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A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.

The aim of this study was to investigate if reconstructed felid embryos obtained by intraspecies or intergeneric cloning can develop in vitro. Fibroblast cells (f) from a domestic cat (DCf), marbled cat (MCf) and bovine (Bf) were used as donor cells, and oocytes (o) from domestic cats (DCo) and bovine (Bo) were used as recipient cytoplasts. There were two intraspecies (donor cell + recipient cytoplast: DCf + DCo and Bf + Bo) and three intergeneric (MCf + DCo, DCf + Bo and MCf + Bo) cloning groups in the study. In Experiment 1, the effects of manipulation media, modified TCM-199 (199H) or Emcare holding medium (EHM), on in vitro development of DCf + DCo embryos were investigated. The blastocyst formation rate (BFR) of the embryos manipulated in EHM (33.3%) was higher (P<0.05) compared with those manipulated in 199H (18.1%). In Experiment 2, DCf + DCo and MCf + DCo embryos were cocultured with or without domestic cat oviductal epithelium cells. Irrespective of coculture, the same BFR was obtained for DCf + DCo embryos (44.4 vs. 38.0%), while MCf + DCo embryos could not develop beyond the morula stage. In experiment 3, although the development of MCf + DCo and DCf + Bo embryos was arrested at the morula stage, 8.6% of MCf + Bo embryos were able to develop to the blastocyst stage. These results demonstrated that EHM was superior to 199H as an embryo manipulation medium and that the DCo and Bo could support the early embryonic development of intergeneric cloned marbled cat embryos up to the morula stage. However, postimplantation development still needs to be investigated.

The present study examined transcription levels of the Oct4, DNMT1, DNMT3a, DNMT3b, HAT1 and HDAC1 genes in cloned felid embryos developing from single one-cell to blastocyst stages. IVF, cloned domestic and leopard cat embryos had low Oct4 and HAT1 levels during the early stages, but transcript expression increased at the eight-cell and blastocyst stages. In contrast, expression in the cloned marble cat embryos was low at all stages. Transcription patterns of HDAC1 were altered in cloned embryos compared with IVF embryos. Transcription levels of DNMT1 decreased markedly throughout development of both IVF and cloned embryos. In IVF embryos, DNMT3a transcripts rarely appeared in the four- to eight-cell stages, but levels increased in the morula to blastocyst stages. In contrast, in cloned embryos, DNMT3a transcript levels were high at the one- to two-cell stages, decreased during subsequent cell division and then increased again at the blastocyst stage. The IVF and cloned embryos showed similar DNMT3b transcription patterns, starting with low levels at the two-cell to morula stages and reaching a maximum at the blastocyst stage. These results suggest that the low level of Oct4 transcripts may be responsible, in part, for the failure of blastocyst production in the cloned marbled cat. However, higher transcription of the DNA methylation genes and lower transcription of the histone acetylation genes were observed in cloned compared with IVF embryos, suggesting that the felids' donor nucleus could not completely reprogramme the nuclear genome and so the re-establishment of embryonic totipotency was not achieved.