Dolores Baxa - Academia.edu (original) (raw)

Papers by Dolores Baxa

Research paper thumbnail of Detection of Nucleospora salmonis in cutthroat trout ( Oncorhynchus clarki) and rainbow trout ( Oncorhynchus mykiss) by loop-mediated isothermal amplification

Aquaculture, Mar 1, 2009

Nucleospora salmonis is an intranuclear microsporidian parasite of salmonids that causes a chroni... more Nucleospora salmonis is an intranuclear microsporidian parasite of salmonids that causes a chronic lymphoblastosis and a leukemic-like condition in both cultured and wild salmonids. Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. Using the LAMP method, a protocol for detection of N. salmonis was developed. A set of four primers, two inner and two outer were designed based on the sequence of ribosomal RNA intermediate spacer of this parasite. Optimum time and temperature conditions for detection of N. salmonis by LAMP were 60 min at 63°C, respectively. The detection-limit (DL) using LAMP was found to be similar to polymerase chain reaction (PCR). In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of N. salmonis infections in cutthroat trout(Oncorhynchus clarki) and rainbow trout (Oncorhynchus mykiss).

Research paper thumbnail of Anthropogenic debris in seafood: Plastic debris and fibers from textiles in fish and bivalves sold for human consumption

Scientific reports, 2015

The ubiquity of anthropogenic debris in hundreds of species of wildlife and the toxicity of chemi... more The ubiquity of anthropogenic debris in hundreds of species of wildlife and the toxicity of chemicals associated with it has begun to raise concerns regarding the presence of anthropogenic debris in seafood. We assessed the presence of anthropogenic debris in fishes and shellfish on sale for human consumption. We sampled from markets in Makassar, Indonesia, and from California, USA. All fish and shellfish were identified to species where possible. Anthropogenic debris was extracted from the digestive tracts of fish and whole shellfish using a 10% KOH solution and quantified under a dissecting microscope. In Indonesia, anthropogenic debris was found in 28% of individual fish and in 55% of all species. Similarly, in the USA, anthropogenic debris was found in 25% of individual fish and in 67% of all species. Anthropogenic debris was also found in 33% of individual shellfish sampled. All of the anthropogenic debris recovered from fish in Indonesia was plastic, whereas anthropogenic debr...

Research paper thumbnail of Effect of water temperature on infections with the microsporidian Enterocytozoon salmonis in chinook salmon

The effect of water temperature on the progress of infections associated with Enterocytozoon salm... more The effect of water temperature on the progress of infections associated with Enterocytozoon salmonis Chilmonczyk, Cox, Hedrick 1991 was examined in chinook salmon Oncorhynchus tshawytscha after intraperitoneal injections of mononuclear leukocytes infected with the microsporidlan parasite. Experimentally infected and control fish were held at water temperatures of 9, 12, 15, 18 and 21°C for 12 wk and then one half of the exposed and control groups of fish a t 9 and 12°C were shifted to 15°C and held for a n additional 8 wk. Among fish held a t constant water temperatures, severe infections occurred among exposed fish a t 15 and 18°C resulting in 90.0% cumulative mortality in both groups. Disease and significant mortality was also observed at 21°C (47.5 %). The parasite and signs of the disease slo\vly developed over time at 12'C and the cumulative mortality reached 73.7% between 13 and 20 wk. Although the development of the microspondian was not arrested at a water temperature of 9"C, infections in chinook salmon were not severe and cumulative mortalities were low (10.0%). However, parallel groups of exposed chinook salmon at g0C which were shifted to 15°C showed a cumulative mortality of 60 0% by 8 wk after transfer to the hlgher water temperature. Shifting the exposed fish from 12 to 15°C did not increase the mortality rate from that of fish kept constantly at 12OC. The control fish (not exposed to E. salmonis) in all temperature groups did not show signs of the disease nor mortality throughout the study.

Research paper thumbnail of A novel and versatile flash-freezing approach for evaluating the health of Delta Smelt

Aquatic toxicology (Amsterdam, Netherlands), 2016

A common approach used to assess environmental impacts in aquatic environments is to measure indi... more A common approach used to assess environmental impacts in aquatic environments is to measure indicators of stress (biomarkers) and condition of fish within ecosystems. Particularly in estuarine ecosystems with multiple stressors, it is often desirable to quantify a suite of biological endpoints that (1) reflect fish condition at several levels of biological organization and time scales and (2) are sensitive to a range of environmental stressors. However, established methods of preservation and processing of fish for specific endpoints are often incompatible. Here, we developed a novel flash-freezing approach for assessing the health of a small, sensitive fish, the endangered Delta Smelt (Hypomesus transpacificus) after collections from the San Francisco Estuary (SFE). We assess whether flash-freezing the entire fish ensures effective preservation of multiple tissues for subsequent biomarker analyses by comparing measurements of fresh to frozen tissue. Tissues included brain, gill, a...

Research paper thumbnail of Detection of early developmental stages of Myxobolus cerebralis in fish and tubificid oligochaete hosts by in situ hybridization

The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in t... more The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in their respective hosts, salmonid ®shes and a tubi®cid oligochaete. Using a modi®ed, non-radioactive in situ hybridization protocol, the parasite, which exhibits radically dierent structural forms during its development in each host, could be speci®cally detected in paran-embedded tissues of both ®sh and oligochaetes. Our study aims to demonstrate the application of the technique for detection of early stages of M. cerebralis in both hosts.

Research paper thumbnail of Chemotherapy of Flexibacter maritimus infections

Research paper thumbnail of In vitro and in vivo activities of Flexibacter maritimus toxins

Research paper thumbnail of NOTE Detection of Rickettsiales-like Prokaryotes by in Situ Hybridization in Black Abalone, Haliotis cracherodii, with Withering Syndrome

Journal of Invertebrate Pathology

Research paper thumbnail of Effects of the corticosteroid kenalog on the carrier state of juvenile channel catfish exposed to Edwardsiella ictaluri

Studies were conducted on the carrier state following experimentally induced infections of Edward... more Studies were conducted on the carrier state following experimentally induced infections of Edwardsiella ictaluri in juvenile channel catfish Ictalurus punctatus. The cumulative mortality of fish exposed to the bacterium at 1.0 x 10 8 colony-forming units/mL for 30 s was 71% after 3 weeks at a water temperature of 25°C The bacterium could not be recovered from the posterior kidneys of 10 fish examined 1, 2, or 3 months after initial exposure, but antibodies were still evident at each sampling. However, the bacterium was isolated from 6 of 20 fish at 4 months postexposure, 14 d after intraperitoneal injection with the corticosteroid Kenalog (0.8 mg/g fish).

Research paper thumbnail of Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebrali... more A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3Ј end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.

Research paper thumbnail of Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

Parasitology Research

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD)... more Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 lm and height of 14 lm, and possessed two soft valves surrounding two spherical polar capsules (2 lm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 lm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.

Research paper thumbnail of Subclinical Mycobacterium infections in wild delta smelt

This study describes the occurrence and magnitude of latent mycobacterial infections and other pa... more This study describes the occurrence and magnitude of latent mycobacterial infections and other pathogens in delta smelt (Hypomesus transpacificus), an endemic fish in the San Francisco Estuary (SFE). Our current knowledge on subclinical infections is characterized by the presence of mycobacterial DNA and absence of clinical signs and Mycobacterium. In our previous studies however, we showed that handling and increased water temperature, stress factors frequently encountered in captivity, have altered infections from benign to severe mycobacteriosis. In the current study, sub adult and adult smelt captured from August 2011 to May 2012 in the SFE were analyzed for presence of bacterial and viral agents (n=741) using standard isolation and culture techniques and Mycobacterium qPCR (n=237). Although mycobacteriosis, Mycobacterium, and virus were not observed in all of the fish examined, 96% of the smelt were positive for mycobacterial DNA. Higher pathogen scores and mycobacterial gene c...

Research paper thumbnail of Nucleic Acid — Based Methods for Detection of Myxobolus Cerebralis

Reviews: Methods and Technologies in Fish Biology and Fisheries, 2002

ABSTRACT Abstract Much of our current knowledge of myxosporean parasites stems from the study of ... more ABSTRACT Abstract Much of our current knowledge of myxosporean parasites stems from the study of M. cerebralis. It was the work of Maria Markiw and Ken Wolf that produced the revelation that alternate hosts are required for the completion of the parasitic life cycle for myxozoans. Spores produced in the oligochaete host, Tubifex tubifex, were found to be infectious for the alternate host, a salmonid fish (Markiw and Wolf, 1983; Wolf and Markiw, 1984, Wolf et al., 1986). The degree to which the disease develops in the fish is dependent on the species of salmonid (see below, Table 1). It is the mature spores of the myxosporean stage that are required for specific diagnosis of diseases of myxosporean etiology. The time required to complete sporogenesis in either host generally takes about 90 days, but can be influenced by temperature (El-Matbouli et al., 1999). This has ramifications for those testing exposed fish by PCR. Wild fish tested after an unknown elapsed time after exposure can not be sampled in the same way as sentinel fish left in the water for brief durations. This is discussed in more detail below.

Research paper thumbnail of Experimental Horizontal Transmission of Enterocytozoon salmonis to Chinook Salmon, Oncorhynchus tshawytscha

The Journal of Protozoology, 1992

Research paper thumbnail of Production of viable cultures of Flavobacterium psychrophilum: approach and control

Research in Microbiology, 1999

Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid ... more Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid hatcheries, few studies have been conducted on its pathogenicity. Difficulties are often experienced when trying to control or quantify standard procedures for in vitro culture of the bacterium. Plate enumeration and counting chamber enumeration combined with epifluorescent microscopy with fluorescent dyes determined that no more than 25% of the bacterial cells present in the cultures were able to produce colonies on agar media. This was strongly dependent upon different medium components. Tryptone-enriched Anacker and Ordal medium proved more suitable than tryptone-yeast extract-salts with skimmed milk. Adding horse serum and trace elements in controlled proportions offered the most reproducible results. Viable but nonculturable forms were apparently not responsible for the difficulties in production of F. psychrophilum, but the cells were highly susceptible to osmotic conditions. Improvements in the media and careful handling of the bacteria in isotonic suspension media resulted in predictable production of viable bacteria and allowed an absorbance/colony-forming-units relation curve to be established. © Elsevier, Paris Flavobacterium psychrophilum / bacterial growth / microbial nutrition / viability

Research paper thumbnail of Identification of harmful cyanobacteria in the Sacramento-San Joaquin Delta and Clear Lake, California by DNA barcoding

SpringerPlus, 2013

Accurate identification of cyanobacteria using traditional morphological taxonomy is challenging ... more Accurate identification of cyanobacteria using traditional morphological taxonomy is challenging due to the magnitude of phenotypic plasticity among natural algal assemblages. In this study, molecular approach was utilized to facilitate the accurate identification of cyanobacteria in the Sacramento-San Joaquin Delta and in Clear Lake in Northern California where recurring blooms have been observed over the past decades. Algal samples were collected from both water bodies in 2011 and the samples containing diverse cyanobacteria as identified by morphological taxonomy were chosen for the molecular analysis. The 16S ribosomal RNA genes (16S rDNA) and the adjacent internal transcribed spacer (ITS) regions were amplified by PCR from the mixed algal samples using cyanobacteria generic primers. The obtained sequences were analyzed by similarity search (BLASTN) and phylogenetic analysis (16S rDNA) to differentiate species sharing significantly similar sequences. A total of 185 plasmid clones were obtained of which 77 were successfully identified to the species level: Aphanizomenon flosaquae, Dolichospermum lemmermannii (taxonomic synonym: Anabaena lemmermannii), Limnoraphis robusta (taxonomic synonym: Lyngbya hieronymusii f. robusta) and Microcystis aeruginosa. To date, Dolichospermum and Limnoraphis found in Clear Lake have only been identified to the genus lavel by microscopy. During the course of this study, morphological identification and DNA barcoding confirmed A. flos-aquae as the predominant cyanobacterium in the Sacramento-San Joaquin Delta indicating a shift from M. aeruginosa that have dominated the blooms in the past decade. Lastly, the species-specific identification of Limnoraphis robusta in Clear Lake is another significant finding as this cyanobacterium has, thus far, only been reported in Lake Atitlan blooms in Guatemala.

Research paper thumbnail of Henneguya sp. in yellowfin goby Acanthogobius flavimanus from the San Francisco Estuary

SpringerPlus, 2013

Myxozoan spores were observed in yellowfin goby Acanthogobius flavimanus collected from Suisun Ma... more Myxozoan spores were observed in yellowfin goby Acanthogobius flavimanus collected from Suisun Marsh, San Francisco Estuary (SFE). Although histopathological changes associated with the parasite were not observed, the spores formed plasmodia that partially blocked the gastric and intestinal mucosa and gut lumen and may affect the perfomance and survival of the yellowfin goby. Morphological features of the spores resembled Henneguya sp. and molecular analysis of the 18S ribosomal DNA (Domain III) confirmed close similarity to H. rhinogobii and H. pseudorhinogobii isolated from the Japanese freshwater goby. The yellowfin goby myxozoan however, is likely an undescribed species based on phylogenetic analysis and morphologic features. Detailed description of vegetative and spore stages are currently lacking for proposal to a new species of Henneguya. A specific PCR test was developed, which confirmed a 100% prevalence of the parasite among randomly collected gobies in group 1 (N = 30) and group 2 (N = 15) at termination of the study at one month in captivity. The myxozoan was also detected from 18 gobies (12%) that died in the first group within two weeks in captivity. Apparently healthy gobies that served as controls did not reveal the presence of the myxozoan by PCR. This study documents the occurrence of a potentially new species of myxozoan in the yellowfin goby and underscores the detection of a parasitic infection in an introduced fish in the SFE. Although the pathogenesis of the myxozoan was not assessed and the prevalence as reported here is restricted to a comparatively small collection site in Suisun slough, the reemergence, identification, and ecological relevance of the parasite on goby populations in the SFE may be investigated in the future using the specific diagnostic tool developed in this study.

Research paper thumbnail of Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia (Oreochromis niloticus) cultured in net cages in Lake Sentani, Papua, Indonesia

SpringerPlus, 2014

Infections with Streptococcus spp. were observed in Nile tilapia cultured in net cages in Lake Se... more Infections with Streptococcus spp. were observed in Nile tilapia cultured in net cages in Lake Sentani, Papua, Indonesia. Clinical signs included exophthalmia, erratic swimming, ascites in abdominal cavity, and external hemorrhages. Four types of bacterial colonies (SK, K10, P20, and M12) were isolated from the brain, kidney, and eyes. Based on phenotypic and genetic (16S rDNA sequencing) characteristics, the isolates were identified as Streptococcus iniae (SK), Streptococcus agalactiae (K10 and P20) and Lactococcus garvieae (M12). The latter species has not been previously isolated or reported from fish streptococcosis in Indonesia. Intraperitoneal injection of healthy tilapia with the bacterial species caused significant morbidity (70%) within 3 days and 100% mortality at 6 days post injection. Experimental infections and reisolation of the bacteria from morbid and dead fish suggest they are the causative agents of streptococcosis, which rendered high mortality among cage cultured Nile tilapia in Lake Sentani. Our results suggest the need for developing diagnostic tools for accurate identification of the agents of streptococcosis. As tilapia aquaculture continues to expand as a means of food production and livelihood in Indonesia, it becomes crucial to ensure that fish resources are monitored and protected from the adverse effects of infectious diseases.

Research paper thumbnail of Sublethal dietary effects of microcystin producing Microcystis on threadfin shad, Dorosoma petenense

Toxicon, 2012

Please cite this article as: Acuña, S., Baxa, D., Teh, S., Sublethal dietary effects of microcyst... more Please cite this article as: Acuña, S., Baxa, D., Teh, S., Sublethal dietary effects of microcystin producing Microcystis on threadfin shad, Dorosoma petenense, Toxicon (2012),

Research paper thumbnail of Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

Parasitology Research, 2004

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD)... more Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 lm and height of 14 lm, and possessed two soft valves surrounding two spherical polar capsules (2 lm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 lm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.

Research paper thumbnail of Detection of Nucleospora salmonis in cutthroat trout ( Oncorhynchus clarki) and rainbow trout ( Oncorhynchus mykiss) by loop-mediated isothermal amplification

Aquaculture, Mar 1, 2009

Nucleospora salmonis is an intranuclear microsporidian parasite of salmonids that causes a chroni... more Nucleospora salmonis is an intranuclear microsporidian parasite of salmonids that causes a chronic lymphoblastosis and a leukemic-like condition in both cultured and wild salmonids. Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. Using the LAMP method, a protocol for detection of N. salmonis was developed. A set of four primers, two inner and two outer were designed based on the sequence of ribosomal RNA intermediate spacer of this parasite. Optimum time and temperature conditions for detection of N. salmonis by LAMP were 60 min at 63°C, respectively. The detection-limit (DL) using LAMP was found to be similar to polymerase chain reaction (PCR). In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of N. salmonis infections in cutthroat trout(Oncorhynchus clarki) and rainbow trout (Oncorhynchus mykiss).

Research paper thumbnail of Anthropogenic debris in seafood: Plastic debris and fibers from textiles in fish and bivalves sold for human consumption

Scientific reports, 2015

The ubiquity of anthropogenic debris in hundreds of species of wildlife and the toxicity of chemi... more The ubiquity of anthropogenic debris in hundreds of species of wildlife and the toxicity of chemicals associated with it has begun to raise concerns regarding the presence of anthropogenic debris in seafood. We assessed the presence of anthropogenic debris in fishes and shellfish on sale for human consumption. We sampled from markets in Makassar, Indonesia, and from California, USA. All fish and shellfish were identified to species where possible. Anthropogenic debris was extracted from the digestive tracts of fish and whole shellfish using a 10% KOH solution and quantified under a dissecting microscope. In Indonesia, anthropogenic debris was found in 28% of individual fish and in 55% of all species. Similarly, in the USA, anthropogenic debris was found in 25% of individual fish and in 67% of all species. Anthropogenic debris was also found in 33% of individual shellfish sampled. All of the anthropogenic debris recovered from fish in Indonesia was plastic, whereas anthropogenic debr...

Research paper thumbnail of Effect of water temperature on infections with the microsporidian Enterocytozoon salmonis in chinook salmon

The effect of water temperature on the progress of infections associated with Enterocytozoon salm... more The effect of water temperature on the progress of infections associated with Enterocytozoon salmonis Chilmonczyk, Cox, Hedrick 1991 was examined in chinook salmon Oncorhynchus tshawytscha after intraperitoneal injections of mononuclear leukocytes infected with the microsporidlan parasite. Experimentally infected and control fish were held at water temperatures of 9, 12, 15, 18 and 21°C for 12 wk and then one half of the exposed and control groups of fish a t 9 and 12°C were shifted to 15°C and held for a n additional 8 wk. Among fish held a t constant water temperatures, severe infections occurred among exposed fish a t 15 and 18°C resulting in 90.0% cumulative mortality in both groups. Disease and significant mortality was also observed at 21°C (47.5 %). The parasite and signs of the disease slo\vly developed over time at 12'C and the cumulative mortality reached 73.7% between 13 and 20 wk. Although the development of the microspondian was not arrested at a water temperature of 9"C, infections in chinook salmon were not severe and cumulative mortalities were low (10.0%). However, parallel groups of exposed chinook salmon at g0C which were shifted to 15°C showed a cumulative mortality of 60 0% by 8 wk after transfer to the hlgher water temperature. Shifting the exposed fish from 12 to 15°C did not increase the mortality rate from that of fish kept constantly at 12OC. The control fish (not exposed to E. salmonis) in all temperature groups did not show signs of the disease nor mortality throughout the study.

Research paper thumbnail of A novel and versatile flash-freezing approach for evaluating the health of Delta Smelt

Aquatic toxicology (Amsterdam, Netherlands), 2016

A common approach used to assess environmental impacts in aquatic environments is to measure indi... more A common approach used to assess environmental impacts in aquatic environments is to measure indicators of stress (biomarkers) and condition of fish within ecosystems. Particularly in estuarine ecosystems with multiple stressors, it is often desirable to quantify a suite of biological endpoints that (1) reflect fish condition at several levels of biological organization and time scales and (2) are sensitive to a range of environmental stressors. However, established methods of preservation and processing of fish for specific endpoints are often incompatible. Here, we developed a novel flash-freezing approach for assessing the health of a small, sensitive fish, the endangered Delta Smelt (Hypomesus transpacificus) after collections from the San Francisco Estuary (SFE). We assess whether flash-freezing the entire fish ensures effective preservation of multiple tissues for subsequent biomarker analyses by comparing measurements of fresh to frozen tissue. Tissues included brain, gill, a...

Research paper thumbnail of Detection of early developmental stages of Myxobolus cerebralis in fish and tubificid oligochaete hosts by in situ hybridization

The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in t... more The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in their respective hosts, salmonid ®shes and a tubi®cid oligochaete. Using a modi®ed, non-radioactive in situ hybridization protocol, the parasite, which exhibits radically dierent structural forms during its development in each host, could be speci®cally detected in paran-embedded tissues of both ®sh and oligochaetes. Our study aims to demonstrate the application of the technique for detection of early stages of M. cerebralis in both hosts.

Research paper thumbnail of Chemotherapy of Flexibacter maritimus infections

Research paper thumbnail of In vitro and in vivo activities of Flexibacter maritimus toxins

Research paper thumbnail of NOTE Detection of Rickettsiales-like Prokaryotes by in Situ Hybridization in Black Abalone, Haliotis cracherodii, with Withering Syndrome

Journal of Invertebrate Pathology

Research paper thumbnail of Effects of the corticosteroid kenalog on the carrier state of juvenile channel catfish exposed to Edwardsiella ictaluri

Studies were conducted on the carrier state following experimentally induced infections of Edward... more Studies were conducted on the carrier state following experimentally induced infections of Edwardsiella ictaluri in juvenile channel catfish Ictalurus punctatus. The cumulative mortality of fish exposed to the bacterium at 1.0 x 10 8 colony-forming units/mL for 30 s was 71% after 3 weeks at a water temperature of 25°C The bacterium could not be recovered from the posterior kidneys of 10 fish examined 1, 2, or 3 months after initial exposure, but antibodies were still evident at each sampling. However, the bacterium was isolated from 6 of 20 fish at 4 months postexposure, 14 d after intraperitoneal injection with the corticosteroid Kenalog (0.8 mg/g fish).

Research paper thumbnail of Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebrali... more A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3Ј end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.

Research paper thumbnail of Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

Parasitology Research

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD)... more Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 lm and height of 14 lm, and possessed two soft valves surrounding two spherical polar capsules (2 lm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 lm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.

Research paper thumbnail of Subclinical Mycobacterium infections in wild delta smelt

This study describes the occurrence and magnitude of latent mycobacterial infections and other pa... more This study describes the occurrence and magnitude of latent mycobacterial infections and other pathogens in delta smelt (Hypomesus transpacificus), an endemic fish in the San Francisco Estuary (SFE). Our current knowledge on subclinical infections is characterized by the presence of mycobacterial DNA and absence of clinical signs and Mycobacterium. In our previous studies however, we showed that handling and increased water temperature, stress factors frequently encountered in captivity, have altered infections from benign to severe mycobacteriosis. In the current study, sub adult and adult smelt captured from August 2011 to May 2012 in the SFE were analyzed for presence of bacterial and viral agents (n=741) using standard isolation and culture techniques and Mycobacterium qPCR (n=237). Although mycobacteriosis, Mycobacterium, and virus were not observed in all of the fish examined, 96% of the smelt were positive for mycobacterial DNA. Higher pathogen scores and mycobacterial gene c...

Research paper thumbnail of Nucleic Acid — Based Methods for Detection of Myxobolus Cerebralis

Reviews: Methods and Technologies in Fish Biology and Fisheries, 2002

ABSTRACT Abstract Much of our current knowledge of myxosporean parasites stems from the study of ... more ABSTRACT Abstract Much of our current knowledge of myxosporean parasites stems from the study of M. cerebralis. It was the work of Maria Markiw and Ken Wolf that produced the revelation that alternate hosts are required for the completion of the parasitic life cycle for myxozoans. Spores produced in the oligochaete host, Tubifex tubifex, were found to be infectious for the alternate host, a salmonid fish (Markiw and Wolf, 1983; Wolf and Markiw, 1984, Wolf et al., 1986). The degree to which the disease develops in the fish is dependent on the species of salmonid (see below, Table 1). It is the mature spores of the myxosporean stage that are required for specific diagnosis of diseases of myxosporean etiology. The time required to complete sporogenesis in either host generally takes about 90 days, but can be influenced by temperature (El-Matbouli et al., 1999). This has ramifications for those testing exposed fish by PCR. Wild fish tested after an unknown elapsed time after exposure can not be sampled in the same way as sentinel fish left in the water for brief durations. This is discussed in more detail below.

Research paper thumbnail of Experimental Horizontal Transmission of Enterocytozoon salmonis to Chinook Salmon, Oncorhynchus tshawytscha

The Journal of Protozoology, 1992

Research paper thumbnail of Production of viable cultures of Flavobacterium psychrophilum: approach and control

Research in Microbiology, 1999

Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid ... more Although the fish pathogen Flavobacterium psychrophilum is a major source of concern in salmonid hatcheries, few studies have been conducted on its pathogenicity. Difficulties are often experienced when trying to control or quantify standard procedures for in vitro culture of the bacterium. Plate enumeration and counting chamber enumeration combined with epifluorescent microscopy with fluorescent dyes determined that no more than 25% of the bacterial cells present in the cultures were able to produce colonies on agar media. This was strongly dependent upon different medium components. Tryptone-enriched Anacker and Ordal medium proved more suitable than tryptone-yeast extract-salts with skimmed milk. Adding horse serum and trace elements in controlled proportions offered the most reproducible results. Viable but nonculturable forms were apparently not responsible for the difficulties in production of F. psychrophilum, but the cells were highly susceptible to osmotic conditions. Improvements in the media and careful handling of the bacteria in isotonic suspension media resulted in predictable production of viable bacteria and allowed an absorbance/colony-forming-units relation curve to be established. © Elsevier, Paris Flavobacterium psychrophilum / bacterial growth / microbial nutrition / viability

Research paper thumbnail of Identification of harmful cyanobacteria in the Sacramento-San Joaquin Delta and Clear Lake, California by DNA barcoding

SpringerPlus, 2013

Accurate identification of cyanobacteria using traditional morphological taxonomy is challenging ... more Accurate identification of cyanobacteria using traditional morphological taxonomy is challenging due to the magnitude of phenotypic plasticity among natural algal assemblages. In this study, molecular approach was utilized to facilitate the accurate identification of cyanobacteria in the Sacramento-San Joaquin Delta and in Clear Lake in Northern California where recurring blooms have been observed over the past decades. Algal samples were collected from both water bodies in 2011 and the samples containing diverse cyanobacteria as identified by morphological taxonomy were chosen for the molecular analysis. The 16S ribosomal RNA genes (16S rDNA) and the adjacent internal transcribed spacer (ITS) regions were amplified by PCR from the mixed algal samples using cyanobacteria generic primers. The obtained sequences were analyzed by similarity search (BLASTN) and phylogenetic analysis (16S rDNA) to differentiate species sharing significantly similar sequences. A total of 185 plasmid clones were obtained of which 77 were successfully identified to the species level: Aphanizomenon flosaquae, Dolichospermum lemmermannii (taxonomic synonym: Anabaena lemmermannii), Limnoraphis robusta (taxonomic synonym: Lyngbya hieronymusii f. robusta) and Microcystis aeruginosa. To date, Dolichospermum and Limnoraphis found in Clear Lake have only been identified to the genus lavel by microscopy. During the course of this study, morphological identification and DNA barcoding confirmed A. flos-aquae as the predominant cyanobacterium in the Sacramento-San Joaquin Delta indicating a shift from M. aeruginosa that have dominated the blooms in the past decade. Lastly, the species-specific identification of Limnoraphis robusta in Clear Lake is another significant finding as this cyanobacterium has, thus far, only been reported in Lake Atitlan blooms in Guatemala.

Research paper thumbnail of Henneguya sp. in yellowfin goby Acanthogobius flavimanus from the San Francisco Estuary

SpringerPlus, 2013

Myxozoan spores were observed in yellowfin goby Acanthogobius flavimanus collected from Suisun Ma... more Myxozoan spores were observed in yellowfin goby Acanthogobius flavimanus collected from Suisun Marsh, San Francisco Estuary (SFE). Although histopathological changes associated with the parasite were not observed, the spores formed plasmodia that partially blocked the gastric and intestinal mucosa and gut lumen and may affect the perfomance and survival of the yellowfin goby. Morphological features of the spores resembled Henneguya sp. and molecular analysis of the 18S ribosomal DNA (Domain III) confirmed close similarity to H. rhinogobii and H. pseudorhinogobii isolated from the Japanese freshwater goby. The yellowfin goby myxozoan however, is likely an undescribed species based on phylogenetic analysis and morphologic features. Detailed description of vegetative and spore stages are currently lacking for proposal to a new species of Henneguya. A specific PCR test was developed, which confirmed a 100% prevalence of the parasite among randomly collected gobies in group 1 (N = 30) and group 2 (N = 15) at termination of the study at one month in captivity. The myxozoan was also detected from 18 gobies (12%) that died in the first group within two weeks in captivity. Apparently healthy gobies that served as controls did not reveal the presence of the myxozoan by PCR. This study documents the occurrence of a potentially new species of myxozoan in the yellowfin goby and underscores the detection of a parasitic infection in an introduced fish in the SFE. Although the pathogenesis of the myxozoan was not assessed and the prevalence as reported here is restricted to a comparatively small collection site in Suisun slough, the reemergence, identification, and ecological relevance of the parasite on goby populations in the SFE may be investigated in the future using the specific diagnostic tool developed in this study.

Research paper thumbnail of Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia (Oreochromis niloticus) cultured in net cages in Lake Sentani, Papua, Indonesia

SpringerPlus, 2014

Infections with Streptococcus spp. were observed in Nile tilapia cultured in net cages in Lake Se... more Infections with Streptococcus spp. were observed in Nile tilapia cultured in net cages in Lake Sentani, Papua, Indonesia. Clinical signs included exophthalmia, erratic swimming, ascites in abdominal cavity, and external hemorrhages. Four types of bacterial colonies (SK, K10, P20, and M12) were isolated from the brain, kidney, and eyes. Based on phenotypic and genetic (16S rDNA sequencing) characteristics, the isolates were identified as Streptococcus iniae (SK), Streptococcus agalactiae (K10 and P20) and Lactococcus garvieae (M12). The latter species has not been previously isolated or reported from fish streptococcosis in Indonesia. Intraperitoneal injection of healthy tilapia with the bacterial species caused significant morbidity (70%) within 3 days and 100% mortality at 6 days post injection. Experimental infections and reisolation of the bacteria from morbid and dead fish suggest they are the causative agents of streptococcosis, which rendered high mortality among cage cultured Nile tilapia in Lake Sentani. Our results suggest the need for developing diagnostic tools for accurate identification of the agents of streptococcosis. As tilapia aquaculture continues to expand as a means of food production and livelihood in Indonesia, it becomes crucial to ensure that fish resources are monitored and protected from the adverse effects of infectious diseases.

Research paper thumbnail of Sublethal dietary effects of microcystin producing Microcystis on threadfin shad, Dorosoma petenense

Toxicon, 2012

Please cite this article as: Acuña, S., Baxa, D., Teh, S., Sublethal dietary effects of microcyst... more Please cite this article as: Acuña, S., Baxa, D., Teh, S., Sublethal dietary effects of microcystin producing Microcystis on threadfin shad, Dorosoma petenense, Toxicon (2012),

Research paper thumbnail of Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

Parasitology Research, 2004

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD)... more Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T. bryosalmonae found in the bryozoan host. Spores found in the urine were subspherical, with a width of 16 lm and height of 14 lm, and possessed two soft valves surrounding two spherical polar capsules (2 lm in diameter) and a single sporoplasm. The absence of hardened valves is a distinguishing characteristic of the newly established class Malacosporea that includes T. bryosalmonae as found in the bryozoan host. The parasite in the urine of rainbow trout possessed only two polar capsules and two valve cells compared to the four polar capsules and four valves observed in the spherical spores of 19 lm in diameter from T. bryosalmonae from the bryozoan host. Despite morphological differences, a relationship between the spores in the urine of rainbow trout and T. bryosalmonae was demonstrated by binding of monoclonal and polyclonal antibodies and DNA probes specific to T. bryosalmonae.