PubMed Health⌕ Search

Biomedical subjects

M A Fernando

Publications and source records attributed to M A Fernando.

At least 19 recordsLinked to original sources
PubMed2006

Differential display analysis of gene expression in two immunologically distinct strains of Eimeria maxima.

Gene expression during sporulation and sporozoite excystation of two strains of Eimeria maxima was analyzed using the mRNA differential display technique. The two strains, the Guelph strain (GS) and a single sporocyst-derived strain (M6) from Florida, have been shown to be immunologically distinct. We isolated and cloned a 453-bp complimentary DNA (cDNA) fragment (GS-453) found only in GS sporozoites. In GS, this mRNA begins to be expressed during the earliest stages of oocyst sporulation and is continuously expressed up to and including in the excysted sporozoite. In all Northern blots, digoxigenin (DIG)-labeled GS-453 probe recognized an mRNA of approximately 1.6 kb from GS but not from RNA of M6. Southern blots using various endonucleases and probed with DIG-labeled GS-453 demonstrated that the genomes of both strains contained sufficiently similar sequences to permit hybridization with the probe, but the pattern of hybridization differed between the two strains. Extensive searches of the GenBank, European Molecular Biology Laboratory, and various apicomplexan expressed sequence tag databases using the DNA or inferred amino acid sequences of GS-453 cDNA clone did not identify similarity to any existing sequences.

Animals↗
PubMed2001

A comparison of sporozoite transport after homologous and heterologous challenge in chickens immunized with the Guelph strain or the Florida strain of Eimeria maxima.

The two strains of Eimeria maxima, Guelph and Florida, used in this study were previously shown to only partially cross-protect immunologically with respect to lesion scores, weight gains and feed conversions after heterologous challenge. In this paper, we provide evidence that this partial lack of cross-protection is manifested at the level of sporozoite transport. In birds immunized and challenged with the homologous strain, sporozoites accumulated in the lamina propria and were blocked from further movement into the crypts by 72 h post-challenge, unlike the situation observed in naive birds. Fewer than 5% of sporozoites were found in the crypts by 72 h post-challenge. In immunized birds challenged with the heterologous strain, fewer sporozoites reached the crypts than in naive birds but at least four times as many sporozoites successfully migrated to the crypts, when compared with birds challenged with the homologous strain. The degree of cross-protection afforded by the heterologous strain as measured by sporozoite transport success was not equally reciprocal.

Animals↗
PubMed2001

Involvement of CD 8+ and CD 3+ lymphocytes in the transport of Eimeria necatrix sporozoites within the intestinal mucosa of chickens.

The phenotype of cells transporting sporozoites of Eimeria necatrix during a primary infection was determined using a panel of six monoclonal antibodies to various chicken lymphocyte surface markers. Sporozoites and cells harboring them were examined at 8, 12 and 18 h postinfection using two-color immunoflorescence and confocal microscopy. The majority of parasites observed within lymphocytes were found in CD 8 + (15%) or CD 3+ (13-22%) cells at all time periods examined. Smaller numbers were found within deltagamma TCR+ (5%) and alphabeta TCR+ (5%) lymphocytes. No sporozoites were found within CD 4+ or IgM+ lymphocytes at any of the time periods.

Animals↗
PubMed2000

Viral double-stranded RNAs of Eimeria spp. of the domestic fowl: analysis of genetic relatedness and divergence among various strains.

Genetic relatedness was examined among putative viral double-stranded RNA (dsRNA) genomes of Eimeria acervilina, E. maxima, and E. necatrix using Northern hybridization. No cross-hybridization was found among these dsRNAs, revealing little sequence homology, if any. In addition, dsRNAs from eight E. acervulina strains collected from different localities in the United States were also examined for genetic relatedness. The putative viral dsRNAs from these eight strains, including the Guelph strain, hybridized with one another to varying degrees, indicating that they are related but divergent.

Animals↗
PubMed1999

RNA-dependent RNA polymerase activity associated with virus-like dsRNA in Eimeria maxima and E. necatrix of the domestic fowl.

RNA-dependent RNA polymerase (RDRP) activity was identified in lysates of Eimeria maxima sporozoites and E. necatrix sporozoites and merozoites. Pretreatment of cell lysates with DNase I, RNase A, proteinase K and actinomycin D prior to RDRP assay was employed to characterize RDRP activity. DNase I and actinomycin D had little effect, while proteinase K abolished RDRP activity in both species. RNase A at a concentration of 1 mg/ml also reduced the polymerase activity in E. maxima and E. necatrix sporozoite lysates to 2% and 0%, respectively. Gel electrophoresis of RDRP products revealed that while most migrated at sizes less than 3 kb, a proportion of labelled products of E. necatrix and E. maxima also migrated to the sizes of their respective putative viral genomes. The RDRP products of E. necatrix were shown to be single-stranded by digestion with RNase in both low- and high-salt solutions and by methylmercuric hydroxide treatment. Moreover, the RDRP products of E. necatrix only hybridized to the 5.6-kb dsRNA of E. necatrix but not to the 4.5-kb dsRNAs of E. necatrix or E. maxima.

Animals↗
PubMed1999

RNA-dependent RNA polymerase activity associated with Eimeria necatrix virus particles containing either double-stranded or single-stranded RNA.

Single-stranded (ss) RNA containing and double-stranded (ds) RNA containing virus particles of Eimeria necatrix were isolated by centrifugation through a CsCl gradient. RNA from the gradient fractions was identified as single-stranded or double-stranded by probing northern blots with digoxigenin-labeled riboprobes. These probes were generated with SP6 and T7 RNA polymerases from a partial cDNA clone derived from 5.6-kb viral dsRNA of E. necatrix. RNA-dependent RNA polymerase (RDRP) activity was identified in these CsCl-purified virus particles. The polymerase products of the ssRNA particles consisted of dsRNA indicating replicase activity, whereas the polymerase products of the dsRNA particles consisted of ssRNA indicating transcriptase. activity. RNase treatment in high salt solution (0.3 M NaCl) of the pooled RDRP products revealed that the products consisted of both RNase-resistant dsRNA and RNase-sensitive ssRNA. These results show that both replicase and transcriptase activities were present in the purified virus. The digoxigenin-labeled products hybridized to both SP6 and T7 transcripts confirming the presence of both activities.

Animals↗
PubMed1999

A case of eosinophilic meningitis.

A case of eosinophilic meningitis is reported, a condition not previously reported from Sri Lanka. We propose Angiostrongylus cantonesis to be the most likely causative agent in this patient.

Adult↗
PubMed1998

Intracellular localization of viral RNA in Eimeria necatrix of the domestic fowl.

The intracellular localization of viral RNA in three different stages of Eimeria necatrix, namely, the first- and second-generation meronts and the macrogamonts, were examined at the light microscopy level by in situ hybridization. Digoxigenin-labeled riboprobes generated from partial cDNA clones from 5.6-kb and 4.5-kb dsRNA (pzenv1 and pzenv2) were used in this study. Viral RNA was found to be confined to the cytoplasm of the eimerian host; no viral RNA was detected in chicken tissue. The intense hybridization signal observed in the cytoplasm of the immature meronts with the SP6 riboprobe of pzenv1 or the T7 riboprobe of pzenv2 was probably due to their hybridization to positive strands that had been extruded from the virus during transcription. In mature meronts the intensity of the signal is lower, signifying a decrease in transcription activity. Viral replicase activity may thus be synchronized with the growth phase of the eimerian host.

Animals↗
PubMed1998

Analysis of infraspecific variation among five strains of Eimeria maxima from North America.

Two laboratory strains from the eastern shore of Maryland 15 years ago and from an Ontario broiler house 23 years ago and three recent field strains of Eimeria maxima (isolated in Maryland, North Carolina and Florida) were examined for phenotypic and genotypic variation using protein profiles, random amplified polymorphic DNA-PCR analysis and DNA sequences obtained from the internal transcribed spacer regions of the rRNA genes. Staining profiles obtained by one-dimensional SDS-PAGE of sporozoite proteins were identical in all five strains. Using random amplified polymorphic DNA-PCR analysis with high %G-C content decamers as primers, we were able to confirm that the five strains are all E. maxima, but were unable to discern any relationships among them because of the limited number of shared polymorphisms identified. In contrast, cloning and sequencing of the internal transcribed spacer-1, 5.8S rDNA and internal transcribed spacer-2 regions of the rRNA genes provided sufficient sequence information to infer phylogenetic relationships among the strains. Almost all of the infraspecific variation was located in the internal transcribed spacer regions. Only two base changes were identified within the 5.8S rRNA gene. Evolutionary relationships among the strains inferred using parsimony analysis of the aligned internal transcribed spacer sequences were well supported, but the hypothesised relationships did not correlate well with the demonstrated immunological cross-reactivities of these strains.

Animals↗
PubMed1997

Isoenzymes of Eimeria from the domestic fowl: electrophoretic variants among species, strains and clones.

Electrophoretic variation of enzymes in five Eimeria spp. of the domestic fowl, including nine strains, ten single-sporocyst clones and two single-sporozoite clones of E. acervulina, three strains each of E. maxima and E. tenella, two strains of E. praecox and one strain of E. necatrix, were assayed using cellulose acetate electrophoresis. Ten enzymes [aldehyde oxidase (AO), alkaline phosphatase (ALP), amylase (AMY), fumarate hydratase (FUM), glucose-6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase (GPI), glutamate-oxaloacetate transferase (GOT), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and phosphoglucomutase (PGM)] were analyzed for their ability to distinguish between these species and strains. Enzymatic activity of G6PDH, GPI, IDH, MDH and PGM was detected in all the Eimeria spp. examined. Strains within each species were characterized by the same electrophoretic variant of G6PDH. Electrophoretic variants of GPI and PGM were the most valuable in the identification of inter- and intra-specific variation, particularly in the field strains of E. acervulina and E. tenella. These two enzymes were used to examine single-sporocyst and single-sporozoite clones derived from two strains of E. acervulina. The enzymes in E. maxima appeared to be conserved, showing no variation among strains with the five enzymes detected. Relative mobilities, calculated as described in this paper, were found to be consistent between different electrophoresis runs and may serve as a reference when this medium is used.

Animals↗
PubMed1997

Analysis of immunological cross-protection and sensitivities to anticoccidial drugs among five geographical and temporal strains of Eimeria maxima.

Two laboratory strains (USDA strain No. 68 isolated from the eastern shore of Maryland 15 years ago and a University of Guelph strain isolated from an Ontario broiler house 23 years ago) and 3 recent field strains of Eimeria maxima [isolated in Maryland (MD), North Carolina (NC) and Florida (FL)] were tested for their ability to induce cross-protective immunity and their sensitivities to a variety of anticoccidial compounds. To assess immunological cross-protection, 1-day-old chicks were inoculated and subsequently challenged at 10 days of age, testing all possible combinations of initial inoculating (immunizing) and subsequent challenge strain. Six days post-challenge, chicks were killed and weight gains and lesion scores were determined and compared to sham inoculated and challenged, and sham challenged age-matched controls. The 2 laboratory strains and the NC strain were fully cross-protective against each other by both these measures. In contrast, the MD and FL strains induced complete protection only against the homologous strain. Reciprocally, no other strains protected chicks completely against the FL and MD strains. Drug sensitivity studies using 10 different anticoccidial formulations at prescribed drug levels showed significant differences between the 2 laboratory strains and the 3 recently isolated field strains; more recent isolates from commercial broiler houses demonstrated complete or partial resistance to a wider range of anticoccidial compounds. No correlation was seen between cross-protection and sensitivities to anticoccidials.

Animals↗
PubMed1997

Ventilatory function in brass workers of Gadaladeniya, Sri Lanka.

A cross sectional study was conducted to determine the respiratory hazards of brass workers. The study group was selected randomly. The control group was selected from the general population matched for age by cluster sampling. There was a total of 154 pairs for the final analysis. A questionnaire was administered to determine the prevalence of respiratory symptoms. Forced vital capacity (FVC), forced expiratory volume in the first second (FEV1.0), forced expiratory flow rate in the mid 50% of the FVC(FEF25%-75%) and peak expiratory flow rate(PEFR) were measured. Chest radiography was performed on those with 5 or more years of service. Cough, phlegm, chronic bronchitis and dyspnoea were significantly higher among brass workers. The ventilatory capacity was significantly lower in all the indicators except FVC. Smoking had no significant effect and a dose response relationship could not be demonstrated after inclusion of age in the regression model. Five point five per cent had evidence of septal lines while 6.4% had emphysema.

Adolescent↗
PubMed1996

dsRNA associated with virus-like particles in Eimeria spp. of the domestic fowl.

RNA segments, identified as double-stranded, were found in sporozoites of the Guelph strains of Eimeria acervulina, E. brunetti, E. maxima and E. necatrix and in 8 of 11 strains of E. acervulina obtained from poultry houses across the United States. These RNAs were resistant to RNase A digestion in the presence of high salt concentrations (0.3 M NaCl). On agarose-gel electrophoresis, E. acervulina had one obvious band at 1.7 kb and a faint band at 3.5 kb; E. brunetti had two bands at 2.1 and 3.3 kb, respectively; E. maxima had one band at 4.5 kb; and E. necatrix had two major bands at 4.5 and 5.6 kb, respectively. No dsRNA band was seen in the three strains of E. tenella examined. Virus-like particles were purified by cesium chloride density centrifugation of homogenates of E. necatrix sporulated oocysts. The fraction at peak virus concentration had a buoyant density of 1.39 g ml-1. These virus-like particles were icosahedral, had no envelope and measured 42-44 nm in diameter. Only one RNA band at 5.6 kb was observed when nucleic acids from gradient fractions containing virus were subjected to electrophoresis. The 4.5-kb dsRNA segment of E. necatrix was not associated with a virus-like particle.

Animals↗
PubMed1996

Effects of the intestinal flagellate, Cochlosoma anatis, on intestinal mucosal morphology and disaccharidase activity in Muscovy ducklings.

Newly hatched female Muscovy ducklings were randomly separated into 2 groups of 12 and 1 group of 13. Ducklings in the first 2 groups were each orally inoculated with 0.5 ml of sterile normal saline containing 0 and 3 x 10(6) trophozoites of Cochlosoma anatis, respectively. Birds in the third group were each orally inoculated with 3 x 10(6) trophozoites for 5 consecutive days. Birds were weighed daily for the first 5 days and then on days 7, 14 and 21 post-inoculation (p.i.). On days 6, 7, 8, 13, 14, 15, 20, 21 and 22 p.i., 1 bird from each group was killed and samples of intestine at 7 levels were taken for trophozoite counts, mucosal disaccharidase analyses and morphometric analysis. Body weights did not differ among treatment groups at any time during the experiment. Trophozoite numbers did not change over the period 6-22 days p.i. Trophozoite numbers were lowest in the anterior small intestine and increased distally, but very few were observed in the caecum. Crypt depth was greater in all regions of the small intestine in inoculated groups compared to uninoculated controls, and was significantly increased in the duodenum, proximal jejunum and mid-jejunum (P < 0.05). Villus height was greater in inoculated groups compared to controls at all levels of the intestine and was significantly increased in the duodenum, proximal jejunum and ileum (P < 0.05). Mucosal palatinase and maltase activity in the small intestine were reduced in inoculated groups compared to uninoculated controls; palatinase activities were significantly reduced in the proximal and mid-jejunum and maltase activities were significantly reduced in the mid-jejunum (P < 0.05). Sucrase activities were significantly increased at all levels of the small intestine in inoculated ducklings compared to uninoculated controls (P < 0.05). Although no clinical signs were evident, Cochlosoma infection significantly altered intestinal morphometrics and mucosal enzyme concentrations in ducklings, in several cases in a counter-intuitive direction.

Animals↗
PubMed1995

Eimeria spp. of the domestic fowl: analysis of genetic variability between species and strains using DNA polymorphisms amplified by arbitrary primers and denaturing gradient-gel electrophoresis.

The genetic relatedness of 5 Eimeria spp. of the domestic fowl, including 11 strains of E. acervulina, 2 strains of E. tenella and 1 precocious line of E. acervulina, was assayed by means of random amplified polymorphic DNA (RAPD) and denaturing gradient-gel electrophoresis (DGGE). Seven different oligonucleotides were used to generate similarity coefficients for the species and strains of Eimeria infecting chickens. Between 1 and 13 DNA segments, depending on the species/strain-primer combination, were amplified with the various primers. Amplification products ranged in size from 0.16 to 3.8 kb. E. acervulina strains demonstrated two to four major common bands unique to the species. These strains also exhibited major and minor differences in their DNA patterns. Band-match analyses from both polyacrylamide and denaturing gradient gels were used to calculate similarity coefficients for the Eimeria spp. and strains tested. Species differences, readily detected upon examination of DNA banding patterns, gave similarity coefficients of 4%-38% and 3%-18% when analyzed by polyacrylamide- and denaturing gradient-gel electrophoresis, respectively. A similar analysis of E. acervulina strains, yielded similarity coefficients of 55%-95% and 51%-85%, respectively. The differences observed between both species and strains were greater when the RAPD-assay products were analyzed via DGGE, indicating that a combination of these two techniques may provide a more stringent analysis of the genetic relatedness of these coccidia.

Animals↗