Jay F. Levine

Prevalence of Salmonella spp. in Oysters in the United States

ABSTRACT Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.

The Dog as a Sentinel for Human Infection: Prevalence of Borrelia burgdorferi C6 Antibodies in Dogs from Southeastern and Mid-Atlantic States

Lyme disease is the most frequently reported human vector-associated disease in the United States. Infection occurs after the bite of an Ixodid tick that is infected with Borrelia burgdorferi. Dogs have often been reported to serve as effective sentinel animals to assess the risk of human B. burgdorferi infection. Based on published data of human Lyme disease case numbers and our clinical impressions, we hypothesized that canine exposure to B. burgdorferi would be lower in North Carolina when compared to the exposure in Virginia, Maryland, and Pennsylvania. To address this hypothesis, we evaluated B. burgdorferi exposure status utilizing a specific and sensitive C6 peptide-based enzyme-linked immunosorbent assay. Our convenience sample included 1,666 canine serum samples submitted to the Vector Borne Disease Diagnostic Laboratory from North Carolina (n = 987), Virginia (n = 472), Maryland (n = 167), and Pennsylvania (n = 40). Comparisons among states were made using the Chi-square test or the Fishers exact test; p-values were adjusted for multiple comparisons using the Bonferroni correction. A Chi-square test for trend was used to determine if there was an increase in the frequency of seroreactors associated with the geographical origin of the samples. The proportion of seroreactive dogs in North Carolina was markedly lower (p < 0.008) than that observed in dogs from Virginia, Maryland, and Pennsylvania. These results support the hypothesis that B. burgdorferi transmission seems to occur infrequently in North Carolina dogs as compared to dogs residing in other southeastern and mid-Atlantic states. Furthermore, they support the utility of dogs as a sentinel to characterize the risk of B. burgdorferi transmission to humans in a defined geographical location.

Relative utilization of reptiles and rodents as hosts by immature Ixodes scapularis (Acari: Ixodidae) in the coastal plain of North Carolina, USA

The interaction of immature black-legged ticks, Ixodes scapularis, with reptiles and rodents was investigated in various woodland habitats in the coastal plain of North Carolina. Reptiles were sampled from April 1 to September 30, 1991. No ticks were found on 95 specimens representing 16 species of snakes. Ticks were found on 54 (36.7%) of 147 lizards. I. scapularis was the only tick recovered from lizards. Some lizards were collected in drift fence traps each month of the study except August. Capture rates averaged one lizard per 16 trap-days. Larvac and nymphs of I. scapularis were removed from the southeastern five-lined skink (Eumeces inexpectatus), the ground skink (Scincella lateralis), the broad-headed skink (E. laticeps) and the eastem glass lizard (Ophisaurus ventralis), but ticks were not found on three other lizard species. Tick infestation rates and loads for parasitized species are presented. Ticks were almost exlusively attached at the base or in the axils of forelimbs of skinks and in the lateral grooves of eastern glass lizards. Rodents were live-trapped at sites where lizards were sampled and at other sites from 1 July, 1990 to 30 January, 1992. Capture rates averaged one rodent per 47 trap-nights. Ticks were found on 23 (17.8%) of 129 animals inspected. Five species of rodents were examined but only four species were found to be tick-infested. In contrast to lizards, few I. scapularis were collected. Rodents, principally the white-footed mouse (Peromyscus leucopus) and cotton mouse (P. gossypinus) were most frequently infested with immature American dog ticks, Dermacentor variabilis, during winter and early spring months. Burdens of D. variabilis on these rodents averaged 0.3 ticks per rodent. Effects of the diversion of ticks from feeding on Peromyscus mice on the transmission of the Lyme disease spirochete are discussed.

Characterization of the Aphanomyces species involved with ulcerative mycosis (UM) in Menhaden.

Aphanomyces and Saprolegnia were isolated from characteristic lesions of Ulcerative Mycosis (UM) on Atlantic menhaden and other fish. One of the Aphanomyces isolates was selected for cultural studies which revealed that the fungus was stimulated to increased vegetative growth and improved zoosporulation by low levels of NaCl. The salt tolerance exhibited by this fungus surpassed the known recorded limits for Oomycetes in regard to zoosporogenesis. The relationship of the fungal isolates to the lesions found on fish and the association of prevailing climatological conditions with the disease suggest a complex interaction between the fungus, possibly pre-stressed fish, and the salinity of the estuary systems of North Carolina.

Effects of opioids and anesthetic drugs on body temperature in cats.

OBJECTIVE To determine which class of opioid alone or in conjunction with other anesthetic drugs causes post-anesthetic hyperthermia in cats. STUDY DESIGN Prospective, randomized, crossover study. ANIMALS Eight adult, healthy, cats (four spayed females and four castrated males weighing 3.8 +/- 0.6 kg). METHODS Each cat was instrumented with a wireless thermistor in the abdominal cavity. Temperature in all phases was recorded every 5 minutes for 5 hours. Population body temperature (PBT) was recorded for approximately 8 days. Baseline body temperature is the final 24 hours of the PBT. All injectable drugs were given intramuscularly. The cats were administered drugs in four phases: 1) hydromorphone (H) 0.05, 0.1, or 0.2 mg kg(-1); 2) morphine (M) (0.5 mg kg(-1)), buprenorphine (BUP) (0.02 mg kg(-1)), or butorphanol (BUT) (0.2 mg kg(-1)); 3) ketamine (K) (5 mg kg(-1)) or ketamine (5 mg kg(-1)) plus hydromorphone (0.1 mg kg(-1)) (KH); 4) isoflurane in oxygen for 1 hour. Fifteen minutes prior to inhalant anesthetic, cats received either no premed (I), hydromorphone (0.1 mg kg(-1)) (IH), or hydromorphone (0.1 mg kg(-1)) plus ketamine (5 mg kg(-1)) (IHK). RESULTS Mean PBT for all unmedicated cats was 38.9 +/- 0.6 degrees C (102.0 +/- 1 degrees F). The temperature of cats administered all doses of hydromorphone increased from baseline (p < 0.03) All four opioids (H, M, BUP and BUT) studied increased body temperature compared with baseline (p < 0.005). A significant difference was observed between baseline temperature values and those in treatment KH (p < 0.03). Following recovery from anesthesia, temperature in treatments IH and IHK was different from baseline (p < 0.002). CONCLUSIONS AND CLINICAL RELEVANCE All of the opioids tested, alone or in combination with ketamine or isoflurane, caused an increase in body temperature. The increase seen was mild to moderate (<40.1 degrees C (104.2 degrees F) and self limiting.

Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L

Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 degrees C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10(1) TCID50 mL(-1). The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV.

TICK-RACCOON ASSOCIATIONS AND THE POTENTIAL FOR LYME DISEASE SPIROCHETE TRANSMISSION IN THE COASTAL PLAIN OF NORTH CAROLINA

Raccoons (Procyon lotor) were live-trapped and examined for ticks from July 1990 to July 1993 in the coastal plain of North Carolina on Marine Corps Base, Camp Lejeune, North Carolina (USA). Five species of ixodid ticks were found on 351 (78%) of 449 raccoons. Amblyomma americanum was the most abundant tick found on raccoons. Dermacentor variabilis, Ixodes texanus, and Ixodes scapularis were frequently collected, while Ixodes cookei were rarely collected from raccoons. Tick burdens were not affected by the age, sex, or trap location of captured raccoons. Ticks parasitizing raccoons had varying seasonal patterns of abundance. Amblyomma americanum were generally collected from raccoons year around, but infestation intensities were greatest in summer from June to September. Dermacentor variabilis adults were most abundant in mid-summer while peak numbers of larvae were collected in the fall. Infestation intensities of Ixodes texanus larvae were greatest in fall and winter months while nymphs were most abundant in winter and spring. No males were collected from raccoons, but females were most frequently collected in the spring and declined in abundance in the summer with no specimens collected in the fall or winter. Numbers of I. scapularis adults appeared to reach peak numbers in the fall while larvae and nymphs were most abundant on raccoons in winter. Spirochetes, Borrelia burgdorferi, were identified in a small percentage (0.2%) of host-seeking A. americanum nymphs and adults, and I. scapularis adults by immunofluorescent antibody assays. Similarly, a small percentage (1.9%) of host-associated A. americanum, D. variabilis, I. texanus and I. cookei contained B. burgdorferi. Borrelia burgdorferi spirochetes were cultured from the blood of 23 (26%) of 87 raccoons.

Effect of hyperthermia in vitro on stress protein synthesisand accumulation in oyster haemocytes

Abstract Haemocytes comprise a major component of the non-specific defence mechanismsin marine bivalves. Induction of stress protein (SP) synthesis and accumulation of SPs was studied in vitro to define the metabolic response of oyster (C. virginica) haemocytes to acute temperature changes. An acute cold shock to near freezing had no significant effect on protein synthesis. However, a comparable heat shock of 20–28° C above the acclimation temperature of 20° C provoked a robust increase in synthesis of several SPs, especially those of about 70 (SP70), 37, 34 and 32 kDa. This response persisted for at least 24 h, during which time both isoforms of SP70-like immunoreactivity accumulated. Concomitantly, there was a decrease in the synthesis, but not in the level, of an actin-like protein of about 45 kDa. The extent of SP synthesis induction also was directly dependent on the duration of the preceding hyperthermia. Extending the duration of heat shock necessitated a longer recovery period, during which time amino acid incorporation returned towards or beyond the initial control values and cell viability was retained. After a severe heat shock at 46° C for 1 h, the predominant protein made for several days was SP70, which is known to be essential for stress tolerance in other biological systems. The results suggest that oyster haemocytes are remarkably resilient, and that SPs may contribute to their ability to resist or repair heat-evoked damage. This molecular adaptability could permit them to maintain immune surveillance during or immediately following serious threats to survival of these sessile ectotherms.

An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts

The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod‐borne disease of a variety of vertebrates and the most prevalent arthropod‐borne disease of humans in the United States. In order to understand better the normal life cycle of B. burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages. By examining populations of B. burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B. burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined. Distinct B. burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B. burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations. These two profiles constituted the phenotypical signature of the population. In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection. At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC. In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full‐length and presumably transcriptionally active state. In the case of OspB, two alleles were again identified. However, their differences were minor and their relationship to OspB antigenic variation more complicated. In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted. Selection of distinct populations in the tick may be responsible for the microorganisms ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.

Sea Cucumber in the Mediterranean: A Potential Species for Aquaculture in the Mediterranean

Sea cucumbers are now a global product known by the Chinese as “beche-de-mer,” “trepang,” or “haisom” and are almost unexploited resources in the Mediterranean region. They are currently harvested in the Mediterranean region and exported from Turkey, and the sea cucumber species is currently the most expensive seafood in Catalonia and the Balearic Islands. Considered a delicacy in many Asian countries, there is also growing interest in their medicinal and nutraceutical value. Their successful utilization in the Mediterranean Sea will require continued refinement of aquaculture techniques and development of processes for enhancing the scale of production. Even though the majority of Mediterranean species are not considered high-value products, it is clear that the future of sea cucumber farming has interesting lucrative potential in the Mediterranean and that turning sea cucumbers into aquaculture value-added products could have profitable economic benefits in this region.