at curcumin concentrations of 100 and 200 μM by 41.6% and 72.8%, respectively. Negative effects of curcumin on MDBK cells were not seen at these concentrations; however, curcumin at concentrations of 1,800, 600 and 400 μM was toxic to MDBK cells and affected cell proliferation. In conclusion, curcumin exhibited a marked inhibitory effect in vitro on E. tenella sporozoites inducing morphological changes and reducing sporozoite viability and infectivity.     

Effects of curcumin on Cryptosporidium parvum in vitro

Cryptosporidium parvum is a zoonotic protozoan parasite having peculiarities among the apicomplexa that could be responsible for its resistance to some drugs and disinfectants against coccidia. The awareness of Cryptosporidium as a health problem in man and animal is increasing and potent drugs are urgently needed. Curcumin, a natural polyphenolic compound, has been found to be active against a variety of diseases including anticarcinogenic, antimicrobial, and antiprotozoal effects. We investigated the effects of curcumin on infectivity and development of C. parvum in a recently established in vitro system combining infection of human ileocecal adenocarcinoma cell cultures with quantification of intracellular parasites by quantitative polymerase chain reaction. Curcumin was found to be effective (>95% inhibition of parasite growth) at 50 μM for 24 h when infected cultures were exposed for more than 12 h. Withdrawal of curcumin after 24 h of exposure did not result in a significant resumption of C. parvum growth. The invasion of host cells by sporozoites (infectivity) was found to be inhibited at least 65% in the presence of 200 μM curcumin. No significant reduction of viability of C. parvum oocysts after incubation with curcumin was recorded. Altogether, curcumin showed promising anticryptosporidial effects under in vitro conditions and deserves further exploration.     

Use of random amplified polymorphic DNA-polymerase chain reaction for the definition of genetic markers for species and strains of porcine Oesophagostomum

Nodular worms are common parasites of pigs, and research has recently started to focus on the biology of these nematodes. However, the methods for delineation of species at immature developmental stages and␣for␣differentiation of various lines of the same species␣remain limited. For differentiation of porcine Oesophagostomum species and strains by genomic fingerprinting, random amplified polymorphic DNA-polymerase chain reaction was performed on DNA derived from 20 larval batches of anthelmintic-susceptible and resistant strains and isolates of these nematodes and 2 ruminant Oesophagostomum spp. Polymorphic DNA markers could be amplified with 9 of the 33 primers tested. In all, 13 markers were species-specific and 6 markers could differentiate between strains or groups of strains. With a combination of the latter, artificially selected anthelmintic-resistant strains and the susceptible mother strain of O. dentatum could be delineated. When single adult worms were compared, considerable variations between strains of the same species and between individuals from the same strain could be detected. The differentiation of Oesophagostomum strains and species at all parasitic stages on the basis of genetic markers could greatly facilitate studies on the biology of these parasites. Received: 6 January 1997 / Accepted: 24 March 1997     

Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R ², 0.98–0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 μM) and monensin (0.00225 to 0.144 μM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10⁵ oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 μM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 μM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 μM or more, inhibition was at least 90%; 0.05 μM still yielded 80% inhibition, whereas at concentrations below 0.00625 μM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.     

Investigations into the production and function of leukotrienes during histotropic development of Oesophagostomum dentatum

 The dynamics of production and excretion of leukotrienes by parasitic larvae of Oesophagostomum dentatum and their role for the development of the larvae were studied. Larvae were cultured in vitro to the fourth stage (L4). Leukotriene B4 (LTB4) was detected in homogenates and in supernatant of larvae, with the homogenate-protein-based values steadily decreasing during development. The homogenate-protein-based values of peptidyl leukotrienes (pepLT) remained fairly stable in both homogenates and supernatants, whereas the worm-count-based pepLT values increased significantly. The addition of diethylcarbamazine (DEC) to the culture medium straight from the beginning of culturing (12.8 or 25.5 mmol/l) reversibly hampered growth and development to L4. Application of DEC at 12.8 mmol/l beginning on day 13 of in vitro cultivation exerted no significant effect on further development to L4. LTB4 appeared to counteract the inhibition of development by DEC. The results of this study indicate that endogenous LTs participate in regulation of the growth and development of O. dentatum. Received: 7 September 1995 / Accepted: 12 December 1995     

Prevalence of <Emphasis Type="Italic">Eimeria bovis</Emphasis> and <Emphasis Type="Italic">Eimeria zuernii</Emphasis> in German cattle herds and factors influencing oocyst excretion

The present study was designed to investigate the prevalence of the pathogenic coccidia species Eimeria bovis and Eimeria zuernii in shed-reared animals in German dairy and fattening facilities. Samples were obtained from 65 cattle farms distributed randomly across all the regions of Germany regardless of the occurrence of clinical problems. The samples were obtained rectally. Faecal consistency and the total number of oocysts per gram of faeces (OPG) were determined, along with the OPG values for E. bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management, along with individual animal data. Eimeria oocysts were detected in 62 of these farms, which give a prevalence of 95.4%. The farm prevalence of the pathogenic species was 76.9% for E. bovis and 83.1% for E. zuernii. The number of oocysts excreted could not be correlated significantly with farm type or farm management but depended on the floor type, the age of the calves and the time after rehousing. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea. E. zuernii had a greater influence on the occurrence of diarrhoea than E. bovis. This study confirms that herd management frequently does not meet the requirements of effective coccidia control despite the fact that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations.