Route Of Challenge Research Articles

Pathogenic Potential of Edwardsiella tarda Strains Isolated from Turbot

In this study, pathogenicity of Edwardsiella tarda isolates from turbot Scophthalmus maximus as well as other strains from different hosts were tested against turbot, sole Solea senegalensis and sea bass Dicentrarchus labrax. In addition, the influence of challenge route and temperature in the pathogenic potential of E. tarda strains from turbot were also examined. The results obtained showed that E. tarda did not have host specificity in virulence and that the turbot isolates were highly virulent regardless of the inoculation route and temperature. Moreover, inoculation experiments performed in mice suggest that these isolates are virulent for mammals. All these findings suggest that this emergent turbot pathogen constitutes an important risk factor for the marine aquaculture.

Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge

We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131–326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.

Comparison of Plasmodium berghei challenge models for the evaluation of pre-erythrocytic malaria vaccines and their effect on perceived vaccine efficacy

BackgroundThe immunological mechanisms responsible for protection against malaria infection vary among Plasmodium species, host species and the developmental stage of parasite, and are poorly understood. A challenge with live parasites is the most relevant approach to testing the efficacy of experimental malaria vaccines. Nevertheless, in the mouse models of Plasmodium berghei and Plasmodium yoelii, parasites are usually delivered by intravenous injection. This route is highly artificial and particularly in the P. berghei model produces inconsistent challenge results. The initial objective of this study was to compare an optimized intravenous (IV) delivery challenge model with an optimized single infectious mosquito bite challenge model. Finding shortcomings of both approaches, an alternative approach was explored, i.e., the subcutaneous challenge.MethodsMice were infected with P. berghei sporozoites by intravenous (tail vein) injection, single mosquito bite, or subcutaneous injection of isolated parasites into the subcutaneous pouch at the base of the hind leg. Infection was determined in blood smears 7 and 14 days later. To determine the usefulness of challenge models for vaccine testing, mice were immunized with circumsporozoite-based DNA vaccines by gene gun.ResultsDespite modifications that allowed infection with a much smaller than reported number of parasites, the IV challenge remained insufficiently reliable and reproducible. Variations in the virulence of the inoculum, if not properly monitored by the rigorous inclusion of sporozoite titration curves in each experiment, can lead to unacceptable variations in reported vaccine efficacies. In contrast, mice with different genetic backgrounds were consistently infected by a single mosquito bite, without overwhelming vaccine-induced protective immune responses. Because of the logistical challenges associated with the mosquito bite model, the subcutaneous challenge route was optimized. This approach, too, yields reliable challenge results, albeit requiring a relatively large inoculum.ConclusionsAlthough a single bite by P. berghei infected Anopheles mosquitoes was superior to the IV challenge route, it is laborious. However, any conclusive evaluation of a pre-erythrocytic malaria vaccine candidate should require challenge through the natural anatomic target site of the parasite, the skin. The subcutaneous injection of isolated parasites represents an attractive compromise. Similar to the mosquito bite model, it allows vaccine-induced antibodies to exert their effect and is, therefore not as prone to the artifacts of the IV challenge.

Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector.

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

Assessment of the role of brine shrimp Artemia in white spot syndrome virus (WSSV) transmission

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.

Pathogenicity of Streptococcus ictaluri to Channel Catfish

The pathogenicity of a Streptococcus ictaluri isolate in channel catfish Ictalurus punctatus at the fry (0.5 g), fingerling (15 g), and juvenile (55 g) stages was determined by experimental bath immersion and injection experiments. Channel catfish were exposed in 1-L immersion baths containing 10(8), 10(9), 10(10), 10(11) or 10(12) colony-forming units (cfu) of S. ictaluri. Fish were also injected intraperitoneally with 0.1 mL of bacterial solution for final doses of 10(4), 10(5), 10(6), 10(7), or 10(8) cfu of S. ictaluri per fish. Streptococcus ictaluri caused mortality in fry, fingerling, and juvenile channel catfish within 21 d postinfection. When mortalities were calculated based on size and challenge route, the cumulative percent mortalities were 11% for fry and 0% for fingerlings by the bath immersion route and 14% for fingerlings and 6% for juveniles by the injection route. Isolation of S. ictaluri from moribund and dead catfish was confirmed by the newly established BIOLOG profile (MicroLog3 system). The results indicate that channel catfish were only susceptible to high concentrations of S. ictaluri and that juvenile channel catfish were less susceptible, possibly explaining why little mortality has been attributed to S. ictaluri infection in catfish aquaculture.

Infectious haematopoietic necrosis virus genogroup‐specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control.

Susceptibility of Atlantic cod Gadus morhua juveniles to different routes of experimental challenge with infectious pancreatic necrosis virus (IPNV)

Atlantic cod Gadus morhua L. juveniles weighing 40 g were challenged with infectious pancreatic necrosis virus (IPNV) by intraperitoneal (i.p.) or intramuscular (i.m.) injection or by bath. The amount of infectious virus was determined over 6 wk in head kidney, heart and pylorus tissues. No mortality or clinical signs were observed in either of the challenged groups. However, 6 wk after challenge virus was still present in the fish, which shows that IPNV can persist asymptomatically in cod. I.p. and i.m. injections were the most efficient routes of challenge giving the highest virus recovery. The prevalence of individuals with a viral titre > or = 500 infectious units g(-1) tissue was lower in the group of fish challenged by bath; thus bath was a less efficient route of challenge than injection. Our data also show that pylorus and head kidney are target organs for IPNV in cod, and levels of virus recovery were not considerably different between these 2 organs. Challenged by injection, the cod heart is also a target organ for IPNV. Compared to head kidney and pylorus, the heart seems to have a minor role in virus multiplication. Virus was also recovered from cohabiting fish, demonstrating that covertly infected cod may represent a reservoir of infectious IPNV for surrounding fish populations. Expression analysis of selected cod immune genes showed that i.p. injection of IPNV induced gene expression of ISG15 and LGP2, markers for the innate antiviral defence, while expression of markers for the inflammatory response (interleukins IL-1 beta, IL-8, IL-10) was not significantly increased.

Vaccine-Induced Protection from Egg Production Losses in Commercial Turkey Breeder Hens Following Experimental Challenge With A Triple-Reassortant H3N2 Avian Influenza Virus

Infections of avian influenza virus (AIV) in turkey breeder hens can cause a decrease in both egg production and quality, resulting in significant production losses. In North Carolina in 2003, a triple-reassortant H3N2 AIV containing human, swine, and avian gene segments was isolated from turkey breeder hens (A/turkey/NC/16108/03). This viral subtype was subsequently isolated from both turkeys and swine in Ohio in 2004, and in Minnesota in 2005, and was responsible for significant losses in turkey production. The objective of this study was to determine if currently available commercial, inactivated avian influenza H3 subtype oil-emulsion vaccines would protect laying turkey hens from egg production losses following challenge with the 2003 H3N2 field virus isolate from North Carolina. Laying turkey hens were vaccinated in the field with two injections of either a commercial monovalent (A/duck/Minnesota/79/79 [H3N4]) or autogenous bivalent (A/turkey/North Carolina/05 (H3N2)-A/turkey/North Carolina/88 [H1N1]) vaccine, at 26 and 30 wk of age, and subsequently challenged under BSL 3-Ag conditions at 32 wk of age. Vaccine-induced efficacy was determined as protection from a 50% decrease in egg production and from a decrease in egg quality within 21 days postchallenge. Results indicate that, following a natural route of challenge (eye drop and intranasal), birds vaccinated with the 2005 North Carolina H3N2 subtype were significantly protected from the drop in egg production observed in both the H3N4 vaccinated and sham-vaccinated hens. The results demonstrate that groups receiving vaccines containing either H3 subtype had a decreased number of unsettable eggs, increased hemagglutination inhibition titers following challenge, and decreased virus isolations from cloacal swabs as compared to the sham-vaccinated group. Phylogenetic analysis of the nucleotide sequence of the HA1 gene segment from the three H3 viruses used in these studies indicated that the two North Carolina turkey isolates had 90.4% similarity in HA1 nucleotide sequence, but had only 77.4% and 76.1% sequence similarity to the HA1 of the H3N4 duck isolate. This study provides the first detailed description of the clinical protection afforded to laying turkey hens by vaccination against challenge with a circulating field isolate of a H3N2 triple-reassortant AIV.

Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.

Oropharyngeal aspiration: An alternative route for challenging in a mouse model of chemical-induced asthma

Background To assess the importance of the route of challenge in an existing mouse model of chemical-induced asthma, we replaced intranasal instillation by oropharyngeal aspiration. To our knowledge, oropharyngeal aspiration as a challenge route has not yet been investigated in a mouse model of chemical-induced asthma. Methods On days 1 and 8, mice were dermally sensitized with toluene diisocyanate (TDI) (0.3%) [or vehicle (acetone/olive oil)] and on day 15 they received a single challenge, via oropharyngeal aspiration, with TDI (0.01%) or vehicle. One day after challenge, airway reactivity to methacholine was measured by a forced oscillation technique (FlexiVent) and total and differential cell counts, as well as levels of KC, IL-5, IL-17 and TNF-α, were assessed in the bronchoalveolar lavage (BAL) fluid. Lymphocytes from the auricular and mediastinal lymph nodes were cultured to determine the concanavaline A-induced secretion of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN-γ. Total serum IgE was measured. Results In TDI-sensitized mice, a significant increase in airway reactivity was found after a single oropharyngeal challenge with TDI. BAL neutrophils and eosinophils were increased 7- and 5-fold, respectively. An upregulation of Th1 (IFN-γ), Th2 (IL-4, IL-10, IL-13) and Th17 (IL-17) cytokines was found in the auricular lymph nodes, in the mediastinal lymph nodes only IL-4 was upregulated. The total serum IgE level in TDI-sensitized mice was significantly increased when compared to control mice. Conclusion We conclude that challenging mice via oropharyngeal aspiration mimics the characteristics of human asthma well, without the possible drawbacks of other techniques.

Effect of dose and challenge routes of Vibrio spp. on co-infection with white spot syndrome virus in Penaeus vannamei

This study was conducted to investigate the effect of dose and challenge routes of Vibrio spp. on co-infection with white spot syndrome virus (WSSV) in specific pathogen-free (SPF) Penaeus vannamei shrimp. Juvenile shrimp were first injected with WSSV at a dose of 30 SID 50 shrimp − 1 (SID 50 = shrimp infectious dose with 50% endpoint) and 24 h later with 10 3, 10 4, 10 5 or 10 6 CFU shrimp − 1 of V. campbellii. Controls did not die during the experiment, except the ones that received 10 6 CFU shrimp − 1 (35–65%). In WSSV-inoculated shrimp, the 100% cumulative mortality were reached at 144–360 h post injection (hpi). WSSV-infected shrimp died much faster when injected with at least 10 4 CFU of V. campbellii with the 100% cumulative mortality reached at 48–96 hpi of virus. The density of V. campbellii in haemolymph of co-infected moribund shrimp collected 6 h after V. campbellii injection was significantly higher than that in shrimp injected with V. campbellii only. There was no difference in the number of WSSV-infected cells between shrimp inoculated with WSSV only, compared to dually inoculated ones. Shrimp which were first injected with WSSV and 24 h (or 48 h) later exposed to 10 6, 10 7, or 10 8 CFU ml − 1 of V. campbellii by immersion did not show any accelerated mortality. When WSSV-infected shrimp were challenged with another Vibrio species, V. harveyi BB120, no accelerated mortality was noted in WSSV-infected shrimp injected with 10 6 CFU shrimp − 1 of V. harveyi BB120. In conclusion, it can be stated that the synergistic effect between WSSV and Vibrio is influenced by the dose, species and infection route of inoculation of the Vibrio bacteria.

Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28

White spot syndrome virus (WSSV) is the causative agent of white spot disease (WSD), one of the most serious diseases affecting global shrimp farming. We compared WSSV infection induction in kuruma shrimp Marsupenaeus japonicus by oral, immersion, and intramuscular injection (IM) exposure methods and evaluated the oral vaccine prepared from the recombinant WSSV proteins rVP26 and rVP28. The 50% lethal doses (LD50) of WSSV by oral, immersion, and IM challenges were 10(-0.4), 109-4.4), and 10(-7.7) g shrimp(-1), respectively, indicating that WSSV infection efficiency by oral challenge was significantly less than the other 2 challenge routes. However, in shrimp farms it is believed that WSSV infection is easily and commonly established by the oral route as a result of cannibalization of WSSV-infected shrimp. Kuruma shrimp vaccinated orally with WSSV rVP26 or rVP28 were challenged with WSSV by oral, immersion, and IM routes to compare protection efficacy. The relative percent survival values were 100% for oral challenge, 70 to 71% for immersion, and 34 to 61% for IM. Thus, the protection against WSSV-infection that was induced in kuruma shrimp by oral vaccination with rVP26 or rVP28 seemed equivalent to that obtained through IM vaccination.

The “Classical” Ovalbumin Challenge Model of Asthma in Mice

Ovalbumin challenge models of asthma offer many opportunities for increasing our understanding of the pathogenetic mechanisms underlying this disease, as well as for identifying novel therapeutic targets. There is no single "classical" model, because numerous alternatives exist with respect to the choice of mouse strain, method of sensitisation, route and duration of challenge, and approach to assessing the host response. Moreover, the limitations of these models need to be recognised when attempting to interpret experimental findings. Nevertheless, careful use of well-defined models allows investigators to answer specific questions that are otherwise difficult to address.

Differential Antigen Requirements for Protection against Systemic and Intranasal Vaccinia Virus Challenges in Mice

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.

Molecular immunology of experimental primary tularemia in mice infected by respiratory or intradermal routes with type A Francisella tularensis

The type A subspecies of Francisella tularensis is a highly virulent facultative intracellular bacterial pathogen, and a potential biological weapon. Recently, there has been renewed interest in developing new vaccines and therapeutics against this bacterium. Natural cases of disease, tularemia, caused by the type A subspecies are very rare. Therefore, the United States Food and Drug Administration will rely on the so-called Animal Rule for efficacy testing of anti- Francisella medicines. This requires the human disease to be modeled in one or more animal species in which the pathogenicity of the agent is reasonably well understood. Mice are natural hosts for F. tularensis, and might be able to satisfy this requirement. Tularemia pathogenesis appears to be primarily due to the host inflammatory response which is poorly understood at the molecular level. Additionally, the extent to which this response varies depending on host and pathogen genetic background, or by pathogen challenge route or dose is unknown. Therefore, the present study examined sera and infected tissues from C57BL/6 and BALB/c mice challenged by natural intradermal (ID) and respiratory routes with one of two distinct type A strains of the pathogen for cytokine and chemokine responses that might help to explain the morbidity associated with tularemia. The results show that the molecular immune response was mostly similar regardless of the variables examined. For instance, mRNA for the proinflammatory cytokine IL-6, and chemokines KC, and IP-10 was consistently upregulated at all sites of infection. Upregulation of mRNA for several other cytokines and chemokines occurred in a more tissue restricted manner. For instance, IFN-γ was highly upregulated in the skin of BALB/c, but not C57BL/6 mice after ID inoculation of the pathogen, whilst IL-10 mRNA upregulation was only consistently seen in the skin and lungs.

Antigenic analysis of some Nigerian street rabies virus using monclonal antibodies

The authors studied 12 street rabies virus isolates from 3 states of Nigeria using both the anti-nucleocapsid and anti-glycoprotein monoclonal antibodies and cross-protection tests. It was observed that all the viruses were rabies having divergent antigenic presentation. Also noticed was an antigenic shift when the viruses were passed through different hosts. Two antirabies vaccines used did not protect animals when challenged through the orthodox route and only traces of protection by one of the vaccines were observed when the route of challenge was intramuscular. In the virus isolation and adaptation, the BHK 21 cell lining was found to be only 37% efficient while the Murine neuroblastoma cells were about 75% efficient. Keywords:Antigenic, Nigerian street rabies virus, monoclonal antibodies. Nigerian Veterinary Journal Vol. 27 (2) 2006: pp. 41-53

Route of challenge is critical in determining the clinical outcome of infection with a very virulent oncogenic herpesvirus, Marek's disease virus

The majority of experimental studies examining Marek's disease virus infection have used parenteral injection of cell-associated virus. The aim of this study was to examine whether the route of entry of virus was critical in determining the outcome of infection. Susceptible (L7) and resistant (L6) White Leghorn chickens were infected with a very virulent Marek's disease virus, RB1B, by either the intra-abdominal or intra-tracheal route. Birds infected by the intra-tracheal route had earlier, higher or more sustained blood, spleen and lung viral concentrations than those infected by the intra-abdominal route. L7 birds had higher viral loads than L6 birds infected by the same route. Clinical outcomes reflected these data. Resistant birds infected by the intra-tracheal route had an increased prevalence of tumours and shorter survival times compared with those infected by the intra-abdominal route. Susceptible birds infected by the intra-tracheal route became paralysed 10 days after infection. L7 birds had shorter survival times and increased prevalences of tumours than L6 birds. The pathology and viraemia seen with intra-tracheal infection could not be fully replicated by increasing the dose in intra-abdominal infections. We conclude that instillation of infective dust produces a more aggressive infection that depends on the route of entry and form of virus, and not just on the challenge dose.

Mortality after IPNV challenge of Atlantic salmon ( Salmo salar L.) differs based on developmental stage of fish or challenge route

The mortality of Atlantic salmon parr and post-smolts was studied after experimental challenge using a virulent strain of infectious pancreatic necrosis virus (IPNV). No mortality was obtained in parr challenged in freshwater, but challenge three weeks after seawater transfer resulted in high mortalities (up to 64%) in cohabitants compared to the intraperitoneally injected fish (up to 19%). No non-infected control fish died. In parr, challenge was performed using intraperitoneal injection (ip) or bath, but even after stressing the fish three times by lowering the water level no mortality occurred. The parr had low virus titres and almost undetectable levels of anti-IPNV antibodies five weeks post-challenge. Post-smolts of the same origin as the parr and challenged by ip injection and/or cohabitation with the same batch of virus, died after challenge. The surviving post-smolts had low virus levels and low antibody levels. Clinically diseased post-smolts had higher IPNV titres in internal organs and virus was detected in all fish that died during the experiment. The IPN virus re-isolated from challenged fish had the same VP2 gene virulence motif as the virus used for challenge. The mortality profiles show that the cohabitant challenge is a reliable challenge model for post-smolts. The parr and post-smolts were fed four different diets, but no difference in mortality was observed between the diet groups. The feed consumption varied between the diets, and fish fed with immunostimulant had eaten less than the others and did not grow well. As there were no differences in mortalities between the diet groups, this verifies the stability of the challenge model for post-smolts.

A primary subcutaneous infection with Paracoccidioides brasiliensis leads to immunoprotection or exacerbated disease depending on the route of challenge

In human and experimental paracoccidioidomycosis the severe disease is characterized by depressed cellular immunity whereas the mild disease is associated with persistent T cell immunity. Since the subcutaneous route of antigen inoculation is an efficient inducer of cellular immunity, we decided to study this route of infection and verify its effect on a lethal secondary infection of susceptible hosts. It was observed that the s.c. infection induces positive delayed type hypersensitivity (DTH) responses in 9 different mouse strains, is a self healing process and susceptible mice develop more intense DTH reactions than resistant mice to Paracoccidioides brasiliensis infection. Unexpectedly, the previous s.c. infection of susceptible mice led to immunoprotection or disease exacerbation depending on the route of fungal challenge. Immunoprotection was achieved after intraperitoneal challenge and was associated with persistent cell-mediated immunity and a mixed type-1/type-2 immunity. Exacerbated disease was found after intravenous challenge, was associated with cellular immunity anergy and prevalent type-2 immune response. As a whole, our work demonstrates that susceptibility to P. brasiliensis infection cannot be ascribed to intrinsic inability to mount cellular immune responses, that a single immunization procedure can result in opposite disease outcomes and immunoprotection can be achieved by a balanced Th1/Th2 immunity.