David Solanes

Experimental Model of Tuberculosis in the Domestic Goat after Endobronchial Infection with Mycobacterium caprae

ABSTRACT Caprine tuberculosis (TB) has increased in recent years, highlighting the need to address the problem the infection poses in goats. Moreover, goats may represent a cheaper alternative for testing of prototype vaccines in large ruminants and humans. With this aim, a Mycobacterium caprae infection model has been developed in goats. Eleven 6-month-old goats were infected by the endobronchial route with 1.5 × 103 CFU, and two other goats were kept as noninfected controls. The animals were monitored for clinical and immunological parameters throughout the experiment. After 14 weeks, the goats were euthanized, and detailed postmortem analysis of lung lesions was performed by multidetector computed tomography (MDCT) and direct observation. The respiratory lymph nodes were also evaluated and cultured for bacteriological analysis. All infected animals were positive in a single intradermal comparative cervical tuberculin (SICCT) test at 12 weeks postinfection (p.i.). Gamma interferon (IFN-γ) antigen-specific responses were detected from 4 weeks p.i. until the end of the experiment. The humoral response to MPB83 was especially strong at 14 weeks p.i. (13 days after SICCT boost). All infected animals presented severe TB lesions in the lungs and associated lymph nodes. M. caprae was recovered from pulmonary lymph nodes in all inoculated goats. MDCT allowed a precise quantitative measure of TB lesions. Lesions in goats induced by M. caprae appeared to be more severe than those induced in cattle by M. bovis over a similar period of time. The present work proposes a reliable new experimental animal model for a better understanding of caprine tuberculosis and future development of vaccine trials in this and other species.

Experimental infection of young adult European breed sheep with Rift Valley fever virus field isolates.

The increasing interest in Rift Valley fever virus (RVFV) and its potential impact on naive animal populations deserve revisiting experimental reproduction of RVFV infection, particularly in those animal breeds for which no data about their susceptibility to RVFV infection have ever been recorded. In this study we show the susceptibility of 9-10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and virus shedding (variable in length depending on the RVFV isolate used) were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in adult lambs and no other clinical symptoms were observed, with the exception of corneal opacity (blue eye) found in 3 out of 16 subcutaneously inoculated sheep. In conclusion, adult sheep from this European breed are readily infected with RVFV without apparent clinical manifestations.

Correlation between clinico-pathological outcome and typing of Haemophilus parasuis field strains.

Haemophilus parasuis is the etiologic agent of Glässers disease in pigs, which is pathologically characterized by serofibrinous polyserositis and arthritis. H. parasuis include virulent and non-virulent strains and confirmation of virulence in H. parasuis is still dependent on experimental reproduction of the disease. Since the variability in virulence is supported by serotyping and genotyping (particularly, multilocus sequence typing [MLST]), we examined the relationship between the classification of 8 field strains by these methods and their capacity to cause disease in snatch-farrowed, colostrum-deprived piglets. The severity of clinical signs and lesions produced by the different strains correlated with the quantity of H. parasuis recovered from the lesions. However, the virulence of the strains in the animal model did not show a total correlation with their serovar or their classification by MLST. More studies are needed to identify a virulence marker that could substitute animal experimentation in H. parasuis. In addition, we reproduced disease in domestic pigs with a strain isolated from the nasal cavity of wild boars. This result indicates the existence of virulent strains of H. parasuis in wild suids, which could produce disease under appropriate circumstances, and suggests a possible source of infection for domestic pigs.

Lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray-dried porcine plasma.

An experiment was conducted to determine whether spray-dried porcine plasma containing 2·47 × 105 dna copies of porcine circovirus type 2 (pcv-2) could infect weanling pigs when fed to them. Five specific pathogen-free (spf) weanling pigs were fed ad libitum for 45 days a control diet and six pigs were fed a test diet containing 8 kg sdpp per 100 kg feed. The two groups were housed in separate biosecurity level-3 rooms. None of the pigs in either group developed any clinical signs or became pcv-2 viraemic or seroconverted.

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.

Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

Livestock susceptibility to infection with middle east respiratory syndrome coronavirus

Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs. To determine whether other animals are potential reservoirs, we inoculated MERS-CoV into llamas, pigs, sheep, and horses and collected nasal and rectal swab samples at various times. The presence of MERS-CoV in the nose of pigs and llamas was confirmed by PCR, titration of infectious virus, immunohistochemistry, and in situ hybridization; seroconversion was detected in animals of both species. Conversely, in sheep and horses, virus-specific antibodies did not develop and no evidence of viral replication in the upper respiratory tract was found. These results prove the susceptibility of llamas and pigs to MERS-CoV infection. Thus, the possibility of MERS-CoV circulation in animals other than dromedaries, such as llamas and pigs, is not negligible.

Experimental West Nile virus infection in Gyr-Saker hybrid falcons.

West Nile disease (WND) has become a major public and veterinary health concern since the appearance of West Nile virus (WNV) in New York in 1999. The following panzootic spread in the U.S. and the recent WNV outbreaks in Europe and the Mediterranean Basin have increased interest in WND. Despite considerable investigation of WNV infection in birds, the effects of WNV on avian populations are still largely unknown. In Europe, raptors have been found to be particularly susceptible to WNV infection, but studies in birds of prey are lacking. To our knowledge, the present study is the first to report an experimental infection with WNV in Gyr-Saker hybrid falcons. We show that 10-week-old captive-reared Gyr-Saker (Falco rusticolus × Falco cherrug) hybrid falcons are susceptible to WNV infection. Neither morbidity nor mortality was observed after subcutaneous WNV inoculation with mixed extracts of non-infected mosquito salivary glands. Both the macroscopic and microscopic lesions observed were similar to those previously reported in naturally and experimentally infected North American raptors. The results obtained in the present study demonstrate that although Gyr-Saker hybrid falcons do not seem to be a good reservoir for WNV transmission via mosquito, they can become infected with WNV, develop viremia and antibodies, and are able to shed the virus.

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.

Standardization of pathological investigations in the framework of experimental ASFV infections

African swine fever is still one of the major viral diseases of swine for which a commercial vaccine is lacking. For the design and development of such preventive products, researchers involved in African swine fever virus (ASFV) vaccinology need standardized challenge protocols and well characterized clinical, pathological and immunological responses of inbreed and outbreed pigs to different viral strains and vaccine-like products. The different approaches used should be assessed by immunologist, virologist and pathologist expertise. The main objectives of this guideline are to (1) briefly contextualize the clinical and pathological ASFV presentations focusing on points that are critical for pathogenesis, (2) provide recommendations concerning the analysis of clinical, gross and microscopic observations and (3) standardize the pathological report, the terminology employed and the evaluation of the severity of the lesions between the ASFV research groups for comparing inter-group data. The presented guidelines establish new approaches to integrate such relevant pathological data with virological and immunological testing, giving support to the global interpretation of the findings in the future experiments of ASFV-related vaccinology and immunology.