R.P.V.J. Rajapakse

Prevalence of Cryptosporidium infection in goats in selected locations in three agroclimatic zones of Sri Lanka.

The prevalence of Cryptosporidium oocysts in the faeces of 1020 goats in three age categories was examined during 1999 in selected locations of three agroclimatic zones of Sri Lanka. The oocysts were demonstrated using the Sheathers sucrose flotation method followed by staining with the modified Ziehl Neelsen technique. Cryptosporidium oocysts were detected in animals from all agroclimatic zones with the highest prevalence of infection in the dry zone (33.6%) compared with 24.7 and 21.7% in the intermediate zones and wet, respectively (P<0.001). Overall, Cryptosporidium oocyst counts were significantly higher in goats of <6 months and 7-12 months of age groups compared with goats of >12 months of age (P<0.001). Cryptosporidium oocysts were detected in 291/1020 (28.5%) animals, while 194/1020 animals (19%), 84/1020 animals (8.2%) and 13/1020 animals (1.3%) excreted low (1-1000 oocysts per gram of faeces), moderate (1000-5000 oocysts per gram of faeces) and high (>5000 oocysts per gram of faeces) counts, respectively. The mean Cryptosporidium count was 383 oocysts per gram of faeces. The majority of the infected goats were asymptomatic. These animals are likely to play an important role in the epidemiology of cryptosporidiosis in goat kids and humans.

The complete mitochondrial genome of Setaria digitata (Nematoda: Filarioidea): Mitochondrial gene content, arrangement and composition compared with other nematodes

In the present study, we determined the complete mitochondrial (mt) genome sequence (13,839bp) of parasitic nematode Setaria digitata and its structure and organization compared with Onchocerca volvulus, Dirofilaria immitis and Brugia malayi. The mt genome of S. digitata is slightly larger than the mt genomes of other filarial nematodes. S. digitata mt genome contains 36 genes (12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs) that are typically found in metazoans. This genome contains a high A+T (75.1%) content and low G+C content (24.9%). The mt gene order for S. digitata is the same as those for O. volvulus, D. immitis and B. malayi but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of S. digitata are TTT, ATT, TTG, ATG, GTT and ATA. Interestingly, the initiation codon TTT is unique to S. digitata mt genome and four protein-coding genes use this codon as a translation initiation codon. Five protein-coding genes use TAG as a stop codon whereas three genes use TAA and four genes use T as a termination codon. Out of 64 possible codons, only 57 are used for mitochondrial protein-coding genes of S. digitata. T-rich codons such as TTT (18.9%), GTT (7.9%), TTG (7.8%), TAT (7%), ATT (5.7%), TCT (4.8%) and TTA (4.1%) are used more frequently. This pattern of codon usage reflects the strong bias for T in the mt genome of S. digitata. In conclusion, the present investigation provides new molecular data for future studies of the comparative mitochondrial genomics and systematic of parasitic nematodes of socio-economic importance.

Excretion of Cryptosporidium oocysts by goats in relation to age and season in the dry zone of Sri Lanka

Pattern of Cryptosporidium oocyst shedding in relation to age and season was investigated monthly from May 1999 to April 2000 in three groups (24 goats per group) of naturally infected goats (from 1 month of age). The three groups designated 1, 2 and 3 were studied for 12, 6 and 3 months, respectively. An association between Cryptosporidium oocyst counts and age was demonstrated. In Group 1, oocyst excretion in the first, second and fourth months of age were significantly higher than that in 6, 7, 8, 9 and 12 months of age (p<0.01), whereas in Group 2, oocyst excretion in the first month of age was significantly higher than that in 2, 4, 5 and 6 months of age (p<0.01). The 3-month observations made in Group 3 showed high oocyst excretion during 1 and 3 months of age. The mean maximum count for Group 1 was 592 oocyst per gram of feces when the animals were 2 months old, while in Groups 2 and 3, this was observed at 3 months of age and the oocyst counts were 787 and 520, respectively. A close association between the prevalence of the Cryptosporidium infection and age of the animal was also observed (p<0.01). At least one-third of the Group 1 animals were excreting Cryptosporidium oocysts during the first 5 months of age. Goats excreted Cryptosporidium oocysts irrespective of the dry or rainy season. The long periods of Cryptosporidium oocyst shedding by goats may have implications for the epidemiology of the disease in susceptible hosts.

Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids

An experimental study was carried out in neonatal goat kids to examine the infectivity of Cryptosporidium oocysts, pattern of oocyst shedding and morphological changes in the intestine during the infection. Cryptosporidium oocysts isolated from adult asymptomatic goats, and identified as C. parvum by polymerase chain reaction (PCR) were used in this study. Of three 4-day-old goat kids, two were orally infected with C. parvum oocysts (10(5) oocysts in 10 ml PBS/kid). One goat kid given 10 ml PBS only by the oral route served as a control. Cryptosporidium oocysts were detected in the faeces of one infected kid on day 3 post-inoculation (pi) whereas in the other 6 days pi. The faecal oocyst counts gradually increased and the peak counts in the two kids were 2 x 10(6)g(-1) (on day 12 pi) and 3.2 x 10(6)g(-1) (on day 14 pi). The increase in faecal oocyst output coincided with diarrhoea in an infected kid from days 10-17 pi. Although the oocyst excretion declined gradually after the peak, both infected kids excreted oocysts until euthanized on days 20 and 22 pi. Light and scanning electron microscopic investigations of the ileum revealed the endogenous stages on the brush border of the enterocytes, infiltration of neutrophils and mononuclear cells into the lamina propria, atrophy, stunting and fusion of villi. For purposes of comparison, goat Cryptosporidium oocysts were inoculated orally (10(3) oocysts/mouse) to eight, 1-week-old mice. All experimental mice excreted oocysts from day 3 pi, and four infected mice continued to excrete oocysts up to day 42 pi. The experimental infection described in goat kids resembled the natural disease in terms of oocyst excretion, clinical signs and intestinal pathology. The ability of oocysts excreted by asymptomatic goats, to infect goat kids and mice is likely to have a major impact on the epidemiology of cryptosporidiosis in livestock and man.

Ancient divergence of Paragonimus westermani in Sri Lanka

Adult flukes of Paragonimus species were obtained from lungs of experimental animals infected with metacercariae found in field-collected freshwater crabs in Sri Lanka. Morphological studies of adult worms under a scanning electron microscope as well as a ordinary microscope were performed in the present study. All of morphological features observed clearly indicated that this species is P. westermani. On the other hand, the shapes of metacercariae were found to be mainly oval, but semioval and spherical ones also coexisted. In spite of the variety of their morphology of the metacercariae, there is no correlation between their shapes of metacercariae and the deoxyribonucleic acid (DNA) sequences. Molecular phylogenetic analyses using two DNA regions (partial mitochondrial cytochrome c oxidase subunit 1 and second internal transcribed spacer of the nuclear ribosomal gene repeat) placed the adult flukes of P. westermani from Sri Lanka basal in a tree including all specimens of P. westermani from various areas in Asia and P. siamensis from Thailand. The present study showed that P. westermani from Sri Lanka is an ancestral form.

Toxocara vitulorum: maternal transfer of antibodies from buffalo cows (Bubalis bubalis) to calves and levels of infection with T vitulorum in the calves

The levels of antibody to the excretory/secretory antigens of the infective larvae and adults of Toxocara vitulorum were measured by gel precipitation and ELISAs in the serum and colostrum of 12 buffalo cows naturally infected with T vitulorum and in the serum of their calves. The antibody levels were compared with the extent of T vitulorum infection as judged by faecal egg counts in the calves. The patterns of bands of the larval antigens and gel precipitating antibodies in cow serum taken one month before calving, in cow colostrum and in calf serum were very similar. Nine cows and their calves had gel precipitating antibodies but the remaining three cows and their calves did not. The ELISA detected anti-larval antibodies in the colostrum of all 12 cows and calves. With the exception of one calf there was a strong correlation (r = 0.902) between the antibody titre in cow colostrum and the titre of passively acquired antibody in calf serum. The titres of these passively acquired antibodies declined to their lowest levels in calves 12 to 25 days of age; the antibody concentrations then began to increase up to day 42 and remained stable for the remainder of the experiment (105 days). The titres of antibodies to the antigens of the adult worms, examined in four cows and their calves, were lower than the titres to the larval antigens; the calves absorbed this anti-adult antibody from the colostrum and the antibody levels reached a plateau between days 12 and 30 and remained stable for the rest of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxocara vitulorum (Ascaridida: Nematoda): mitochondrial gene content, arrangement and composition compared with other Toxocara species.

Partial mitochondrial (mt) genome sequence (10,486bp) from the parasitic nematode Toxocara vitulorum was determined and its organization and structure compared with those of T. cati, T. canis and T. malaysiensis. The obtained mt genome sequence of T. vitulorum contains 10 protein-coding genes (cytochrome c oxidase subunits 1-3, Nicotinamide adenine dinucleotide dehydrogenase subunits 1-5, ATP synthase subunit 6 and cytochrome b), 14 transfer RNA genes and the large ribosomal RNA gene (rrnL), non-coding regions. ORF encoding for ATPase subunit 8 is not found in this partial mtDNA sequence. Five translation initiation codons were inferred, ATT, ATG, GTG, GTT and TTG. Most of the genes used TAG or TAA as a stop codon and two genes ended with a T. The gene arrangement and composition of the T. vitulorum mt genome is very similar to that of other Toxocara species mitochondrial genomes sequenced thus far. All genes are transcribed in the same direction, as other Toxocara species. This genome has a high A+T content (67.5%) and low G+C content (32.5%). Phylogenetic reconstruction based on aligned nucleotide sequences of seven taxa provided strong support that Toxocara vitulorum is more closely related to T. malaysiensis than to T. canis and T. cati.

The effect of serum and colostrum immunoglobulins from buffaloes infected withToxocara vitulorum onT. vitulorum larvae in vitro and in vivo in mice

Serum and colostrum were collected from adult buffalo cows naturally infected withToxocara vitulorum. When injected into mice, the colostrum reduced the number of larvae ofT. vitulorum that migrated in the mice. Injection of particularly the IgG-containing fraction but also the IgM-containing fraction of Sephadex G200-chromatographed colostrum also passively protected mice. When incubated for 6 h in buffalo serum or colostrum or fractions of these from Sephadex G200 and diethylaminoethanol Sephadex,T. vitulorum larvae had their activity in vitro curtailed. When the larvae were then fed to mice, their ability to migrate was markedly inhibited as compared with that of larvae that had been incubated in fetal calf serum or in helminthfree sheep colostrum. Fractions of serum and colostrum containing IgG1 had greater inhibitory effects both on the larvae in vitro and on their subsequent migration in mice than did IgG2-containing fractions. IgM also inhibited the larvae as 2-mercaptoethanol reduction of IgM in the IgM-containing peak eluted from Sephadex G200 reduced the inhibitory activity of this peak, although the activity was not completely abrogated.

Antigen-induced protection against infection withToxocara vitulorum larvae in mice

Larvae ofToxocara vitulorum hatched and migrated in the tissues of normal mice. Larvae survived in reasonable numbers, particularly in the liver and, to a lesser extent, in the lungs and kidneys, for at least 4–7 days and in muscles, albeit only in low numbers, for at least 3 weeks. Oral infection of mice on three or more occasions withT. vitulorum eggs induced protection against a challenge infection with eggs ofT. vitulorum. Prior parenteral immunization of mice with a variety ofT. vitulorum soluble antigens (extracts, excretions/secretions, or perienteric fluid and their fractions) from adult parasites and/or infective larvae induced statistically significant protection against infection. The most effective protective immunogens were three or more injections with perienteric fluid from adults (100% protection) and excretions/secretions from infective larvae ofT. vitulorum (>92% protection).

Identities of two Paragonimus species from Sri Lanka inferred from molecular sequences.

Metacercariae of Paragonimus spp. were obtained from field-collected freshwater crabs in Sri Lanka. Genomic DNA was extracted from single metacercariae. Two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified using the polymerase chain reaction. Two differing sequences were obtained for each of these gene regions. Phylogenetic analyses placed the type 1 sequences as sister to a clade containing P. westermani and P. siamensis whereas the type 2 sequences were close to published sequences of P. siamensis from Thailand. The possible taxonomic status of these two types are discussed. This is the first report of molecular data about Paragonimus from Sri Lanka.