Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Occurrence and Molecular Typing of Giardia psittaci in Parakeets in Germany-A Case Study.

A grey-hooded parakeet (Psilopsiagon aymara) and two budgerigars (Melopsittacus undulatus) from different owners presented with decreased activity, vomitus, and diarrhea. A microscopic examination of feces showed trophozoites of the protozoan flagellate Giardia. A commercial immunochromatographic dipstick test for Giardia sp. antigens confirmed the infection. These findings were assured by PCR of the small subunit ribosomal RNA (SSU rRNA) gene and coproantigen ELISA. Sequencing of PCR products of the SSU rRNA (292 bp) and ß-giardin genes (511 bp) identified Giardia psittaci as the species involved. Therefore, our results show that a GSA 65-based coproantigen ELISA, which was established for diagnosis of Giardia duodenalis is applicable for the detection of G. psittaci. A treatment with ronidazole was started. Additionally, fecal examination and dissection of the dead birds revealed coinfection with the fungal pathogen Macrorhabdus ornithogaster. One budgerigar survived and repeatedly tested negative after treatment with ronidazole. The described cases indicate that a single infection with G. psittaci has a good prognosis, whereas the prognosis is poor when coinfections occur, especially with M. ornithogaster.

Reporte de caso- Presentación y tipificación molecular de Giardia psittaci en periquitos en Alemania: Un estudio de caso. Un periquito catita aimará (Psilopsiagon aymara) y dos periquitos australianos (Melopsittacus undulatus) de diferentes propietarios presentaron actividad disminuida, vómito y diarrea. El examen microscópico de las heces mostró trofozoitos del protozoo flagelado Giardia. Una prueba de tira reactiva inmunocromatográfica comercial para antígenos de Giardia sp. confirmó la infección. Estos resultados fueron confirmados por PCR para el gene de ARN de la subunidad pequeña ribosomal (SSU rRNA) y por ELISA de coproantígeno. La secuenciación de los productos de PCR del ARNr de SSU (292 pb) y los genes de ß-giardina (511 pb) identificaron a Giardia psittaci como la especie involucrada. Por lo tanto, estos resultados muestran que el método de ELISA de coproantígeno basado en GSA 65, que se estableció para el diagnóstico de Giardia duodenalis, es aplicable para la detección de G. psittaci. Se inició un tratamiento con ronidazol. Además, el examen fecal y la disección de las aves muertas revelaron coinfección con el patógeno fúngico Macrorhabdus ornithogaster. Un periquito australiano sobrevivió y dio negativo repetidamente después del tratamiento con ronidazol. Los casos descritos indican que la infección única con G. psittaci tiene un buen pronóstico, mientras que el pronóstico es malo cuando ocurren coinfecciones, especialmente con M. ornithogaster. Abbreviations: GSA = Giardia-specific antigen; OD = optical density; rRNA = ribosomal ribonucleic acid; SSU = small subunit.

Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in Germany.

A total of 18,259 feline faecal samples from cats in Germany were collected and analysed for the presence of Toxoplasma gondii oocysts between June 2007 and December 2008. The proportion of T. gondii-positive samples collected between January and June was significantly lower than between July and December. The age of cats shedding T. gondii oocysts was not significantly different from the age of negative control cats. Forty-six T. gondii-positive samples were genetically characterised using nine PCR-restriction fragment length polymorphism (RFLP) markers which included newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. In addition, 22 isolates that had already been partially characterised in a previous study were further typed using PCR-RFLP markers c22-8, c29-2, L358, PK1 and Apico. Genotyping of the 68 isolates revealed that the majority of T. gondii isolates (n=54) had Type II patterns at all loci but displayed a Type I pattern at the Apico locus. Three isolates displayed Type II patterns at all loci, including the Apico locus. In addition, we detected one isolate with clonal Type III patterns at all loci and three isolates with atypical and mixed genotypes. Seven isolates could not be fully genotyped. One of those isolates displayed alleles of both Types I and II at the Apico locus. To our knowledge this is the first description of the presence of T. gondii genotypes different from the clonal Types I, II and III in the faeces of naturally infected cats.

First isolation of Neospora caninum from the faeces of a dog from Portugal.

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of gamma-interferon knockout mice with 10(2) sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers.

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries.

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.