Effects of Bacillus amyloliquefaciens Instead of Antibiotics on Growth Performance, Intestinal Health, and Intestinal Microbiota of Broilers.

The aim of this study was to evaluate the dietary effects of Bacillus amyloliquefaciens SC06 (SC06) instead of antibiotics on the growth performance, intestinal health, and intestinal microbiota of broilers. A total of 360 30-day-old Lingnan yellow broilers were randomly allocated into two groups with six replicates per group (30 birds per replicate). The broilers were fed either a non-supplemented diet or a diet supplemented with 108 colony-forming units lyophilized SC06 per kilogram feed for 30 days. Results showed that SC06 supplementation had no effect on the growth performance compared with that of the control group. SC06 treatment significantly (P <0.05) increased the total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) activity in the liver, and the activities of trypsin, α-amylase (AMS), and Na+K+-ATPase in the ileum, whereas it decreased (P < 0.05) lipase, gamma glutamyl transpeptidase (γ-GT), and maltase activities in the ileum. Meanwhile, SC06 treatment also improved the immune function indicated by the significantly (P < 0.05) increased anti-inflammatory cytokine [interleukin (IL)-10] level and the decreased (P < 0.05) pro-inflammatory cytokine [IL-6 and tumor necrosis factor (TNF)-α] levels in the ileum. Furthermore, we also found that SC06 enhanced the intestinal epithelial intercellular integrity (tight junction and adhesion belt) in the ileum. Microbial analysis showed that SC06 mainly increased the alpha diversity indices in the jejunum, ileum, and cecum. SC06 treatment also significantly (P < 0.05) increased the abundances of Bacteroidetes, Bacteroidales, Bacteroides, Fusobacteria, Clostridiaceae, and Veillonellaceae in the cecum and simultaneously decreased the abundances of Planococcaceae in the duodenum, Microbacteriaceae in the jejunum, and Lachnospiraceae, [Ruminococcus] and Ruminococcus in cecum. In conclusion, these results suggested that B. amyloliquefaciens instead of antibiotics showed a potential beneficial effect on the intestinal health of broilers.

The role of iron homeostasis and iron-mediated ROS in cancer.

As an important trace element, iron plays an essential role in many biology processes like cell proliferation, metabolism, and mitochondrial function. However, the disruption of iron homeostasis tends to cells death and human diseases due to it servers as mediator to promote the production of reactive oxygen species (ROS). In this review, first we introduced the mechanism of complex iron-mediated ROS involved in apoptosis, necroptosis, ferroptosis and pyroptosis. Next, we discussed the controversial role of excess iron and iron deficiency in tumor. Finally, we discussed the anti-cancer effects of iron on both sides, and novel iron-related strategies. This review outlined the mechanisms and regulation of iron homeostasis and iron-mediated ROS in tumors, and discussed the iron-related treatments.

Oral administration of Lactobacillus rhamnosus GG to newborn piglets augments gut barrier function in pre-weaning piglets.

To understand the effects of Lactobacillus rhamnosus GG (ATCC 53103) on intestinal barrier function in pre-weaning piglets under normal conditions, twenty-four newborn littermate piglets were randomly divided into two groups. Piglets in the control group were orally administered with 2 mL 0.1 g/mL sterilized skim milk while the treatment group was administered the same volume of sterilized skim milk with the addition of viable L. rhamnosus at the 1st, 3rd, and 5th days after birth. The feeding trial was conducted for 25 d. Results showed that piglets in the L. rhamnosus group exhibited increased weaning weight and average daily weight gain, whereas diarrhea incidence was decreased. The bacterial abundance and composition of cecal contents, especially Firmicutes, Bacteroidetes, and Fusobacteria, were altered by probiotic treatment. In addition, L. rhamnosus increased the jejunal permeability and promoted the immunologic barrier through regulating antimicrobial peptides, cytokines, and chemokines via Toll-like receptors. Our findings indicate that oral administration of L. rhamnosus GG to newborn piglets is beneficial for intestinal health of pre-weaning piglets by improving the biological, physical, and immunologic barriers of intestinal mucosa.

Glycyrrhizic acid activates chicken macrophages and enhances their Salmonella-killing capacity in vitro.

OBJECTIVE: Salmonella enterica remains a major cause of food-borne disease in humans, and Salmonella Typhimurium (ST) contamination of poultry products is a worldwide problem. Since macrophages play an essential role in controlling Salmonella infection, the aim of this study was to evaluate the effect of glycyrrhizic acid (GA) on immune function of chicken HD11 macrophages. METHODS: Chicken HD11 macrophages were treated with GA (0, 12.5, 25, 50, 100, 200, 400, or 800 µg/ml) and lipopolysaccharide (LPS, 500 ng/ml) for 3, 6, 12, 24, or 48 h. Evaluated responses included phagocytosis, bacteria-killing, gene expression of cell surface molecules (cluster of differentiation 40 (CD40), CD80, CD83, and CD197) and antimicrobial effectors (inducible nitric oxide synthase (iNOS), NADPH oxidase-1 (NOX-1), interferon-γ (IFN-γ), LPS-induced tumor necrosis factor (TNF)-α factor (LITAF), interleukin-6 (IL-6), and IL-10), and production of nitric oxide (NO) and hydrogen peroxide (H2O2). RESULTS: GA increased the internalization of both fluorescein isothiocyanate (FITC)-dextran and ST by HD11 cells and markedly decreased the intracellular survival of ST. We found that the messenger RNA (mRNA) expression of cell surface molecules (CD40, CD80, CD83, and CD197) and cytokines (IFN-γ, IL-6, and IL-10) of HD11 cells was up-regulated following GA exposure. The expression of iNOS and NOX-1 was induced by GA and thereby the productions of NO and H2O2 in HD11 cells were enhanced. Notably, it was verified that nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways were responsible for GA-induced synthesis of NO and IFN-γ gene expression. CONCLUSIONS: Taken together, these results suggested that GA exhibits a potent immune regulatory effect to activate chicken macrophages and enhances Salmonella-killing capacity.

Pidotimod exacerbates allergic pulmonary infection in an OVA mouse model of asthma.

Pidotimod is a synthetic dipeptide with biological and immuno­modulatory properties. It has been widely used for treatment and prevention of recurrent respiratory infections. However, its impact on the regulation of allergic pulmonary inflammation is still not clear. In the current study, an ovalbumin (OVA)­induced allergic asthma model was used to investigate the immune­modulating effects of pidotimod on airway eosinophilia, mucus metaplasia and inflammatory factor expression compared with dexamethasone (positive control). The authors determined that treatment with pidotimod exacerbated pulmonary inflammation as demonstrated by significantly increased eosinophil infiltration, dramatically elevated immunoglobulin E production, and enhanced T helper 2 response. Moreover, treatment failed to attenuate mucus production in lung tissue, and did not reduce OVA­induced high levels of FIZZ1 and Arg1 expression in asthmatic mice. In contrast, administration of dexamethasone was efficient in alleviating allergic airway inflammation in OVA­induced asthmatic mice. These data indicated that pidotimod as an immunotherapeutic agent should be used cautiously and the effectiveness for controlling allergic asthma needs further evaluation and research.

Effect of dietary supplementation of Bacillus subtilis B10 on biochemical and molecular parameters in the serum and liver of high-fat diet-induced obese mice.

While a high-fat diet (HFD) is assumed to be related to fat-mediated oxidative stress decreasing antioxidant enzyme activity, probiotics are believed to have positive effects on the regulation of HFD-induced obesity as well as lipid metabolism, energy homeostasis, and anti-oxidation. Because Bacillus subtilis B10 has beneficial effects on the abnormal lipid metabolism and the oxidative stress in HFD-induced obese mice, ICR mice were randomly assigned into an HFD group and the HFD was supplemented with 0.1% (w/w) Bacillus subtilis B10 (HFD+B10 group). Thereafter, 30-d treatments were run, and then hepatic lipid level and antioxidant status were measured. The expression of genes related to lipid metabolism and oxidative stress in the liver was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). We found that HFD-induced obese mice treated with B10 showed a decrease in weight gain, serum glucose activity as well as hepatic triglyceride (TG), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) activities. In addition, the gene expressions of antioxidant genes, glutathione reductase (GR), xanthine oxidase (XO), heat-shock protein 90 (Hsp90), and lipid synthesis gene 3ß-hydroxysteroid-∆24 reductase (DHCR24) in the HFD+B10 group were down-regulated, suggesting alleviation of oxidative stress, while the lipolysis gene 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), energy metabolism gene peroxisome proliferator-activated receptor α (PPARα) and the gene encoding tumor-suppressor protein p53 were up-regulated. The regulatory and positive effect of dietary supplementation of probiotic B10 suggests that it has a beneficial effect on the homeostasis of the lipid metabolism and on alleviating oxidative stress in HFD-induced obese mice.

Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a critical role to activate immune response. They may be targeted for immunomodulation by microbes, including probiotics. In this study, chicken bone marrow dendrite cells (chi-BMDCs) were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10 (Bs), co-culture of Sb + Bs and phosphate buffer saline (PBS) as a control group (Ctr) at 3, 6, and 12 h intervals. Results revealed that treatment groups modulated the phenotype and biological functions of chi-BMDCs. Scan electron microscopy showed attachment of probiotics on the surface of chi-BMDCs. Additionally transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC-II, CD40, CD80 and CD86 up-regulated in stimulated groups. Furthermore, toll-like receptors TLR1, TLR2, TLR4, and chicken specific TLR15 expressions were improved and downstream associated factors MyD88, TRAF6, TAB1, and NFκ-B mRNA levels increased in all treatment groups as compared to control. Surprisingly, NFκ-B response was noted significant higher in LPS treatment among all groups. Moreover, IL-1ß, IL-17, IL-4, TGF-ß, and IL-10 production levels were found higher, and lower concentration of INF-γ and IL-8 were observed in Sb, Bs, and Sb + Bs treatment groups. In contrast, LPS groups showed prominent increase in IL-12, INF-γ, and IL-8 concentration levels as compared to control group. Altogether, these results emphasize a potentially important role of Saccharomyces boulardii and Bacillus subtilis B10 in modulating immunological functions of chi-BMDCs by targeting specific toll like receptors (TLRs) and associated factors. The role of probiotics on chi-BMDCs functionality in a non-mammalian species have been presented for the first time.

Probiotic Bacillus amyloliquefaciens mediate M1 macrophage polarization in mouse bone marrow-derived macrophages.

Depending on the microenvironment, macrophages can acquire distinct functional phenotypes, referred to as classically activated M1 and M2. M1 macrophages are considered potent effector cells that kill intracellular pathogens, and M2 macrophages promote the resolution of wound healing. In this study, we are interested to know whether probiotic Bacillus amyloliquefaciens (Ba) can induce macrophages polarization. Real-time fluorescence PCR analysis demonstrated that the expression of IL-1ß, iNOS, TNF-α and IL-6 genes for M1 macrophages was significantly increased at 1.5 h after probiotic Ba treatment compared to the probiotic Ba-free treatment (P < 0.01), whereas the expression of M2 macrophage marker genes (Arg1, Fizz1, MR, Ym1) was decreased (P < 0.05). Furthermore, the phagocytic activity was dramatically increased in the Ba-treated BMDMs using a FITC-dextran endocytosis assay. Together, these findings indicated that probiotic Ba facilitated polarization of M1 macrophages and enhanced its phagocytic capacity. The results expanded our knowledge about probiotic function-involved macrophage polarization.

Effects of Bacillus subtilis†B10 spores on viability and biological functions of murine macrophages.

This study was conducted to investigate the effects of Bacillus subtilis B10 spores on the viability and biological functions of murine macrophage. RAW 264.7 cells were stimulated both with and without B. subtilis B10 spores for 12 h. Then cell viability was determined to evaluate the cytotoxic effect of B. subtilis B10 spores to the cells, and the activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH), the production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and the secretion of inflammatory cytokines were measured to analyze the functions of macrophages. The results showed that B. subtilis B10 spores were not harmful to RAW 264.7 cells and they also strongly enhanced the activities of ACP and LDH (P < 0.01), remarkably increased NO and iNOS production (P < 0.01), and significantly stimulated the secretion of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-1 beta (IL-1ß), IL-6, IL-8 and IL-12 (P < 0.01) while they reduced anti-inflammatory cytokine IL-10 (P < 0.01). The outcomes suggest that B. subtilis B10 spores are not only safe for murine macrophages, but also can activate these cells and enhance their immune function. The above findings suggest that B. subtilis B10 spores are potentially probiotic.

Protective effects of the aqueous extract of Scutellaria baicalensis against acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells.

CONTEXT: Scutellaria baicalensis Georgi (Labiatae) (SbG), one of the fifty fundamental herbs of Chinese herbology, has been reported to have anti-asthmatic, antifungal, antioxidative, and anti-inflammatory activities. OBJECTIVE: This study was designed to determine the protective effects of the extract of SbG against the acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The MTT reduction assay was employed to determine cell viability. The total cellular glutathione (GSH) level was detected using a colorimetric GSH assay kit. Cellular GSH production was conducted by detecting the mRNA expression levels of γ-glutamylcysteine ligase catalytic subunit and modifier subunit. RESULTS: Concentration-dependent cytotoxic effects of acrolein were observed while SbG could effectively protect the acrolein-induced oxidative damage. The protective mechanism was investigated, showing that the increased GSH content in the SbG-incubated HUVE cells was associated with the protective effects of SbG-treated cells. Further RT-PCR data confirmed the elevated mRNA expressions of GSH synthesis enzymes. DISCUSSION AND CONCLUSION: The current study strongly indicated that SbG could be a potential antioxidant against oxidative stress in treating cardiovascular diseases.

[Identification and denitrification characteristics of an aerobic denitrifier].

An aerobic denitrifier named F1 was isolated from grass fish pond water by BTB culture medium preliminary screening and denitrification activity analysis. The isolate was identified as Pseudomonas stutzeri through morphological feature, biochemical characteristics and 16S rDNA sequence analysis. Further studies showed that the optimal carbon resources for F1 denitrification were sodium acetate, sodium citrate, glucose and sucrose, with which the nitrate removal rate could reach 100%. When C/N ratio was above 10, the nitrogen removal rate of strain F1 was more than 96% and no nitrite was accumulated. The optimum condition for F1 growth and aerobic denitrification was temperature 30 degrees C and pH 7.0. The F1 could tolerate dissolved oxygen level of 3.4-7.2 mg/L, and its nitrogen removal rate was more than 85% in 24 hours. Denitrification process of F1 mainly occurred in the exponential phase with NaNO3 or NaNO2 as nitrogen resource, and its denitrification rate reached 92.51% and 82.73% , respectively after 32 h of culture. These results suggest that strain F1 can denitrify NO3(-) or NO2(-) directly, and can tolerant a high dissolved oxygen level, and these characteristics make it a good candidate for aquaculture water quality treatment.

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum.

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10-25th and C-terminal 1-9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1-7th and C-terminal 1-3rd residues were not necessary to maintain enzymatic activity.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice.

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.

Nisin-controlled extracellular production of apidaecin in Lactococcus lactis.

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations.

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.

Functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments.

Two aspects were studied to elucidate the functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments: (i) Functions of the N-terminal and C-terminal fragments of apidaecin were first studied by measuring their antibacterial activity, their ability to enter Escherichia coli cells and their effects on the activities of beta-galactosidase and alkaline phosphatase. The results indicate that neither the N-terminal nor the C-terminal of apidaecin contains intracellular delivery unit or active segment. (ii) The effect of apidaecin on the ATPase activity of DnaK, and the interactions of apidaecin with E.coli lidless DnaK and DnaK D-E helix were studied. Results showed that apidaecin could interact with the E.coli lidless DnaK protein and stimulate its ATPase activity, but not with E.coli DnaK D-E helix. This indicated that the antimicrobial activity of apidaecin may be shown by stimulating the ATPase activity of DnaK by binding to its conventional substrate-binding site, to decrease its cellular concentration of DnaK by competing with natural substrates and inhibit the enzymes' activities of E. coli cells. It is the first study to suggest that the apidaecin-binding site of DnaK is the conventional substrate binging site.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides.

The objectives of this study were to assess the potential of two photosynthetic bacteria (PSB), Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions (pH 2.0, pH 3.0 and pH 4.0 gastric juices) and in simulated small intestinal transit conditions (pH 8.0, with or without 0.3% bile salts) was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochromis nilotica, were also detected. All the treatments were determined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference (P>0.05) among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

Protection of Procambarus clarkii against white spot syndrome virus using recombinant oral vaccine expressed in Pichia pastoris.

The potential for oral vaccination of crayfish against white spot syndrome virus was investigated. The envelope proteins VP19 and VP28 were expressed in yeast (Pichia pastoris). The expressed proteins were used as oral vaccines in different forms viz., in whole culture form, whole culture sonicated form, whole culture centrifuged supernatant form, and cell residue form. The recombinant proteins were mixed with food pellets and fed to crayfish for 25 days. The vaccinated groups were divided into two even groups and challenged on the 3rd and 21st day of post vaccination. Among different vaccine groups the relative percent survival (RPS) values of sonicated form and supernatant form vaccines were found the best and met the criterion (>RPS 60%) of effective vaccine even after 21st day of post vaccination. Development of vaccine by using recombinant proteins VP19 and VP28 in yeast as expression vector was feasible with significant effects.

Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii.

The effect of hyperthermia on the development of white spot syndrome virus (WSSV) in the crayfish Procambarus clarkii was studied by competitive PCR. Crayfish were exposed to different temperatures (24 +/- 1 and 32 +/- 1 degrees C) after WSSV injection. No mortality was observed when crayfish were held at 32 +/- 1 degrees C, but mortality reached 100% when crayfish were transferred to 24 +/- 1 degrees C. Competitive PCR showed that viral levels at 32 +/- 1 degrees C remained at 10(5) copies mg(-1) tissue, while at 24 +/- 1 degrees C levels were significantly higher, rising from 10(4) to 10(10) copies mg(-1) tissue. These results suggest that hyperthermia reduces viral replication, but does not eliminate viral particles from WSSV-infected crayfish.