Fernando Fernandez

West Nile virus isolation from equines in Argentina, 2006.

West Nile virus (WNV) was isolated from the brains of 3 horses that died from encephalitis in February 2006. The horses were from different farms in central Argentina and had not traveled outside the country. This is the first isolation of WNV in South America.

Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines

Abstract Heterotypic passive immunity to IND (P[5]G6) bovine rotavirus (BRV) was evaluated. Three groups of calves (n = 5 per group) were fed 1% pooled colostrum supplements (birth to 7 days of age) from BRV seropositive cows vaccinated with recombinant SA11(P[2]G3) rotavirus-like particles (VLPs), recombinant SA11 rotavirus core-like particles (CLPs), or inactivated SA11 rotavirus (SA11). Control calves (n = 5 per group) received either pooled colostrum from unvaccinated (BRV field exposure seropositive) control cows, or no colostrum. IgG1 antibody titers to IND BRV for the pooled colostrum were: 1048576 (VLP); 1048576 (CLP); 262144 (SA11); and 16384 (control colostrum). Elevated titers of BRV neutralizing (VN) antibodies were present in VLP colostrum (98 000), and SA11 colostrum (25 000), but not in CLP colostrum (1400), compared to colostrum from nonvaccinates (2081). Calves were orally inoculated with virulent IND BRV at 2 days of age and challenged at post-inoculation day (PID) 21. Calves were monitored daily for diarrhea and faecal BRV shedding through PID 10 and post-challenge day (PCD) 10. After colostrum feeding, the IgG1 antibody titers were highest in serum and faeces of calves fed VLP and CLP colostrum, but VN and IgA antibodies were highest in calves fed VLP colostrum. After BRV inoculation, calves fed colostrum from vaccinated cows had significantly fewer days of BRV-associated diarrhea and BRV shedding than control calves. All calves fed VLP colostrum were protected from diarrhea after BRV inoculation; two calves shed BRV. In the CLP colostrum group, one calf developed BRV-associated diarrhea and all calves shed virus. In the SA11 colostrum group, three calves developed BRV-associated diarrhea and four calves shed virus. BRV-associated diarrhea and shedding occurred in 9 of 10 control calves. Active IgM antibody responses occurred in faeces and/or serum of most calves after BRV inoculation. However, the highest active antibody responses (IgM and IgG1 in serum, and IgM, IgG1 or IgA in faeces) after BRV inoculation were in calves fed control or no colostrum, in association with clinical diarrhea in most of these calves. After challenge at PID 21, BRV-associated diarrhea and shedding were of short duration or absent, in all groups. These results demonstrate the efficacy of colostrum from VLP vaccinated cows to provide heterologous, passive protection against BRV diarrhea and shedding in calves. In comparison, calves fed CLP or SA11 colostrum were only partially protected against BRV diarrhea or shedding.

Modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus

Abstract The effect of colostral maternal antibodies (Abs), acquired via colostrum, on passive protection and development of systemic and mucosal immune responses against rotavirus was evaluated in neonatal calves. Colostrum-deprived (CD) calves, or calves receiving one dose of pooled control colostrum (CC) or immune colostrum (IC), containing an IgG1 titer to bovine rotavirus (BRV) of 1:16,384 or 1:262,144, respectively, were orally inoculated with 105.5 FFU of IND (P[5]G6) BRV at 2 days of age. Calves were monitored daily for diarrhea, virus shedding and anti-BRV Abs in feces by ELISA. Anti-rotavirus Ab titers in serum were evaluated weekly by isotype-specific ELISA and virus neutralization (VN). At 21 days post-inoculation (dpi), all animals were euthanized and the number of anti-BRV antibody secreting cells (ASC) in intestinal and systemic lymphoid tissues were evaluated by ELISPOT. After colostrum intake, IC calves had significantly higher IgG1 serum titers (GMT=28,526) than CC (GMT=1195) or CD calves (GMT<4). After BRV inoculation, all animals became infected with a mean duration of virus shedding between 6 and 10 days. However, IC calves had significantly fewer days of diarrhea (0.8 days) compared to CD and CC calves (11 and 7 days, respectively). In both groups receiving colostrum there was a delay in the onset of diarrhea and virus shedding associated with IgG1 in feces. In serum and feces, CD and CC calves had peak anti-BRV IgM titers at 7 dpi, but IgA and IgG1 responses were significantly lower in CC calves. Antibody titers detected in serum and feces were associated with circulation of ASC of the same isotype in blood. The IC calves had only an IgM response in feces. At 21 dpi, anti-BRV ASC responses were observed in all analyzed tissues of the three groups, except bone marrow. The intestine was the main site of ASC response against BRV and highest IgA ASC numbers. There was an inverse relationship between passive IgG1 titers and magnitude of ASC responses, with fewer IgG1 ASC in CC calves and significantly lower ASC numbers of all isotypes in IC calves. Thus, passive anti-BRV IgG1 negatively affects active immune responses in a dose-dependent manner. In ileal Peyer’s patches, IgM ASC predominated in calves receiving colostrum; IgG1 ASC predominated in CD calves. The presence in IC calves of IgG1 in feces in the absence of an IgG1 ASC response is consistent with the transfer of serum IgG1 back into the gut contributing to the protection of the intestinal mucosa.

Molecular characterization of the first isolation of rotavirus in guanacos (Lama guanicoe)

Summary.Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and numerous animal species. We report G-type, P-type and phylogenetic analysis of two RV strains isolated from newborn guanacos (Lama guanicoe) with acute diarrhoea in Argentina. Isolates were group A G8 RV. Phylogenetic analysis showed a close relationship to other G8 bovine RV from Japan, U.S.A. and Switzerland. P-types were P[1] and an unusual P[14], related to human and goat P[14] strains. This is the first report of a P[14]G8 strain in our country and the first study on molecular epidemiology of rotavirus found in a new species.

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

Abstract The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30–200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P <0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6–100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were ∼2–10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary.We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4 µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.

Foot-and-mouth disease virus (FMDV) experimental infection: Susceptibility and immune response of adult mice

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.

Equine herpesvirus 2 (EHV-2) infection in thoroughbred horses in Argentina

BackgroundEquine herpesvirus 2 is a gamma-herpesvirus that infects horses worldwide. Although EHV-2 has been implicated in immunosuppression in foals, upper respiratory tract disease, conjunctivitis, general malaise and poor performance, its precise role as a pathogen remains uncertain. The purpose of the present study was to analyse the incidence of EHV-2 in an Argentinean horse population and correlate it with age and clinical status of the animals.ResultsA serological study on 153 thoroughbred racing horses confirmed the presence of EHV-2 in the Argentinean equine population. A virus neutralization test showed a total of 79.7 % animals were sero-positive for EHV-2. An increase in antibodies titre with age as well as infection at earlier ages were observed.EHV-2 was isolated from 2 out of 22 nasal swabs from horses showing respiratory symptoms. The virus grew slowly and showed characteristic cytopathic effect after several blind passages on RK13 cells. The identity of the isolates was confirmed by nested PCR and restriction enzyme assay (REA).ConclusionThis is the first report on the presence of EHV-2 in Argentina and adds new data to the virus distribution map. Though EHV-2 was isolated from foals showing respiratory symptoms, further studies are needed to unequivocally associate this virus with clinical symptoms.

Reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus — Escherichia coli vaccine in the dam

Abstract An outbreak of neonatal diarrhea occurred among beef calves (2000 animals) from one large Argentinian farm in 1985. Rotavirus was detected in 78% ( 106 136 ) and enterotoxigenic Escherichia coli in 1.5% of the samples ( 2 136 ) obtained from sick calves. In comparison rotavirus was identified in only 1.6% ( 1 63 ) of the samples from clinically healthy calves. The rotavirus strain responsible for the outbreak was characterized as serotype 6 belonging to group A. In the following three years the protective capacity of a combined rotavirus—E. coli inactivated vaccines administered to the dams during the last third of the gestation period was evaluated on this farm by comparison of morbidity due to diarrhea in calves from vaccinated vs. placebo cows within the same year. The morbidity due to diarrhea among calves from dams in the vaccinated and placebo groups was 34% and 77%, respectively in 1986; 23% and 47% in 1987, and 15% and 34%, in 1988. In 1987 morbidity of diarrhea in calves born from vaccinated heifers was 54% and 74% in calves from placebo heifers. In 1988 morbidity from diarrhea was 41% and 54%, respectively among calves in these two groups. In all experiments, calves from heifers showed significantly greater morbidity than calves from cows. Differences in diarrhea morbidity between the vaccinated and placebo groups were statistically significant (P<0.05). Additional studies showed that the diarrhea had a significant influence (P<0.05) on the average live weight of the calves at weaning (5 to 7 months) with an average weight loss of 7.8 kg per calf among the calves affected with diarrhea.