Efficacy of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV and BRSV in experimentally infected calves.

The efficacy of a quadrivalent vaccine against viral bovine respiratory diseases (BRD) was assessed in four experimental studies. Calves between 2 and 9 months of age were allocated to one of two treatment groups (n=9-15) and then received either the vaccine or sterile saline in two doses approximately 3 weeks apart. Three to 5 weeks after the second injection, animals were challenged experimentally with one of the viruses, bovine herpes-virus-1 (BHV-1), parainfluenza type-3 virus (PI(3)V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV) and were then monitored for at least 2 weeks. The administration of the vaccine was associated with enhanced antibody response to all four viruses post-challenge, with the reduction of the amount or duration (or both) of virus shedding in the BHV-1, PI(3)V, BVDV and BRSV studies and with an improvement of some clinical signs in the BHV-1 (nasal discharge, and rectal temperature) and the PI(3)V studies (abnormal respiration, and depression).

Duration of immunity of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV, and BRSV in experimentally infected calves.

Several laboratory studies assessed the duration of immunity of a quadrivalent vaccine (Rispoval 4, Pfizer Animal Health) against bovine respiratory diseases (BRD) caused by bovine herpes-virus type-1 (BHV-1), parainfluenza type-3 virus (PI3V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV). Calves between 7 weeks and 6 months of age were allocated to treatment and then were injected with two doses of either the vaccine or the placebo 3 weeks apart. Six to 12 months after the second injection, animals were challenged with BHV-1 (n=16), PI3V (n=31), BVDV (n=16), or BRSV (n=20) and the course of viral infection was monitored by serological, haematological (in the BVDV study only), clinical, and virological means for > or =2 weeks. Infection induced mild clinical signs of respiratory disease and elevated rectal temperature in both vaccinated and control animals and was followed by a dramatic rise in neutralising antibodies in all treatment groups. Titres reached higher levels in vaccinated calves than in control calves after challenge with BHV-1, BVDV, or BRSV. On day 3 after PI3V challenge, virus shedding was reduced from 3.64 log10TCID50 in control animals to 2.59 log10TCID50 in vaccinated animals. On days 6 and 8 after BRSV challenge, there were fewer vaccinated animals (n=2/10 and 0/10, respectively) shedding the virus than control animals (n=8/10 and 3/10, respectively). Moreover, after challenge, the mean duration of virus shedding was reduced from 3.8 days in control animals to 1 day in vaccinated animals in the BVDV study and from 3.4 days in control animals to 1.2 days in vaccinated animals in the BRSV study. The duration of immunity of >or =6 months for PI3V, BHV-1 and BVDV, and 12 months for BRSV, after vaccination with Rispoval 4, was associated mainly with enhanced post-challenge antibody response to all four viruses and reduction of the amount or duration of virus shedding or both.

Localization of foot-and-mouth disease virus RNA by in situ hybridization within bovine tissues.

Foot-and-mouth disease is a highly contagious disease of cloven hooved animals. In cattle, both acute and long-term persistent infections occur. Foot-and-mouth disease virus (FMDV), a picornavirus, has been shown, using virus isolation procedures, to replicate in the pharynx and soft palate of cattle. In this study, in situ hybridization has been used to detect FMDV RNA within the cells of tissues removed from infected bovines. A digoxigenin-labelled anti-sense RNA probe was prepared corresponding to a region of the FMDV genome encoding part of the RNA-dependent RNA polymerase (3D). The efficacy and specificity of this probe for in situ hybridisation was determined using virus-infected cells in tissue culture. Strong cytoplasmic staining was only detected in FMDV-infected cells. Various tissue samples were collected from FMDV-infected cattle between 5 and 17 days post-infection. Viral RNA was detected by in situ hybridisation within cells of the soft palate, tonsil and pharynx up to 17 days post-infection. This technique is useful for the study of FMDV localization in cattle both during and after the acute clinical phase of disease and may assist in identifying specific sites of virus persistence.

Emergency vaccination of sheep against foot-and-mouth disease: protection against disease and reduction in contact transmission.

The ability of several emergency FMD vaccine formulations to elicit early protective immunity in sheep was examined. All vaccine formulations were shown to protect sheep against airborne challenge with homologous FMDV within 4 days of vaccination. Protection was associated in part with the induction of serum antibody responses but was also demonstrated in the absence of any detectable antibody response at the time of challenge. Aqueous Al(OH)3/saponin vaccine formulations and oil emulsion vaccines based on Montanide ISA 206 adjuvant reduced virus replication and the numbers of animals subclinically infected up to 28 days post-challenge, when compared with non-vaccinated animals, consequently limiting transmission of the disease or infection to in-contact susceptible animals.

Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.

A recombinant live vector vaccine was produced by insertion of cDNA encoding the structural proteins (P1) of foot-and-mouth disease virus (FMDV) into a replication-competent human adenovirus type 5 vaccine strain (Ad5 wt). Groups of cattle (n = 3) were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunizations. In the absence of a detectable antibody response to FMDV, significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. The observed partial protection against clinical disease was not associated with a reduction in titre of persistent FMDV infections in the oropharynx of challenged cattle.

Donkeys as reservoirs of African horse sickness virus.

Investigations have been carried out to elucidate the possible role of the donkey in the epidemiology of African horse sickness (AHS). These studies have shown that despite the absence of pyrexia or other observable clinical signs, donkeys become infected with virulent AHS virus serotype 4 (AHSV 4) and that they develop a viraemia which can persist for at least 12 days, albeit at a comparatively lower titre than that recorded for similarly infected ponies. AHSV 4 showed a similar tissue tropism in the pony and donkey but the virus appeared to replicate less efficiently in donkey tissues. The only gross pathological changes observed in the donkeys post mortem were increased fluid accumulation in the serosal lined compartments, particularly the peritoneal cavity, and petechial and ecchymotic haemorrhages on the left hepatic ligament. The absence of infectious virus or viral antigens in any of the tissues collected at 14 and 19 days post inoculation (dpi) from 6 experimental donkeys suggest that, though susceptible to infection, the donkey is unlikely to be a long term reservoir for AHSV. Although AHSV 4 was detected in all 6 donkeys following the primary inoculation, no virus could be isolated from blood collected from two donkeys subsequently challenged with a second virulent virus, AHSV 5. Data generated from virus neutralisation tests showed a second primary antibody response, against AHSV 5, in these donkeys at 12 dpi. In contrast, the boost in antibody levels detected from 5 dpi, as measured by ELISA, was probably due to an anamnestic response against the AHSV group-specific viral proteins. Homogenised spleen tissue, collected post mortem from a donkey 7 dpi with AHSV 4, caused a lethal, cardiac form of AHS when inoculated into a susceptible pony.

Immunohistochemical demonstration of African horse sickness viral antigen in tissues of experimentally infected equines.

African horse sickness virus (AHSV) antigen was demonstrated immunohistochemically in formalin-fixed, paraffin-embedded sections of tissues collected from three ponies suffering from the peracute form of the disease and from one pony affected by the fever form. The pattern of the antigen distribution indicated a particular organ tropism characterised by an accumulation of AHSV antigen in cardio-pulmonary tissues of the animals with the peracute disease and in the spleen of the pony with the fever form. AHSV antigen was identified in endothelial cells of small blood vessels, particularly capillaries and in large mono-nuclear cells resembling macrophages or reticular cells of lymphatic tissues. Occasional circulating mononuclear cells with the morphology of monocytes were also positively stained within the larger vessels. The immunohistochemical results confirm earlier work suggesting that AHSV may have different tropisms to particular organs during various forms of the disease and that different target cell populations exist in vivo. Immunohistochemistry may be an additional useful method for diagnostic and research purposes in AHS.

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.

OBJECTIVE: To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep. DESIGN: A clinical and pathological study. PROCEDURE: Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies. RESULTS: All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection. CONCLUSIONS: The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus.

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.

Antibody to the nonstructural proteins of foot-and-mouth disease virus in vaccinated animals exposed to infection.

Cattle which have been infected with foot-and-mouth disease (FMD) virus can be differentiated from those that have been vaccinated on the basis of the detection of antibody to one or more of the non-structural (NS) proteins of the virus. Cattle which have been protected by vaccination can become persistently infected with FMD virus (FMDV) without ever showing clinical signs. Vaccinated, protected cattle which are persistently infected cannot be distinguished from animals that merely have been vaccinated on the basis of serological tests for antibody to the structural proteins of FMDV. Sera were collected from groups of cattle for varying periods after exposure to infection under experimental conditions. On the basis of isolation of virus from probang samples collected during the course of the experiments it was possible to classify the cattle according to the following criteria; naive, infected and eliminated the virus (convalescent), infected and persistently infected with FMDV (carriers), vaccinated alone, vaccinated and either convalescent or carrier. Sera were examined for antibody to the NS proteins Lb, 2C, 3A, 3D, and 3ABC by an indirect profiling ELISA using E. coli-expressed fusion proteins as antigens. Considerable variation was observed in the antibody response to NS proteins of both naive and vaccinated animals following infection. The extent of individual variation was so great that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined. The majority of vaccinated, protected animals showed an antibody response to NS proteins, particularly 3ABC, following exposure to infection. However, the carrier state was demonstrated in some vaccinated, protected animals in which no antibody response to any of the NS proteins could be detected. The detection of antibody to NS proteins can therefore be used on a group, or herd, basis to detect circulation of virus in a vaccinated population but further investigations in the field are required to determine the sampling level necessary for statistical acceptance. On an individual animal basis, however, freedom from antibody to NS proteins in a vaccinated animal, or an animal of unknown history, does not necessarily imply that the animal is free from infection with FMD virus and, furthermore, the titre of antibody to NS proteins is not a useful predictive measure of whether or not an infected animal has successfully eliminated the virus.

Differentiating foot-and-mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins.

We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.

Emergency vaccination of pigs against foot-and-mouth disease: protection against disease and reduction in contact transmission.

The protective ability of two novel oil-based FMD vaccines in pigs was examined. Both vaccine formulations were shown to protect pigs against airborne challenge with homologous FMDV within four days of vaccination, but not at two and three days post-vaccination. Protection was associated with the induction of variable and low titre serum antibody responses. A transmission study showed that protective immunisation resulted in reduced virus excretion. Vaccination at seven days, but not at four days, prior to challenge prevented contact transmission of FMD. The two formulations tested in this study have the favourable characteristics of low viscosity, low reactivity and high potency emergency FMD vaccines for use in strategic vaccination campaigns to assist the control of outbreaks of FMD.

Immunohistochemical demonstration of African horse sickness viral antigen in formalin-fixed equine tissues.

The distribution of viral antigen was studied in various tissues of three ponies, aged 3-4 years, infected experimentally with a virulent strain of African horse sickness virus (AHSV) serotype 4. Tissues were collected from the animals in the terminal stage of the peracute form of the disease and from one noninfected horse, included as a control. A polyclonal antibody with specificity for AHSV, plus the nonstructural protein NS2, was used in a sensitive avidin-biotin-peroxidase-complex (ABC) method performed on formalin-fixed, paraffin-embedded tissue sections. AHSV antigen was located primarily in endothelial cells of capillaries and small venous and arteriolar vessels, particularly of cardiopulmonary tissues. Viral antigen was also identified in cells resembling macrophages and in reticular cells of spleen and lymph nodes. The pattern of viral antigen labeling in some lymph nodes along the mantle zone of lymphoid follicles was compatible with the morphology of cellular processes of follicular dendritic cells. In some tissues, viral antigen was detected occasionally in circulating cells, probably monocytes, within the larger vessels. These findings suggest that endothelial cells, and to a lesser extent mononuclear cells, are the main target cells of AHSV infection during the late stage of the peracute form of the disease.

Dexamethasone inhibits virus production and the secretory IgA response in oesophageal-pharyngeal fluid in cattle persistently infected with foot-and-mouth disease virus.

Cattle persistently infected with foot-and-mouth disease virus were treated with dexamethasone to suppress the immune system in an attempt to influence the level of virus recovery from oesophageal pharyngeal (probang) samples. Twelve carrier cattle were assigned to one of three groups: control; 0.1 mg/kg dexamethasone; and 0.5 mg/kg dexamethasone. Groups 2 and 3 were injected intramuscularly three times weekly for 3 weeks with dexamethasone between days 33 and 56 post-infection with foot-and-mouth disease virus (FMDV). Cattle in both groups developed a leucocytosis, neutrophilia and lymphopenia. The secretory IgA response to FMDV infection was inhibited following, but not during, dexamethasone treatment between days 70 and 98 post-infection (P < 0.05). FMDV recovery from probang samples was reduced between days 40 and 64 post-infection (P < 0.05) during treatment with either 0.1 or 0.5 mg/kg dexamethasone. Following cessation of dosing with dexamethasone virus recovery returned to control levels. These observations suggest dexamethasone inhibits shedding of FMDV in a reversible manner which may be related to its immunosuppressive, anti-inflammatory or physiological actions.

Isotype-specific antibody responses to foot-and-mouth disease virus in sera and secretions of "carrier' and "non-carrier' cattle.

Isotype-specific antibody responses to foot-and-mouth disease virus (FMDV) were measured in the sera and upper respiratory tract secretions of vaccinated and susceptible cattle challenged with FMDV by direct contact or by intranasal inoculation. A comparison was made between cattle that eliminated FMDV and those that developed and maintained a persistent infection. Serological and mucosal antibody responses were detected in all animals after challenge. IgA and IgM were detected before the development of IgG1 and IgG2 responses. IgM was not detected in vaccinated cattle. Challenge with FMDV elicited a prolonged biphasic secretory antibody response in FMDV "carrier' animals only. The response was detected as FMDV-specific IgA in both mucosal secretions and serum samples, which gained statistical significance (P < 0.05) by 5 weeks after challenge. This observation could represent the basis of a test to differentiate vaccinated and/or recovered convalescent cattle from FMDV "carriers'.

Antigenic analysis of type O foot-and-mouth disease virus in the persistently infected bovine.

The antigenic profiles of serotype O strains of FMDV collected from the oropharynx of persistently infected cattle were defined with a panel of monoclonal antibodies (mAb's) in an indirect antigen-trapping ELISA. The mAb profiling showed no significant loss of reactivity in two neutralising antigenic sites of persistent FMDV isolates collected over a period of eight months. Early and late serum taken from a carrier animal showed similar neutralising activity against early and late carrier isolates. The antigenic stability of serotype O strains of FMDV collected during the carrier state suggests that antigenic variation facilitated by pressure from the host humoral immune response is not critical to the establishment or maintenance of a persistent infection with FMDV.

The interference by maternally-derived antibody with active immunization of farm animals against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a highly contagious disease affecting ruminants and pigs. In countries in which control of FMD relies predominantly on vaccination, young stock ingest specific anti-FMD virus antibodies in the colostrum. This maternally-derived antibody (MDA) provides immediate protection against infection with FMD virus, but also interferes with the development of active immunity following vaccination. However, susceptibility to infection precedes the ability to respond to vaccination in the presence of MDA. Currently available vaccines cannot overcome this inhibitory effect of MDA, and protection of young stock can only be provided by their isolation from FMD virus.

Optimization of an in situ hybridization technique for the detection of foot-and-mouth disease virus in bovine tissues using the digoxigenin system.

An in situ hybridization technique has been optimised for use on paraffin-embedded sections of tissues collected from cattle infected experimentally with foot-and-mouth disease virus type O1BFS. Tissue was collected 5 days after infection by direct contact. In situ hybridization was carried out using an RNA probe corresponding to a region of the 3D gene which codes for the RNA polymerase, and labelled with digoxigenin. Consistent, reproducible signal was detected within the epithelial layers of the palatine tonsil, soft palate and pharyngeal tissue studied. This is the first time that a digoxigenin-based system has been used successfully for FMD virus RNA detection with bovine tissue samples.

The carrier state in foot and mouth disease--an immunological review.

The carrier state in foot and mouth disease (FMD) is characterized by the asymptomatic low-level excretion of foot and mouth disease virus (FMDV) from the oropharynx of ruminants for periods that are species and virus strain-dependent. Persistent infection with FMDV readily occurs following the failure of virus elimination at the acute stage of infection, a process thought to be mediated through the phagocytosis of antibody/virus immune complexes. Recent evidence supports the view that carrier cattle are important in the epidemiology of FMD in the field. The absence of histopathological change in persistently infected tissues and the reduced cytopathology of carrier virus isolates in tissue culture suggest that less lytic FMDV variants are generated or selected in the carrier animal. Altered virus replication, due to attenuation or interference, rather than antigenic variation may therefore allow evasion of the exaggerated FMDV-specific systemic and local humoral immune responses that occur in the carrier state. Although cell-mediated immune mechanisms for FMDV clearance have not been described, the eventual elimination of many persistent virus infections involves this arm of the immune system. Thus, further investigation of cellular elements of the immune response, more particularly the local interaction of mononuclear cell infiltrates with persistently infected cells, represents an area of research that has the potential to elucidate the carrier state problem.