Immune response following a vaccination campaign against rabies in dogs from northwestern Spain.

A cross-sectional study was carried out to determine the seroprevalence of antibodies to the rabies virus in 156 vaccinated dogs from two provinces in the Castilla y León Autonomous Community (northwest Spain). An obligatory anti-rabies programme is currently in place in this region. Seroprevalence was established by an indirect enzyme-linked immunosorbent assay. Of the 156 animals tested, 91 (58.3%) were positive (titres of 0.5 IU ml-1 or above). However, Soria province showed a significantly higher seroprevalence (77.1%) than León province (50%). Age, sex, habitat and use were evaluated with regard to the response obtained after vaccination: no significant differences were discovered for any of these factors. However, guard and herding dogs in León province tended to have lower seroprevalence than dogs not used in these ways. In general, there is a limited response to the vaccination programme in dogs from Castilla y León-especially in León province.

Role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in Spain.

Faeces samples from diarrhoeic and non-diarrhoeic lambs and goat kids aged 1-45 days were examined for enteric pathogens. Cryptosporidium parvum was detected in both diarrhoeic lambs (45%) and goat kids (42%) but not in non-diarrhoeic animals. F5+ (K99+) and/or F41+ Escherichia coli strains were isolated from 26% and 22% of the diarrhoeic lambs and goat kids, respectively, although these strains, which did not produce enterotoxins ST I or LT I, were found with similar frequencies in non-diarrhoeic animals. A F5-F41-ST I+ E. coli strain was isolated from a diarrhoeic lamb (0.6%). Verotoxigenic E. coli was isolated from both diarrhoeic and non-diarrhoeic lambs (4.1% and 8.2%, respectively) and there was no association between infection and diarrhoea. The prevalence of group A rotavirus infection in diarrhoeic lambs was very low (2.1%). Groups A and B rotaviruses were detected in three (8.1%) and five (13.5%) diarrhoeic goat kids from two single outbreaks. Group C rotaviruses were detected in four non-diarrhoeic goat kids. An association of diarrhoea and infection was demonstrated only for group B rotavirus. Clostridium perfringens was isolated from 10.8% of the diarrhoeic goat kids but not from non-diarrhoeic goat kids or lambs. Salmonella arizonae was isolated from a diarrhoeic goat kid (2.7%) and the clinical characteristics of the outbreaks where these two latter enteropathogens were found different from the rest. Picobirnaviruses were detected in a diarrhoeic lamb. No coronaviruses were detected using a bovine coronavirus ELISA. No evidence was found of synergistic effect between the agents studied. Enteric pathogens were not found in four (8.7%) and three (20%) outbreaks of diarrhoea in lambs and goat kids, respectively.

Relationship between somatic cell count and intramammary infection of the half udder in dairy ewes.

A total of 1382 milk samples from half udders of three breeds of ewes (686 Assaf, 422 Churra, and 274 Castellana) were collected aseptically at midlactation from 18 flocks in the Castile-León region of Spain. Bacteriological tests were carried out on the samples to ascertain the status and type of IMI. Somatic cell counts were determined for all samples after bacteriological analysis. Analysis of variance showed significant effects of breed, IMI status, flock nested within breed by milking type, organism within IMI status, and interactions of breed by milking type and of breed by organism. The most frequent bacterial groups were staphylococci, especially coagulase-negative staphylococci, followed by streptococci, micrococci, and yeasts. The SCC response was related to the bacterial group involved in the IMI; coagulase-negative staphylococci showed significantly lower SCC values than those for coagulase-positive staphylococci and streptococci. Highest percentages of IMI by major pathogens and highest SCC, both in the absence of IMI and in staphylococcal IMI, were in Assaf ewes. The threshold of 300,000 cells/ml allowed 81% of the half udders to be correctly classified according to IMI status. However, the existence of breed differences in SCC response and IMI type could make the use of specific SCC thresholds for each breed advisable.

Canine seroprevalence of Rickettsia conorii infection (Mediterranean spotted fever) in Castilla y León (northwest Spain).

A seroepidemiological study was conducted in 308 dogs to determine the presence of antibodies to Rickettsia conorii, using an indirect immunofluorescence assay (IFA). Seven of the provinces of the Castilla y León region (Burgos, León, Palencia, Salamanca, Soria, Valladolid, and Zamora) were covered by the study. Of the 308 dogs analysed, 72 (23.4%) showed significant titers by IFA (1/40 or higher). Seroprevalences were significantly different between provinces of origin of the animals. These were below 30% in almost all the provinces studied, except for Salamanca province, where the percentage of seropositive dogs was much greater (93.3%). Potential risk factors (presence of ticks on the animals, age, sex, use, habitat, and season) relating to the presence of Mediterranean spotted fever, or Boutonneuse fever, were evaluated. Animals used for guard or pastor activities and those living in rural areas (these factors are closely linked), together with those suffering from tick infestation, had significantly higher seroprevalence than the remainder. The frequency of seropositive dogs increased during the summer months, and these coincide with the period of greatest activity by the vector. Sex and age variables were not identified as risk factors.

An outbreak of diarrhoea associated with atypical rotaviruses in goat kids.

The presence of rotaviruses was investigated in the faeces of 31 goat kids in a dairy herd that experienced an outbreak of severe diarrhoea which caused dehydration, anorexia and prostration in seven (22.6 per cent) of them. All the affected animals were two to three days old. A group A-specific ELISA failed to detect rotaviruses in any of the samples but the characteristics electropherotype of group B rotaviruses was observed by electrophoresis in polyacrylamide gels in six of the animals. A highly significant statistical association between the shedding of rotavirus and the occurrence of diarrhoea was demonstrated. All the rotaviruses were detected in animals three to four days old. Cryptosporidium parvum, Clostridium perfringens and enterotoxigenic Escherichia coli were not detected in the seven diarrhoeic animals.

Prevalence of neutralizing antibodies to 9 rotavirus strains representing 7 G-serotypes in sheep sera.

Neutralizing antibodies to 9 rotavirus strains representing serotypes G1, G3, G5, G6, G8, G9, and G10 were investigated in 212 ovine serum samples from 3 age groups, 1-week-old lambs, 2- to 3-months-old lambs and adult sheep. All sera from 1-week-old lambs had neutralizing antibodies to all 9 rotavirus strains. Both neutralizing antibody titers and prevalences to all 9 strains markedly decreased in the 2- to 3-months-old lamb group and increased again in the adult sheep group. Also, adult sheep sera neutralized a larger number of rotavirus strains than 2- to 3-months-old lamb sera. The highest neutralizing antibody titers and prevalences were found to strains B223 and K923, representing serotype G10, to strain RRV, representing serotype G3, and to strain NCDV, representing serotype G6, indicating that these could be the predominant 3 rotavirus serotypes in Spanish sheep. The rotavirus serotypes infecting sheep observed by us differ from those described for cattle, where G6 is the most prevalent serotype followed by G10, and G3 has been seldom found. Very low prevalences were observed for strains WA and OSU representing serotypes G1 and G5 respectively, suggesting that they probably do not infect sheep and neutralizing antibodies found are derived from heterotypic responses to other serotypes. Intermediate prevalences and titers were found to strains UK (serotype G6), 69M (serotype G8) and WI61 (serotype G9). Neutralizing antibodies distinguished between different strains sharing their VP7 specificity: B223 and K923, a bovine and an ovine serotype G10 strains, and NCDV and UK, two serotype G6 bovine rotavirus strains with different VP4 antigen.

Seroepidemiological survey for Borrelia burgdorferi (Lyme disease) in dogs from northwestern of Spain.

A random epidemiological study was undertaken to estimate the prevalence of canine borreliosis (Lyme disease) in Castilla y León, the largest region in Spain. The presence of antibodies was determined by indirect immunofluorescence assay (IFA), using the Borrelia burgdorferi B31 strain as antigen. Sera from 308 dogs from 7 provinces in the region were tested. Of all the animals sampled, 37 (21%) were seropositive (titres of 1/64 or above). Almost all the provinces had seroprevalence of 20% or below, except for an important focus of borreliosis in the Zamora province (42.8%). Potential risk factors (age, sex, use, habitat, season, and presence of ticks on the animals) relating to the presence of antibodies were evaluated. Those dogs which had at some time had ticks were more often seropositive, at 24.2%, than those which had never had them, at only 6.2% (p < 10-n). No significant differences were discovered for the remaining factors studied. This work indicates that dogs in Castilla y León, even if in most cases they do not develop the disease, are exposed to the Lyme disease agent.

Serpulina hyodysenteriae challenge of fattening pigs vaccinated with anadjuvanted bivalent bacterin against swine dysentery.

An experimental fattening period was performed with 36 pigs in order to evaluate the safety and efficacy of a bivalent adjuvanted Serpulina hyodysenteriae bacterin. The pigs originated from three different farms and were split into six groups. Eighteen pigs were vaccinated twice, two weeks apart and the rest served as controls. One unvaccinated pig in each group was inoculated intragastrically with 4 x 10(8) active S. hyodysenteriae spirochaetes, B 78 strain. Weight evolution, clinical symptoms, shedding of S. hyodysenteriae and serum antibody titres were monitored for 12 weeks in all pigs. The severity of clinical symptoms and shedding of spirochaetes in faeces were significantly reduced in vaccinated pigs as compared to controls. Statistically significant differences in weight gain between vaccinates and controls were detected coinciding with the maximum shedding of S. hyodysenteriae in faeces. All vaccinated pigs seroconverted, as measured by indirect ELISA, and had a good anamnestic response after the second vaccine dose. The bacterin proved to be useful for both the reduction of clinical symptoms and the number of infected animals in a fattening unit. Adverse post-vaccinal reactions were not observed in any pig.

Evaluation of an enzyme-linked immunosorbent assay for the detection of sheep infected and vaccinated with Brucella melitensis.

An indirect enzyme-linked immunosorbent assay (ELISA), using the lipopolysaccharide of the cell wall as an antigen, was used to detect Brucella melitensis antibodies in ovine serum. The test was carried out on 703 samples of field serums, which were also analyzed by the complement fixation (CF) test and the rose Bengal (RB) test. The ELISA results were more similar to those of the CF test (kappa = 0.89) than to the results of the RB test (kappa = 0.73). The ELISA also had high sensitivity (94.7%) and a somewhat lower specificity (90.4%). One group of 139 young brucellosis-free animals 3-6 months of age were vaccinated with B. melitensis rev. 1 at a dose of 1.2 x 10(9) live organisms. The ELISA detected a significantly lower number of reactors than the CF and RB tests (P < 0.001). The ELISA values remained below the cutoff level during the 9 months following vaccination.

Evaluation of a blocking ELISA using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies.

Two blocking enzyme-linked immunosorbent assays (ELISAs) involving the use of monoclonal antibodies (MAb) as capture and detecting agents and an indirect fluorescence test (IFT) were used for the detection of porcine epidemic diarrhea virus (PEDV) antigen or its antibodies. In the ELISA for viral antigen detection, the blocking step is accomplished by incubation of fecal samples, in duplicate wells, with PEDV-specific positive or negative serum, whereas in the ELISA for antibody detection the blocking step is accomplished by incubation of serum samples with a gut-origin virus suspension. All the methods developed were used to monitor an experimental infection in piglets with the CV-777 strain of PEDV and a natural PED outbreak in a swine fattening unit. The antigen-detection ELISA was able to detect PEDV shedding for a longer time than have previously described methods. The blocking ELISA for antibody detection was able to detect the serum antibody response sooner after PEDV infection than did IFT.

Rotavirus excretion by kids in a naturally infected goat herd.

A cross-sectional study was carried out in a dairy goat herd, investigating the presence of rotavirus by means of ELISA, polyacrylamide gel electrophoresis (PAGE) and two latex agglutination tests in feces of 63 goat kids younger than 1 month, with and without diarrhea, and in feces of 19 adult goats during the first few days after parturition. All animals belonged to a herd located in the mountains of the León province (NW Spain). Rotaviruses were found in 18 out of 63 goat kid fecal samples but no significant association between shedding of rotavirus and presence of diarrhea could be established. Rotaviruses were found in kids aged 6 to 21 days, and more frequently between 6 and 10 days. No shedding of virus was detected in any of the adults. Considering ELISA as the reference test, PAGE and both latex agglutination tests were less sensitive. One of the latex tests was also highly non-specific. All PAGE-positive samples showed the typical electropherotype of group A rotavirus. Feces were also screened for other pathogens including Escherichia coli, Clostridium perfringens and Cryptosporidium parvum. C. parvum oocysts were detected in the feces of six out of 45 goat kids tested, all six suffering from diarrhea. This paper represents the first description of rotavirus infections in goats in Spain. The possible mechanisms of viral diffusion within the herd and its role as pathogen in goats are discussed.

Se realizó un estudio de corte en una explotación de ganado caprino lechero investigando la presencia de rotavirus mediante ELISA, electroforesis en gel de poliacrilamida (PAGE) y 2 pruebas de aglutinación en látex en las heces de 63 cabritos menores de un mes, con y sin diarrea, y en las heces de 19 cabras durante los primeros días tras el parto. Todos los animales pertenecían a una explotación situada en la montaña de la provincia de León (NO de España). Se encontraron rotavirus en 18 de las 63 muestras fecales de cabritos analizadas, aunque no pudo demostrarse la existencia de asociación estadísticamente significativa entre la eliminación del virus y la presencia de diarrea. Las edades de los cabritos en que se detectó eliminación de rotavirus estaban comprendidas entre los 6 y los 21 días y más frecuentemente entre los 6 y los 10. Considerando ELISA como la prueba de referencia tanto PAGE como ambas pruebas de aglutinación en látex fueron menos sensibles. Una de las pruebas de aglutinación en látex fue también muy inespecífica. En todas las muestras positivas mediante electroforesis se observó el electroferotipo característico de los rotavirus del grupo A. También se estudió la presencia en las heces de otros patógenos como Escherichia coli, Clostridium perfringens y Cryptosporidium parvum. Se detectaron ooquistes de C. parvum en las heces de 6 de 45 cabritos investigados, los 6 padecían diarrea. Este trabajo representa la primera descripción de la infección por rotavirus en la especie caprina en España. Se discuten los posibles mecanismos de difusión del virus dentro de la explotación así como su papel como patógeno en cabras.

Comparison of a monoclonal antibody capture ELISA (MACELISA) to indirect ELISA and virus neutralization test for the serodiagnosis of transmissible gastroenteritis virus.

An enzyme-linked immunosorbent assay (ELISA) in which the antigen is captured to the plate by monoclonal antibodies (MACELISA) was developed for the detection of antibodies to transmissible gastroenteritis virus (TGEV). The viral antigen was semipurified from TGEV-infected cells by simple ultracentrifugation. MACELISA results with 258 field sera were compared with those of a standard indirect ELISA and with the virus neutralization test (VNT). Sensitivity, specificity, and kappa values of MACELISA indicated a strong correlation with VNT results, whereas an indirect ELISA was less sensitive and much less specific than VNT. The serologic response of 4 pigs orally inoculated and intraperitoneally boostered with TGEV was compared using the 3 tests. Its sensitivity, specificity, and ability to use unpurified antigen make the MACELISA the advisable first step in TGEV serodiagnosis.