Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction.

Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level.

Lymphocyte subsets in the small intestine of piglets fed with probiotic and zinc: a qualitative and quantitative micro-anatomical study.

BACKGROUND: Piglet mortality is a real concern to the pig farmers. The major cause is due to the late maturation of the immune system and dietary changes in postweaned piglets. The potential role of probiotic and zinc in the stimulation of the immune system is well established. Hence, the present study was undertaken to evaluate alterations of T and B cells in the small intestine after dietary inclusion of probiotic and zinc in pre and post-weaned piglets. MATERIALS AND METHODS: A total of 18 healthy Large White Yorkshire (LWY) piglets, irrespective of sex obtained from 3 litters at the age-group of 20, 30 and 60 days. They were divided into a control group fed with basal diet and a treatment group fed with probiotic and zinc supplement along with the basal diet, consisting of three animals in each group. The piglets were weaned at 28 days of age. After sacrificing the animals at day 20, 30 and 60 from both the groups, the abdominal cavity was opened and small intestinal tissue samples were collected, processed and stained by indirect immunofluorescence technique. The slides were evaluated under the fluorescent light microscope. The data were statistically analysed. RESULTS: The different T and B cell subsets were recorded in the lining epithelium, core of villus, crypt area of lamina propria and Peyer's patch area. The number of CD4+, CD8+, IgA+ and IgM+ cells was higher in the treated piglets than the control group of animals, irrespective of segments of intestine and age-group. CONCLUSIONS: It can be concluded that the dietary supplementation of probiotic and zinc was found to be good additives as they can stimulate the immune response in piglets, especially during the critical early post-weaning period.

Multidrug resistant staphylococci isolated from pigs with exudative epidermitis in North eastern Region of India.

Exudative epidermatitis or greasy pig disease (GPD) is a contagious disease of pig and endemic worldwide caused by toxigenic strains under genus Staphylococcus. The present study reported an outbreak of GPD in Champhai district of Mizoram adjoining to the southern border of Myanmar. A total of 60 samples were collected from 22 clinically affected animals and processed for isolation and identification of Staphylococcus spp. All the isolates were subjected to antimicrobial sensitivity assay, biofilm production assay and detection of virulence genes, biofilm genes and mec genes followed by cloning and sequencing for phylogenetic analysis. A total of 44 staphylococci belonged to four species (S. sciuri, S. aureus,S. lentus, and S. hyicus) were isolated. Majority of the isolates were multidrug resistant with maximum resistance against ampicillin, penicillin including vancomycin. None of the S. hyicus isolates was methicillin resistant (MRSH) but 66·67% isolates were MRSA. By PCR, mecA gene was detected in S. aureus (n = 2), S. sciuri (n = 4) and S. lentus (n = 3). Biofilm associated gene icaD was detected in S. aureus (n = 3), S. sciuri (n = 5), S. hyicus (n = 4) and S. lentus (n = 6). The exfoliative toxin genes (ehxB, shetA and tsst1) were detected in S. hyicus (n = 3) and S. aureus (n = 1) isolates. All the isolates were closely related with the isolates from pigs of China, Germany, Japan and USA. The pathogens might be transmitted through illegal migration of pigs from Myanmar to India.

Small intestinal mucosal cells in piglets fed with probiotic and zinc: a qualitative and quantitative microanatomical study.

BACKGROUND: Probiotics and zinc are commonly used and beneficial in pig production. This work aimed to assess the effects of probiotic and zinc on the mucosal cells of the small intestine in respect to digestive capacity and immunity in pre- and post-weaned piglets. MATERIALS AND METHODS: Eighteen Large White Yorkshire piglets were divided equally into control and treatment groups. The piglets were maintained in standard management conditions and were weaned at 28 days of age. The treatment group of piglets fed a mixture of probiotics orally at 1.25 × 109 CFU/day and zinc at 2000 ppm/day from birth to 10 days of age. At three different age-groups viz. day 20 (pre-weaning) and, day 30 and day 60 (post-weaning), the animals were sacrificed. For histomorphology, the tissue samples were processed and stained with Mayer's haematoxylin and eosin for routine study, combined periodic acid-Schiff-Alcian blue for mucopolysaccharides and Masson-Hamperl argentaffin technique for argentaffin cells. The stained slides were observed under the microscope. The samples were processed as per the standard procedure for scanning and transmission electron microscopy. The statistical analysis of the data using the appropriate statistical tests was also conducted. RESULTS: The mucosal epithelium of villi and crypts were lined by enterocytes, goblet cells, argentaffin cells, microfold (M-cell) cells, tuft cells and intraepithelial lymphocytes. The multipotent stem cells were located at the crypt base. The length of the enterocyte microvilli was significantly longer (p < 0.05) in the treatment group of piglets. The number of different types of goblet cells and argentaffin cells was more in treated piglets irrespective of segments of intestine and age. The intraepithelial lymphocytes were located in apical, nuclear and basal positions in the lining epithelium of both villus tip and base with their significant increase in the treatment group of piglets. The transmission electron microscopy revealed the frequent occurrence of tuft cells in the lining mucosa of the small intestine in treated piglets. CONCLUSIONS: Dietary supplementation of probiotic and zinc induced the number of different mucosal cells of villi and crypts in the small intestine that might suggest the greater absorptive capacity of nutrients and effective immunity in critical pre and post-weaned piglets.

Higher prevalence of multidrug-resistant extended-spectrum ß-lactamases producing Escherichia coli in unorganized pig farms compared to organized pig farms in Mizoram, India.

AIM: The present study was conducted to record the prevalence of multidrug-resistant (MDR), extended-spectrum ß-lactamases (ESBLs) producing Escherichia coli from pig population of organized and unorganized farms of Mizoram and to record the presence of ESBLs, non-ESBLs, and integrons. MATERIALS AND METHODS: Fecal samples were collected from pigs under organized (n=40) and unorganized (n=58) farms of Mizoram. Samples were processed for isolation and identification of E. coli by conventional techniques, BD Phoenix™ automated bacterial system, and polymerase chain reaction (PCR) based confirmatory test. All the isolates were subjected to antimicrobial sensitivity test by disk diffusion assay and ESBLs production by double-disk synergy test (DDST). The ESBLs producing isolates were subjected to PCR for determination of ESBLs genes and all the isolates were screened for non-ESBLs genes and integrons by PCR. RESULTS: A total of 258 E. coli was isolated and identified from organized (n=120) and unorganized farms (n=138). Majority of the E. coli isolates exhibited high level of resistance against amoxicillin (Ax) (81.78%), cefalexin (85.42%), co-trimoxazole (50.78%), sulfafurazole (69.38%), tetracycline (65.89%), and trimethoprim (TR) (51.94%). Statistically highly significant (p<0.01) variations in resistance among the isolates from organized and unorganized farms were recorded in case of Ax, ampicillin, cephalexin, ciprofloxacin, co-trimoxazole, gentamicin, piperacillin, and TR. By DDST, 65.89% isolates were recorded as ESBLs producer, of which 82/120 (68.33%) and 88/138 (63.77%) were from organized and unorganized farms, respectively. A total of 29/258 (11.24%) isolates were positive for at least one ESBLs gene. blaTEM was most frequently (9.69%) gene, followed by blaCTX -M (5.04%) and blaCMY (0.78%). Altogether, 6 (5.00%), 4 (3.33%), and 2 (1.67%) isolates from the organized farms were positive for blaCTX-M , blaTEM , and blaCMY genes, respectively. Similarly, 21 (15.22%) and 7 (5.07%) isolates from the unorganized farms were positive for blaTEM and blaCTX-M genes, respectively. None of them were positive for blaSHV genes. Altogether 57 (22.09%), 9 (3.49%), 66 (25.58%), 78 (30.23%), 21 (8.14%), and 18 (6.98%) isolates were positive for tetA, tetB, sul1, sul2, aadA, and dfrla genes, respectively. The prevalence of non-ESBLs genes was higher in the E. coli isolates from the unorganized farms than organized farms. CONCLUSION: MDR and ESBLs producing E. coli are circulating among the pigs and their environment in Mizoram. Pigs under unorganized farms exhibited higher level of resistance against majority of the antimicrobials, including third-generation cephalosporins, which might be an indication of overuse or misuse of antibiotics under the unorganized piggery sectors in Mizoram.

Prevalence and molecular characterization of Salmonella species associated with piglet diarrhea in North East India.

Salmonellosis is a public health concern worldwide and also causes huge losses to the piggery industry. A total of 457 fecal samples were collected from organized and unorganized farms including indigenous and crossbreed piglets of North East India. Salmonella isolates were serotyped, screened for their virulence genes, characterized for drug resistance pattern and representative isolates were cloned and sequenced for their partial length enterotoxin (stn) gene. A total of 8.31% Salmonella were identified with higher prevalence observed in unorganized compared to organized farms and higher detection level in cross breed compared to indigenous piglets. Salmonella typhimurium (65.78%) was found to be the predominant serovar and irrespective of serovars high number of isolates (68.4%) harboured enterotoxin gene. The isolates were multidrug resistant showing highest resistance against cefalexin (77.31%). Sequence analysis of stn gene showed two isolates having diverse sequence compared to other isolates. Our study revealed the significance of Salmonella as important pathogen with zoonotic potential between porcine and human populations. This is probably the first systematic study of Salmonella species associated with piglet diarrhea in India.

Extended spectrum beta-lactamase producing Shiga-toxin producing-Escherichia coli in piglets, humans and water sources in North East region of India.

Extended spectrum ß-lactamases (ESBL) producing Shiga toxin producing E. coli (STEC) are posing a constant threat to public health throughout the world leading to serious infections and raising key therapeutic issues. A total of 219 fecal samples were collected from piglets with diarrheoa, pig farmers and water sources in North East India; and were processed for isolation of Escherichia coli. The isolates were screened for antimicrobial resistance and suspected isolates for ESBLs production by double-disk synergy test (DDST). Escherichia coli isolates positive for DDST were subjected for detection of selected ESBL/beta-lactamase genes and virulence associated genes by PCR. By DDST, 337 (67·94%) E. coli isolates were detected as ESBLs producer, of which 211 (66·98%), 117 (70·91%) and 9 (56·25%) isolates were from piglets, humans and water sources respectively. A total of 64 (12·90%) isolates were recorded as STEC, of which 48 (9·68%), 6 (1·21%) and 10 (2·02%) were from human, piglets and water respectively. Majority of the STEC isolates (64·06%) possessed multiple virulence genes, of which 59·38% also harboured ESBL/beta-lactamase genes with 32·81% STEC isolates being positive for multiple ESBL/beta-lactamase genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Multidrug resistant (MDR) enteric bacteria are global concern. Association of MDR traits in STEC isolates are another rising issue in human and animal health perspective. The interaction of such organisms among the human, domestic animals and adjoining water sources require to be analysed systematically. The present study exhibited the possible transmission of MDR-STEC among the human, domestic animals and water sources in the North eastern states of India. To the best of our knowledge, this is the first report of such kind in India.

Extended-spectrum ß-lactamases producing multidrug resistance Escherichia coli, Salmonella and Klebsiella pneumoniae in pig population of Assam and Meghalaya, India.

AIM: The present study was conducted to record the prevalence of extended spectrum ß-lactamases (ESBLs) producing Escherichia coli, Salmonella spp., and Klebsiella pneumoniae from pig population of Assam and Meghalaya and to record the ability of the resistant bacteria to transfer the resistance genes horizontally. MATERIALS AND METHODS: Fecal samples (n=228), collected from pigs of Assam (n=99) and Meghalaya (n=129), were processed for isolation and identification of E. coli and Salmonella spp. All the isolates were tested for ESBLs production by double disc synergy test (DDST) followed by screening for ESBLs producing genes (blaTEM, blaSHV, blaCTX-M, and blaCMY) by polymerase chain reaction (PCR). Possible transfer of resistance encoding genes between enteric bacterial species was carried out by in vitro and in vivo horizontal gene transfer (HGT) method. RESULTS: A total of 897 enteric bacteria (867 E. coli and 30 Salmonella) were isolated and identified. Altogether 25.41% isolates were confirmed as ESBL producers by DDST method. Majority of the isolates were E. coli followed by Salmonella. By PCR, 9.03% isolates were found positive for at least one of the target resistance genes. blaSHV was absent in all the isolates. blaCMY was the most prevalent gene. All the E. coli isolates from Assam were negative for blaTEM. A total of 2.76% isolates were positive for blaTEM + blaCMY. On the other hand, 0.67% isolates were positive for blaCTX-M + blaCMY genes. Only 0.33% isolates carried all the three genes. Altogether, 4.68% bacteria carried the resistance encoding genes in their plasmids. blaTEM gene could be successfully transferred from Salmonella (donor) to E. coli (recipient) by in vitro (5.5-5.7×10-5) and in vivo (6.5×10-5 to 8.8×10-4) methods. In vivo method was more effective than in vitro in the transfer of resistance genes. CONCLUSION: The pig population of Assam and Meghalaya are carrying multidrug resistance and ESBLs producing E. coli and Salmonella. The isolates are also capable to transfer their resistance trait to other bacterial species by HGT. The present finding could be considered as a serious public health concern as similar trait can also be transmitted to the human commensal bacteria as well as pathogens.

Prevalence and molecular characterization of porcine Picobirnavirus in piglets of North East Region of India.

Picobirnaviruses (PBVs) have been recognized as one of the important causal viral agents of gastroenteritis in several animal species especially in young immunocompromised hosts. In this study, we report the prevalence and molecular epidemiology of porcine PBVs from North Eastern Hilly region of India. A total of 457 fecal samples from piglets were collected from local (n = 130) and cross (n = 327) breed piglets in different seasons for 2 years. All the samples were subjected to RNA-PAGE and RT-PCR analysis for detection of PBVs. A total of 4.59 and 11.15% samples were recorded as positive for PBVs by RNA-PAGE and RT-PCR, respectively. Rate of detection was higher from diarrhoeic animals (13.56%) compared to non-diarrhoeic (4.23%) animals. Higher prevalence rate was observed from unorganized farms (14.22%) compared to organized farms (8.0%) with slightly higher detection from cross breed (11.62%) compared to local breed (10.0%). Maximum cases of piglet diarrhea associated with PBVs were detected during summer (16.4%) and winter (14.39%) seasons compared to autumn (4.80%) and spring (6.45%). All the samples were positive for PBV genogroup I only. Based upon the sequence analysis, the isolates were unique and placed in separate clad and were not closely associated with any other Indian isolates of PBVs so far. Two isolates were closely related with one Chinese isolate recovered from sewage water. This is the first systematic study of prevalence of PBVs associated with piglet diarrhea.

Evaluation of specific humoral immune response in pigs vaccinated with cell culture adapted classical swine fever vaccine.

AIM: To determine an efficient vaccination schedule on the basis of the humoral immune response of cell culture adapted live classical swine fever virus (CSFV) vaccinated pigs and maternally derived antibody (MDA) in piglets of vaccinated sows. MATERIALS AND METHODS: A cell culture adapted live CSFV vaccine was subjected to different vaccination schedule in the present study. Serum samples were collected before vaccination (day 0) and 7, 14, 28, 42, 56, 180, 194, 208, 270, 284 and 298 days after vaccination and were analyzed by liquid phase blocking enzyme-linked immunosorbent assay. Moreover, MDA titre was detected in the serum of piglets at 21 and 42 days of age after farrowing of the vaccinated sows. RESULTS: On 28 days after vaccination, serum samples of 83.33% vaccinated pigs showed the desirable level of antibody titer (log10 1.50 at 1:32 dilution), whereas 100% animals showed log10 1.50 at 1:32 dilution after 42 days of vaccination. Animals received a booster dose at 28 and 180 days post vaccination showed stable high-level antibody titre till the end of the study period. Further, piglets born from pigs vaccinated 1 month after conception showed the desirable level of MDA up to 42 days of age. CONCLUSION: CSF causes major losses in pig industry. Lapinised vaccines against CSFV are used routinely in endemic countries. In the present study, a cell culture adapted live attenuated vaccine has been evaluated. Based on the level of humoral immune response of vaccinated pigs and MDA titer in piglets born from immunized sows, it may be concluded that the more effective vaccination schedule for prevention of CSF is primary vaccination at 2 months of age followed by booster vaccination at 28 and 180 days post primary vaccination and at 1 month of gestation.

Predominance of genotype 1.1 and emergence of genotype 2.2 classical swine fever viruses in north-eastern region of India.

Classical swine fever (CSF) is a highly contagious and the most important disease of pigs worldwide.CSF is enzootic in pig herds in India and continues to cause huge economic losses to pig farmers. Nearly 40% of the total pig population of India is present in the north-eastern (NE) states where pig husbandry plays an important role in the socio-economic development. Pigs reared in the backyards are the only source of livelihood for a majority of poor tribal population in the region. Hardly any CSF vaccination is currently being undertaken in the unorganized pig farming in the NE region due to economic reasons and vaccine unavailability. A thorough understanding of the current epidemiological status of CSF is essential for the effective control of the disease in the NE region. Hence, we carried out molecular characterization of CSFV isolates from field outbreaks during 2011-2012 in the entire north-eastern region of India to establish the genetic groups of prevalent CSF viruses in the region. A total of 17 CSFV isolates obtained from different parts of the NE region were characterized by comparing the sequences of three partial genomic regions of the virus, that is 150 nt of 5' UTR, 190 nt of E2 and 409 nt of NS5B. Of the 17 CSFV isolates, 15 isolates belonged to 1.1 (88.2%) and two isolates (11.8%) belonged to 2.2 subgenogroup. The genogroup 2.2 CSFV were associated with outbreaks in Arunachal Pradesh that shares international borders with Bhutan, Myanmar and China. Genogroup 2.2 CSFV isolated in the present study shared high level of sequence similarity with 2.2 viruses form China, raising the possibility of virus incursion from this region. In summary, we found a continued predominance of 1.1 subgroup and an emergence of 2.2 subgroup CSFV in NE region of India.

Detection & characterization of Shiga toxin producing Escherichia coli (STEC) & enteropathogenic Escherichia coli (EPEC) in poultry birds with diarrhoea.

BACKGROUND & OBJECTIVES: Limited information is available on shiga toxin producing Escherichia coli (STEC) in animals and birds from India. An outbreak of acute diarrhoea in poultry birds at Aizawl, Mizoram was investigated for detection and characterization of STEC and enteropathogenic E. coli (EPEC). METHODS: E. coli was isolated and identified from rectal swabs, intestinal contents, heart blood and spleen of 19 poultry birds that died due to acute diarrhoea during the outbreak. Phenotypic characterization was done by standard bacteriological and biochemical techniques. All the isolates were serotyped based on their somatic antigens. Virulence genes (stx 1, stx 2, eaeA and hlyA) were detected by multiplex PCR assay. RESULTS: A total of 42 E. coli isolates were obtained, of which 24 belonged to 3 serogroups (O64, O89 and O91) and the remaining 18 were untypable (UT). Altogether, 14 (33.33%) isolates carried at least 1 virulence gene, of which 10 (23.81%) and 4 (9.52%) were recorded as STEC and EPEC, respectively. Of the 10 STEC isolates, one carried only stx2 , one carried stx 2 and hlyA, four carried stx1 , stx2 and hlyA, two carried stx 1, eaeA and hlyA genes and two carried stx 1 and eaeA. Of the four EPEC isolates, two carried eaeA and hlyA, one carried only eaeA gene and 1 carried only hlyA gene. INTERPRETATION & CONCLUSIONS: This is the first report on the involvement of STEC in poultry in India.

Molecular Characterization of Classical swine fever virus Involved in the Outbreak in Mizoram.

Classical swine fever is the most insidious and devastating disease of pigs and wild boars. The virus is closely related to the other members of the genus Pestivirus. The outbreak recorded in Mizoram, India was strategically important as the state shares porous international boundary with East Asian countries. Both immunodiagnostic and molecular techniques were used to confirm the involvement of Classical swine fever virus (CSFV) in this outbreak. Sandwich ELISA and direct FAT could detect CSFV in the tissue samples. RT-nPCR specifically amplified E2 and 5'NTR product of 271 bp. Phylogenetic analysis showed, that the Mizoram isolate (MZ4/69) was very close to the Chinese strain Shimen-HVRI (93.0%) rather than other Indian isolate (CSF-30-03). Present study provides a valuable sequence based molecular data on Indian isolate of CSFV and will be useful in investigation on transmission of such disease from neighbour countries.

Purification of dibenzothiophene monooxygenase from a recombinant Escherichia coli.

Dibenzothiophene monooxygenase is the first enzyme involved in the degradation of dibenzothiophene. This gene was expressed via the pET28a vector in E. coli and was purified in a single step using affinity chromatography. The protein was purified 39-fold with a specific activity of 38 U/mg.

Biotechnology of desulfurization of diesel: prospects and challenges.

To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to make a huge investment to bring down the sulfur content in diesel to the desired level, using conventional hydrodesulfurization (HDS) technology, by which sulfur is catalytically converted to hydrogen sulfide in the presence of hydrogen. These reactions proceed rapidly only at high temperature and pressure and therefore the capital cost as well as the operating cost associated with HDS very high. Biological desulfurization has the potential of being developed as a viable technology downstream of classical HDS. Various attempts have been made to develop biotechnological processes based on microbiological desulfurization employing aerobic and anaerobic bacteria. However, there are several bottlenecks limiting commercialization of the process. This review discusses various aspects of microbial desulfurization and the progress made towards its commercialization.

A potential resource for bioconversion of domestic wastewater sludge.

Twenty seven filamentous fungal strains representing five genera; Aspergillus, Penicillium, Trichoderma, Myriodontium and Pleurotus were isolated from four sources; domestic wastewater sludge cake (SC) from IWK (Indah Water Konsortium) wastewater treatment plant, palm oil mill effluent compost from Sri Ulu palm Oil Processing Mill, compost of plant debris, and fungal fruiting bodies from a rotten wood stump. Thirty-three strains/isolates were tested for their ability to convert domestic wastewater sludge into compost by assessing biomass production and growth rate on sludge enriched media. The strains/isolates Aspergillus niger, SS-T2008, WW-P1003 and RW-P1 512 produced the highest dry biomass at higher sludge supplemented culture media from their respective group (Aspergillus, Trichoderma, Penicillium and Basidiomycetes, respectively). This implied these strains are better adapted for growth at higher sludge rich substances, and subsequently may be efficient in bioconversion/biodegradation of sludge. The fungi isolated from ecological closely related sources were more amendable to adaptation in a sludge rich culture media.

Treatment of wastewater sludge by liquid state bioconversion process.

This study was conducted to evaluate the effect of an eminent decay fungus, Phanerocheate chrysosporium of organic residues on wastewater sludge for its improvement through decomposition and separation of waste particles by Liquid State Bioconversion (LSB). The effect of fungal treatment was compared to uninoculated (Control) at three different harvests 7, 14 and 21 days after inoculation (DAI). The observed results showed that the weight loss and solid content of wastewater sludge were significantly influenced by Phanerocheate chrysosporium. Both parameters were highly influenced at 7 DAI. The COD and pH of wastewater sludge were also highly influenced by fungal treatment.

Biodesulphurization of coal: mechanism and rate limiting factors.

The pyrite sulphur removal from coal by Thiobacillus ferrooxidans was studied in batch reactor. A combination of SEM, IR and XRD was used to study the presence of superficial phases and the changes in solid surface during biodesulphurization. Biodesulphurization was found to be a three-step process. In the first step (0-4 days), direct oxidation of pyrite by bacteria brought about 28% pyritic sulphur removal. Both direct and indirect oxidation contributed to the second step (4-10 days) resulting in 51% pyrite removal. The deposition of elemental sulphur, jarosite and ferric sulphate precipitates in the third step reduced the pyrite availability and ferric iron concentration in the leachate and brought the process of biodesulphurization to an end.