Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.     

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.     

Actinobacillus pleuropneumoniae serotype 5, subtypes a and b: cross protection experiments

Vaccination of pigs with a killed culture of A. pleuropneumoniae serotype 5, strain K17 (subtype a) afforded a high degree of protection against challenge with strains L20 and T928 (subtype b). The reverse experiment showed that strain L20 gave good protection against challenge with strain K17 whereas strain T928 did not afford an acceptable protection against challenge with this strain.The considerable cross immunity shown to exist between strains K17 and L20 indicates a high degree of homogeneity of the antigenic determinants of the two strains involved in induction of protective immunity and suggest that antibodies to capsular subtype specific determinants may not play a significant role in the specific defence against A. pleuropneumoniae strains belonging to serotype 5. The finding that a vaccine prepared from strain T928 did not afford an acceptable protection against challenge with strain K17 indicates a variable expression among serotype 5 strains of the antigenic determinants which induce protective immunity against A. pleuropneumoniae infection.     

Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains

Background

Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their in vitro and in vivo properties is currently available.

Methods

For the in vitro comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed.

Results

The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the in vivo properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle.

Conclusions

Our results show that the A663 strain used in this study is less adapted to in vitro replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.     

BHV-1 vaccine induces cross-protection against BHV-5 disease in cattle

Protection against BHV-5 disease induced by inactivated BHV-1 or BHV-5 based vaccines was analysed. Two groups of calves were subcutaneously immunized with an inactivated BHV-1 or BHV-5 based vaccine. A third group was not vaccinated and used as control. In the post-vaccination period, we studied the humoral and cellular immune response resulting similar to both groups. The efficacy of the vaccines was tested after intranasal challenge of the calves with a virulent Argentinean BHV-5 isolate (A-663). All control animals developed neurological signs associated with BHV-5 infection and high levels of virus shedding. Calves immunized with the BHV-1 and BHV-5 inactivated vaccines were protected against BHV-5 disease. Our study provides evidence that strongly support the existence of cross-protection between BHV-1 and BHV-5 in calves. Even though this has already been suggested by previous works, this is the first time an exhaustive study of the immune response is performed and typical clinical BHV-5 meningoencephalitis signs are reproduced in an experimental BHV-5 challenge trial.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in Argentina.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Comparative studies of strains of infectious bovine rhinotracheitis virus isolated from latently infected calves

Three strains (479 C, 778 TL, 982 LE) of infectious bovine rhinotracheitis (IBR) virus isolated from latently infected calves were compared with the prototype strain of IBR virus (LA strain) in studies which included restriction endonuclease analysis, experimental infection, and reciprocal cross protection tests in cattle. From the restriction endonuclease analysis it appeared that the 3 "latent" viruses were derived from the same isolate, and that it differed slightly from the LA strain. However, latency does not seem to have affected the pathogenicity or the immunogenicity of the virus. This is demonstrated by the identical clinical and virologic response of calves subjected to experimental infection with the various strains under study, and by the finding that when the LA strain and a "latent" strain (982 LE) were tested in cross protection tests in cattle, they proved to be mutually protective.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in argentina

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Comparative study of two strains of Bovid herpesvirus-4

Two strains of Bovid herepesvirus-4 (BHV-4), i.e. the prototype strain DN-599, obtained from a steer suffering of a respiratory disease, and the strain 85/BH 16TV, originated from a cow with vulvovaginitis, were compared in studies which included restriction endonuclease analysis, experimental infection and reciprocal cross protection tests. The restriction endonuclease analysis revealed that the resultant DNA patterns of the isolates were generally similar with only a difference in one fragment. The two strains were capable of causing respiratory tract infection in calves, even if they displayed a different level of virulence: the strain 85/BH 16TV being the most virulent while the strain DN-599 the least. The two viral strains were mutually protective in that the calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. Finally, both viral strains failed to evoke the production of neutralizing antibody in the experimental calves.     

FP1株番鸭源禽Ⅰ型副粘病毒抗原性分析

为明确FP1株番鸭源禽Ⅰ型副粘病毒强毒与鸡新城疫强毒标准株F48E9之间的抗原性差异,本研究通过交互血凝抑制试验、血清交叉中和试验及雏番鸭免疫攻毒保护试验比较了FP1株番鸭源禽I型副粘病毒强毒与鸡新城疫强毒标准株F48E9之间的抗原性.结果显示同源阳性血清对病毒的血凝抑制效价比异源阳性血清对病毒的血凝抑制效价高2.66～4个滴度;两者之间的抗原相关值R为0.35;对于5日龄经肌注途径免疫接种1羽份量La Sota弱毒疫苗14d后的雏番鸭,以FP1株番鸭源禽Ⅰ型副粘病毒强毒、鸡新城疫强毒标准株F48E9分别攻击后,其保护指数分别为54.5、92.9.以上结果表明,FP1株番鸭源禽Ⅰ型副粘病毒与鸡新城疫强毒标准株F48E9存在较明显的抗原性差异,为鸭副粘病毒病疫苗的研制和该病的防控提供了依据.     

A study of some biologic properties of Bovid herpesvirus-4.

This article summarizes the results of a study on several strains of Bovid herpesvirus-4 (BHV-4), isolated from cattle. The study had several objectives, namely, to verify (a) the disease-causing potential of the virus, (b) the possibility by BHV-4 to induce a latent infection in the natural host and (c) the entity of the relationships among strains of the virus isolated from different disease syndromes. The following data were obtained: (1) All strains tested were able to replicate in experimentally infected calves; however, only one strain (85/BH 16TV) caused an overt systemic disease. (2) The nervous system as well as the lymphoid structures appeared to be the target organs for replication of the virus. (3) BHV-4, like other herpesviruses, was able to establish latent infection in cattle. (4) When two strains of the virus, isolated from cattle affected by different disease syndromes, i.e. respiratory disease (strain DN-599) or vulvovaginitis (strain 85/BH 16TV), respectively, they resulted to be closely related to each other. In particular, they revealed a similar DNA pattern and both strains were able to cause respiratory disease in calves. Moreover, the two viral strains were mutually protective in that calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. (5) All BHV-4 strains tested generally failed to evoke a significant production of neutralizing antibody in the experimental calves.     

Biological diversity among serotype 2 Marek's disease viruses

Selected biological characteristics were determined for 14 low-passage serotype 2 Marek's disease virus (MDV) isolates. Four of these isolates were also tested after extensive serial passage in chicken embryo fibroblast cultures. Observations were made on replication in vitro and in vivo, pathogenicity by in ovo inoculation, antigenicity, and protection against virulent MDV challenge. Among the low-passage isolates, there were some differences in pathogenicity after in ovo inoculation but relatively little difference in other characteristics, with the exception of the HN-1 strain, which replicated more rapidly in cell culture but produced generally lower in vivo responses than other isolates. After extended in vitro passage, isolates replicated much more readily in cell culture and produced lower pathologic responses in vivo than low-passage isolates, as has been reported for serotype 1 isolates. No antigenic differences among isolates were detected, but high-passage isolates induced lower levels of precipitating antibodies than low-passage isolates, indicating a possible reduction in A antigen production. The observed diversity associated with strain and passage level may be of value in the selection of optimum vaccine strains.     

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.     

Limited heterogeneity between strains of Eimeria tenella isolated from Britain and Bangladesh

Single-oocyst-derived field strains of Eimeria tenella isolated from Rugby in the United Kingdom (E tenella R) and from Mymensingh and Dhaka in Bangladesh (E tenella M and D, respectively) and a laboratory strain (E tenella, Houghton, H) were compared by isoenzyme electrophoresis, reactivity with antisporozoite monoclonal antibodies and, for some pairs of strains, cross-protection in vivo. The three field strains conformed to one zymodeme with respect to six isoenzymes. For glucose phosphate isomerase (GPI) all field strains were characterised by GPI-9. A panel of six different monoclonal antibodies raised against sporozoites of E tenella H did not discriminate between strains by titration in an immunofluorescence assay against air-dried, acetone fixed sporozoites. In cross-protection experiments involving immunisation and challenge of young chickens, two immunisation schedules were used which, after homologous challenge, provided complete immunity either by the criterion of oocyst output, or by the criterion of weight gain (and more than 94 per cent protection by the criterion of oocyst output). While strain heterogeneity was minimal in the former situation, there was poor cross protection between some strains in the latter case. Under those conditions, heterologous challenge with E tenella M resulted in dysentery and in significantly (P less than 0.05) increased oocyst output and decreased weight gain. The results suggested that E tenella M was immunologically superior to E tenella R and H strains. The results show that a limited degree of immunogenic variability exists between these strains of E tenella and that, unless homologous strain immunity is complete by the criterion of oocyst output, challenge with heterologous strains may result in depressed weight gain.     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

An attenuated Salmonella Enteritidis strain derivative of the main genotype circulating in Uruguay is an effective vaccine for chickens

We have recently reported that Salmonella enterica serovar Enteritidis (S. Enteritidis) strains circulating in Uruguay, are unevenly distributed among different genetic subtypes, with a predominant genotype that is a common contaminant of poultry-derived food and that accounts for the vast majority of human cases of food-borne disease. Herein, we describe the construction of a genetically-defined aroC derivative (LVR02) of a local strain of S. Enteritidis belonging to the major genetic type. We demonstrated the attenuation and the immunogenicity of that strain in a mouse model, and evaluated it as a vaccine for commercial layer chickens. LVR02 proved to be stable, attenuated, innocuous, immunogenic and to induce protective immunity against a S. Enteritidis challenge when used for oral vaccination. A single oral dose of LVR02 administered to newly hatched chickens induced protection against oral challenge with the parental virulent strain, preventing systemic and persistent intestinal infection and significantly reducing the shedding of the challenge strain in birds' feces. A second vaccine dose at 15 days post-hatching boosted the immunogenicity of the vaccine, and strengthened the protection achieved with a single dose. This strain may represent the basis of a live vaccine to be included in national control programs to reduce circulation of this pathogen in the country.     

Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs.

In an attempt to establish if cross protection can be induced by different strains of Haemophilus parasuis, three groups of 12 gnotobiotic pigs were immunized each with an aluminum hydroxide adsorbed whole cell bacterin of one of three H. parasuis strains. Two weeks later, four pigs within each vaccinated group were challenged with aerosols of live cultures of each of the three test strains and observed for response. Two virulent strains V1 and V2 protected all the vaccinated pigs, while all nonvaccinated controls succumbed to Glasser's disease when challenged with these strains. Vaccination with strain LV (of low virulence) protected the pigs against challenge with strain V2, but not against strain V1. Strain LV did not cause disease in the immunized animals and only in one of ten nonimmunized pigs upon second challenge. The results suggest that strains may differ in antigenicity and that virulence and immunoprotection are positively related. Strains to be used in commercial vaccines should therefore be selected carefully. Antibodies detected in the sera of vaccinated pigs were to outer membrane proteins of the bacteria, but not to lipopolysaccharides or capsular polysaccharides. This would suggest that for gnotobiotic pigs outer membrane proteins are more immunogenic than lipopolysaccharide or capsular antigens. Further work is needed to determine if outer membrane proteins also contribute protective immunogens.     

Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV- 1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.     

Avirulence and immunogenicity in mice of a bovine mastitis Staphylococcus aureus mutant.

An avirulent mutant, designated RC122, was derived from Staphylococcus aureus bovine mastitis strain RC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutant RC122, which was isolated on the basis of reduced colony size, showed diminished virulence in mice (LD50 of RC122: 3.1 x 10(10) cfu vs LD50 of RC108: 2.3 x 10(7) cfu). Mutant RC122 grew more slowly than its parental strain and showed decreased production of several exoproteins, such as alpha- and beta-hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo growth conditions. Clearance studies performed in the mouse kidney revealed that the kinetics of disappearance of the mutant was similar to that of its parental strain. Protection experiments carried out by intraperitoneal administration in mice showed that mutant RC122 conferred a good degree of protection from challenge with homologous and heterologous strains.