Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids

Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics.     

Specific serum antibody responses in channel catfish (Ictalurus punctatus) provide limited protection against Streptococcus ictaluri challenge

Passive immunization studies were conducted to determine the role of specific antibodies in immunity to Streptococcus ictaluri. Adult channel catfish (Ictalurus punctatus) were injected i.p. with tryptic soy broth as control or with 1.5 × 10(7)colony-forming units (cfu) S. ictaluri/fish at 0, 30, and 60 d, and serum was collected 90 d after the original challenge. Fish were passively immunized by i.p. injection with serum from the tryptic soy broth (TSB) control group, anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (SSI), or heat-inactivated anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (HISSI). These passively immunized fish were then challenged 72 h later with 1.5 × 10(8)cfu S. ictaluri/fish. Over 21 d, the mean cumulative percent survival was 43.3 (TSB), 63.3 (SSI), and 50.0 (HISSI). A significant difference in cumulative percent survival was noted between the TSB and the HISSI groups, and significant differences were noted between these groups and the SSI group. Serum obtained from immunized fish 72 h after passive immunization exhibited increased anti-S. ictaluri antibody levels. Twenty-one days after the challenge, the HISSI and SSI group antibody levels significantly increased above their corresponding pre-challenge levels. No significant (r(2)=0.0806; P<0.5985) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results indicate that both specific anti-S. ictaluri antibodies and non-specific immune responses are important for protection against S. ictaluri.     

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.     

Leukokinesis in bovine ostertagiasis: stimulation of leukocyte migration by Ostertagia

A blind-well chemotaxis chamber method was used to indicate migration stimulation of bovine neutrophil and eosinophil polymorphonuclear leukocytes and macrophages as related to ostertagiasis. Live exsheathed Ostertagia ostertagi 3rd-stage larvae (L3) and soluble L3 antigen (SLA), prepared by freeze thawing and sonic disruption of L3, enhanced cellular migration for eosinophils, but not for neutrophils and macrophages. Products of lymphocytes cultured with SLA for 3 to 6 hours were also examined, using lymphocytes from peripheral blood of helminth-free cattle and cattle infected with O ostertagi or Trichostrongylus axei. Lymphokines that enhanced cellular migration of neutrophils, eosinophils, and macrophages were present in culture supernatants of SLA-stimulated lymphocytes from O ostertagi-infected cattle, but not from cattle infected with T axei or helminth-free cattle. Seemingly, L3 and SLA were stimulants of eosinophil migration. Further, neutrophil, eosinophil, and macrophage migration was modulated by lymphokines produced by SLA-stimulated lymphocytes from cattle with ostertagiasis.     

The effect of selenium deficiency in goats on lymphocyte production of leukocyte migration inhibitory factor

The effect of dietary selenium on caprine leukocyte migration inhibitory factor (LMIF) production was examined $\text{in vitro}$ using lymphocytes from goats fed a diet deficient in selenium. Selenium deficiency was determined by decreased plasma glutathione peroxidase (GSH-Px). The ability of peripheral blood lymphocytes to produce LMIF induced by concanavalin A (Con A) was significantly (P < 0.05) inhibited when cells from selenium-deficient and selenium-adequate goats were compared. In contrast, no significant (P > 0.05) differences were found between lymphocytes from selenium-deficient and selenium-adequate goats for Interleukin-2 (IL-2) production and blastogenesis induced by Con A. These data suggest that selenium deficiency may selectively impair LMIF production and hence the ability of lymphocytes to modulate neutrophil migration.     

Phytophthora disease of alder (Alnus glutinosa) in France: investigations between 1995 and 1999

A severe decline of alder associated with an undescribed Phytophthora species was identified for the first time in England in 1993. No generalized decline of alder was reported in France before 1990. The first diebacks and mortalities of common alder were observed at the beginning of the 1990s, but the so‐called alder Phytophthora was not isolated in France until 1996. First, a synthesis about alder declines that were known in France before 1995 is presented. Then, a survey was established in north‐eastern France; 108 sites were visited and the alder Phytophthora was isolated from 57 of them. All the main rivers were found to be affected and damage levels are significant along some of them. The frequency of the alder Phytophthora and other fungi isolated from declining alders is discussed. Finally, information on other alder declines in France is presented region by region, and a map summarizes the known distribution of the disease. The alder Phytophthora is quite common and widespread in France, with western and north‐eastern France being especially affected; however, the number of diseased or dead trees varies greatly from one site to another. All records are from Alnus glutinosa; other Alnus species were seldom seen in the surveys.     

Organization and sequence of four flagellin-encoding genes of Edwardsiella ictaluri

Edwardsiella ictaluri , the cause of enteric septicaemia in channel catfish ( Ictalurus punctatus ), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments from a λ-ZAP phage genomic library of E. ictaluri . Four flagellin genes ( fliC1, fliC2, fliC3 and fliC4 ) are arranged in tandem within 6 kb in the E. ictaluri genome. Each flagellin-coding sequence is preceded by a σ²⁸ recognition site consensus sequence. The predicted amino acid sequences of all four flagellin proteins (between 36 and 37.5 kDa) are similar in the N-terminal (1–160 aa) and C-terminal (last 74 aa) portions and are divergent in the central portion of the proteins. Proteins encoded by flC1, fliC2 and fliC3 are more similar to each other (88–90% aa identity) than to the protein encoded by fliC4 (76–78% aa identity). basic local alignment search tool analysis of GenBank sequences showed that all flagellin aa sequences are more similar to those of Serratia marcescens (72–74% identity) than to those of Edwardsiella tarda (≤64% identity). Primary determination of E. ictaluri flagellin gene sequences facilitate advanced studies on the role of flagella in host–pathogen interaction.