The Canine Vaginal Flora: A Large-Cohort Retrospective Study.

Microbiological examinations are frequently performed as part of breeding management examinations in the bitch, but also in case of (suspected) reproductive tract problems. As most bacteria are opportunistic pathogens, evaluation of bacterial findings is challenging for veterinarians. Besides, breeders might request antimicrobial treatment in breeding bitches, fearing conception failure-even without medical indication. Considering the rising threat of antimicrobial resistance, gaining deeper insights into the bacterial findings from the vagina of healthy and (suspected) reproductive-diseased bitches might contribute to the knowledge of the canine aerobic vaginal flora and consequently improve the responsible use of antibiotics. We analyzed results from bacteriological cultures of 23,254 vaginal swabs sent in to three commercial laboratories in Germany between 2015 and 2021, where standard aerobic microbiological examination was carried out. We found a variety of 319 bacterial species that mostly grew in mixed cultures of two or more bacterial species. Commonly found species were Escherichia coli, beta-hemolytic Streptococci, coagulase-positive Staphylococci, Pasteurellales, and aerobic sporulators, as well as other Streptococcus spp. Our results showed a large diversity of the canine vaginal flora in healthy and (suspected) reproductive-diseased bitches. They largely support earlier findings of small studies on the physiological canine vaginal flora, emphasizing that solely the results of a bacterial evaluation should not be the base for antimicrobial treatment. Instead, bacterial findings should be evaluated with the results of a clinical gynecological examination.

First detection of autochthonous Lamanema chavezi infections in llamas (Lama glama) in Europe.

Lamanema chavezi is one of the most pathogenic nematode species of South American camelids (SAC), with a homoxenous life cycle involving enterohepatic migration of its larvae in the host. So far, it has been found in the Americas and New Zealand. The first autochthonous L. chavezi infections in SAC in Europe are reported here. On a SAC farm in Germany, a 15-month-old male llama with a short history of diarrhoea died in September 2017, followed nine months later by a three-year-old female llama with a history of emaciation, apathy, anorexia, anaemia and tetraparesis with retained sensorium. Both animals were born and raised on the farm, which had imported three llamas directly from Chile 4-14 years earlier. At necropsy, the main lesions in both cases were numerous white-yellow to dark red foci, up to 3 mm in size, close to the Glisson's capsule and deep in the parenchyma of the liver. Histologically, the livers showed haemorrhagic tracks by and with nematode larvae and a necro-haemorrhagic to fibrinous inflammation with a predominantly lymphohistiocytic infiltration. The larvae were 30-50 µm in diameter and had external longitudinal cuticular ridges. Larvae extracted from unfixed liver tissue were 1800-2000 µm long and about 80 µm in diameter, with a terminal spine at the posterior end, which is characteristic of female L. chavezi stages. The ribosomal DNA including the almost complete 18S rRNA gene, the first internal transcribed spacer, the 5.8S RNA gene, the second internal transcribed spacer (ITS) and the partial 28S rRNA gene from isolated larvae were amplified using nematode-specific oligonucleotide primers and then sequenced. The assembled nematode sequence of 3448 bp showed an identity of 99.4% to previously published L. chavezi sequences in the BLASTN search. Low numbers of L. chavezi-like eggs were found in the faeces of seven (29%) of 24 llamas and alpacas in the herd, including some farm-born crias, tested two years after the last fatal case. The results show for the first time that L. chavezi has not only been imported into Europe from South America, but has also completed its life cycle locally, resulting in autochthonous infections of SAC. This was also suspected to be the cause of the fatal disease in two llamas.

[Ocular thelaziosis in a dog in Germany - an autochthonous case?] / Okuläre Thelaziose bei einem Hund in Deutschland ­ ein autochthoner Fall?

Thelazia callipaeda is a vector-borne nematode transmitted by Phortica spp. (fruit flies) and one of the causes of mild to severe conjunctivitis and keratitis in dogs, cats, rabbits and humans. It has been formerly known as the oriental eye worm based on its geographic occurrence. By now, it has been shown to be endemic in several southern and eastern European countries as well as extending its geographic distribution further throughout Europe. In the present case report, T. callipaeda infection was diagnosed in a female dog from Germany. The dog was referred by a local veterinarian due to a treatment-resistant conjunctivitis. A comprehensive ophthalmologic examination revealed 3 adult eye worms in the conjunctival sac as well as on the bulbar side of the nictitating membrane of the left eye. These were identified by morphological features and molecular techniques as T. callipaeda and represented the primary cause of the dog's unilateral blepharospasm, hyperemic conjunctiva and epiphora. Treatment consisted of manual collection of the adult worms and the administration of moxidectin/imidacloprid as spot on (Advocate®, Bayer). All clinical signs resolved within one week after treatment. In Germany, ocular thelaziosis still represents a rare disease. Usually, it is diagnosed in cats and dogs either imported from abroad or accompanying the owners on holiday travels to endemic countries. The dog presented in this case report was born in Germany. Except for a one-week stay in the Netherlands 11 months before the symptoms began, it had never traveled abroad. Considering the prepatency of T. callipaeda, an autochthonous transmission in this case is highly probable.

Urinary shedding of leptospires and presence of Leptospira antibodies in healthy dogs from Upper Bavaria.

Leptospirosis is classified as a re-emerging zoonotic disease with global impor- tance. The aim of this study was to determine urinary shedding of leptospires in healthy dogs and to identify the shedded leptospire species. Furthermore, antibody presence against leptospires was evaluated. In a prospective study urine samples of 200 healthy dogs from Upper Bavaria were randomly collected and evaluated by real-time polymerase chain reaction (PCR) specific for the lipL32 gene of pathogenic Leptospira (L) spp. Positive samples were further character- ized via multilocus sequence typing (MLST) to identify the Leptospira species. Microagglutination test (MAT) was performed to determine serum antibody titers. Three of 200 urine samples were found to be PCR-positive resulting in a urinary shedding prevalence of 1.5% (95% confidence interval 0.3-4.5%). All three dogs had been vaccinated before with a bivalent vaccine, covering the serogroups Canicola and lcterohaemorrhagiae. One dog shed leptospires of the species L. borgpetersenii, and two of the species L. interrogans. Of all dogs, 17.0% had antibody titers ≥ 1:100, and 3.5% titers ≥ 1:400 to serovars of non-vaccinal sero- groups. Healthy dogs that shed leptospires represent a possible risk for humans and other animals. The study emphasizes the importance of general hygiene measures in veterinary practice while handling urine of all dogs, and the use of vaccines that protect against a broader range of serogroups and that prevent urinary shedding.

No evidence of Dirofilaria repens infection in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) from Brandenburg, Germany.

Dirofilaria (D.) repens is a nematode causing dirofilariasis in dogs, cats and in humans. Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) are well-known wildlife reservoirs for zoonotic diseases. These two species are highly abundant in Germany, frequently exposed to vector mosquitoes and potentially susceptible to Dirofilaria infections. To obtain data about D. repens infections in these animals, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and examined for the presence of Dirofilaria DNA by means of D. repens-specific PCR. D. repens-specific DNA could not be amplified from the lungs of red foxes (n = 122; 0%) nor from the lungs of raccoon dogs (n = 13; 0%), suggesting a limited role if a role at all in the natural transmission cycle of D. repens in Brandenburg.

Detection of Angiostrongylus vasorum in Red Foxes (Vulpes vulpes) from Brandenburg, Germany.

Angiostrongylus (A.) vasorum is a nematode that causes angiostrongylosis in domestic and wild canids. Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) are suspected of providing a wildlife reservoir for A. vasorum infections in pet dogs. To obtain data on the occurrence of A. vasorum in wildlife, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and examined for the presence of A. vasorum DNA by means of real-time PCR. A. vasorum DNA was detected in 11 out of 122 (9.0 %) lungs of red foxes and in none of the lung samples of raccoon dogs. These data suggest that red foxes are a reservoir of A. vasorum infections for pet dogs in this area.

Transmission of Anaplasma phagocytophilum from endothelial cells to peripheral granulocytes in vitro under shear flow conditions.

Anaplasma phagocytophilum (Ap) is a tick-borne pathogen, which can cause granulocytic anaplasmosis in humans and animals. In vivo this obligate intracellular pathogen is primarily located in circulating mature granulocytes, but it also infects endothelial cells. In order to study the interaction between Ap-infected endothelial cells and human granulocytes under conditions similar to those found naturally in the infected host, an in vitro model that mimics physiological flow conditions in the microvasculature was established. Cell-to-cell interactions were then visualized by microscopy, which showed that granulocytes adhered strongly to Ap-infected endothelial cells at a shear stress of 0.5 dyne/cm(2). In addition, Ap-transmission assays under flow conditions showed that the bacteria transferred from infected endothelial cells to circulating granulocytes and were able to establish infection in constantly moving granulocytes. Cell surface analysis showed that Ap induced up-regulation of the cell adhesion molecules ICAM-1 and VCAM-1 on infected endothelial cells in a dose-dependent manner. Furthermore, IL-8 secretion by endothelial cells indicated that the presence of Ap induced a pro-inflammatory response. In summary, the results of this study suggest that endothelial cells of the microvasculature (1) provide an excellent site for Ap dissemination to peripheral blood granulocytes under flow conditions and therefore may play a crucial role in the development of persistent infection, and (2) are stimulated by Ap to express surface molecules and cytokines that may lead to inflammatory responses at the site of the infection.

CDPKs of Cryptosporidium parvum--stage-specific expression in vitro.

Cryptosporidium parvum is a zoonotic agent that bears a high risk for the health of particularly immunocompromised humans and animals. As currently available drugs and therapies against cryptosporidiosis do not turn out satisfactory, more intensive research on the control of this parasite is necessary. The genus Cryptosporidium is unique within the phylum Apicomplexa as its localisation is intracellular but extracytoplasmatic. Infection of host cells is initially a parasite-driven process, but the signalling events and their downstream actions within Cryptosporidium are poorly understood. Calcium-dependent protein kinases (CDPKs) are probably involved in the regulation of invasion and egress. Previously described in plants, algae and other Apicomplexa, CDPKs are not found in vertebrates. They are thus promising targets for pharmaceutical intervention. While CDPK1 is well characterised in Toxoplasma gondii (TgCDPK1) and Plasmodium falciparum (PfCDPK1), only little information exists about the expression and function of CDPK in C. parvum. Here, we describe results of the in silico analysis of seven CpCDPKs. Five CpCDPKs contain potential sites for N-myristoylation and N-palmitoylation. In a nested 3' rapid amplification of cDNA ends (RACE)-PCR, expression of six CpCDPKs resulted in distinct bands in infected cell cultures and extracts of freshly excysted sporozoites. The length of the 3' untranslated region (3' UTR) is described as well. Our results indicate CDPK expression to be stage specific on the mRNA level.

Analysis of the population structure of Anaplasma phagocytophilum using multilocus sequence typing.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.

Detection of Anaplasma phagocytophilum in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) from Brandenburg, Germany.

Anaplasma phagocytophilum is an obligate intracellular and tick-transmitted bacterium, which causes granulocytic anaplasmosis in animals and humans. Although infection with A. phagocytophilum in domestic animals and vector ticks is documented, there is sparse information on the occurrence of A. phagocytophilum in wild animals. Red foxes (Vulpes vulpes) as well as raccoon dogs (Nyctereutes procyonoides) are wildlife species highly abundant in certain areas of Germany and represent a potential wildlife reservoir for zoonotic diseases. To obtain data about the occurrence of A. phagocytophilum in these animals, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and were examined for the presence of A. phagocytophilum DNA by means of real-time PCR. Anaplasma phagocytophilum was detected in 10 out of 122 (8.2%) lungs of red foxes and in 3 out of 13 (23%) lungs of raccoon dogs. To the best of our knowledge, A. phagocytophilum was detected for the first time in red foxes and raccoon dogs in Germany.

Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.

Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum. Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium. In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells.

The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST).

BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.

Mitochondrial genes and genomes support a cryptic species of tapeworm within Taenia taeniaeformis.

Taenia taeniaeformis is a globally distributed cestode, which uses felids as definitive and rodents as intermediate hosts. The complete mitochondrial DNA (mtDNA) of T. taeniaeformis from Germany (Tt-GER) was sequenced, and compared with that of another isolate from China (GenBank NC_014768; Tt-CHN), both taken from cats. Analysis of the two mtDNAs indicated that the isolates are significantly different from one another with 12.6% and 9.9% nucleotide and amino acid divergence between them, for concatenated protein-coding genes; overall difference based on a pairwise nucleotide alignment of complete mtDNAs was 11.8%. A phylogenetic analysis based on the 12 protein-coding genes of all available taeniid mtDNAs confirmed the two T. taeniaeformis isolates as sister taxa (likely separate species) and early divergent members of the genus, as suggested previously by morphology. Phylogenetic analysis of published fragments of mt genes rrnS, cox1 and nad1, which represent multiple geographic isolates of T. taeniaeformis also resolve two distinct clades that at present do not seem to be geographically isolated. Mean pairwise (nucleotide) differences between the two clades of T. taeniaeformis were approximately 11%, 10% and 13% in partial rrnS (182bp), cox1 (371bp) and nad1 (459bp) genes, respectively. Differences between entire mtDNAs and partial mt genes of the two T. taeniaeformis isolates are of a similar magnitude between established taeniid sister species. Tt-CHN differs from all other Taenia mtDNAs in lacking a short (∼69bp) non-coding region between trnY and trnL1. Partial mt fragment analysis highlighted likely misidentifications of T. taeniaeformis on GenBank.

First case of Anaplasma platys infection in a dog from Croatia.

BACKGROUND: It is known that Anaplasma (A.) platys, the causative agent of infectious canine cyclic thrombocytopenia, is endemic in countries of the Mediterranean basin. However, few reports are available from the Balkans. This case report describes a dog, which was imported from Croatia to Germany in May 2010. One month later the dog was presented to a local veterinarian in Germany due to intermittent/recurrent diarrhoea. Diagnostic tests were performed to identify infections caused by Anaplasma spp., Ehrlichia spp., Hepatozoon canis, Babesia spp., Leishmania spp., Borrelia burgdorferi and/or Dirofilaria immitis. FINDINGS: Haematological examination of a blood smear revealed basophilic inclusions in thrombocytes, which were confirmed as A. platys with a species-specific real-time PCR. Additionally, an infection with Babesia (B.) vogeli was also detected (PCR and serology). No specific antibodies against Anaplasma antigen were detectable. Although the dog showed no specific clinical signs, thrombocytopenia, anaemia and elevated C-reactive protein (CRP) were observed. Sequencing of a 1,348-bp partial ribosomal RNA gene revealed highest homology to A. platys sequences from Thailand, Japan and France. CONCLUSIONS: A. platys was detected for first time in a dog imported from Croatia. As the dog was also co-infected by B. vogeli, unique serological and haematological findings were recorded. Thrombocytopenia, anaemia and elevated values of C-reactive protein were the laboratory test abnormalities observed in this case. A. platys infections should be considered in dogs coming from Croatia and adjacent regions.

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 (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 colorant 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.

Cross-reactivity of anti-Eimeria tenella antibody fragments on merozoites and sporozoites of different chicken Eimeria species.

Eimeria tenella-specific antibodies were examined for the cross-reactivity on the sporozoites and merozoites of E. tenella, Eimeria maxima, Eimeria acervulina and Eimeria brunetti in an indirect fluorescence antibody test. Two of nine antibodies showed cross-reactivity with sporozoites of E. maxima, E. acervulina and E. brunetti; however, the localization of specific fluorescence differed between species. No antibody binding was observed on merozoites. The suitability of these antibodies to alter the infectivity of Eimeria sporozoites and/or merozoites must be verified in cell culture models and in vivo experimental infections.

Current surveys on the prevalence and distribution of Dirofilaria spp. in dogs in Germany.

The Central Upper-Rhine (Baden-Württemberg, Germany) is one of the warmest regions in Germany and also harbours abundant numbers of mosquitoes. Case reports on presumably autochthonous occurrence of Dirofilaria spp. were reported previously and were a reason for a further investigation into the occurrence of vector-borne pathogens. For this purpose, 44 hunting dogs from the Central Upper-Rhine region were tested between 4(th) and 29(th) June 2007. The blood samples were tested using the Knott's test and IDEXX SNAP 4Dx test. The Knott's test revealed unsheathed microfilaria identified as Dirofilaria repens by PCR in 3 dogs with no history of travelling (6.8%; 95% CI: 2.4-18.2%). The seroprevalence for Anaplasma phagocytophilum was 43.2% (95% CI: 29.7-57.8%), but only 4.5% (95% CI: 1.3-15.1%) for antibodies to Borrelia C6 peptide. Dirofilaria immitis antigen was not detected in any of the samples. A further 288 blood samples from non-hunting companion dogs of the Central Upper-Rhine region were tested negative for heartworm antigen between February and August 2007.