Arwid Daugschies

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 400xa0μM showed considerable effects on sporozoite morphology and viability in a dose-dependent manner after incubation over 3, 6, 18 and 24xa0h while lower curcumin concentrations (6.25 and 12.5xa0μM) were not effective. In comparison to the untreated control, sporozoite infectivity was reduced at curcumin concentrations of 100 and 200xa0μM 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 400xa0μM 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.

Diagnosis of Imported Canine Filarial Infections in Germany 2008 – 2010

Filarial infections of dogs are attracting attention across Europe because of the risk of spread into previously non-endemic areas (e.g. Dirofilaria repens with Culicidae as vectors) and as emerging zoonotic agents. The occurrence of filarial infections in German dogs has been analysed based on 8,545 samples collected either from imported animals or following travel into endemic regions. All samples were tested by means of modified Knott’s test and heartworm antigen assay within the period 2008 – 2010. Heartworm antigen was detected in 127 samples (1.49 %; 95 % CI: 1.25 – 1.77 %), but only 38 dogs also had microfilariae in their blood samples. On the other hand, 125 animals (1.46 %; 95 % CI: 1.23 – 1.74 %) were only positive in the Knott’s test. For discrimination by means of PCR and sequencing a total of 73 blood samples as well as two samples of adult worms were included, which have been sent by veterinarians during 2008 – 2010. A mono-infection caused by D. repens was detected in 35 cases, while D. immitis was proven in 15 samples, with 6 of these showing a combination of D. immitis and D. repens. Imported Dipetalonema dracunculoides (transmitted by Rhipicephalus sanguineus or Hippobosca longipennis) or Acanthocheilonema reconditum (fleas and lice serve as intermediate hosts) infections were diagnosed in 24 cases and in a single sample a co-infection of A. reconditum and D. repens was evident. D. repens was the most common filarial infection imported and it was introduced into Germany from eleven European countries. Slovenia and Hungary are reported for the first time as endemic for D. repens and A. reconditum, respectively. Furthermore this study reports, to the best of our knowledge, for the first time import of D. dracunculoides from the Canary Islands, A. reconditum from Majorca, D. immitis from Corfu and a co-infection of D. repens and A. reconditum from Spain as well as mixed infections of D. repens and D. immitis from Corfu, Sardinia and Bulgaria. Co-infections with other arthropod-borne infections as well as therapeutical follow-up were also considered. Selamectin (as spot-on formulation) was not able to clear microfilaraemia in dogs infected with either D. repens, A. reconditum or D. dracunculoides, whereas a topical moxidectin/imidacloprid formulation was able to eliminate microfilariae in one dog infected with A. reconditum.

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 50xa0µM for 24xa0h when infected cultures were exposed for more than 12xa0h. Withdrawal of curcumin after 24xa0h 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 200xa0µM 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.

Combination of cell culture and quantitative PCR for screening of drugs against Cryptosporidium parvum

Cryptosporidium parvum is a zoonotic pathogen causing self-limiting diarrhea in immunocompetent patients. An assay combining cell culture and real time quantitative PCR (qPCR) is reported here to verify drug efficacy against C. parvum in vitro. The monolayers of Human ileocecal adenocarcinoma cells (HCT-8) were infected by sporozoites excysted directly on the cells and were incubated with monensin, halofuginone bromide and hexadecylphosphocholine until 45h post infection (p.i.). The genomic DNA was extracted at 3, 27 and 45h p.i. and subjected to qPCR targeting the 70kDa heat shock protein gene to quantify the development of C. parvum. The reliability of the method was validated by testing of monensin and halofuginone bromide, which are well known to be effective in vitro. With the dose dependency monensin and halofuginone showed a maximum inhibition of 98.15% and 98.05% at 0.144 and 25microM, respectively, compared with non-treated controls at the endpoint incubation, confirming previous reports. The reduction of the parasite DNA reproduction over 27h p.i. compared with 3h p.i. was found to be as 97-99% in 0.144microM monensin and 99% in 25microM halofuginone treated cells. The new antileishmanial compound hexadecylphosphocholine (24.5microM, Miltefosine) showed 78-98% inhibition at 45h p.i., however, the reproduction of parasite DNA was reduced to 96-98% over 27h p.i. The method has the potential to easily and reliably assess anticryptosporidial compounds in adequately equipped routine laboratories.

Prevalence of Eimeria bovis and Eimeria zuernii in German cattle herds and factors influencing oocyst excretion

AbstractThe present study was designed to investigate the prevalence of the pathogenic coccidia species E. bovis and E. 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 for Eimeria spp., along with the separate OPG values for Eimeria (E.) bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management together with individual animal data.n Eimeria oocysts, regardless of the kind of Eimeria spp., were detected in 62 of these farms, which gives 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 average oocyst excretion level was 2,950 OPG in terms of total Eimeria spp. oocyst excretion, 700 OPG for E. bovis and 1,500 OPG 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 which influences the infection pressure, on the age of the calves and the time after rehousing. In general, higher oocyst excretion rates were found in calves kept on litter compared to rearing on slatted floor. Younger calves and calves sampled early after housing shed higher amounts of oocysts than older calves and calves stabled a longer period before sampling, respectively. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea, defined as observation of a loose to liquid faecal consistency. Excretion of E. zuernii oocysts was more closely linked to the occurrence of diarrhoea than E. bovis oocyst excretion. This study confirms that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations and a significant cause of diarrhoeal disease in calf rearing.

Occurrence and molecular characterization of Cryptosporidium spp. genotypes in European hedgehogs ( Erinaceus europaeus L.) in Germany

Juvenile hedgehogs having insufficient body weight are often brought for overwintering to hedgehog rehabilitation centres. Faecal samples of juvenile hedgehogs and overwintering hedgehogs (n=188) collected prior to releasing them back into the wilderness were examined for the presence of Cryptosporidium coproantigen and oocysts. Altogether 56 (29.8%) submitted samples were positive for coproantigen. Forty-five (39.5%, n=114) of the positive samples originated from newly rescued hedgehogs, while 11 (14.8%, n=74) positive samples were from animals that spent several months at the station. Fifteen samples subjected to PCR-RFLP analysis on the partial 18S rRNA locus suggested the presence of C. parvum. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA, actin gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new, proposed as VIIa subtype family. Cryptosporidium sp. genotype belonging to VIIa subtype family is closely related to C. parvum but is genetically distinct being probably a hedgehog-specific Cryptosporidium sp. genotype with unknown zoonotical potential. Hedgehogs excreting Cryptosporidium oocysts represent a potential source for human infections, but also an anthroponotic nature of the IIc subtype family should be reviewed.

Therapy and prevention of cryptosporidiosis in animals

Cryptosporidiosis is a common gastro-intestinal illness in animals and man worldwide. The disease is devastating in immune-suppressed individuals but self-limiting in competent hosts. The infectious stages of the organism (oocysts) are shed in the faeces of affected individuals, survive in adverse environmental conditions and spread by direct contact or through contaminants (food, water). Due to the robustness of the oocysts, their tenacity, tiny size, and resistance to common disinfectants, the parasite is difficult to eradicate from contaminated environments. To obtain sufficient control both treatment of infected hosts and inactivation of oocysts are necessary. Several drugs are commonly used to treat cryptosporidiosis in man and very few in animals but none of them are completely effective in terms of both clinical and parasitological response. Only a few chemical agents are able to inactivate oocysts in the environment including water treatment plants but their application has certain limitations. Therefore, control of cryptosporidiosis remains a global challenge in both veterinary and human medicine. Extensive research has been performed on suitable drugs and disinfectants. Thousands of agents have been tested both in vivo and in vitro. Some are excitingly active in vitro but exhibit poor or no response in clinical trials. Currently, no single or combined drug therapy has proven to be completely effective against this disease. This article will focus on therapy and prevention of cryptosporidiosis in animals including perspectives for new drugs.

Study of the Comparative Efficacy of Toltrazuril and Diclazuril against Ovine Coccidiosis in Housed Lambs

A blinded, controlled and randomised field study was conducted on a sheep farm with a known history of coccidiosis and a high prevalence mainly of the pathogenic coccidium Eimeria ovinoidalis. The efficacy of treatment with toltrazuril (Baycox® 5 % suspension) against natural infections with Eimeria crandallis and/or Eimeria ovinoidalis in housed lambs was investigated in comparison with diclazuril and untreated controls. Both drugs were administered either metaphylactically (i.e., in the prepatency of Eimeria spp.) or therapeutically (after onset of oocyst excretion). A total of 145 animals aged 1 to 5 days at the start of the study were included. Examination of faecal samples was performed every second day between days 13 and 49 of the study. The assessment of treatment efficacy was based mainly on total oocyst excretion and the number of E. crandallis and E. ovinoidalis oocysts (OPG) shed throughout the study. Oocyst excretion was reduced significantly in both groups treated with toltrazuril compared with the untreated control group and with both diclazuril-treated groups. The most prevalent and most severe diarrhoea was observed in the untreated control group. In this study, toltrazuril proved to be highly effective in controlling ovine coccidiosis both metaphylactically and therapeutically. The efficacy of toltrazuril was significantly higher than the efficacy of the control substance with regard to the duration and amount of oocyst excretion, both for the comparison of metaphylactic as well as therapeutic treatment.

Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8.

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 μg per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.

Combination of cell culture and quantitative PCR (cc-qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions.

Oocysts of Cryptosporidium parvum are resistant to environmental conditions and many disinfectants. A combination of cell culture and quantitative real time PCR (cc-qPCR) is established for evaluation of anticoccidial disinfectants against C. parvum. C. parvum oocysts were treated with disinfectants, washed and oocysts were incubated with HCT-8 cell monolayers in the presence of excystation medium for 3h. Subsequently, unbound parasites were removed by washing with growing medium and the infected monolayers were further maintained in fresh growing medium for 48h. Genomic DNA was extracted from each sample and qPCR performed targeting a specific sequence of the 70kDa heat shock protein gene in order to quantify development. Treatment of oocysts with cresolic disinfectants demonstrated dose dependent reduction of viability of oocysts. More than 98% inactivations were recorded with at least 2% concentration of cresolic disinfectants after 2h of treatment. Bleach (sodium hypochlorite) at 6% solution induced 92.7% inactivation of C. parvum oocysts after 2h. Thermally treated oocysts (56 and 70 degrees C for 20min) demonstrated complete inactivation, whereas at 38 degrees C no inactivation was observed. Application of Neopredisan((R)) 135-1 and Aldecoc((R)) TGE (4% for 2h) as recommended according to the current guidelines stipulated by DVG (German Veterinary Society) consistently inactivated more than 99.5% of oocysts. The suggested cc-qPCR method appeared to be suited for standardized testing of inactivation measures, particularly for evaluation of chemical disinfectants and thus cc-qPCR is proposed as an alternative to the established chicken infectivity model for Eimeria tenella for testing anticoccidial disinfectants. A minimum inactivation of 99.5% in cc-qPCR model is claimed as a suitable threshold for certification of chemical products for disinfection of coccidia oocysts.