Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic wer... more Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic were screened for Cryptosporidium by microscopy and PCR/sequencing at loci coding small-subunit rRNA, Cryptosporidium oocyst wall protein, actin and 70 kDa heat shock protein. Cryptosporidium infections were detected in 74 voles (22.6%). Rates of infection did not differ between males and females nor between juveniles and adults. Phylogenetic analysis revealed the presence of eight Cryptosporidium species/genotypes including two new species, C. alticolis and C. microti. These species from wild-caught common voles were able to infect common and meadow voles under experimental conditions, with a prepatent period of 3-5 days post-infection (DPI), but they were not infectious for various other rodents or chickens. Meadow voles lost infection earlier than common voles (11-14 vs 13-16 DPI) and had significantly lower infection intensity. Cryptosporidium alticolis infects the anterior small intest...
Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chicke... more Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chickens (Gallus gallus), domestic ducks (Anas platyrhynchos) and ring-necked pheasants (Phasianus colchicus) under experimental conditions. Cryptosporidium avium was not infectious for ring-necked pheasants, but it was infectious for ducks and chickens at all age categories. The course of infection in ducks did not differ among age categories, but 365-day-old chickens had less severe infections than 1- and 21-day-old chickens. The patent period in chickens and ducks was >30 DPI, but ducks started to shed oocysts of C. avium earlier (5-6 DPI) and at a lower intensity (accumulated value of infection intensity of 58,000-65,000 OPG) than chickens (9-11 DPI and accumulated value of infection intensity of 100,000-105,000 OPG). Experimentally infected birds showed no clinical signs of cryptosporidiosis.
The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill tu... more The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill turkey carcasses was investigated. Aerobic plate counts from a 50-cm2 area of the breast sampled by excision and by swabbing were compared. Escherichia coli and Salmonella recoveries were determined from turkeys sampled by a carcass rinse (CR), a modified rinse with the carcass supported in a swing (MCR), a two-site swab of 50 cm2 at the back and thigh (2S), a one-site swab of 50 cm2 beneath the wing (1S), a whole-carcass swab of the inner and outer carcass surface (WS), and excision of 25 g of neck skin tissue (NE). The effect of diluent volume (25, 50, and 100 ml) on E. coli counts from swab samples was also assessed. The aerobic plate count from breast tissue sampled by excision was greater than that by swabbing (P < 0.05). E. coli recoveries by the MCR method were similar to those by CR. E. coli counts from 1S and WS samples were higher when swabs were stomached in 50 rather than 25...
The Science of the total environment, Jan 10, 2016
This study investigated the effects of the alginate and polyvinyl alcohol (PVA) entrapment on the... more This study investigated the effects of the alginate and polyvinyl alcohol (PVA) entrapment on the viability of Escherichia coli cells exposed to single wall carbon nanotubes (SWCNTs) with a diameter of 1-2nm. Viability was examined using a galactosidase enzyme assay, LIVE/DEAD BacLight assay, and total ribonucleic acid quantity. Variables studied included SWCNT concentration (5, 10, 20, 50, 100, 200, 500, and 1000μg/ml), SWCNT length (0.5-2μm for short SWCNTs and 5-30μm for long SWCNTs), and initial bacterial concentration (6.5 log10 CFU and 9 log10 CFU per test). Results showed that both alginate and PVA entrapments mitigate the bactericidal effect of SWCNTs. At the highest SWCNT concentration tested (1000μg/ml), the viability of the cells relative to controls (systems with only E. coli, no SWCNTs), was 0-60% for free cells and 60-90% for alginate and PVA entrapped cells. The bactericidal effect depended on SWCNT type and concentration, and bacterial concentration. In general, long...
Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoe... more Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoeniceus; order Passeriformes) were examined for the presence of Cryptosporidium by PCR amplification and sequence analysis of the 18S ribosomal RNA (18S rRNA), actin, and 70-kDa heat shock protein (HSP70) genes. Twelve birds (17.1Â %) were positive for the Cryptosporidium 18S rRNA gene: six birds were positive at the proventriculus site only and six birds were positive at the proventriculus and intestinal sites. Sequence analysis of the 18S rRNA, actin and HSP70 genes showed the presence of the gastric species Cryptosporidium galli in a single proventriculus sample and a closely related genotype, which we have named Cryptosporidium avian genotype VI, in all other positive samples. These findings contribute to our understanding of Cryptosporidium diversification in passerines, the largest avian order.
A total of 269 faecal samples of various game animals, including 136 red deer (Cervus elaphus Lin... more A total of 269 faecal samples of various game animals, including 136 red deer (Cervus elaphus Linnaeus), 64 European fallow deer (Dama dama [Linnaeus]), 26 white-tailed deer (Odocoileus virginianus [Zimmermann]), and 43 mouflon sheep (Ovis orientalis musimon Pallas) were collected at 15 game preserves across the Czech Republic and examined for infection with species of Cryptosporidium Tyzzer, 1910 using microscopy (following aniline-carbol-methyl violet staining) and molecular tools. Oocysts of Cryptosporidium spp. were detected in one faecal sample originating from red deer. Ten positive cases of infection with cryptosporidia, including the case that was positive by microscopy, were detected using nested PCR. No associations between infection with cryptosporidia and diarrhoea were detected. Phylogenetic analyses based on the small subunit of the rRNA gene revealed the presence of three Cryptosporidium species/genotypes in ten positive samples: Cryptosporidium ubiquitum Fayer, SantĂn et Macarisin, 2010 was identified in five red deer, C. muris Tyzzer, 1907 in three samples (from a red deer, white-tailed deer and mouflon sheep), and Cryptosporidium deer genotype in two white-tailed deer. Subtyping of isolates of C. ubiquitum based on sequence analysis of the 60-kDa glycoprotein gene revealed that they belong to the XIId family. Finding C. muris and C. ubiquitum XIId for the first time in various wild cervids and caprines broadens their host range.
The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causi... more The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The
A combined enricher reactor (ER)-permeable reactive biobarrier (PRBB) .system was developed to tr... more A combined enricher reactor (ER)-permeable reactive biobarrier (PRBB) .system was developed to treat groundwater with contaminants that appear in batches. An enricher reactor is an offline reactor used to enrich contaminant degraders by supplying necessary growth materials, and the enriched degraders are used to augment PRBB to increase its performance after a period of contaminant absence. Benchscale experiments on PRBBs with and without bacterial supply from the enricher reactor were conducted to evaluate PRBB removal performances for benzene, which was used as a model contaminant. Benzene absence periods of 10 and 25 days were tested in the presence and absence of ethanol. The PRBBs without the bioaugmentation from the enricher reactor experienced a decrease in perfonnance from approximately 65% to 30% after benzene reappeared. The presence of ethanol accelerated the benzene removal performance recovery of PRBBs. The 25-day benzene absence period cau.sed greater changes in the bacterial community structure, regardless of the ethanol availability. Water Environ. Res. 83, 603 (2011).
Proceedings of the Water Environment Federation, 2010
Permeable reactive biobarrier (PRBB) is a flow-through zone where microorganisms degrade contamin... more Permeable reactive biobarrier (PRBB) is a flow-through zone where microorganisms degrade contaminants in groundwater. Discontinuous presence of contaminants in groundwater causes performance loss of a PRBB in removing the target contaminant. A novel enricher reactor (ER)-PRBB system was developed to treat groundwater with contaminants that reappear after an absence period. ER is an offline reactor for enriching contaminant degraders, which were used for augmenting PRBB to maintain its performance after a period of contaminant absence. The ER-PRBB concept was initially applied to remove benzene that reappeared after absence periods of 10 and 25 days. PRBBs without ER augmentation experienced performance losses of up to 15% higher than ER-PRBBs. The role of inducer compounds in the ER to enrich bacteria that can degrade a mixture of benzene, toluene, ethylbenzene, and xylene (BTEX) was investigated with an objective to minimize the use of toxic chemicals as inducers. Three inducer types were studied: individual BTEX compounds, BTEX mixture, and benzoate (a non toxic and a common intermediate for BTEX biodegradation). Complete BTEX removal was observed for degraders enriched on all three inducer types; however, the removal rates were dependent on the inducer type. Degraders enriched on toluene and BTEX had the highest degradation rates for BTEX of 0.006 to 0.014 day-1 and 0.006 to 0.012 day-1 , respectively, while degraders enriched on benzoate showed the lowest degradation rates of 0.004 to 0.009 day-1. The ER-PRBB technique was finally applied to address the performance loss of a PRBB due to inhibition interactions among BTEX, when the mixture reappeared after a iv 10 day absence period. The ER-PRBBs experienced minimal to no performance loss, while PRBBs without ER augmentation experienced performance losses between 11% and 35%. Presence of ethanol during the BTEX absence period increased the performance loss of PRBB for benzene removal. PRBBs augmented with degraders enriched on toluene alone overcame the inhibition interaction between benzene and toluene indicating that toluene can be used as a single effective inducer in an ER. The ER-PRBB was demonstrated to be a promising remediation technique and has potential for applications to a wide range of organic contaminants. v ACKNOWLEDGEMENTS I would like to thank my major advisors, Drs. Eakalak Khan and G. Padmanabhan, for their guidance, advice, and support during my doctoral research at North Dakota State University (NDSU). I am extremely thankful to Dr. Khan for being a great listener, providing valuable suggestions, and critiquing my work constantly with long hours of discussion. I am highly indebted to him for allowing and encouraging me to try new things and experiments which helped me develop as an independent researcher. I would also wish to express my sincere gratitude to Dr. Padmanabhan for providing me with this opportunity and guiding me through various difficult times during the research. I would also like to express my gratitude to Drs. John McEvoy and Achintya Bezbaruah for their valuable comments and suggestions. Additionally, I would like to thank Dr. McEvoy for providing me unlimited access to his laboratory and computational facilities at the Veterinary and Microbiological Sciences Department. I would like to express my appreciation to many of my past and present colleagues for helping me solve various issues with experimental setup and instrumentation, and above all for creating a friendly and cooperative environment in our Environmental Engineering Laboratory at NDSU. I am greatly indebted to Mr. Tanush Wadhawan for his unconditional support throughout my research. Thanks to Dr. Sumana Siripattanakul for her valuable insights in helping me formulate the concepts and developing the research ideas during the beginning of this study. Thanks to Nathan Derby and Nayan Reddy for helping me build reactor columns for my experiments, and Drs. Thunyalux Ratpukdi, Qigang Chang, Sita Krajangpan, Chaiwat Rongsayamanont, vi and Francis Casey for helping me with the operation of the reactor columns for tracer studies and continuous flow experiments. I would also like to thank Dr. Sudipta Pramanik who had trained me in various microbiological studies and Mr. Halis Simsek for helping with in the analyses of samples for total organic carbon, cultivating the bacterial cultures and preparing samples for gas chromatography (GC) analysis. Special thanks for Ms. Catherine Giddings who helped me during various parts of microbiological work and untiringly trained me in various molecular techniques. Sincere thanks to Mr. Dean Sletten, who helped me with troubleshooting GC and mass selective detector (MSD) on numerous occasions regardless of his busy schedule. I would like to express my deep gratitude to the Dean of the Graduate School, Dr. David Wittrock, for his support and advice throughout my study at NDSU. Moreover, I would like to thank Janis Bork, Cathy Marks, Melissa Selders-Ortez, Melissa Ostby, and Jindallay Warne for their affection and constant support that greatly helped me focus on the research. I would like to thank my wife, Dr. Lakshmi Harini Mallavarapu, for all her support, love and patience. I am also thankful to my brothers Subramanyam Kasisomayajula and Viswanath Kasisomayajula and my friends for their encouragement and support during my entire doctoral study at NDSU. Last but not the least, I would like to thank my parents Padmavathi Kasisomayajula and Somanatham Kasisomayajula for their everlasting love, continued support, patience, and above all their belief in me during all my endeavors. vii
h i g h l i g h t s • Biofilm stages in static batch conditions were similar to dynamic condition... more h i g h l i g h t s • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs.
The impact of single-walled carbon nanotubes (SWCNTs) on Escherichia coli ATCC 8739 was investiga... more The impact of single-walled carbon nanotubes (SWCNTs) on Escherichia coli ATCC 8739 was investigated using four indicators of viability: enzyme activity, membrane integrity, plate count, and total RNA. The study examined the effects of SWCNT concentration (5, 10, 20, 50, 100, 200, 500, and 1,000 lg/ml), SWCNT length (0.5-2 and 5-30 lm), and bacterial density (6.5 log 10 CFU and 9 log 10 CFU per treatment) on E. coli ATCC 8739 viability. Results show that anti-bacterial activity is dependent on both the length and concentration of SWCNTs. Long SWCNTs (5-30 lm) were more toxic for E. coli than short SWCNTs (0.5-2 lm). The susceptibility of E. coli to SWCNTs was dependent on the initial density of cells in the treatment, with cells at the higher density being more resistant. Estimates of viability reductions were generally similar for the four assays examined; however, the beta galactosidase and LIVE/ DEAD assays were more conservative than the plate count as indicators of viability reductions.
A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screene... more A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screened for the presence of Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi DNA by genus-specific nested PCR. Isolates were genotyped by sequence analysis of SSU rRNA, GP60, TRAP-C1, COWP, and HSP70 loci in Cryptosporidium, and the ITS region in microsporidia. Cryptosporidium spp. was detected on 3/18 horse farms and 1/15 farms where donkeys were kept. Overall, five (2.3%) horse and two (1.6%) donkey specimens were PCR positive for Cryptosporidium. Genotyping at SSU and GP60 loci revealed that three isolates from horses and donkeys were C. parvum subtype family IIaA16G1R1, one isolate from a horse was, C. muris RN66, and one isolate from a donkey was C. muris TS03. An isolate from a horse shared 99.4% and 99.3% similarity with Cryptosporidium hominis and C. cuniculus, respectively, at the SSU locus. This isolate shared 100% identity with C. hominis at the TRAP-C1, COWP,...
Bench-scale sand column breakthrough experiments were conducted to examine atrazine remediation i... more Bench-scale sand column breakthrough experiments were conducted to examine atrazine remediation in agricultural infiltrate by Agrobacterium radiobacter J14a (J14a) immobilized in phosphorylated-polyvinyl alcohol compared to free J14a cells. The effects of cell loading and infiltration rate on atrazine degradation and the loss of J14a were investigated. Four sets of experiments, i) tracers, ii) immobilized dead cells, iii) immobilized cells, and iv) free cells, were performed. The atrazine bioremediation at the cell loadings of 300, 600, and 900 mg dry cells l−1 and the infiltration rates of 1, 3, and 6 cm d−1 were tested for 5 column pore volumes (PV). The atrazine breakthrough results indicated that the immobilized dead cells significantly retarded atrazine transport. The atrazine removal efficiencies at the infiltration rates of 1, 3, and 6 cm d−1 were 100%, 80–97%, and 50–70% respectively. Atrazine remediation capacity for the immobilized cells was not significantly different fro...
We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig g... more We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81-5.96 µm (mean = 5.16) × 4.23-5.29 µm (mean = 4.83) with a length to width ratio of 1.07 ± 0.06 (n = 400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4-6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250-4000 oocysts per gram of faeces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on Small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species.
From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa... more From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa) 29 randomly selected localities (both forest areas and enclosures) across the Central European countries of Austria, the Czech Republic, Poland, and the Slovak Republic were investigated. Cryptosporidium oocysts were microscopicaly detected in 11 out of 460 faecal samples examined using aniline-carbol-methyl violet staining. Sixty-one Cryptosporidium infections, including the 11 infections that were detected by microscopy, were detected using genus-or species-specific nested PCR amplification of SSU rDNA. This represents a 5.5 fold greater sensitivity for PCR relative to microscopy. Combining genus-and species-specific PCR tools significantly changes the perspective on the occurrence of Cryptosporidium spp. in wild boars. While RFLP and direct sequencing of genus specific PCR-amplified products revealed 56 C. suis (20) and C. scrofarum (36) monoinfections and only 5 mixed infections of these species, species-specific molecular tools showed 44 monoinfections and 17 mixed infections with these species. PCR analysis of the gp60 gene did not reveal any other Cryptosporidium infections.
Salmonella present on the feathers of live birds could be a source of contamination to carcass sk... more Salmonella present on the feathers of live birds could be a source of contamination to carcass skin during defeathering. In this study, the possibility of transfer of Salmonella from the feathers of live turkeys to carcass tissue during the defeathering process at a commercial turkey processing plant was investigated. The contribution of scald water and the fingers of the picker machines to cross contamination were also examined. Over 4 visits, swab samples were collected from 174 randomly selected tagged birds before and after defeathering. Two swab samples from the fingers of the picker machines and a sample of scald water were also collected during each visit. Detection of Salmonella was carried out following standard cultural and identification methods. The DNA fingerprints obtained from pulsed field gel electrophoresis of Salmonella serotypes isolated before and after defeathering, from scald water, and from the fingers of the picker machines were compared to trace cross contamination routes. Salmonella prevalence was similar before and after defeathering during visits 2 and 3 and significantly
From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveye... more From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1-6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.
The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses... more The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses, was identified as the cause of the diarrheal disease cryptosporidiosis in an immunocompetent man in the Czech Republic. This is the first report of human illness caused by the Cryptosporidium hedgehog genotype.
We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvu... more We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum with an unusually high frequency of liquid stools. Wild mice were the most likely source of infection, demonstrating the potential for wild-mouse-borne Cryptosporidium to infect humans and highlighting the health risks associated with synantropic rodents.
Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic wer... more Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic were screened for Cryptosporidium by microscopy and PCR/sequencing at loci coding small-subunit rRNA, Cryptosporidium oocyst wall protein, actin and 70 kDa heat shock protein. Cryptosporidium infections were detected in 74 voles (22.6%). Rates of infection did not differ between males and females nor between juveniles and adults. Phylogenetic analysis revealed the presence of eight Cryptosporidium species/genotypes including two new species, C. alticolis and C. microti. These species from wild-caught common voles were able to infect common and meadow voles under experimental conditions, with a prepatent period of 3-5 days post-infection (DPI), but they were not infectious for various other rodents or chickens. Meadow voles lost infection earlier than common voles (11-14 vs 13-16 DPI) and had significantly lower infection intensity. Cryptosporidium alticolis infects the anterior small intest...
Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chicke... more Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chickens (Gallus gallus), domestic ducks (Anas platyrhynchos) and ring-necked pheasants (Phasianus colchicus) under experimental conditions. Cryptosporidium avium was not infectious for ring-necked pheasants, but it was infectious for ducks and chickens at all age categories. The course of infection in ducks did not differ among age categories, but 365-day-old chickens had less severe infections than 1- and 21-day-old chickens. The patent period in chickens and ducks was >30 DPI, but ducks started to shed oocysts of C. avium earlier (5-6 DPI) and at a lower intensity (accumulated value of infection intensity of 58,000-65,000 OPG) than chickens (9-11 DPI and accumulated value of infection intensity of 100,000-105,000 OPG). Experimentally infected birds showed no clinical signs of cryptosporidiosis.
The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill tu... more The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill turkey carcasses was investigated. Aerobic plate counts from a 50-cm2 area of the breast sampled by excision and by swabbing were compared. Escherichia coli and Salmonella recoveries were determined from turkeys sampled by a carcass rinse (CR), a modified rinse with the carcass supported in a swing (MCR), a two-site swab of 50 cm2 at the back and thigh (2S), a one-site swab of 50 cm2 beneath the wing (1S), a whole-carcass swab of the inner and outer carcass surface (WS), and excision of 25 g of neck skin tissue (NE). The effect of diluent volume (25, 50, and 100 ml) on E. coli counts from swab samples was also assessed. The aerobic plate count from breast tissue sampled by excision was greater than that by swabbing (P < 0.05). E. coli recoveries by the MCR method were similar to those by CR. E. coli counts from 1S and WS samples were higher when swabs were stomached in 50 rather than 25...
The Science of the total environment, Jan 10, 2016
This study investigated the effects of the alginate and polyvinyl alcohol (PVA) entrapment on the... more This study investigated the effects of the alginate and polyvinyl alcohol (PVA) entrapment on the viability of Escherichia coli cells exposed to single wall carbon nanotubes (SWCNTs) with a diameter of 1-2nm. Viability was examined using a galactosidase enzyme assay, LIVE/DEAD BacLight assay, and total ribonucleic acid quantity. Variables studied included SWCNT concentration (5, 10, 20, 50, 100, 200, 500, and 1000μg/ml), SWCNT length (0.5-2μm for short SWCNTs and 5-30μm for long SWCNTs), and initial bacterial concentration (6.5 log10 CFU and 9 log10 CFU per test). Results showed that both alginate and PVA entrapments mitigate the bactericidal effect of SWCNTs. At the highest SWCNT concentration tested (1000μg/ml), the viability of the cells relative to controls (systems with only E. coli, no SWCNTs), was 0-60% for free cells and 60-90% for alginate and PVA entrapped cells. The bactericidal effect depended on SWCNT type and concentration, and bacterial concentration. In general, long...
Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoe... more Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoeniceus; order Passeriformes) were examined for the presence of Cryptosporidium by PCR amplification and sequence analysis of the 18S ribosomal RNA (18S rRNA), actin, and 70-kDa heat shock protein (HSP70) genes. Twelve birds (17.1Â %) were positive for the Cryptosporidium 18S rRNA gene: six birds were positive at the proventriculus site only and six birds were positive at the proventriculus and intestinal sites. Sequence analysis of the 18S rRNA, actin and HSP70 genes showed the presence of the gastric species Cryptosporidium galli in a single proventriculus sample and a closely related genotype, which we have named Cryptosporidium avian genotype VI, in all other positive samples. These findings contribute to our understanding of Cryptosporidium diversification in passerines, the largest avian order.
A total of 269 faecal samples of various game animals, including 136 red deer (Cervus elaphus Lin... more A total of 269 faecal samples of various game animals, including 136 red deer (Cervus elaphus Linnaeus), 64 European fallow deer (Dama dama [Linnaeus]), 26 white-tailed deer (Odocoileus virginianus [Zimmermann]), and 43 mouflon sheep (Ovis orientalis musimon Pallas) were collected at 15 game preserves across the Czech Republic and examined for infection with species of Cryptosporidium Tyzzer, 1910 using microscopy (following aniline-carbol-methyl violet staining) and molecular tools. Oocysts of Cryptosporidium spp. were detected in one faecal sample originating from red deer. Ten positive cases of infection with cryptosporidia, including the case that was positive by microscopy, were detected using nested PCR. No associations between infection with cryptosporidia and diarrhoea were detected. Phylogenetic analyses based on the small subunit of the rRNA gene revealed the presence of three Cryptosporidium species/genotypes in ten positive samples: Cryptosporidium ubiquitum Fayer, SantĂn et Macarisin, 2010 was identified in five red deer, C. muris Tyzzer, 1907 in three samples (from a red deer, white-tailed deer and mouflon sheep), and Cryptosporidium deer genotype in two white-tailed deer. Subtyping of isolates of C. ubiquitum based on sequence analysis of the 60-kDa glycoprotein gene revealed that they belong to the XIId family. Finding C. muris and C. ubiquitum XIId for the first time in various wild cervids and caprines broadens their host range.
The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causi... more The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The
A combined enricher reactor (ER)-permeable reactive biobarrier (PRBB) .system was developed to tr... more A combined enricher reactor (ER)-permeable reactive biobarrier (PRBB) .system was developed to treat groundwater with contaminants that appear in batches. An enricher reactor is an offline reactor used to enrich contaminant degraders by supplying necessary growth materials, and the enriched degraders are used to augment PRBB to increase its performance after a period of contaminant absence. Benchscale experiments on PRBBs with and without bacterial supply from the enricher reactor were conducted to evaluate PRBB removal performances for benzene, which was used as a model contaminant. Benzene absence periods of 10 and 25 days were tested in the presence and absence of ethanol. The PRBBs without the bioaugmentation from the enricher reactor experienced a decrease in perfonnance from approximately 65% to 30% after benzene reappeared. The presence of ethanol accelerated the benzene removal performance recovery of PRBBs. The 25-day benzene absence period cau.sed greater changes in the bacterial community structure, regardless of the ethanol availability. Water Environ. Res. 83, 603 (2011).
Proceedings of the Water Environment Federation, 2010
Permeable reactive biobarrier (PRBB) is a flow-through zone where microorganisms degrade contamin... more Permeable reactive biobarrier (PRBB) is a flow-through zone where microorganisms degrade contaminants in groundwater. Discontinuous presence of contaminants in groundwater causes performance loss of a PRBB in removing the target contaminant. A novel enricher reactor (ER)-PRBB system was developed to treat groundwater with contaminants that reappear after an absence period. ER is an offline reactor for enriching contaminant degraders, which were used for augmenting PRBB to maintain its performance after a period of contaminant absence. The ER-PRBB concept was initially applied to remove benzene that reappeared after absence periods of 10 and 25 days. PRBBs without ER augmentation experienced performance losses of up to 15% higher than ER-PRBBs. The role of inducer compounds in the ER to enrich bacteria that can degrade a mixture of benzene, toluene, ethylbenzene, and xylene (BTEX) was investigated with an objective to minimize the use of toxic chemicals as inducers. Three inducer types were studied: individual BTEX compounds, BTEX mixture, and benzoate (a non toxic and a common intermediate for BTEX biodegradation). Complete BTEX removal was observed for degraders enriched on all three inducer types; however, the removal rates were dependent on the inducer type. Degraders enriched on toluene and BTEX had the highest degradation rates for BTEX of 0.006 to 0.014 day-1 and 0.006 to 0.012 day-1 , respectively, while degraders enriched on benzoate showed the lowest degradation rates of 0.004 to 0.009 day-1. The ER-PRBB technique was finally applied to address the performance loss of a PRBB due to inhibition interactions among BTEX, when the mixture reappeared after a iv 10 day absence period. The ER-PRBBs experienced minimal to no performance loss, while PRBBs without ER augmentation experienced performance losses between 11% and 35%. Presence of ethanol during the BTEX absence period increased the performance loss of PRBB for benzene removal. PRBBs augmented with degraders enriched on toluene alone overcame the inhibition interaction between benzene and toluene indicating that toluene can be used as a single effective inducer in an ER. The ER-PRBB was demonstrated to be a promising remediation technique and has potential for applications to a wide range of organic contaminants. v ACKNOWLEDGEMENTS I would like to thank my major advisors, Drs. Eakalak Khan and G. Padmanabhan, for their guidance, advice, and support during my doctoral research at North Dakota State University (NDSU). I am extremely thankful to Dr. Khan for being a great listener, providing valuable suggestions, and critiquing my work constantly with long hours of discussion. I am highly indebted to him for allowing and encouraging me to try new things and experiments which helped me develop as an independent researcher. I would also wish to express my sincere gratitude to Dr. Padmanabhan for providing me with this opportunity and guiding me through various difficult times during the research. I would also like to express my gratitude to Drs. John McEvoy and Achintya Bezbaruah for their valuable comments and suggestions. Additionally, I would like to thank Dr. McEvoy for providing me unlimited access to his laboratory and computational facilities at the Veterinary and Microbiological Sciences Department. I would like to express my appreciation to many of my past and present colleagues for helping me solve various issues with experimental setup and instrumentation, and above all for creating a friendly and cooperative environment in our Environmental Engineering Laboratory at NDSU. I am greatly indebted to Mr. Tanush Wadhawan for his unconditional support throughout my research. Thanks to Dr. Sumana Siripattanakul for her valuable insights in helping me formulate the concepts and developing the research ideas during the beginning of this study. Thanks to Nathan Derby and Nayan Reddy for helping me build reactor columns for my experiments, and Drs. Thunyalux Ratpukdi, Qigang Chang, Sita Krajangpan, Chaiwat Rongsayamanont, vi and Francis Casey for helping me with the operation of the reactor columns for tracer studies and continuous flow experiments. I would also like to thank Dr. Sudipta Pramanik who had trained me in various microbiological studies and Mr. Halis Simsek for helping with in the analyses of samples for total organic carbon, cultivating the bacterial cultures and preparing samples for gas chromatography (GC) analysis. Special thanks for Ms. Catherine Giddings who helped me during various parts of microbiological work and untiringly trained me in various molecular techniques. Sincere thanks to Mr. Dean Sletten, who helped me with troubleshooting GC and mass selective detector (MSD) on numerous occasions regardless of his busy schedule. I would like to express my deep gratitude to the Dean of the Graduate School, Dr. David Wittrock, for his support and advice throughout my study at NDSU. Moreover, I would like to thank Janis Bork, Cathy Marks, Melissa Selders-Ortez, Melissa Ostby, and Jindallay Warne for their affection and constant support that greatly helped me focus on the research. I would like to thank my wife, Dr. Lakshmi Harini Mallavarapu, for all her support, love and patience. I am also thankful to my brothers Subramanyam Kasisomayajula and Viswanath Kasisomayajula and my friends for their encouragement and support during my entire doctoral study at NDSU. Last but not the least, I would like to thank my parents Padmavathi Kasisomayajula and Somanatham Kasisomayajula for their everlasting love, continued support, patience, and above all their belief in me during all my endeavors. vii
h i g h l i g h t s • Biofilm stages in static batch conditions were similar to dynamic condition... more h i g h l i g h t s • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs.
The impact of single-walled carbon nanotubes (SWCNTs) on Escherichia coli ATCC 8739 was investiga... more The impact of single-walled carbon nanotubes (SWCNTs) on Escherichia coli ATCC 8739 was investigated using four indicators of viability: enzyme activity, membrane integrity, plate count, and total RNA. The study examined the effects of SWCNT concentration (5, 10, 20, 50, 100, 200, 500, and 1,000 lg/ml), SWCNT length (0.5-2 and 5-30 lm), and bacterial density (6.5 log 10 CFU and 9 log 10 CFU per treatment) on E. coli ATCC 8739 viability. Results show that anti-bacterial activity is dependent on both the length and concentration of SWCNTs. Long SWCNTs (5-30 lm) were more toxic for E. coli than short SWCNTs (0.5-2 lm). The susceptibility of E. coli to SWCNTs was dependent on the initial density of cells in the treatment, with cells at the higher density being more resistant. Estimates of viability reductions were generally similar for the four assays examined; however, the beta galactosidase and LIVE/ DEAD assays were more conservative than the plate count as indicators of viability reductions.
A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screene... more A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screened for the presence of Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi DNA by genus-specific nested PCR. Isolates were genotyped by sequence analysis of SSU rRNA, GP60, TRAP-C1, COWP, and HSP70 loci in Cryptosporidium, and the ITS region in microsporidia. Cryptosporidium spp. was detected on 3/18 horse farms and 1/15 farms where donkeys were kept. Overall, five (2.3%) horse and two (1.6%) donkey specimens were PCR positive for Cryptosporidium. Genotyping at SSU and GP60 loci revealed that three isolates from horses and donkeys were C. parvum subtype family IIaA16G1R1, one isolate from a horse was, C. muris RN66, and one isolate from a donkey was C. muris TS03. An isolate from a horse shared 99.4% and 99.3% similarity with Cryptosporidium hominis and C. cuniculus, respectively, at the SSU locus. This isolate shared 100% identity with C. hominis at the TRAP-C1, COWP,...
Bench-scale sand column breakthrough experiments were conducted to examine atrazine remediation i... more Bench-scale sand column breakthrough experiments were conducted to examine atrazine remediation in agricultural infiltrate by Agrobacterium radiobacter J14a (J14a) immobilized in phosphorylated-polyvinyl alcohol compared to free J14a cells. The effects of cell loading and infiltration rate on atrazine degradation and the loss of J14a were investigated. Four sets of experiments, i) tracers, ii) immobilized dead cells, iii) immobilized cells, and iv) free cells, were performed. The atrazine bioremediation at the cell loadings of 300, 600, and 900 mg dry cells l−1 and the infiltration rates of 1, 3, and 6 cm d−1 were tested for 5 column pore volumes (PV). The atrazine breakthrough results indicated that the immobilized dead cells significantly retarded atrazine transport. The atrazine removal efficiencies at the infiltration rates of 1, 3, and 6 cm d−1 were 100%, 80–97%, and 50–70% respectively. Atrazine remediation capacity for the immobilized cells was not significantly different fro...
We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig g... more We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81-5.96 µm (mean = 5.16) × 4.23-5.29 µm (mean = 4.83) with a length to width ratio of 1.07 ± 0.06 (n = 400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4-6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250-4000 oocysts per gram of faeces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on Small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species.
From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa... more From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa) 29 randomly selected localities (both forest areas and enclosures) across the Central European countries of Austria, the Czech Republic, Poland, and the Slovak Republic were investigated. Cryptosporidium oocysts were microscopicaly detected in 11 out of 460 faecal samples examined using aniline-carbol-methyl violet staining. Sixty-one Cryptosporidium infections, including the 11 infections that were detected by microscopy, were detected using genus-or species-specific nested PCR amplification of SSU rDNA. This represents a 5.5 fold greater sensitivity for PCR relative to microscopy. Combining genus-and species-specific PCR tools significantly changes the perspective on the occurrence of Cryptosporidium spp. in wild boars. While RFLP and direct sequencing of genus specific PCR-amplified products revealed 56 C. suis (20) and C. scrofarum (36) monoinfections and only 5 mixed infections of these species, species-specific molecular tools showed 44 monoinfections and 17 mixed infections with these species. PCR analysis of the gp60 gene did not reveal any other Cryptosporidium infections.
Salmonella present on the feathers of live birds could be a source of contamination to carcass sk... more Salmonella present on the feathers of live birds could be a source of contamination to carcass skin during defeathering. In this study, the possibility of transfer of Salmonella from the feathers of live turkeys to carcass tissue during the defeathering process at a commercial turkey processing plant was investigated. The contribution of scald water and the fingers of the picker machines to cross contamination were also examined. Over 4 visits, swab samples were collected from 174 randomly selected tagged birds before and after defeathering. Two swab samples from the fingers of the picker machines and a sample of scald water were also collected during each visit. Detection of Salmonella was carried out following standard cultural and identification methods. The DNA fingerprints obtained from pulsed field gel electrophoresis of Salmonella serotypes isolated before and after defeathering, from scald water, and from the fingers of the picker machines were compared to trace cross contamination routes. Salmonella prevalence was similar before and after defeathering during visits 2 and 3 and significantly
From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveye... more From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1-6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.
The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses... more The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses, was identified as the cause of the diarrheal disease cryptosporidiosis in an immunocompetent man in the Czech Republic. This is the first report of human illness caused by the Cryptosporidium hedgehog genotype.
We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvu... more We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum with an unusually high frequency of liquid stools. Wild mice were the most likely source of infection, demonstrating the potential for wild-mouse-borne Cryptosporidium to infect humans and highlighting the health risks associated with synantropic rodents.
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