Occurrence of Cryptosporidium parvum and bacterial pathogens in faecal material in the red fox (Vulpes vulpes) population.
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Animals↗
Biomedical subjects
J R Rao
Publications and source records attributed to J R Rao.
Increased erythromycin resistance in clinical Campylobacter in Northern Ireland--an update.
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Animals↗
Detection of toxoplasmosis in experimentally infected goats by PCR.
PCR was used to diagnose toxoplasmosis in two pairs of Barbari goats infected by oral administration of doses of either 10(4) or 10(5) oocysts of Toxoplasma gondii. Blood and lymph node aspirates were collected from the infected goats and control goat at intervals, and tissues were also collected from a fetus that was aborted and a doe that died during the trial. Both processed and unprocessed samples were used for the PCR, using primers directed to the multicopy B1 gene. None of the blood samples was positive, but a specific signal was obtained from the lymph node aspirates after partial DNA extraction. Direct PCR of the lung, muscle and mesenteric lymph node of the doe and lung tissue of the aborted fetus yielded the target fragment. The simplified PCR protocols, including partial DNA extraction and direct assay of lung tissue, were effective for the diagnosis of toxoplasmosis.
Aborted Fetus↗
Investigation of faecal pathogens in a flock of mute swans (Cygnus olor).
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Animals↗
Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land.
A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material (n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp, Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log10 7.01 colony forming units (cfu) per gram SMC material (range: log10 6.53-7.52 cfu/g). Fungal counts gave a mean count of log(10) 4.57 cfu per gram SMC material (range: log10 3.93-4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe-microbe and plant-microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10(18) cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.
Agaricales↗
In vitro generation of cloned populations of Toxoplasma gondii.
Cloned populations were generated from Indian isolates of Toxoplasma gondii by transferring single tissue cysts from the brains of chronically infected mice to confluent murine macrophage (J774A.1) monolayers. The clones were then maintained continuously as tachyzoites in culture. Physical rupture of the tissue cysts and release of bradyzoites prior to seeding was found to be necessary for establishment of the parasite in culture. Although intact tissue cysts seeded over monolayers released bradyzoites spontaneously, they did not succeed in setting up an infection in the monolayers. Random amplified polymorphic DNA (RAPD)-PCR, which revealed distinct patterns for a clone and its progenitor, further confirmed the efficiency of the technique. The cloning technique was found to be simple and rapid compared to those involving limiting dilutions.
Animals↗
A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata.
A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.
Animals↗
Prevalence of bacterial faecal pathogens in separated and unseparated stored pig slurry.
AIMS: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. METHODS: A total of 43 stored slurry specimens originating from a fattening house over the period February-April 2002 were analysed, consisting of unseparated (n = 14) slurry, separated solids (n = 16) and separated liquid (n = 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. RESULTS: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twenty-nine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51.7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84.6%), furazolidone (38.5%), nalidixic acid (15.4%) and streptomycin (15.4%). The majority (57.7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34.6% of isolates being resistant to three agents and the remainder (7.7%) being resistant to four antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.
Animal Husbandry↗
Biohazards and ecotoxicological considerations of landspreading of spent compost wastes.
Spent mushroom compost (SMC) is a major waste of the mushroom industry with low economic value. SMC arises after mushroom production in phase II compost (pIIC), predominantly comprising straw and chicken litter as principal raw ingredients. The majority of SMC waste is disposed off by application to agricultural land. It is an attractive proposition for utilising SMC as soil inorganic fertiliser supplementation. However, there is limited data available as to the consequences of this method of disposal either in terms of microbiological loading of food-borne pathogens and those of concern to mushroom industry itself. The resulting imbalance of the natural flora of the agricultural land has not been properly audited. This study aims to initially examine SMC for prevalence of faecal bacterial pathogens including Campylobacter spp., Salmonella spp. and Listeria monocytogenes that may arise from chicken litter. At another level, it aims to ascertain the pathogenic bacteria (Pseudomonas syringae, pv phaseolicola or tolasii) and fungal populations (Trichoderma, Verticillium species) originating mainly from the straw component of the SMC, which are of concern to the mushroom industry. Lastly, the study would also qualitatively identify the diversity of bacterial populations within SMC. This was largely accomplished through employment of rDNA, PCR and direct sequencing strategies on the culturable microflora. However, for specific mushroom pathogens, nucleic acids (DNA or RNA) were directly extracted from composts before subjecting to sequence analysis. In accordance with the current legislation (ABP 02/02, Animal By Products wastes disposal EC No. 1774/2002), it is imperative to regulate the farm wastes carrying residues from animal sources including SMC before they are regarded safe for land spreading operations. The ecological microbe-microbe and plant-microbe interactions that potentially occur between the native bacterial soil flora and those added annually (approximately 10(18) cells) needs to be reviewed with caution. The above study highlights the ecological consequences involved in the disposal of SMC wastes on agricultural land and its implications for plant, animal and human health.
Agaricales↗
Biosorption of phenol from an aqueous solution by Aspergillus niger biomass.
Phenols in trace quantities are usually present in the treated effluent of many wastewater-treatment plants. Phenol contamination of drinking water even at 1 microg/l concentration can cause significant taste and odor problems. This study investigates the use of non-viable pretreated cells of Aspergillus niger to remove phenol from an aqueous solution. Five types of non-viable pretreated A. niger biomass powders were used as a biosorbent to remove phenol present in an aqueous solution at a concentration of 1,000 microg/l. Sulfuric acid-treated non-viable biomass powder, which was the most effective, was used as a biosorbent in a further study. The maximum removal of phenol was observed at an initial pH of 5.1 for the sulfuric acid-treated biomass. The adsorption of phenol by pretreated A. niger biomass was best described by the Brunauer Emmet Teller model. Desorption of phenol using distilled deionized water was found to be approximately 5% suggesting a strong biosorption by the biomass. Sulfuric acid-treated biomass beads developed through immobilization in polysulphone were used in a column study. Approximately 66% of phenol was removed in the column operated at an initial pH of 5.1 and an initial concentration of 1,000 microg/l of phenol.
Adsorption↗
Improved molecular identification of Thermoactinomyces spp. associated with mushroom worker's lung by 16S rDNA sequence typing.
Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including Thermoactinomyces spp. It is difficult to distinguish phenotypically the eight species of this genus; therefore, this study sought to develop an improved molecular means of identifying Thermoactinomyces spp. associated with MWL by partial 16S rDNA PCR amplification and direct sequencing. Hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species, were identified and employed with highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicons. A novel 24-mer 16S rDNA oligonucleotide upstream primer was designed from in silico alignments of all Thermoactinomyces spp. and was employed in combination with downstream (reverse) 16S rDNA primers. This permitted the successful identification of all four isolates associated with mushroom workers' lung. The method may be useful in the identification of Thermoactinomyces spp. associated with allergic alveolitis or pneumonitis associated with occupational exposure in agricultural and horticultural environments.
Agricultural Workers' Diseases↗
DNA polymorphism in Trypanosoma evansi isolates defined by randomly amplified polymorphic DNA-PCR.
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Animals↗
Identification of Trypanosoma evansi by DNA hybridisation using a non-radioactive probe generated by arbitrary primer PCR: short communication.
A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer-polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes.
Animals↗
Random amplification of polymorphic DNA fingerprinting of Trypansoma evansi.
The total genomic DNAs from Trypanosoma evansi isolates of bubaline, equine and cameline origin were amplified by polymerase chain reaction using eight arbitrarily selected 10-base primers. Informative band patterns were obtained for all isolates analysed. Depending upon the T. evansi isolate primer combination, between 1 and 11 reproducible DNA fingerprints of 205 to 3016 bp were amplified, suggesting minor and major differences in their RAPD (random amplified polymorphic DNA) profiles.
Animals↗
Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction.
The mechanically transmitted haemoflagellate, Trypanosoma evansi causes 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection of T. evansi is important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed from T. evansi repetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA of T. evansi derived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 microliters crude blood samples. Following experimental infection of calves with 5 x 10(5) T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle, Babesia bigemina and Theileria annulata were not amplified with the primers.
Animals↗
Random amplified polymorphic DNA for the specific detection of bubaline Echinococcus granulosus by hybridization assay.
The polymerase chain reaction (PCR) method to randomly amplify polymorphic DNA (RAPD) was used to differentiate the bubaline and bovine strains of Echinococcus granulosus and buffalo host DNA. Four random oligonucleotide primers of 10-11 mer were analyzed for their ability to direct the amplification of polymorphic DNA fragments from parasites and bovine DNA. Significant DNA polymorphism was observed between the E. granulosus isolates. A selectively amplified DNA fragment 0.9 kilobase (kb) from E. granulosus buffalo isolate by primer AP2 (5'-TGCCGAGCTG-3') was reamplified and used as a DIG-labelled DNA probe. Dot-blot hybridization of total genomic DNA differentiated buffalo E. granulosus isolate from bovine isolate and bubaline host DNA with no detectable cross-hybridization signal.
Animals↗
Effect of immunostimulation on cytotoxic activity of intestinal intraepithelial lymphocytes of chickens in infectious bursal disease and Eimeria tenella infections.
In chickens, intestinal intraepithelial lymphocytes (iIELs) exhibit spontaneous natural killer (NK) cell like activity, by which they are active in the first line of defence on gut epithelial linings. In the present study, the cytotoxic activity of iIELs was found to be drastically suppressed in chickens experimentally infected with infectious bursal disease (IBD) virus at the age of 5 weeks and also in chickens experimentally infected with Eimeria tenella oocysts at the age of 8 weeks (p < 0.01). As nonspecific immunostimulation is gaining importance, immunostimulants such as immunostimulating Mycobacterium phlei (ISMP) and bone marrow culture supernatant (BMCS) were tested for their influence on the functional activity of iIELs of chickens in IBD and E. tenella infections. In chickens primed with ISMP a week prior to respective experimental infections, it was found that the cytotoxic activity of iIELs was restored (p < 0.01) in both IBD and E. tenella infections. At the same time, in chickens primed with BMCS a week prior to respective experimental infections, the cytotoxic activity of iIELs was restored to a certain extent (p < 0.01) in E. tenella but not at all in IBD infection. These results showed that application of immunostimulation helped potentiate and restore the functional activity of iIELs of chickens in IBD and E. tenella infections.
Animals↗
Transmission and protection in leprosy: indications of the role of mucosal immunity.
Recent advances in treatment have achieved a large drop in the prevalence of active leprosy cases, but the incidence is at best decreasing slowly. Most people within leprosy-endemic populations have been exposed to Mycobacterium leprae, but few develop disease and it seems likely that the majority of the population develops protective immunity. If the site of initial infection is in the nose, dissemination of bacilli around the body to skin and nerve implies that the initial infection is bacilliferous and it has been shown that nasal M. leprae are detectable by polymerase chain reaction (PCR) of nasal swabs. Since salivary anti-M. leprae IgA (sMLIgA) levels are correlated with protection, we have surveyed groups of leprosy patients, contacts and the general population for both their sMLIgA and nasal PCR positivity. A total of 304 subjects were enrolled in the study: PCR and mucosal challenge tests were performed in 204 of these individuals. sMLIgA was present in 66% of treated patients, 76% of leprosy workers and 72% of healthy contacts. However, only 33% of indigenous subjects were sMLIgA+, in contrast to the earlier studies showing 74% positivity. PCR for M. leprae was present in both household contacts (2%) and indigenous controls (5%). In a subsequent follow-up study, nasal swabs were taken from 97 of those studied in the first series: three PCR+ individuals followed up after one year became negative, while of the remaining 94 PCR- individuals retested, 2 became positive. Of 112 subjects retested with the mucosal challenge test for sMLIgA: 22 converted from positive to negative and 12 from negative to positive. These results suggest that there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response, despite the fact that few individuals will develop clinical disease. This may explain the current lack of effect of multidrug therapy (MDT) control programmes on incidence, although the reduction in general population immunity is consistent with some effect of MDT on transmission.
Adolescent↗