Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality ... more Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolaye...
Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of t... more Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10 :: himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale . Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isola...
The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Aut... more The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals.
To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). 2 boa c... more To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). 2 boa constrictors with IBD and 1 boa exposed to an affected snake. Snakes were euthanatized, and tissue specimens and blood samples were submitted for virus isolation. Tissue specimens were cultured with or without commercially available viper heart cells and examined by use of transmission electron microscopy (TEM) for evidence of viral replication. Reverse transcriptase activ ty was determined in sucrose gradient-purified virus. Western blotting was performed, using polyclonal antibodies against 1 of the isolated viruses. Specificity of the rabbit anti-virus antibody was evaluated, using an immunogold-labeling TEM technique. 3 viruses (RV-1, RV-2, and RV-3) were isolated. The isolates were morphologically comparable to members of the Retroviridae family. Reverse transcriptase activity was high in sucrose gradient fractions that were rich in virus. Polyclonal antibody against RV-1 reacted with proteins of similar relative mobility in RV-1 and RV-2. By use of immunogold labeling, this antibody also recognized virions of both RV-1 and RV-2. A retrovirus was isolated from boid snakes with IBD or exposed to IBD. Western blot analysis of viral proteins indicated that viruses isolated from the different snakes were similar. Whether this virus represents the causative agent of IBD is yet to be determined. The isolation of retroviruses from boid snakes with IBD is an important step n the process of identifying the causative agent of this disease.
Journal of the science of food and agriculture, Jan 30, 2017
Programmed cell death (PCD) is a part of plant development that has been studied for petal senesc... more Programmed cell death (PCD) is a part of plant development that has been studied for petal senescence and vegetative tissue but has not been thoroughly investigated for fleshy fruits. The purpose of this research was to examine ripening and over-ripening in banana fruit to determine if there were processes in common to previously described PCD. Loss of cellular integrity (over 40%) and development of senescence related dark spot (SRDS) occurred after day 8 in banana peel. Nuclease and protease activity in the peel increased during ripening starting from day 2, and decreased during over-ripening. The highest activity was for proteases and nucleases with apparent molecular weights of 86 kDa and 27 kDa, respectively. Images of SRDS showed shrinkage of the upper layers of cells, visually suggesting cell death. Decrease of electron dense areas was evident in TEM micrographs of nuclei. This study shows for the first time that ripening and over-ripening of banana peel share physiological a...
Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal sys... more Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.
Programmed cell death (PCD) has been well characterized in floral and vegetative tissues but litt... more Programmed cell death (PCD) has been well characterized in floral and vegetative tissues but little has been reported on fleshy fruits. The purpose of this study was to determine if ripening and over-ripening of fleshy fruit are accompanied by classic symptoms of PCD. Bananas were ethylene-treated at green stage and shipped overnight to the UF. Upon arrival they were stored at 20 °C and evaluated for biochemical and molecular changes. Fresh peel tissue was collected and stored in 4% paraformaldehyde in phosphate buffered saline (pH7.0) for microscopy and in situ cell death detection (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL). Fruit arrived at color stage 2, after 4 d they had reached color stage 4 and on day 8 fruit were at stage 7 with presence of senescence-related spots (SRS). Ethylene production increased within 24 h of storage and reached climacteric peak after day 2 (0.55 ± 0.01 ng kg-1 s-1). CO2 production sharply increased on day 2, reaching a maxi...
The genus Megalocytivirus includes large double-stranded DNA viruses within the family Iridovirid... more The genus Megalocytivirus includes large double-stranded DNA viruses within the family Iridoviridae. Megalocytiviruses induce lethal systemic disease at water temperature ranging from 7.5 to 32oC in a wide variety of fresh water and marine fish hosts. Megalocytivirus epizootics have caused significant economic losses for both the food-fish and ornamental fish industries. The objective of the current study was to characterize an iridovirus causing systemic infections in cultured Nile tilapia (Oreochromis niloticus). Fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, and genetic analyses. There was a severe systemic abundance of cytomegalic cells that were further characterized by an increased cytoplasmic to nuclear ratio that resulted in peripheral compression of the nucleus with occasional scattered nuclear fragments and a fine, granular, amphophilic cytoplasm. The cells generally had an intravascular and perivascular distri...
Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality ... more Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolaye...
Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of t... more Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10 :: himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale . Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isola...
The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Aut... more The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals.
To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). 2 boa c... more To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). 2 boa constrictors with IBD and 1 boa exposed to an affected snake. Snakes were euthanatized, and tissue specimens and blood samples were submitted for virus isolation. Tissue specimens were cultured with or without commercially available viper heart cells and examined by use of transmission electron microscopy (TEM) for evidence of viral replication. Reverse transcriptase activ ty was determined in sucrose gradient-purified virus. Western blotting was performed, using polyclonal antibodies against 1 of the isolated viruses. Specificity of the rabbit anti-virus antibody was evaluated, using an immunogold-labeling TEM technique. 3 viruses (RV-1, RV-2, and RV-3) were isolated. The isolates were morphologically comparable to members of the Retroviridae family. Reverse transcriptase activity was high in sucrose gradient fractions that were rich in virus. Polyclonal antibody against RV-1 reacted with proteins of similar relative mobility in RV-1 and RV-2. By use of immunogold labeling, this antibody also recognized virions of both RV-1 and RV-2. A retrovirus was isolated from boid snakes with IBD or exposed to IBD. Western blot analysis of viral proteins indicated that viruses isolated from the different snakes were similar. Whether this virus represents the causative agent of IBD is yet to be determined. The isolation of retroviruses from boid snakes with IBD is an important step n the process of identifying the causative agent of this disease.
Journal of the science of food and agriculture, Jan 30, 2017
Programmed cell death (PCD) is a part of plant development that has been studied for petal senesc... more Programmed cell death (PCD) is a part of plant development that has been studied for petal senescence and vegetative tissue but has not been thoroughly investigated for fleshy fruits. The purpose of this research was to examine ripening and over-ripening in banana fruit to determine if there were processes in common to previously described PCD. Loss of cellular integrity (over 40%) and development of senescence related dark spot (SRDS) occurred after day 8 in banana peel. Nuclease and protease activity in the peel increased during ripening starting from day 2, and decreased during over-ripening. The highest activity was for proteases and nucleases with apparent molecular weights of 86 kDa and 27 kDa, respectively. Images of SRDS showed shrinkage of the upper layers of cells, visually suggesting cell death. Decrease of electron dense areas was evident in TEM micrographs of nuclei. This study shows for the first time that ripening and over-ripening of banana peel share physiological a...
Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal sys... more Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.
Programmed cell death (PCD) has been well characterized in floral and vegetative tissues but litt... more Programmed cell death (PCD) has been well characterized in floral and vegetative tissues but little has been reported on fleshy fruits. The purpose of this study was to determine if ripening and over-ripening of fleshy fruit are accompanied by classic symptoms of PCD. Bananas were ethylene-treated at green stage and shipped overnight to the UF. Upon arrival they were stored at 20 °C and evaluated for biochemical and molecular changes. Fresh peel tissue was collected and stored in 4% paraformaldehyde in phosphate buffered saline (pH7.0) for microscopy and in situ cell death detection (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL). Fruit arrived at color stage 2, after 4 d they had reached color stage 4 and on day 8 fruit were at stage 7 with presence of senescence-related spots (SRS). Ethylene production increased within 24 h of storage and reached climacteric peak after day 2 (0.55 ± 0.01 ng kg-1 s-1). CO2 production sharply increased on day 2, reaching a maxi...
The genus Megalocytivirus includes large double-stranded DNA viruses within the family Iridovirid... more The genus Megalocytivirus includes large double-stranded DNA viruses within the family Iridoviridae. Megalocytiviruses induce lethal systemic disease at water temperature ranging from 7.5 to 32oC in a wide variety of fresh water and marine fish hosts. Megalocytivirus epizootics have caused significant economic losses for both the food-fish and ornamental fish industries. The objective of the current study was to characterize an iridovirus causing systemic infections in cultured Nile tilapia (Oreochromis niloticus). Fixed specimens were processed for histopathologic evaluation, transmission electron microscopic examination, and genetic analyses. There was a severe systemic abundance of cytomegalic cells that were further characterized by an increased cytoplasmic to nuclear ratio that resulted in peripheral compression of the nucleus with occasional scattered nuclear fragments and a fine, granular, amphophilic cytoplasm. The cells generally had an intravascular and perivascular distri...
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