Mosaad Hilali

Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are carnivores and eat variety of foods, including garbage. In the present study, tissues and sera of 51 stray dogs (Canis familiaris) from Giza, Egypt were examined for T. gondii infection. Sera were examined for antibodies to T. gondii by the modified agglutination test (MAT); 50 of 51 (98%) were seropositive with titers of 20 in four, 40 in four, 80 in one, 100 in eight, 200 in 17, 400 in 11, 800 or higher in five. Hearts of 43 seropositive dogs were bioassayed in mice. Viable T. gondii was isolated from 22 dogs; these isolates were designated TgDogEg1 to TgDogEg22. DNA isolated from cell culture derived tachyzoites of 22 isolates was genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed three genotypes and one mixed infection. The three genotypes are ToxoDB PCR-RFLP #2 (type III, four isolates), #3 (type II variant, 11 isolates), #20 (six isolates), 1 mixed infection. These results revealed the dominance of clonal type II, III and ToxoDB #20 lineages of T. gondii in stray dogs from Giza, Egypt.

The definitive hosts of Sarcocystis sinensis in water buffaloes have hitherto been unknown, but the close similarity of this species to the cat-transmitted Sarcocystis bovifelis in cattle suggested they were felids. In a previous study, two domestic cats were fed macroscopic sarcocysts of Sarcocystis fusiformis contained within or dissected from the esophageal muscles of water buffaloes, while no microscopic sarcocysts of S. sinensis were noticed. Both cats started shedding small numbers of sporocysts 8-10 days post infection (dpi) and were euthanized 15 dpi. Using a PCR-based molecular assay targeting the mitochondrial cox1 gene of S. fusiformis, both cats were shown to act as definitive hosts for this species. In the present study, DNA samples derived from oocysts/sporocysts in the intestinal mucosa of both cats were further examined by PCR for the presence of S. sinensis using 2 newly designed primers selectively targeting the cox1 gene of this species. All 6 DNA samples examined from each cat tested positive for S. sinensis. A 1,038-bp-long portion of cox1 was amplified and sequenced as 2 overlapping fragments from 5 of these DNA samples. The 5 sequences shared 99.3-100% identity with 7 previous cox1 sequences of S. sinensis obtained from sarcocysts in water buffaloes. Additionally, amplification of the ITS1 region with primers targeting various Sarcocystis spp., yielded amplicons of 2 different lengths, corresponding to those obtained from sarcocyst isolates of S. sinensis and S. fusiformis, respectively. This is the first study to show that cats act as definitive hosts for S. sinensis.

A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.

Four valid species of Sarcocystis have been reported from the water buffalo (Bubalus bubalis): Sarcocystis fusiformis, Sarcocystis buffalonis, Sarcocystis levinei and Sarcocystis dubeyi. Here, we redescribe structure of S. fusiformis sarcocysts by scanning and transmission electron microscopy (SEM, TEM). Twenty-one macroscopic sarcocysts from oesophagus of the water buffalo in Egypt were examined by light microscopy, SEM and TEM. The sarcocyst wall was up to 9 μm thick, depending on the section and the technique. In 5 μm paraffin-embedded sections, the sarcocyst wall was indistinct, 2-5 μm thick and appeared smooth. In 1 μm plastic-embedded sections stained with toluidine blue, the sarcocyst wall was 2.5-5.2 μm thick and had branched villar protrusions (vp)-like branches of a dead tree. By SEM, the sarcocyst wall had a mesh-like structure with irregularly shaped vp that were folded over the sarcocyst wall. On each vp there were uniform papillomatous structures that were 100 nm wide. By TEM, vp were up to 6 μm long and contained filamentous tubular structures, most of which were parallel to the long axis of the projections; granules were absent from these tubules. By TEM, bradyzoites within the same cyst varied from 11.2 to 16.8 μm in length. By TEM, bradyzoites had a very long (10 μm) convoluted mitochondrion, up to 12 dense granules, but only 2 rhoptries. This redescription should help to differentiate the sarcocysts of S. fusiformis from similar sarcocysts in domestic and wild ruminants.