Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.
We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.