The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.     

Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality

Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human Leukocyte Antigen (HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: C57BL/6 background interferon gamma gene knock out (KO), HLA-A*1101, HLA-A*0201, HLA-B*0702, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Some Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test the response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.     

Micronucleus test with cyclophosphamide administered by intraperitoneal injection and oral gavage

The effect of route of administration on the micronucleus test was examined in 2 laboratories: cyclophosphamide (CYP) was administered by intraperitoneal injection (i.p.) or oral gavage (p.o.) to 2 strains of mice. MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the final micronucleus test was performed with a 48-h sampling time at doses of 25-200 mg/kg i.p. and 50-400 mg/kg p.o. CYP via the i.p. route was more toxic and induced more micronucleated polychromatic erythrocytes (MNPCEs) in MS/Ae mice than in CD-1 mice. Administration-route-related differences were not distinctly shown in the MS/Ae strain. In CD-1, however, higher doses were required for the p.o. route than for the i.p. route to induce about equal amounts of clastogenic damage.     

IFN mimetic as a therapeutic for lethal vaccinia virus infection: possible effects on innate and adaptive immune responses

We have developed small peptide mimetics of IFN-gamma that can bypass the poxvirus virulence factor B8R protein, which binds to intact IFN-gamma and prevents its interaction with receptor extracellular domain. Thus, these peptides inhibit vaccinia virus replication in cell culture where intact IFN-gamma is ineffective. We demonstrate here that the mouse IFN-gamma-mimetic peptide, IFN-gamma(95-132), protects C57BL/6 mice against overwhelming lethal vaccinia virus infection. The mimetic peptide was synthesized with an attached lipophilic group for penetration of cell plasma membrane. Injection of mimetic i.p. before and at the time of intranasal (10(6) PFU) or i.p. (10(7) PFU) challenge with virus resulted in complete protection at 200 microg of mimetic and 40-60% protection at 5 microg of mimetic. Initiation of treatment of mice with IFN-gamma mimetic up to 2 days postinfection resulted in complete protection against death, whereas initiation of treatment at 6 days postinfection resulted in 40% protection. Administration of mimetic by the oral route also completely protected mice against the intranasal route of a lethal dose of vaccinia virus challenge. In addition to its direct antiviral effect, the mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to vaccinia virus. The combination of antiviral and adjuvant effects by the IFN mimetic probably plays a role in its potent anti-vaccinia virus properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses.     

Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.     

Micronucleus test with 2-acetylaminofluorene by intraperitoneal injection and oral administration

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering 2-acetylaminofluorene (2-AAF) by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, the full-scale experiment was performed with a 24-h sampling time at doses ranging from 75 to 600 mg/kg by both routes. The results indicated that 2-AAF induced micronucleated polychromatic erythrocytes (MNPCEs) at all doses tested by both routes. In the MS/Ae strain, higher doses were required by p.o. than by i.p. to reach a similar level of MNPCE incidence. On the other hand, similar responses were recorded by both administration routes with CD-1 mice. Since the LD50 for the p.o. route was higher than that for the i.p. route in both strains, the route-related difference with MS/Ae mice became small when the comparison between i.p. and p.o. was made on the basis of the LD50. Thus both i.p. and p.o. routes are acceptable in the micronucleus test of this chemical.     

Inoculation of the attenuated Coxsackievirus B3 Sabin3-like strain induces a protection against virulent CVB3 Nancy and CVB4 E2 strains in Swiss mice by both oral and intraperitoneal routes

We have previously addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the Coxsackievirus B3 (CVB3). Accordingly, we have described that Sabin3-like mutation (U⁴⁷³→C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we assessed the protection provided by the Sabin3-like mutant against CVB3 infection. For this purpose, we analyzed, in vivo, the Sabin3-like phenotype in Swiss mice inoculated with CVB3 and CVB4 E2 prototype strains either by oral or intraperitoneal (i.p) routes and explored the capacity of this mutant to act as a vaccine vector after the challenge. The Sabin3-like RNA was detected by semi-nested PCR in different organs: heart, pancreas and intestine at 10 days post-inoculation with both oral and i.p routes. Additionally, we did not observe any histological alterations in heart and intestine tissues. RNA was detected in the different organs of all mice immunized with the Sabin3-like strain and challenged with either CVB3 or CVB4 E2 by oral route at 7 days post-challenge. In contrast, no histological alteration of heart or pancreas tissues was observed after challenge with both wild-strains. Interestingly, the detection of viral RNA in heart, pancreas and intestine of mice immunized by i.p route was negative at 7 days post-challenge with CVB3 and CVB4 E2, and mice were protected from myocarditis and pancreatitis.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.     

Experimental transmission and tissue tropism of white spot syndrome virus (WSSV) in two species of lobsters, Panulirus homarus and Panulirus ornatus

The susceptibility of two species of lobsters, Panulirus homarus and Panulirus ornatus to white spot syndrome virus (WSSV) was tested by oral route and intramuscular injection. The results revealed that these lobsters were as highly susceptible as marine shrimp when the WSSV was administered intramuscularly. The WSSV caused 100% mortality in both Panulirus homarus and Panulirus ornatus, at 168 and 120 h, respectively, after intramuscular injection and failed to cause mortality when given orally. The presence of WSSV in moribund lobsters was confirmed by single-step and nested PCR, Western blot, histology, and bioassay test. It was found in eyestalk, gill, head muscle, tail muscle, hemolymph, appendages, and stomach. In lobsters with oral route infection, all tested organs except stomach and head muscle was negative for WSSV by nested PCR at 120 h post-inoculation. The stomach and head muscle was positive by nested PCR at 120 h p.i., but negative at 168 h p.i. Western blot analysis was negative in all the tested organs of both species of lobster at 120 h post-inoculation by oral route.     

Detection of Clostridium botulinum Toxin by Local Paralysis Elicited with Intramuscular Challenge

Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure.     

Effect of route of administration in the micronucleus test with potassium bromate

The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice (MS/Ae and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.     

Murine bone marrow IgA responses to orally administered sheep erythrocytes

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.     

Protection, immune responses and side effects in Atlantic salmon (Salmo salarL.) vaccinated against furunculosis by different procedures

In an experimental trial lasting approximately 6 months, 10 different vaccination regimes against furunculosis were studied in Atlantic salmon pre-smolts. Single and repeated administration of vaccine by the intraperitoneal (i.p.), immersion or oral route, and revaccination by combinations of these methods, were tested. In challenge assays initiated 8 and 16 weeks after vaccination, fish injected once with a trivalent vaccine, and fish injected twice with a monovalent vaccine, both containing aluminium phosphate as adjuvant, were moderately protected. Non-injection vaccination protocols consistently failed to protect the fish. Compared with unvaccinated fish, protected groups showed elevated antibody responses toAeromonas salmonicidaantigens throughout the study. Increasedin vitroproliferation of head kidney leucocytes from i.p. vaccinated fish was found 16 weeks after vaccination. The use of a polyvalent vaccine preparation, and revaccination by injection or the oral route improved both immune responses and survival, compared to a single inoculation of monovalent vaccine. In all groups subjected to i.p. administration of vaccine, minor to moderate intraperitoneal lesions were found. In conclusion, i.p. administration of adjuvanted vaccine, preferably in a polyvalent formulation, is the optimal method of inducing anti-furunculosis immunity in Atlantic salmon, and is apparently necessary for an effective immunoprophylaxis of salmonid fish against furunculosis.     

A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD₅₀) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10⁶ PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10⁸ PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10⁸ PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.     

A novel neurotoxoid vaccine prevents mucosal botulism

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 10(3) LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 microg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.     

Distribution, metabolism, and fetal uptake of pentavalent arsenic in pregnant mice following oral or intraperitoneal administration

Pregnant CD-1 mice were treated with 20 (i.p.) or 40 (p.o.) mg/kg sodium arsenate on gestation day 18 (plug = day 1). Individual fetuses, pooled placentas and maternal blood, urine, liver, and kidneys were obtained from three or more litters at intervals up to 24 hours following treatment. Acid-digested samples were analyzed for total arsenic by hydride generation atomic absorption spectrophotometry. The rate of arsenic elimination from maternal samples was not influenced by administration route. First-order elimination followed a brief period of distribution, and the biological half-life was approximately 10 hours. Arsenic was found in most samples, with mean peak concentrations expressed as micrograms As/gm (wet wt.) or /ml (values listed are post-treatment sampling times in minutes or hours and concentrations for i.p. and for p.o. treated groups, respectively) as follows: fetuses-2, 3.5; 6, 0.8, placentas-2, 9.3; 1, 2.3, blood-10 minutes, 6.9; 1, 2.0, urine-1, 712; 2, 342, kidney-20 minutes, 25.4; 1, 11.0, liver-0.5, 7.9; 1, 11.7. By 24 hours, arsenic levels in fetuses and placentas had declined to 0.22 microgram/gm and 0.74 microgram/gm for i.p. and 0.33 microgram/gm and 0.57 microgram/gm for p.o. treatments, respectively. Fetal arsenic uptake and loss were more rapid following i.p. than p.o. treatments, and although the i.p. dose was only half that used p.o., peak fetal As+5 was almost fivefold higher following i.p. treatment. These results agree with the finding that oral dosing of pregnant mice with arsenate has less effect on the conceptus than does treatment by injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of oral administration of live herpes simplex virus type 1 as a vaccine.

Mice given herpes simplex virus type 1 (HSV-1) (Miyama +GC strain) intragastrically via a stainless-steel cannula were rendered immune to subsequent lethal intraperitoneal (i.p.) challenge with HSV-1. The orally administered HSV-1 was completely inactivated in the stomach within a few minutes of inoculation. However, systemic immunity was established 14 days after oral inoculation with the virus and retained for up to 6 months. The mechanisms of establishing systemic immunity were investigated by means of adoptive transfer comparisons. When splenic cells from HSV-1-immunized mice were transplanted into nonimmunized mice, all of the recipient mice survived after a lethal i.p. challenge with the virus. Immunity was not established in antithymocyte serum-treated mice or by transfer of serum from immunized to nonimmunized mice. In addition, all HSV-1-immunized mice died after lethal challenge with HSV-2 and influenza virus A. These findings suggest that the immunity was virus specific, with T lymphocytes playing a major role in its establishment. The present study therefore supports the possibility of oral immunization with live HSV-1 as a vaccine.     

Micronucleus test with methyl methanesulfonate administered by intraperitoneal injection and oral gavage

The effects of 2 routes of administration, intraperitoneal injection (i.p.) and oral gavage (p.o.), in the micronucleus test were evaluated using methyl methanesulfonate (MMS) and 2 strains of mice (MS/Ae and CD-1). A small-scale acute toxicity study and a pilot micronucleus experiment were carried out first. On the basis of the results obtained, a final micronucleus test was performed at doses of 20, 40, 80, and 160 mg/kg (i.p.) and 40, 80, 160, and 320 mg/kg (p.o.), with a 24-h sampling time. MMS induced micronucleated polychromatic erythrocytes (MNPCEs) in both routes in both mouse strains under the conditions used. At 40 and 80 mg/kg, MMS induced a higher number of MNPCEs by the i.p. route in both strains. A 160 mg/kg MMS dose induced higher numbers of MNPCEs by the p.o. route in MS/Ae mice. The route-related difference with MMS on the basis of mg/kg disappeared when the difference was determined on the basis of a ratio of the LD50. In practice, both i.p. and p.o. routes are acceptable as routes of administration in the micronucleus test using this chemical.     

Experimental Infection of Mice with Yersinia enterocolitica Serotype O9 by Oral and Parenteral Routes: Spreading and Enterotropism of Virulent Yersiniae

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions. Received: 31 August 1998 / Accepted: 13 November 1998     

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.