Anja Joachim

Encephalitozoonosis in rabbits.

Encephalitozoon cuniculi is an obligatory intracellular microsporidian parasite that can infect a wide range of mammals, including rodents, rabbits, horses, carnivores and humans, in which the organism is known as an opportunistic pathogen of immunocompromised individuals. Nevertheless, the main host for E. cuniculi is the rabbit and infections usually have a sub-clinical course. However, severe disease is recognised in pet rabbits more frequently within the last years. As the central nervous system, the kidney and the eye are predilection organs for the organism, predominant histopathological alterations comprise granulomatous meningoencephalitis, chronical interstitial nephritis and phacoclastic uveitis. A definitive diagnosis of encephalitozoonosis in vivo is difficult, but it is important for specific treatment and the determination of possible zoonotic risks. This review article covers epidemiology, pathology, pathophysiology, immunology, clinical signs, differential diagnosis, diagnosis and treatment of encephalitozoonosis in rabbits.

Prevalence and control of bovine cryptosporidiosis in German dairy herds.

Abstract In a 5-year survey regarding its prevalence and importance in five German state veterinary laboratories Cryptosporidium was diagnosed annually in 19–36% of faecal samples either submitted to the laboratories or taken post mortem. In approximately half of the cases no other enteropathogens were detected. However, only 73% of 30 laboratories participating in a questionnaire survey routinely tested for this parasite, and the majority of researchers considered cryptosporidiosis to be of minor importance. In a placebo-controlled field study 152 suckling calves were treated daily against cryptosporidiosis either with sulfadimidine or with halofuginone (Halocur®, Intervet) over 1 week. Treatment by oral drench started at the onset of diarrhoea in the herd. Oocyst excretion, faecal consistency and health status were recorded five times for a 3-week period. Oocyst excretion peaked 7–14 days in the placebo group after the onset of diarrhoea, and during that period prevalence and intensity of excretion were significantly lower in the halofuginone-treated group compared to the sulfadimidine and the placebo control groups. The health status (diarrhoea, dehydration) declined in all groups but was significantly (P<0.05–0.001) better in the halofuginone group in the first 2 weeks. Halofuginone effectively (P<0.05–0.001) reduced oocyst excretion and improved the health status of the treated animals, while sulfadimidine had no effect against Cryptosporidium.

Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits.

Infections with Encephalitozoon cuniculi in rabbits are observed at increasing frequency and are known as opportunistic infections in immunocompromised humans. 191 pet rabbits with suspected encephalitozoonosis, presented at the Animal Hospital of the Veterinary University of Vienna (Austria), were included in this study. Rabbits were serologically examined for antibodies against E. cuniculi (144 positive out of 184 rabbits with suspected encephalitozoonosis compared to 14 positive out of 40 clinically healthy rabbits tested as part of a standard health check) and Toxoplasma gondii (8 positive out of 157). Of the 144 seropositive rabbits with clinical signs, 75% showed neurological symptoms, 14.6% demonstrated phacoclastic uveitis and 3.5% suffered from renal failure. 6.9% of the animals had combined symptoms. Vestibular disease dominated within the rabbits that showed neurological symptoms. Polymerase chain reaction (PCR) could not detect parasite DNA in urine or cerebrospinal fluid (CSF), but did so in 4 out of 5 samples of liquefied lens material in cases with phacoclastic uveitis due to lens capsule rupture. Additionally further diagnostic procedures, such as inspection of the external ear canal (N=69), radiography of the tympanic bullae (N=65) were performed to rule out differential diagnosis. 54.2% of the patients exhibiting neurological symptoms recovered within a few days, while 87.5% of the rabbits suffering from renal failure died or had to be euthanized.

Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in fecal specimens

To optimize routine screening for cryptosporidiosis, 198 stool samples from patients at risk and from calves were examined by enzyme immunoassay (EIA), a direct fluorescent-antibody (DFA) and a modified immunofluorescence assay. Ninety-nine samples were positive in at least one assay, whereas 99 were negative in all three assays. Sensitivity of antigen EIA and DFA were similar (94%, 95% CI: 88-98%, and 91%, 95% CI: 84-95%). The modified immunofluorescence was significantly less sensitive (64%, 95% CI: 55-74%). 149 samples were also examined by two nested PCR assays targeting either the 18S rRNA or Cryptosporidium outer wall protein (COWP) gene. A PCR product was amplified from 86 out of 89 samples being positive in at least one other assay (sensitivity 97%, 95% CI: 91-99%). None was obtained from 60 samples negative in the three other assays. PCR assays did not increase the detection rate. Antigen EIA or DFA appear sufficient for routine Cryptosporidium screening of fecal samples.

A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Eicosanoids in parasites and parasitic infections

Eicosanoids are lipid mediators with multiple functions in vertebrate tissues and invertebrate organisms. In this review the roles of eicosanoids--mostly prostaglandins (PGs), thromboxanes and leukotrienes--in parasite physiology and host-parasite interactions are discussed. PGs are present in the saliva of blood-sucking arthropods facilitating feeding by increasing local blood flow and prolonged attachment of ticks by immune suppression. Release of various eicosanoids has also been demonstrated for a number of protozoan and metazoan endoparasites. These substances appear to play a role in penetration, immune suppression, inflammation or modulation of haemostasis, enabling parasite invasion and establishment. Moreover, endogenous eicosanoids serve various functions in parasite metabolism and physiology. In many parasitic infections eicosanoids are involved in host pathology, e.g. granuloma formation, coagulopathy, secretory diarrhoea, or fever. Immune suppression by induction of PG release, in particular PGE2, by host defence cells appears to be a common feature of many parasitic infections and is though to be important for parasite establishment. Contradictory results have been obtained for gastrointestinal nematode infections, which probably reflect the considerable differences between the various models employed. Although most of the available studies indicate an important role for eicosanoids in parasites and parasitic infections, our current knowledge is still fragmentary and more data are urgently needed.

Occurrence of Isospora suis in larger piglet production units and on specialized piglet rearing farms

Mixed fecal samples of 264 litters from five piglet production farms (155-238 sows/farm) were investigated three times during the suckling period for the occurrence of Isospora suis over the period of 1 year. On all five farms Isopora suis was found to be a common endoparasite with infection rates being highest in litters of 3-4 weeks of age. By the end of the third investigation period the cumulative infection rate was 53.8% of the litters ranging from 20.0% to 81.5% for the single farms. During the suckling period the infection rate increased from 18.6% to 32.6% and then to 37.7%. Diarrhea was present in 66.3% of the sampled litters with the highest rates at the end of the suckling period. 63.4% of the litters which showed diarrhea and 34.8% of those without diarrhea excreted I. suis within the study period. Diarrhea was recorded for 78.2% of the I. suis-positive litters and for 52.5% of the Isospora-negative litters. In summer and fall the occurrence of I. suis was higher (66.3% and 61.0%, respectively) than in spring and winter (47.7% and 37.9%, respectively). In litters with diarrhea and pathogenic E. coli I. suis often occurred simultaneously. Above-average hygiene measures and mainly perforated pen floors seemed to lower the risk of isosporosis. With the exception of Strongyloides ransomi other parasites were not found in the fecal samples of suckling piglets. Two specialized piglet rearing farms, a conventional large-scale rearing unit and a farm managed according to the segregated early weaning (SEW) system were examined three times during the 6-7 week rearing period. In both units I. suis was common, but was not correlated with diarrhea. In the SEW unit the infection rates decreased from 37.5% to 20.2% and to 4.1%, while the infection rate in the conventional unit slightly increased from the first (17.2%) to the second (21.9%) investigation and stayed at this level at the third sampling.

Diagnostic markers for encephalitozoonosis in pet rabbits.

Encephalitozoonosis is a common disease in pet rabbits, routinely diagnosed in vivo by serological examination or post mortem by histopathology. Recently molecular techniques have become increasingly important as diagnostic tools. The application of different diagnostic markers for in vivo and post mortem determination of E. cuniculi in naturally infected pet rabbits were compared. The examined population was divided into 33 rabbits with symptoms of encephalitozoonosis (group I) and 38 animals without symptoms (group II) which were all tested by serological analysis (Indirect Immunofluorescence Test), histological examination including special spore staining (Ziehl-Neelsen, acid-fast trichrome) and conventional and nested PCR (organs, body fluids). Additionally, in group III lens material (n=10) of animals (n=9) with phacoclastic uveitis were examined by conventional PCR. Infections with E. cuniculi could be determined post mortem in 78.8% of the rabbits of group I and in 57.9% of group II by histological examination combined with spore staining. In group I 69.7% and in group II 50.0% showed seroconversion. Conventional PCR was only sufficiently sensitive in samples of eyes with phacoclastic uveitis (n=10; 100%). Therefore nested PCR was performed in tissue samples, urine and cerebrospinal fluid (CSF) with positive results in 63.6% of group I and 42.1% of group II. Positive results in serology, pathohistology (spore detection and histological changes in the brain and/or kidneys) and nested PCR were obtained in 52.1% of the rabbits (group I and II, n=71), whereas 31.0% showed negative results in all three diagnostic techniques. 5.6% of the rabbits were positive in two methods and 11.3% in one method. In 37 rabbits (group I and II) with positive results in nested PCR, E. cuniculi DNA could be detected more frequently in the brain (91.9%) than in the kidney (54.1%). Furthermore nested PCR of urine revealed positive results in 29.7% of the rabbits (n=37) with seroconversion and/or confirmed E. cuniculi infection by spore detection. All 25 samples of CSF tested negative in nested PCR. Conventional PCR of eyes with phacoclastic uveitis was an excellent diagnostic marker in living rabbits, while nested PCR of urine or CSF was not reliable. Histological examination combined with special staining was the most sensitive method in post mortem diagnostics. Nested PCR appears to be a good post mortem method to investigate organs, especially brains, of chronically infected animals.

Parasites of domestic owned cats in Europe: co-infestations and risk factors

BackgroundDomestic cats can be infested by a large range of parasite species. Parasitic infestations may cause very different clinical signs. Endoparasites and ectoparasites are rarely explored in the same study and therefore multiparasitism is poorly documented. The present survey aimed to improve knowledge of the prevalence and risk factors associated with ecto- and endoparasite infestations in owned cats in Europe.MethodsFrom March 2012 to May 2013, 1519 owned cats were included in a multicenter study conducted in 9 veterinary faculties throughout Europe (Austria, Belgium, France, Hungary, Italy, Romania and Spain). For each cat, ectoparasites were checked by combing of the coat surface associated with otoscopic evaluation and microscopy on cerumen samples. Endoparasites were identified by standard coproscopical examinations performed on fresh faecal samples. Risk factors and their influence on parasitism were evaluated by univariate analysis followed by a multivariate statistical analysis (including center of examination, age, outdoor access, multipet status, and frequency of treatments as main criteria) with logistic regression models.ResultsOverall, 50.7% of cats resulted positive for at least one internal or one external parasite species. Ectoparasites were found in 29.6% of cats (CI95 27.3-32.0%). Otodectes cynotis was the most frequently identified species (17.4%), followed by fleas (15.5%). Endoparasites were identified in 35.1% of the cats (CI95 32.7-35.7%), including gastro-intestinal helminths in 25.7% (CI95 23.5-28.0), respiratory nematodes in 5.5% (CI95 4.2-7.0%) and protozoans in 13.5% (CI95 11.8-15.3%). Toxocara cati was the most commonly diagnosed endoparasite (19.7%, CI95 17.8-21.8%). Co-infestation with endoparasites and ectoparasites was found in 14.0% of the cats, and 11.9% harbored both ectoparasites and gastro-intestinal helminths.Age, outdoor access, living with other pets, and anthelmintic or insecticide treatments were significantly associated with the prevalence of various parasites.ConclusionsThis survey demonstrates that parasitism is not a rare event in European owned cat populations. The prevalence of multi-parasitism is significantly greater than expected by chance and hence there is tendency for some individual cats to be more prone to infestation by both endo- and ectoparasites due to common risk factors.

Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation

Encephalitozoon cuniculi is a common infectious agent of rabbits. The aim of this study was to determine the distribution and extent of histological lesions in the brain and in the kidney of naturally infected pet rabbits with or without clinical encephalitozoonosis. In 71 animals (33 with symptoms) which died or were euthanised, histopathological examination including staining of spores (Ziehl–Neelsen, acid-fast trichrome) was performed and changes were described quantitatively. The cerebrum was the most frequently affected brain region (97.5%), whilst the cerebellum (55%) and the vestibular cores (37.5%) were less commonly concerned. Granulomas were found in 77.5% of animals with encephalitis and in 12.5% of rabbits with interstitial nephritis. Although cerebral granulomas were found irrespective of the grade of histological changes, they were significantly correlated with changes at higher grades. There was no correlation between the severity of encephalitis and neurological symptoms. Since severe lesions were also found in clinically inconspicuous animals, histological findings of inflammatory lesions are not indicative of overt encephalitozoonosis as the causative agent for neurological signs. Other diseases causing neurological symptoms, such as suppurative encephalitis, otitis media as well as malignant lymphoma were also detected in the rabbit population that was examined in the present study.