Preliminary Extraction and Identification of the 44.5 kDa Outer Membrane Proteins Isolated from Bovine Fusobacterium necrophorum (AB)

Fusobacterium necrophorum (AB) in the pharynx, respiratory tract, female reproductive tract or urinary system is the causative agent of footrot and hepatic abscesses in animals and acute Lemierre’s syndrome in humans. Current methods do not effectively protect animals and humans against F. necrophorum (AB). The outer membrane proteins (OMP) of F. necrophorum (AB) can be used as new material to protect against the diseases induced by F. necrophorum (AB). The aim of this study was to extract OMP and examine the immunogenic response of OMP. The preliminary extraction of OMP of F. necrophorum (AB) was identified by SDS-PAGE and stained by Coomassie Brilliant Blue R-250 (CB B R-250) and silver staining methods. The results showed that only a major band of 44.5 kDa was observed when staining the gel using CB B R-250. This band represented the target protein. In contrast, many small bands were observed by the silver staining method. The OMP also exhibited immune biological activities according to western blot analysis. The brightest band among the multi-banding observed was the OMP. Thus, the OMP was obtained and had immunogenic activity. The results provide a new direction to protect animals and humans against F. necrophorum (AB) in the clinical setting.     

Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot

Footrot is a debilitating disease of sheep resulting in lameness, production losses and suffering. To study the basic bacteriology of the disease, a survey was initiated across commercial farms and non-commercial research flocks to compare the bacteriology of symptomatic footrot infected sheep with healthy asymptomatic sheep. Of the 80 farmers initially contacted, 14 collected hoof swabs and returned the swabs by post. Following DNA extraction, species-specific PCR was used to identify if Dichelobacter nodosus (D. nodosus) or Fusobacterium necrophorum (F. necrophorum) species were present on each swab. Of the 42 swabs taken from symptomatic footrot infected sheep, 17 were positive for both F. necrophorum and D. nodosus, two were positive for F. necrophorum only, two for D. nodosus only and 23 swabs were negative for both F. necrophorum and D. nod osus. Of the 50 swabs received from healthy asymptomatic sheep, one was positive for F. necrophorum only and 49 were negative for both D. nodosus and F. necrophorum. This suggests that both F. necrophorum and D. nodosus are linked to footrot in the field in a pastoral farming system. If these bacteria are linked together and collectively cause footrot, this may need to be considered when managing a footrot outbreak, or maintaining a quarantine.     

Expression of p24 gag Protein of Bovine Leukemia Virus in Insect Cells and Its Use in Immunodetection of the Disease

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.     

Molecular Characterization and Toxicity Confirmation of LukM/F′-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India

The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F′-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein–Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samples. Being intermediately resistant to vancomycin, they showed characteristic double zone hemolysis on 7 % sheep blood agar and typical type II reaction for coagulase test indicating the pathogenic attributes. Further with RAPD-PCR and 16S rDNA-RFLP, epidemiological specificity and genotypic relatedness of isolates to S. aureus was confirmed. Subsequently, the presence of leukotoxin (lukM) gene in native isolates was detected by leukotoxin gene specific PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay evaluated for secreted leukotoxin in cell free supernatant was estimated to be 223 toxic units which had an LD₅₀ cytotoxic activity on bovine neutrophil. Thus, the data acquired during study can be of prime diagnostic method for timely and accurate analysis of subclinical mastitis samples which goes undetected at consumer level.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.     

Development of Two Murine Antibodies against Neospora caninum Using Phage Display Technology and Application on the Detection of N. caninum

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.     

TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever

Introduction

East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever.

Methods

The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively.

Results

All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites.

Conclusion

It was concluded that TpUB05 is a potential marker of protective immunity in ECF worth investigating further.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Bacteriologic studies of liver abscesses of Awassi sheep in Jordan

A total of 337 liver abscesses of Awassi sheep that were found in different slaughterhouses in Jordan were examined for their causal agents. Fifteen different bacterial species were isolated from 297 liver abscesses. No bacteria were isolated from the remaining 40 liver abscesses. Fusobacterium necrophorum biovar B were isolated from 195 (58%) abscesses. Arcanobacterium pyogenes, F. necrophorum biovar A, Escherichia coli and Clostridium perfringens were isolated from 41 (12%), 34 (10%), 30 (9%) and 17 (5%), respectively, liver abscesses. Our study suggested that F. necrophorum biovar B is the most prevalent bacterium incriminated for liver abscesses in Awassi sheep.     

Edible vaccine development: stability of Mannheimia haemolytica A1 leukotoxin 50 during post-harvest processing and storage of field-grown transgenic white clover

Mannheimia haemolytica is the principal microorganism responsible for bovine pneumonic pasteurellosis, or shipping fever. We have previously expressed a fragment of leukotoxin, a major virulent factor of M. haemolytica A1, as a fusion protein with green fluorescent protein (GFP) in transgenic white clover and demonstrated that this antigen was immunogenic and elicited toxin neutralizing antibodies in rabbits. These previous results showed that using plants to produce M. haemolytica antigen for use as a vaccine against this disease is a viable strategy. In this present study, we examined the stability of the truncated leukotoxin GFP-fusion protein (Lkt50-GFP) in field-grown transgenic white clover. Transgenic clover expressing Lkt50-GFP was clonally propagated and a confined field trial was established. Western immunoblotting showed that the level of Lkt50-GFP expression in field plants was the same as in transgenic plants maintained under optimal conditions in the greenhouse. We also observed that after harvesting and oven drying at 50 °C, the antigen was still present in the dried clover after 1 year of storage at ambient temperature. As special post-harvest conditions (e.g., refrigeration) are not required, the use of transgenic plants to deliver an oral vaccine against shipping fever appears to be economically feasible.     

Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica

The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.     

Development of phage-based single chain Fv antibody reagents for detection of Yersinia pestis

Background

Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)^. F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.

Methods

Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.

Results

Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.

Conclusions

Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolyticaA1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.     

Acute phase response in lame cattle with interdigital dermatitis

This study was undertaken to evaluate acute phase response via assessing the concentration of serum sialic acids (total, lipid-bound and protein-bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins [haptoglobin (Hp) and serum amyloid A (SAA)] in lame cattle with interdigital dermatitis. Fifteen hoof scrapings from lame cows were collected from eight commercial dairy farms. As a consequence of the difficulty in culturing and isolation, a PCR technique was used to detect the organism. None of the colonies on enriched blood agar was identified as Fusobacterium necrophorum. Four (26.6%) out of the 15 hoof scrapings examined tested positive for the presence of the lktA gene (402 bp) of F. necrophorum. It seems that culture cannot be considered as the gold standard method for F. necrophorum isolation. Molecular detection is suggested as an alternative method. In the blood serum of different groups of animals (control, lameness and F. necrophorum-positive lameness) Hp, SAA, total sialic acid, lipid-bound sialic acid, and protein-bound sialic acid, and IFN-γ and TNF-α were measured using validated standard procedures. All parameters were significantly higher in the lameness group and the F. necrophorum-positive lameness group compared with the healthy group (P < 0.01 in all cases). Mean SAA concentrations in the lameness group and the F. necrophorum-positive lameness group was relatively 4.6 and 8.0 times higher than the control group. Corresponding measures for Hp indicate a 3.3 times increase in the lameness group compared to the control. In the lameness group, significant associations were observed for Hp with PBSA, SAA with TSA, TSA with PBSA, TSA with LBSA, PBSA with LBSA, and SAA with IFN-γ.     

Preliminary studies of the complement fixation test to confirm the diagnosis of bovine ephemeral fever

A strain of bovine ephemeral fever (BEF) virus isolated in China in 1976 was adapted to growth in tissue cultures. A baby hamster kidney complement fixing (CF) antigen, stable at -20 degrees C for at least 120 days, was prepared from the BEF virus grown in tissue culture and used to test bovine sera for antibodies to that virus. CF antibodies were detected in all of 31 cattle after convalescence from experimental infection with BEF virus, in 208 (98%) of 213 cattle observed to have shown clinical ephemeral fever in an epidemic, in 96 cattle in these herds which did not show clinical signs of ephemeral fever and 16 cattle from herds in northern China outside the epidemic area. The CF antibodies to BEF virus were found to persist in 34 (89%) of 38 cattle which were bled 6 years after natural exposure to ephemeral fever. The CF antigen is economical to prepare and is suitable to differentiate ephemeral fever from other viral infections with which it could possibly be confused on clinical appearance.     

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A₂ but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A₂ were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.     

Pestivirus Infections in Norway. Serological Investigations in Cattle,Sheep and Pigs

Serum samples from 1133 dairy cows (187 herds), 3712 ewes (103 flocks) and 1317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28 % of the cattle herds and 18 % of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.