John P Hawke | Louisiana State University (original) (raw)

Papers by John P Hawke

Antimicrobial resistance is a major public health concern. Therefore, it is critical to have stan... more Antimicrobial resistance is a major public health concern. Therefore, it is critical to have standard methods and criteria, called epidemiological cutoff values (ECVs, a.k.a. ECOFFs) to monitor for the development of resistance. To develop ECVs for the fish pathogen Flavobacterium columnare, we tested the susceptibility of 229 isolates using the standard broth dilution susceptibility testing method in the Clinical Laboratory and Standards Institute (CLSI) VET04-A2 guideline. Using the method, we determined the minimum inhibitory concentration (MIC) against 10 antibiotics: ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole. We analyzed frequency distributions for each antibiotic to estimate an ECV which separates the wild-type isolates without resistance from the non-wild-type isolates that have developed resistance using two statistical methods: ECOFFinder (Turnidge et al., 2006) and Normalized Resistance Interpretation (Kronvall, 2010). ECVs were estimated for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline. The estimated ECVs showed that the isolates categorized similarly among the quinolones (enrofloxacin, flumequine, and oxolinic acid) as wild-type and non-wild-type. Therefore, laboratories could potentially use one of these compounds to monitor quinolone resistance. An ECV for the potentiated sulfonamides could not be estimated because the potentiators may be masking resistance to sulfonamide. Similarly, an ECV for gentamicin was not evaluated because there are currently no existing quality control ranges to verify the results. The CLSI Veterinary Antimicrobial Susceptibility Testing subcommittee is currently reviewing the data and the ECVs proposed from our analysis. If adopted, the ECVs will be included in the next revision of the VET04-supplement document.

Journal of Aquatic Animal Health, Jun 1, 2015

We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybri... more We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybrid Striped Bass (Striped Bass Morone saxatilis × White Bass M. chrysops) and wild and cultured Gulf Killifish Fundulus grandis from coastal waters of the U.S. Gulf of Mexico (Gulf coast) and compared those isolates to strains from tilapias Oreochromis spp. reared in Mississippi, Thailand, Ecuador, and Honduras and to the original Gulf coast strain identified by Plumb et al. ( 1974 ). The isolates were subjected to phylogenetic, biochemical, and antibiotic susceptibility analyses. Genetic analysis was performed using partial sequence comparison of (1) the 16S ribosomal RNA (rRNA) gene; (2) the sipA gene, which encodes a surface immunogenic protein; (3) the cspA gene, which encodes a cell surface-associated protein; and (4) the secY gene, which encodes components of a general protein secretion pathway. Phylogenies inferred from sipA, secY, and cspA gene sequence comparisons were more discriminating than that inferred from the 16S rRNA gene sequence comparison. The U.S. Gulf coast strains showed a high degree of similarity to strains from South America and Central America and belonged to a unique group that can be distinguished from other group B streptococci. In agreement with the molecular findings, biochemical and antimicrobial resistance analyses demonstrated that the isolates recovered from the U.S. Gulf coast and Latin America were more similar to each other than to isolates from Thailand. Three laboratory challenge methods for inducing streptococcosis in Gulf Killifish were evaluated-intraperitoneal (IP) injection, immersion (IMM), and immersion plus abrasion (IMMA)-using serial dilutions of S. agalactiae isolate LADL 97-151, a representative U.S. Gulf coast strain. The dose that was lethal to 50% of test fish by 14 d postchallenge was approximately 2 CFU/fish via IP injection. In contrast, the fish that were challenged via IMM or IMMA presented cumulative mortality less than 40% by 14 d postchallenge. Received July 31, 2014; accepted March 11, 2015.

Journal of Fish Diseases, Dec 14, 2015

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Is... more The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm-raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive-sequence-mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep-PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep-PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.

Journal of Aquatic Animal Health, Sep 1, 2003

Journal of Clinical Microbiology, Sep 1, 2003

Quality control (QC) ranges for disk diffusion susceptibility testing of aquatic bacterial isolat... more Quality control (QC) ranges for disk diffusion susceptibility testing of aquatic bacterial isolates were proposed as a result of a multilaboratory study conducted according to procedures established by the National Committee for Clinical Laboratory Standards (NCCLS). Ranges were proposed for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 at 22 and 28°C for nine different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, ormetoprim-sulfadimethoxine, and trimethoprim-sulfamethoxazole). All tests were conducted on standard Mueller-Hinton agar. With >95% of all data points fitting within the proposed QC ranges, the results from this study comply with NCCLS guidelines and have been accepted by the NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing. These QC guidelines will permit greater accuracy in interpreting results and, for the first time, the ability to reliably compare susceptibility test data between aquatic animal disease diagnostic laboratories.

PubMed, 1997

Enteric septicaemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most serious diseas... more Enteric septicaemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most serious disease affecting commercial catfish culture in the United States. ESC is generally an acute septicaemia that develops very quickly, especially in the temperature range of 22-28 degrees C, with a more chronic disease presentation outside this range. The ability of E. ictaluri to avoid the host's immune system and proliferate into a systemic infection is impressive. Catfish kidney tissue cultured positive for E. ictaluri as soon as 15 minutes following gastric lavage and signs of disease are observed microscopically within two days of immersion challenge, with reported mortalities as early as five days following immersion challenge. Analysis of E. ictaluri antigens by several investigators using SDS-PAGE and colorimetric western blotting with immune catfish has identified as many as 15 immunogenic bands. Analysis using two-dimensional SDS-PAGE and chemiluminescent western blotting identified 14 bands and 25 spots as consistently immunogenic. The strongest immunodominant antigens were reported as 34-37 KD and 60 KD, depending on the study. Lipopolysaccharide is the only purified component of E. ictaluri tested for fish vaccination, and results indicated that very poor protection was induced unless Freund's Complete Adjuvant was used. Because E. ictaluri strains are serologically homogeneous, most studies on vaccination have emphasized killed whole cell preparations and have delivered equivocal results. Although antibodies are produced to a variety of preparations, a positive antibody response does not correlate with protection unless very high titres are achieved. Efficacy of killed products has been demonstrated in field trials, and an orally delivered product has been licensed. However, protection probably relies on booster exposure of the host to E. ictaluri during non-permissive temperatures. As a facultative intracellular pathogen, further studies on vaccination of catfish against E. ictaluri should target products and delivery methods that favour induction of cell mediated immunity.

Journal of Aquatic Animal Health, Jun 1, 1992

A selective medium was used for the isolation of Cytophaga columnaris in 154 routine cases submit... more A selective medium was used for the isolation of Cytophaga columnaris in 154 routine cases submitted to the Louisiana Aquatic Animal Diagnostic Laboratory. In 46 cases, systemic C. columnaris that would not have been detected with routine cytophaga media was isolated from diseased channel catfish Ictalurus punctatus. Strain variation in susceptibility to Romet (sulfadimethoxine and ormetoprim, 5:1) was noted. Of the 86 strains tested, 83 (97%) were regarded as susceptible. All strains tested were susceptible to Terramycin (oxytetracycline HCl). The minimal inhibitory concentration (MIC) of Romet on representative strains of C. columnaris ranged from 7.81 to 15.62 μg/mL. The MIC of Terramycin on selected strains was 0.195 μg/mL.

Journal of Fish Diseases, Aug 1, 2009

Francisella sp. is an emergent bacterial pathogen that causes acute to chronic disease in warm an... more Francisella sp. is an emergent bacterial pathogen that causes acute to chronic disease in warm and cold water cultured and wild fish species. During the past 3 years, the bacterium has been detected in tilapia, Oreochromis niloticus, cultured in Costa Rica. Infected fish presented non-specific clinical signs, such as erratic swimming, anorexia, anaemia, exophthalmia and high mortality. Upon macroscopic and microscopic examination, several internal organs (mainly spleen and kidney) were enlarged and contained white nodules. Histological examination revealed the presence of multifocal granulomatous lesions, with the presence of numerous small, pleomorphic, cocco-bacilli. The bacteria were isolated from infected tilapia on selective media and grown on several media with and without antibiotics. Specific PCR primers to the Francisella genus were used to confirm the preliminary diagnoses. In comparison with several bacterial 16S rRNA sequences, our isolate was found to share 99% identity with other Fransicella spp. isolated from fish, and more than 97% identity to the human pathogen Francisella tularensis. Koch&amp;#39;s postulates were fulfilled after experimental intraperitoneal and gill exposure challenges.

Journal of Applied Aquaculture, Mar 14, 1994

ABSTRACT Enteric septicemia of cattish (ESC), caused by the bacterium Edwardsiella ictaluri, has ... more ABSTRACT Enteric septicemia of cattish (ESC), caused by the bacterium Edwardsiella ictaluri, has become the most significant disease problem affecting the commercial channel catfish, Ictalurus punctatus, industry in the United States. Although antibiotics are used extensively for the control of ESC, there are inherent problems associated with their use. Consequently, experiments were initiated to evaluate the effectiveness of vaccination program that used immersion and oral delivery methods to administer a killed E. ictaluri vaccine to fry and fingerling channel cafish. In a preliminary pond study with laboratory challenge, mortality in a group vaccinated with a combination of immersion and oral procedures was only 5.0% in both high- and low-dose challenges. This was significantly different (P c 0.01) from non-vaccinated controls, which had 46.7%mortality in the lowdose challenge and the 6 1.7% mortality in the highdose challenge. This corresponds to relative percent survival (RPS) values of 89.3 and 91.9 respectively. Subsequent field trials further indicated the efficacy of a vaccination program for the prevention of ESC in channel catfish. In 1987-1988, a field study was conducted using 12 commercial ponds, with three replicates of four treatments. The four treatments included vaccination by immersion only, oral only, a combination of both immersion and oral procedures, and non-vaccinated conwols. Relative percent survival was 57.4 for the immersion only treatment, 50.3 for the oral only treatment, and 53.5 for the combination immersion and oral treatment. In 1989-1990, no significant difference was found between vaccinated and non-vaccinated fish. However, in 1989-1990, a vaccine-oil emulsion was topcoated on a floating feed, rather than incorporating vaccine in a sinking pellet. In 1990-1991, overall mortality in vaccinated fish was significantly less (P < 0.05) than non-vaccinated fish, with 41.2% mortality in vaccinates compared to 63.5% in non-vaccinated fish, for an RPS of 35.1. In examining RPS values for individual farms, two farms had excellent results, with RPS values of 81.3 and 76.9; two farms had only moderate success, with RPS values of 26.6 and 15.4; and one location had greater mortality in the vaccinated fish than in the non-vaccinated fish. However, that farm had only two ponds in the study and experienced significant losses to proliferative gill disease in the pond with vaccinated fish.

International Journal for Parasitology, 2015

Since June 2012, samples of wild caught white shrimp, Litopenaeus setiferus, from the Gulf of Mex... more Since June 2012, samples of wild caught white shrimp, Litopenaeus setiferus, from the Gulf of Mexico, Plaquemines and Jefferson Parishes (Louisiana, USA) with clinical signs of microsporidiosis have been delivered to the Louisiana Aquatic Diagnostic Laboratory for identification. Infection was limited predominantly to female gonads and was caused by a microsporidium producing roundish pansporoblasts with eight spores (3.6 Â 2.1 lm) and an anisofilar (2-3 + 4-6) polar filament. These features allowed identification of the microsporidium as Agmasoma penaei Sprague, 1950. Agmasoma penaei is known as a microsporidium with worldwide distribution, causing devastating epizootic disease among wild and cultured shrimps. This paper provides molecular and morphological characterisation of A. penaei from the type host and type locality. Comparison of the novel ssrDNA sequence of A. penaei from Louisiana, USA with that of A. penaei from Thailand revealed 95% similarity, which suggests these geographical isolates are two different species. The A. penaei sequences did not show significant homology to any other examined taxon. Phylogenetic reconstructions using the ssrDNA and alpha-and beta-tubulin sequences supported its affiliation with the Clade IV Terresporidia sensu Vossbrink 2005, and its association with parasites of fresh and salt water crustaceans of the genera Artemia, Daphnia and Cyclops.

Journal of Aquatic Animal Health, Jun 1, 2003

ABSTRACT The purpose of this study was to use an in vitro assay to evaluate the ability of Photob... more ABSTRACT The purpose of this study was to use an in vitro assay to evaluate the ability of Photobacterium damselae subsp. piscicida to survive and replicate within macrophages obtained from hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). The results indicated that the number of bacteria recovered from macrophages after 3, 6, 12, and 18 h of incubation increased significantly from time 0 (105, 510, 1,091 and 1,385%, respectively). Replication of the intracellular bacteria was observed at a very low multiplicity of infection (1 bacterium to 100 macrophages). In contrast, the number of Escherichia coli recovered from macrophages declined 47, 80, 91, and 96%, respectively. Light and electron microscopy confirmed the internalization, uptake, and multiplication of bacteria within spacious, clear vacuoles in the macrophages. Using acid phosphatase as a lysosomal marker, we provide evidence that P. damselae subsp. piscicida inhibits phagolysosomal fusion. This study demonstrates that P. damselae subsp. piscicida is a very successful facultative, intracellular pathogen that can survive and multiply within hybrid striped bass macrophages.

Veterinary Pathology, Jun 28, 2023

Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus describ... more Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus described in the Caribbean spiny lobster, Panulirus argus. PaV1 infection in decapod species that commonly co-occur with P. argus, including the spotted spiny lobster Panulirus guttatus, has not been previously described. In 2016, 14 Caribbean and 5 spotted spiny lobsters were collected near Summerland Key, Florida, to supplement the resident population of the Audubon Aquarium of the Americas in New Orleans, Louisiana. After 5 months in quarantine, Caribbean and spotted spiny lobsters began to exhibit clinical signs of lethargy and dying in the molt. Initial histologic evaluation revealed intranuclear inclusion bodies in circulating hemocytes in the spongy connective tissue of the epidermis, suggesting a viral infection. Samples of hepatopancreas and hemolymph from deceased Caribbean and spotted spiny lobsters tested negative for white spot syndrome virus and positive for PaV1 using real-time quantitative polymerase chain reaction (qPCR). Intranuclear, eosinophilic to amphophilic, Cowdry type A inclusion bodies observed primarily within fixed phagocytes and circulating hemocytes in the hepatopancreas of freshly euthanized Caribbean spiny lobsters were consistent with PaV1 infection. Transmission electron microscopy revealed that hemocytes associated with hepatopancreatic tubules contained viral inclusions with location, size, and morphology consistent with previously described PaV1 infection. These findings highlight the significance of using molecular diagnostics in conjunction with histopathology and electron microscopy in the investigation and diagnosis of PaV1 in spiny lobsters. Further study is required to investigate the relationship of PaV1-associated mortality events and microscopic lesions in the spotted spiny lobster.

Genome Announcements, Dec 31, 2015

Here, we report the draft genome sequences of Edwardsiella ictaluri strains LADL11-100 and LADL11... more Here, we report the draft genome sequences of Edwardsiella ictaluri strains LADL11-100 and LADL11-194, two isolates from natural outbreaks of edwardsiellosis in the zebrafish Danio rerio, as well as the sequences of the plasmids carried by the zebrafish strain of E. ictaluri.

gram-negative bacterium Edwardsiella ictaluri, is one of the most important diseases of farm-rais... more gram-negative bacterium Edwardsiella ictaluri, is one of the most important diseases of farm-raised channel catfish (Ictalurus punctatus). ESC accounts for 20 to 30 percent of all disease cases submitted to fish diagnostic laboratories in the southeastern United States. In Mississippi, where catfish make up the majority of case submissions, ESC has accounted for as much as 47 percent of the yearly total. Other members of the genus, Edwardsiella tarda and the newly discovered E. piscicida, make up a much smaller portion of the total bacterial cases (about 2 percent). Economic losses resulting from ESC are in the millions of dollars yearly but have leveled off in recent years with the downsizing of the catfish industry. The causative bacterium of ESC, Edwardsiella ictaluri, is strictly a pathogen of fish and does not infect mammals or humans so there is no concern of zoonotic infection. ESC was first recognized as a new infectious disease of pond-raised channel catfish in 1976 from th...

Journal of Aquatic Animal Health, 2018

Fifteen adult koi (Cyprinus carpio) simultaneously developed white cutaneous proliferations affec... more Fifteen adult koi (Cyprinus carpio) simultaneously developed white cutaneous proliferations affecting up to 30% of their bodies. Onset of lesions (September, 2014) was associated with return to a remodeled backyard water garden after temporarily being maintained in a plastic swimming pool. A single water temperature taken during the outbreak read 21°C on November 17, 2014. The water garden was under no extrinsic heat source, with average ambient temperatures ranging from 26.4-9.4 °C during the course of the outbreak (September 2014-January 2015, respectively). Representative skin biopsies were obtained from two koi; histologic features included severe epidermal hyperplasia, dysplasia, keratinocyte apoptosis, decreased and haphazardly distributed goblet cells with absence of club cells, keratinocyte hydropic degeneration, and moderate infiltration by lymphocytes and eosinophilic granular cells. Ultrastructural findings included intranuclear non-enveloped hexagonal nucleocapsids, and abundant cytoplasmic enveloped virions morphologically consistent with the Alloherpesviridae family. PCR was performed on formalin fixed paraffin embedded shavings from the two biopsied koi targeting the thymidine kinase gene of Cyprinid herpesvirus 1 (CyHV-1). In correlation with the aforementioned findings, results are consistent with an outbreak of CyHV-1 in a population of adult koi.

Protistology, 2016

During the years 2012-2014 samples of Atlantic white shrimp Litopenaeus setiferus collected offsh... more During the years 2012-2014 samples of Atlantic white shrimp Litopenaeus setiferus collected offshore near Plaquemines Parish, Louisiana (USA) were submitted to the Louisiana Aquatic Diagnostic Laboratory with "tumor like" lesions in the exoskeleton and internal organs. Light (LM) and electron microscopy (EM) examination indicated that 19 shrimp were infected with the microsporidium Agmasoma penaei (=Thelohania penaei Sprague, 1950, n. comb. Hazard and Oldacre, 1975). The Louisiana isolate of A. penaei was morphologically and genetically characterized previously (Sokolova et al., 2015). In 15 of 19 shrimp the infection was limited to subcutaneous and ovarian tissue, and in 4 shrimp musculature also seemed to be affected looking unusually whitish and/or opaque. According to our observations, A. penaei did not occur in muscle tissue, however, records on tissue tropism of A. penaei obtained by earlier authors, occasionally included skeletal muscles. Upon examination of skeletal muscles of these 4 shrimp by LM we found out that the tissue contained microsporidia-like individual spores of much smaller (2.0 × 1.1µm) size, than those of A. penaei. Electron microscopy and molecular analysis indicated that L. setiferus was co-infected by another microsporidium, structurally and genetically similar to Perezia nelsoni Sprague 1950 from the Mississippi coast of the Gulf of Mexico (Cheney et al., 2000; Canning et al., 2002; Genebank reference: "Pleistophora sp. LS" Accession # AJ252959). Persistent co-infection of white shrimp with two microsporidia with different tissue tropism reported herein, suggests that infection of muscles previously attributed to A. penaei, might be due to the overlooked P. nelsoni. The paper describes fine morphology and phylogenetic relations of P. nelsoni from a new location. The novel SSUrDNA sequence was deposited in Genebank, under accession number KX85642.

An offshore ocean cage feeding trial was conducted by the American Soybean Association Internatio... more An offshore ocean cage feeding trial was conducted by the American Soybean Association International Marketing (ASA-IM) program in southern Hainan Province, China, in 2007 to compare pompano performance with feeds having standard and low fishmeal inclusion rates. The trial compared pompano growth and feed conversion efficiency with the standard ASA-IM 43/12 pompano growout diet, in which 45% of the protein was supplied by fishmeal, and a nutritionally equivalent diet in which dehulled soybean meal and soy protein concentrate were the major protein contributors, and in which fishmeal supplied only 16% of the protein. Pompano were cultured in 100-m 3 OCAT submersible offshore ocean cages at an average density of approximately 9,600 fish per cage. There was no difference in pompano performance with the two feeds. Pompano in two OCAT cages fed the ASA-IM 43/12 standard feed grew from approximately 19 g to 608 g in 146 days with >99% survival. Pompano in two OCAT cages fed the 43/12 s...

Minitransposons and delivery vehicles .. Transformation and mutagenesis protocol. Selection and e... more Minitransposons and delivery vehicles .. Transformation and mutagenesis protocol. Selection and evaluation of mutant ........ Minimal inhibitory concentration of E D D A .

Francisella asiatica is a Gram-negative, pleomorphic, facultative intracellular, bacterial pathog... more Francisella asiatica is a Gram-negative, pleomorphic, facultative intracellular, bacterial pathogen that causes acute to chronic disease in a wide variety of warm-water cultured and wild fish species. Outbreaks of francisellosis in warm water fish have been documented in Taiwan, Japan, United Kingdom, Hawaii, and Latin America (including Costa Rica) but the organism has only been reported from the United States on one occasion from hybrid striped bass in California. In 2010, the bacterium was detected from diseased tilapia by culture on cystine heart agar supplemented with hemoglobin and by utilizing an F. asiatica–specific real-time polymerase chain reaction assay. The tilapia (Oreochromis niloticus) were cultured in an indoor, closed, recirculating aquaculture facility in the Midwest of the United States. The identity of isolates recovered from diseased fish was confirmed as F. asiatica by amplification and sequence comparison of the 16S ribosomal RNA and intracellular growth locu...