Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds.

Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified.     

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.     

Experimental demonstration of an antigenic relationship between Leptospira and equine cornea

Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. They bound themselves to equine cornea in vivo and in vitro as was proved by immunofluorescence. This antigenic relationship may be in part responsible for pathogenesis of corneal opacity in leptospirosis of horses.     

Biochemical analysis by SDS-PAGE and western blotting of the antigenic relationship between Leptospira and equine ocular tissues.

The antigenic relationship between Leptospira interrogans, equine cornea and lens was previously noted in our studies. Serum antibodies from horses inoculated with serovars wolffi, pomona, icterohaemorrhagiae, and tarassovi, were able to bind to five antigenic fractions from both cornea and lens, as demonstrated by immunoblotting. These antigens seem to be made up of protein and carbohydrates. After treatment with periodate for cleavage of glycoside ring structures, those fractions kept their condition of target for anti-Leptospira antibodies. Nevertheless, all fractions lost that condition after being subjected to peptide hydrolysis.     

Observation on the antigenic affinity of Leptospira and Brucella in the serological examination of cattle]

36,9% bovine serums which showed a non-specific reaction in examination for brucellosis had titre greater than or equal to 1:400 leptospirosis agglutinins. 15.1% of the serums showed a positive reaction (greater than 1:800), mostly those against L. sejroe and L. grippotyphosa. The cross reactions, found in individual cases within up to 39 days since the first reaction to brucellosis in cattle may signalize early Leptospira infections.     

Determination of the antigenic relationships among six serotypes of infectious bronchitis virus using the enzyme-linked immunosorbent assay and serum-neutralization test

The indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect antibody to infectious bronchitis (IB) virus in chickens. A cross-reactivity study was performed to determine the antigenic relationships among six strains: Massachusetts, Connecticut, Beaudette, and three Ontario field isolates. For comparison, the cross-reactivity study was performed in eggs using the serum-neutralization (SN) test for IB. The ELISA proved to be more broadly cross-reactive than the IB-SN test. This suggests that the ELISA for IB will be useful as a rapid diagnostic test for serum profiling in unvaccinated flocks, regardless of the IB serotype involved.     

Studies on the antigenic relationships of six adherent isolates of Bordetella bronchiseptica

Six isolates of Bordetella bronchiseptica recovered from swine with atrophic rhinitis were studied. All hemagglutinated swine red blood cells, autoagglutinated in saline and showed fimbriae by electron microscopy. Hyperimmune sera against each were produced in rabbits and the antigenic relationships between the isolates were studied by cross-absorption and by the determination of the cross-reactivity indices of pairs of sera. Three isolates seemed to be identical by both methods, while 2 others showed close antigenic relationships. Hemagglutination titers with heterologous antigens and cross-reactivity indices greater than 0 suggest some degree of cross-immunity among the isolates studied, even when antigenic heterogeneity was demonstrated.     

Demonstration of parvovirus in Canadian swine and antigenic relationships with isolates from other countries.

A Canadian isolate of porcine parvovirus, isolated from cultured pig thyroid cells, was shown to be antigenically indistinguishable from a British (59e/63) and a German (G10/1) strain when treated by the modified direct complement-fixation, the hemagglutination-inhibition and the fluorescent antibody tests. These tests also revealed that antibodies to parvoviruses were detectable in a large proportion of the conventionally raised pigs in the provinces of Quebec and Ontario. Cell cultures, prepared from tissues collected in a slaughterhouse, were often found to be infected with parvovirus. In cell cultures the infection was demonstrated more effectively by immunofluorescence than by the hemagglutination test.     

Lack of antigenic relationships between avian pneumovirus and four avian paramyxoviruses

Avian paramyxoviruses (PMVs) and avian pneumovirus (APV) belong to the family Paramyxoviridae. Antigenic relationships between PMVs were shown previously, hence, this study was designed to investigate possible antigenic relationships between APV and four avian PMVs (PMV-1, PMV-2, PMV-3, and PMV-7). Enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) test, and virus neutralization (VN) test in chicken embryos and in Vero cells were used. The HI test was performed with the PMVs as antigens against the APV and PMVs antisera. The ELISA and VN test in chicken embryos were performed with PMVs and APV antigens and antisera. The VN test in vero cells was performed with the APV as an antigen against the PMV antisera. All the viruses were isolated in the United States or Canada. No antigenic relationships between APV and the PMVs were detected by the described tests.     

Risk factor analysis for antibodies to Brucella, Leptospira and C. burnetii among cattle in the Adamawa Region of Cameroon: a cross-sectional study

Brucellosis, leptospirosis and Q fever are important livestock diseases, commonly responsible for significant production losses, yet their epidemiology in sub-Saharan Africa is largely unknown. Animal reservoirs pose the main risk of transmission to humans, where serious disease can occur. In the developing world setting, the flu-like symptoms of the acute stages of these diseases can be misdiagnosed as malaria, which can result in the administration of the wrong treatment, prolonged disease and increase in antibiotic resistance. Multivariable mixed-effects logistic regression models in this study revealed potential risk factors associated with the aforementioned pathogens in cattle in the Adamawa Region of Cameroon, with wildlife, namely, buffaloes, playing a major role in both Brucella and Coxiella burnetii seropositivity. Cattle mixing with other herds at night and cattle grazing in an area on a route taken by herds on transhumance appear to be positively associated with Leptospira seropositivity, while female cows and whether buffaloes are seen during grazing or transhumance are positively associated with C. burnetii seropositivity. On the other hand, animals that have been on transhumance in the past year and animals belonging to herdsmen of the Fulbe ethnic group appear to be protected against Leptospira and C. burnetii, respectively. Cattle of more than 2 years old appear to have increased odds of being seropositive to either pathogen. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these three pathogens in Africa, taking particular consideration of the wildlife involvement in the disease transmission.     

Comparative serological study of Leptospira serovar hardjo genotypes for use in the microscopic agglutination test.

A comparative serological study was conducted using the Leptospira microscopic agglutination test (MAT). Genotypes hardjoprajitno (HP), hardjo-bovis A (HA), and hardjo-bovis B (HB) were compared to determine which best detects hardjo antibody in cattle serum. A total of 2,431 cattle sera were tested. Sera were collected from 4 geographic regions of the United States. Samples were obtained without knowledge of breed, age, vaccination history, or herd health status. Of the sera collected, 60.7% (1,475) were negative at the 1 : 100 dilution for all three genotypes. Serological reactivity at the 1 : 100 dilution was identified in 956 (39.3%) of the sera tested. Considering the 956 positive sera, 941 (98.4%) reacted to HP, whereas the remaining 15 sera (1.6%) reacted to only HA and/or HB. A total of 394/941 (41.9%) HP positive sera failed to react to HA or HB. The results of this study support the conclusion that HP antigen was most sensitive in detection of hardjo antibody.     

Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum

Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity.     

Molecular approaches for inferring evolutionary relationships among protistan parasites.

Within the diverse group of parasites broadly recognized as protists, there are limited morphological characters that can be used to distinguish species and even fewer characters that can infer evolutionary relationships among species. For this reason, molecular data are commonly used to infer relationships among species and strains. These studies most commonly rely on sequences associated with the ribosomal RNA genes but increasingly other nuclear, mitochondrial or plastid genes are contributing data. Molecular systematics has been invaluable in expanding the range of characters that are available for inferring relationships among protistan taxa. As an adjunct to morphological characters, sequence data allow us to better understand the evolutionary history of this group of parasites and thereby erect a well-supported taxonomic framework that reflects these historical relationships. Importantly, the predictive nature of such a framework can aid the search for therapeutic compounds (e.g. via shared biochemical pathways) and highlight organisms that should be tested for cross-reactivity in immunological or molecular diagnostic methods (e.g. use of the closest relatives to assess test specificity). For emergent and poorly known parasites, molecular characterization and placement within the broader phylogenetic framework can help predict likely life history traits, including possible or likely definitive hosts.     

Leptospira strains isolated from cattle in the Amazon region,Brazil, evidence of a variety of species and serogroups with a high frequency of the Sejroe serogroup

In Brazil, there have been few leptospires isolated from cattle, especially in the Amazon, implying that the epidemiology of the disease in this region is still largely unclear. In a previous study, 52 Leptospira isolates were obtained from urine of cattle raised in the Brazilian Amazon and, to achieve a greater understanding of Leptospira infection in cattle of this region, the present study aimed to serologically and molecularly characterizes all these isolates. The laboratory assays used were the microscopic agglutination test (MAT) adopting a panel of polyclonal antisera against Leptospira spp. for serogrouping the isolates, DNA sequencing (secY) and multiple locus variable number tandem repeat analysis (MLVA). The isolates belonged to five species: 20/52 were identified as L. borgpetersenii (38.5 %); 18/52 as L. kirschneri (34.6 %); 9/52 as L. santarosai (17.3 %); 3/52 as L. noguchii (5.8 %) and 2/52 as L. interrogans (3.8 %). With serogrouping, nine different serogroups were detected, with a high frequency of the Sejroe serogroup. MLVA showed that all L. borgpetersenii isolates had a profile compatible with serovar Hardjo; moreover, the other isolates demonstrated a diversity of patterns, and some of them may represent strains not yet characterized. In the Brazilian Amazon, the leptospires circulating in cattle revealed the unique aspects of infections in this area which, in addition to a variety of strains, were characterized by a high frequency of the Sejroe serogroup, highlighting the serovar Hardjo, which has not been reported in other regions of Brazil.     

Infertility and abortion among first-lactation dairy cows seropositive or seronegative for Leptospira interrogans serovar hardjo.

OBJECTIVE: To estimate the extent to which exposure to Leptospira hardjo before or at the time of first parturition was associated with infertility and abortion during the first lactation among dairy cows that had not been vaccinated for > or = 12 months. ANIMALS: 207 first-lactation cows from a herd of 2,000 lactating cows. PROCEDURE: Cows were tested for antibodies to L hardjo within 40 days after calving. Time from calving to first breeding, time from calving to conception, number of breedings per conception, and risk of abortion were compared between cows seropositive for L hardjo and cows that were seronegative. RESULTS: For the 9 (4.3%) cows that were seropositive for L hardjo, median time from calving to conception (132.6 days) was significantly longer than time for seronegative cows (95.4 days). Cows that were seropositive were twice as likely (relative risk, 2.07) to fail to conceive as seronegative cows. Mean number of breedings required per conception for seropositive cows (3.4) was significantly higher than that for seronegative cows (2.1). The proportion of seropositive cows that aborted was not significantly different from the proportion of seronegative cows that aborted. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of nonvaccinated dairy cows to L hardjo can be associated with a subsequent reduction in fertility, as indicated by a greater time from calving to conception and higher number of breedings required per conception. The efficacy of leptospiral vaccines should be assessed to determine whether vaccination will minimize herd infertility associated with L hardjo infection.     

Serological comparison and antigenic relationships of seven serotypes of infectious bronchitis virus using the hemagglutination-inhibition test

The antigenic relationships, antigenic spectrum, and immunogenicity of seven isolates of infectious bronchitis virus (IBV) were examined using the hemagglutination-inhibition (HI) test. Because there was a discontinuity of antigenic relationships and a high degree of cross-reactivity among serotypes of IBV in cross-hemagglutination-inhibition tests, the range of antigenic spectrum used to group the serotypes with the HI test should be wider than the limits suggested by the plaque-reduction test. The HI test may provide valuable information in monitoring the immune status of a flock following vaccination when the area has a history of infectious bronchitis infection. It may also be used as a rapid diagnostic test if a flock is experiencing an outbreak of a disease caused by emergence of a new type of IBV. Interpretation of HI titers in evaluating immune status of chickens following infection with IBV depends on further cross-challenge and cross-protection studies of various types of IBV.