Lactococcus lactis Expressing Type I Interferon From Atlantic Salmon Enhances the Innate Antiviral Immune Response In Vivo and In Vitro.

In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.

Identification and assessment of pathogenicity of a naturally reassorted infectious bursal disease virus from Henan, China.

Infectious bursal disease virus (IBDV) is still a vital etiological agent in poultry farms. IBDV outbreaks occasionally occur due to the presence of very virulent, reassortment or variant strains. Vaccine immunization has played crucial roles in IBD control for decades. However, survival pressure of IBDV from the vaccine immunization also increases the reassortments of circulating viruses. In this study, an IBDV strain was isolated from several broiler farms in Henan Province, central part of China, and named IBDV HN strain. Based on the results of RT-PCR, sequencing and phylogenic analyses of VP1 and VP2 genes, the IBDV HN strain is a novel reassortment strain in the Henan region. Segment A of this strain appears to originate from the very virulent IBDV strain, while segment B comes from the other field reassortment strains. This may be the result of natural reassortant of virus circulating in the field. About 60% (6/10) of experimentally infected specific pathogen-free chickens died after 3 to 5 d post-infection with typical symptom and pathological lesions. The IBDV HN strain was prone to horizontal transmission, which poses a serious threat to the chicken industry. Further investigation on the prevalence, virulence, and evolution of HN strain IBDV will provide a foundation for the prevention and control of the disease in this region.

Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada.

Antibiotic-free and safe animal products are most desirable among consumers. However, ensuring safe poultry products is a challenging task when the chicken immune system is compromised. Infectious bursal disease virus (IBDV) causes immunosuppression and predisposes chickens to secondary infections. Breeder vaccination against IBDV is routinely practiced for producing chicks with maternally-derived antibody (MAb) to prevent infection in newly hatched chicks. The majority of IBDV circulating in Canadian farms are variant strains (vIBDV). Whether circulating vIBDV strains are immunosuppressive in chicks or are amenable to current vaccine regimens has not previously been tested through challenge studies. In this study, one-day-old broiler chicks (n=240) carrying MAb were obtained from broiler breeders vaccinated with commercial IBDV vaccines. In the first set of experiments (n=40/group), at six days post-hatch, one group was challenged with a Canadian field isolate, vIBDV (strain-SK09) (3×10(3) EID50). The second and the third groups (controls) were inoculated with non-immunosuppressive IBDV D-78 (10×10(3) TCID50) and saline, respectively. Histopathological examination on days 14 and 30 post-challenge revealed that despite the high level of MAb, vIBDV (SK09) caused severe bursal damage in chicks. Another set of experiments with treatment groups as above, demonstrated that pre-exposure of chicks with vIBDV (SK09) caused immunosuppression resulting in significantly higher mortality and disease severity in chicks challenged with a virulent strain of Escherichia coli (E. coli). Our data provide evidence that IBDV strains circulating in Canada are immunosuppressive, not amenable to current anti-IBDV vaccination strategy, and a potential threat to antibiotic-free chicken farming.

A cooperative approach to animal disease response activities: Analytical hierarchy process (AHP) and vvIBD in California poultry.

Very virulent infectious bursal disease virus (vvIBDv) was first detected in the United States at the end of 2008. Since its detection, Federal and State animal health officials, the poultry industry and the research/academic community have led response activities through a collaborative effort. By June 2011, much still remained unknown regarding the basic epidemiology and ecology of vvIBD in California, although there were a number of potential activities to fill this information gap. Available resources limited the ability to pursue all the activities, and responsible parties and stakeholders recognized the need to prioritize the activities. The analytic hierarchy process (AHP) is a useful multi-criteria decision making methodology that incorporates qualitative information (in the form of judgments) with available quantitative information. This is especially useful when there is very limited quantitative information, such as in the situation with vvIBD in California. A commercial package that allows ready use of the AHP model was utilized for prioritizing activities, incorporating input from members from the three stakeholder groups: State and Federal animal health officials, poultry industry, and research/academia. Based on their inputs on 17 potential activities, the participants identified three priority activities; specifically determination of risk factors for re-emergence or re-introduction at affected premises, development of a laboratory diagnostic test to screen for segment B of the vvIBDV genome and surveillance of other potential reservoirs (mealworms, rodents, beetles). In order to evaluate the ability of the AHP to respond to differences, a sensitivity analysis was done in order to evaluate changes in prioritization of activities. Changes in prioritization were noted demonstrating the plasticity of the model under different conditions. However, a 50% increase or decrease in weighting was necessary to affect the order of the three highest scoring activities. The use of a tool such as the AHP enables the development of a transparent, repeatable and flexible decision process, which can be useful in certain animal health response situations including the re-emergence of a previously eliminated disease or the introduction of a foreign animal disease.

Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L.

Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome.

Immune Response of Salmonella Challenged Broiler Chickens Fed Diets Containing Gallipro®, a Bacillus subtilis Probiotic.

This study was conducted to investigate the effect of feeding a probiotic, Bacillus subtilis, on antibody titers against Newcastle and infectious bursal viruses in broiler chickens challenged with Salmonella enterica serotype Enteritidis. One hundred and sixty 1-day-old broiler chicks were randomly assigned to four treatments in a completely randomized design. The treatments were negative control, probiotic-treated group, challenged group, and challenged probiotic treated group. Salmonella challenging decreased (P < 0.05) the relative weights of spleen and bursa. Inclusion of probiotic to diet of challenged chickens increased the relative weight of spleen, but had no effect on the relative weight of bursa. There were no differences for the antibody titers of chickens between negative control and probiotic-treated group. Salmonella challenging decreased (P < 0.05) antibody titers against Newcastle and infectious bursal viruses. Improvements in the antibody titers were observed by the addition of probiotic to diet of these chickens. The results showed that dietary inclusion of probiotic had no significant effect on immune parameters of chickens at non-contaminated environment, display a greater efficacy at environment contaminated with pathogen and can improve immune responses of infected chickens.

Experimental transmission of infectious pancreatic necrosis virus from the blue mussel, Mytilus edulis, to cohabitating Atlantic Salmon (Salmo salar) smolts.

Integrated multitrophic aquaculture (IMTA) reduces the environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter-feeding particulate-bound organic nutrients. As bioaccumulating organisms, shellfish may also increase disease risk on farms by serving as reservoirs for important finfish pathogens such as infectious pancreatic necrosis virus (IPNV). The ability of the blue mussel (Mytilus edulis) to bioaccumulate and transmit IPNV to naive Atlantic salmon (Salmo salar) smolts was investigated. To determine the ability of mussels to filter and accumulate viable IPNV, mussels were held in water containing log 4.6 50% tissue culture infective dose(s) (TCID50) of the West Buxton strain of IPNV ml(-1). Viable IPNV was detected in the digestive glands (DGs) of IPNV-exposed mussels as early as 2 h postexposure. The viral load in mussel DG tissue significantly increased with time and reached log 5.35 ± 0.25 TCID50 g of DG tissue(-1) after 120 h of exposure. IPNV titers never reached levels that were significantly greater than that in the water. Viable IPNV was detected in mussel feces out to 7 days postdepuration, and the virus persisted in DG tissues for at least 18 days of depuration. To determine whether IPNV can be transmitted from mussels to Atlantic salmon, IPNV-exposed mussels were cohabitated with naive Atlantic salmon smolts. Transmission of IPNV did occur from mussels to smolts at a low frequency. The results demonstrate that a nonenveloped virus, such as IPNV, can accumulate in mussels and be transferred to naive fish.

Risk factors associated with infectious bursal disease in commercial chickens in Bangladesh.

To identify risk factors associated with infectious bursal disease (IBD) in commercial chickens in Bangladesh, we conducted a matched case-control study on 32 commercial farms affected with IBD and 96 IBD unaffected farms taking flock size (1000 vs. >1000 birds) and location of the farms (whether it is in rural or urban area) as matching variables. Epidemiological data from case and control farms were collected through the use of a pretested questionnaire, and analyzed by matched-pair analysis and multivariable conditional logistic regression. In the multivariable analysis, the variables 'receive visitors on the farm premises' (odds ratio [OR]=4.49; 95% confidence interval [CI]=1.50-13.42; p=0.007), 'purchase live poultry from the open market' (OR=7.59; 95% CI=2.69-21.45; p=<0.001), 'workers live outside the farms premises' (OR=5.89; 95% CI=1.87-18.60; p=0.002) and 'access of vendor vehicles on the farm premises' (OR=5.19; 95% CI=1.39-19.31; p=0.014) were identified as risk factors for IBD in commercial chicken farms. The proper management of the risk factors along with knowledge of the disease could help to reduce the incidence of IBDV infection in commercial chickens in Bangladesh.

Comparative susceptibility of chickens, turkeys and ducks to infectious bursal disease virus using immunohistochemistry.

Twenty chicks, 12 turkey poults and 10 ducklings, all 5 weeks old were infected with 2 x 10(3.5) chick LD(50) IBD virus to determine the course of the virus in the 3 poultry species. Uninfected control birds were kept separately. Two infected and 2 control birds/species were euthanized at time intervals between 3 and 168 hours post infection (pi). Sections of thymus, bursa of Fabricius, spleen, liver, kidney, proventriculus and ceacal tonsil were stained for the detection of IBD virus antigen using immunoperoxidase technique. IBD virus antigen positive cells stained reddish-brown and the amount of such cells in tissue sections were noted and scored. Stained cells were present in all organs examined for up to 168 hours pi in the 3 poultry species except ceacal tonsils of ducks at 72 and 120 hours pi. Antigen score was highest in chickens and least in ducks as reflected by average of total scores/sampling time of 12, 10.8 and 8 in chickens, turkeys and ducks respectively. Total antigen score/sampling time in infected chickens peaked twice; 24/48 and 144 hours pi, whereas such bi-phasic peaks were absent in turkeys and ducks. Range of total antigen score at different sampling times was 7-17.5 in chickens, 10-13 in turkeys and 7-10 in ducks indicative of marked viral replication in chickens. Presence of IBD viral antigen in organs of all 3 poultry species is indicative of infections. The innate ability of turkeys and ducks to prevent appreciable replication of IBD virus after infection requires further investigation.

Infectious bursal disease virus-induced immunosuppression exacerbates Campylobacter jejuni colonization and shedding in chickens.

Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in humans in the United States. Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. To analyze a possible role of IBDV-induced immunosuppression in colonization and shedding of C. jejuni, two experiments were conducted. In both experiments, group 1 consisted of noninoculated control chickens, groups 2 and 3 were inoculated with varying doses of C. jejuni, and groups 4 and 5 were inoculated initially with IBDV followed by doses of C. jejuni similar to groups 2 and 3. Campylobacter jejuni was recovered from the cloaca and cecum, but not the small intestines, from all chickens in groups 2 and 3. In groups 4 and 5, C. jejuni was recovered from the small intestines, cecum, and cloaca from all chickens. The amount (colony-forming units/sample) of C. jejuni recovered from chickens in groups 4 and 5 was significantly greater (P < 0.05) than the amount recovered from chickens in groups 2 and 3; and C. jejuni was also present sooner in these groups than in groups 2 and 3. Bursa samples from chickens in groups 4 and 5 were significantly smaller (P < 0.05) than in the other groups. Additionally, real-time polymerase chain reaction results for IBDV were positive in groups 4 and 5 and negative in all other groups. This study indicated that IBDV infection exacerbated colonization and shedding of C. jejuni, presumably through the immune suppression this virus causes in chickens. It highlights the need for further investigation into the role of immunosuppression in preharvest control strategies for food-borne disease-causing agents.

Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection.

Effect of a variant infectious bursal disease virus (E/Del) on Salmonella typhimurium infection in commercial broiler chickens.

The effect of infectious bursal disease virus (IBDV) on Salmonella typhimurium (ST) infections in broilers was investigated in terms of Salmonella shedding and persistence, pathogenicity, and isotype specific humoral immune responses. Thirty-six, 1-day-old, straight-run commercial broiler chickens that were Salmonella negative by polymerase chain reaction (PCR) and culture were divided into two groups of 18 chicks each (ST and ST-IBDV). One group (ST-IBDV) of chicks received the E/Del strain of IBDV (10(5.0) median tissue culture infective dose [TCID50]/ml) through the ocular and cloacal routes divided into doses of 50 microl each at 2 days of age. Both groups were then inoculated with 10(8) colony-forming units (CFU)/ml nalidixic acid-resistant ST in the drinking water at 3 days of age. Environmental Salmonella counts were higher in the ST-IBDV group at 2 and 3 wk postinfection (PI) compared to the ST group. ST carriage in the cecal contents between the ST and ST-IBDV groups was not statistically different. The ST-IBDV group had a single mortality at 10 days postinfection compared to none in the ST group. The ST-IBDV group had significantly lower bursa to body weight ratios at 4 and 6 wk, as well as higher bursal lesion scores than the ST group at 2, 4, and 6 wk PI. The ST group had significant increase in serum IgG from 2 to 6 wk PI in comparison to the ST-IBDV group, which had no significant changes over time. Both IgA and IgM were significantly increased at 4 and 6 wk relative to 2-wk levels. There was an IBDV-induced failure of anti-Salmonella IgG seroconversion over time in ST-IBDV. Both groups continued to shed high levels of Salmonella up to the end of the study despite high antibody levels in the ST group and an unimpaired IgM and IgA production in the ST-IBDV group, indicating a limited influence of humoral immunity on Salmonella clearance.

Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp.

The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (10(3)-10(4) TCID50 IPNV per ml, 10(6)-10(7) colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (10(3)-10(4) TCID50 IPNV per g fish or 10(7) CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers.

Genetic heterogeneity in the VP2 gene of infectious bursal disease viruses detected in commercially reared chickens.

The genetic heterogeneity of infectious bursal disease virus (IBDV) vaccine strains was compared with IBDV detected in bursa tissue of commercially reared chickens. The vaccine strains tested represented classic viruses from the United States, South Africa, England, and France plus variant viruses from the United States. Bursa tissue samples used for the detection of IBDV from commercially reared chickens were from the United States. Genetic heterogeneity was examined using restriction fragment length polymorphisms (RFLP) of a 743-bp fragment of the VP2 gene that was amplified using reverse transcriptase/polymerase chain reaction (RT/PCR). The RT/PCR products were digested using restriction enzymes BstNI and MboI. On the basis of RFLP profiles, viruses were placed into molecular groups. Thirty-eight vaccine and laboratory strains of IBDV were placed into five molecular groups. Groups 1 and 2 contained variant viruses, groups 3 and 4 contained classic viruses, and group 5 contained Lukert/Edgar strain classic viruses. In contrast to these five molecular groups, 22 molecular groups were observed for 70 IBDV-positive samples from U.S. chicken flocks. Twenty-two of the 70 IBDV-positive samples were placed into molecular groups 1 (2 samples), 2 (18 samples), and 5 (2 samples). Nineteen new RFLP profiles that did not match the five molecular groups observed for vaccine strains were detected. No viruses were observed to have RFLP profiles like molecular groups 3 and 4 observed in the vaccine strains. The genetic heterogeneity was greater among IBDV strains circulating in commercially reared poultry compared with vaccine strains of the virus.

Molecular characterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains.

Seven infectious bursal disease virus (IBDV) strains isolated from China have been characterized in this study, including a classical strain CJ801, an attenuated strain GZ911, a variant strain GZ902, and four very virulent strains G9201, G9302, F9502, and HK46. With the use of reverse transcription-polymerase chain reaction, the full-length VP2 genes were amplified and the hypervariable regions were sequenced. Protein sequences of the hypervariable region (a.a. 143-382) of the field isolates confirmed their identities. CJ801 has the highest identity to the classical strains STC and 52/70. GZ902 has the highest identity to the American variant strains A, E, and GLS, and they share unique amino acid residue at positions 249K and 254S, which are not present in standard serotype 1 strains. Attenuated strain GZ911, like other cell culture-adapted strains, has substitutions at positions 279(D to N) and 284 (A to T) as well as in the serine-rich heptapeptide region. Hence, these substitutions may take an important role in the reduced virulence of these strains. The four very virulent strains have the highest identity to the European very virulent strain UK661 and Japanese strain OKYM. These strains share unique amino acid residues at positions 222A, 256I, and 294I, which are not present in other less virulent strains. The very virulent strains isolated in Guangdong (G9201, G9303) and Fujian (F9502) Provinces have one to five amino acid substitutions at the two hydrophilic domains of VP2 comparing with UK661 and OKYM, indicating that new very virulent strains are evolving. Phylogenetic analysis suggests that Chinese very virulent IBDVs and European very virulent strains are derived from similar origin.

Studies on antigenic relatedness of classic and variant strains of infectious bursal disease viruses.

Antigenic relatedness of six classic and variant strains of serotype 1 infectious bursal disease virus (IBDV) and one serotype 2 IBDV was investigated by Western blotting and enzyme-linked immunosorbent assay (ELISA) using polyclonal, monoclonal, and monospecific antibodies to single viral proteins (VP2 and VP3). All virus strains cross-reacted similarly, and the viruses were not distinguishable from each other by ELISA or Western blot analysis performed with polyclonal or non-neutralizing monoclonal and monospecific antibodies. Non-neutralizing antibodies against the VP2 (40 kilodaltons) reacted strongly with VP2 of classic and variant strains of serotype 1 and reacted weakly with VP2 of serotype 2 OH strain. This indicated that common antigens were recognized and that these epitopes were not strictly dependent on the native structure of the virus.

Comparative pathogenicity and serogrouping of three Washington isolates of infectious bursal disease virus.

The pathology of three infectious bursal disease virus (IBDV) isolates of Washington poultry origin (WA-678, WA-770, and WA-994) and seven other known IBDV strains (SAL, D-78, MO, OH, Var-A, 2512, and IM) was studied in 3-week-old specific-pathogen-free chickens. Inoculation with IM and 2512 strains resulted in illness and death. No clinical signs or mortality were present with WA-678, WA-770, and WA-994. Macroscopically, bursae were swollen and gelatinous with occasional hemorrhages. Isolate WA-994 caused marked bursal atrophy. Isolate WA-678 elicited moderate bursal pathology. Isolate WA-770 resulted in minimal atrophy. Strains IM and 2512 caused severe bursal atrophy, and strains Var-A and D-78 caused moderate atrophy. Strains SAL, MO, and OH caused no demonstrable bursal atrophy. Results of the cross-neutralization study showed that the three isolates were more closely related to serotype 1 than to serotype 2 IBDV.

Presence of lesions without virus replication in the thymus of chickens exposed to infectious bursal disease virus.

Specific-pathogen-free (SPF) chickens were exposed to the IM and VA isolates of virulent infectious bursal disease virus (IBDV). Both viruses induced rapidly progressing lymphoid cell depletion in the bursa. The bursal lesions persisted through the observation period of 16 days. The virus-exposed birds also had histologic lesions in the thymus. Thymic lesions peaked at 3-4 days postinoculation (PI) and then subsided. Immunofluorescence (IF) and antigen-capture enzyme-linked immunosorbent assay (ELISA) detected abundant viral antigen in the bursa, but not in the thymus, of chickens during the first week after infection with IM-IBDV or VA-IBDV. This result indicated that the presence of histologic lesions in the thymus was not associated with active infection and replication of the virus in thymic cells. Inoculation of homogenates of bursal and thymic tissues from virus-exposed chickens into embryonated chicken eggs revealed the presence of infectious virus from both tissues. We speculated that the virus recovered from thymus may have been contributed by virus-infected cells that were circulating through the thymus at the time when this tissue was homogenized.