Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.

The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs.     

Immunoglobulin M (IgM) indirect enzyme-linked immunosorbent assay and the involvement of IgM-rheumatoid factor in the serodiagnosis of BHV-1 infection.

An IgM enzyme-linked immunosorbent assay (IgM-ELISA) for the detection of antibodies to bovine herpesvirus-1 (BHV-1) was developed. Its applicability was examined by serological studies in two calves experimentally infected with virulent BHV-1 over a period of 60 days. IgM antibodies were detected by ELISA on day 6 after infection, and there was an increase in IgG antibodies on day 9. Serum neutralizing (SN) antibodies were detected only on day 13, confirming the higher sensitivity of the ELISAs. A similar study of four calves treated with a commercial inactivated virus vaccine indicated no detectable IgM-ELISA response, and late SN and IgG-ELISA reactivity. Thus IgM-ELISA appears to be of value in assessing recent infection, whereas IgG-ELISA and SN cannot distinguish between infection and vaccination. The possible limitations imposed on the specific IgM-ELISA by the presence of IgM rheumatoid factor (IgM-RF) in bovine serum were examined. IgM-RF levels were determined in bovines of various ages. Elevated values of IgM-RF were found in the sera of older animals; their occurrence may lead to false IgM-positive diagnosis (16%) of BHV-1 infection. This was examined in 113 serum specimens from suspected cases of BHV-1 infection and in 32 bulls at an insemination center. Pretreatment of serum samples with an antibovine IgG serum eliminated false positivity of the IgM-ELISA. It is concluded that IgM-ELISA should be of particular value in the diagnosis of recent infection with BHV-1, mainly in calves.     

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively.     

Serological and molecular diagnosis of bovine viral diarrhoea virus and evidence of other viral infections in dairy calves with respiratory disease in Venezuela

An investigation based on 2 studies was carried out to assess the involvement of bovine virus diarrhoea virus (BVDV), bovine herpesvirus type 1 (BHV-1), and bovine respiratory syncytial virus (BRSV) in calf respiratory disease in dairy farms in Venezuela. In the first study, 8 farms were selected and paired serum samples from 42 calves with respiratory disease were tested by ELISA for antibodies to the 3 viruses. Seroconversion to BVDV, BHV-1, and BRSV was found to 5, 2, and 6 farms out of the 8, respectively. The proportion of calves that showed seroconversion to BVDV, BHV-1, and BRSV were 19%, 14%, and 26%, respectively. In the second study, another farm having previous serological evidence of BVDV infection was selected. The decline of maternal antibodies against BVDV was monitored in 20 calves and the half-life of maternal antibodies was 34 +/- 12 days presumably indicating an early natural infection with BVDV. Furthermore, sera free of BVDV antibodies that were collected in studies 1 and 2 and were assayed for the presence of BVDV by nested RT-PCR. Two BVDV strains were detected and compared to those of ruminant and porcine pestiviruses. Both strains were assigned to subgroup Ib of type I BVDV. This investigation provides information on BVDV genotypes circulating in Venezuela and may contribute to the establishment of official control programmes against the viruses studied.     

Fatal, generalized bovine herpesvirus type-1 infection associated with a modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine administered to neonatal calves.

Generalized bovine herpesvirus 1 (BHV-1) infection was diagnosed in six Salers calves from the same herd. The calves had received an intramuscular injection of modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine between birth and three days of age. The purpose of this study was to determine if the outbreak was associated with the vaccine strain of BHV-1. Analysis of epidemiological data and BHV-1 DNA for restriction fragment length polymorphism was undertaken. Multifocal necrosis in multiple organs was observed on pathological examination, and the presence of BHV-1 in tissues was confirmed by immunohistochemistry. Forty-three calves (aged birth to thirty days) were vaccinated over an 11-day interval. The 10 deaths recorded for vaccinated calves were clustered over a subsequent 14-day interval. Mortality in calves vaccinated between birth and three days of age was significantly higher than in nonvaccinated calves (chi-square test; p < or = 0.025), and this mortality was characterized by a greater age at death and duration of illness for vaccinated calves (t test; p < or = 0.001). The patterns of the restriction fragments, generated by six restriction endonucleases, of BHV-1 isolated from a necropsied calf and from the vaccine were identical, and different from that of a laboratory strain of BHV-1 (P8-2). These findings support the conclusion that newborn calves were susceptible to an intramuscularly injected vaccine strain of BHV-1, and that administration of an intramuscular modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine to neonatal calves may not be an innocuous procedure.     

Serological evaluation of relationship between viral pathogens (BHV-1, BVDV,BRSV, PI-3V,and Adeno3) and dairy calf pneumonia by indirect ELISA

In this study, viral pathogens associated with nine outbreaks of naturally occurring dairy calf pneumonia in Mashhad area of Khorasan Razavi province from September 2008 to May 2009 were assessed. Five diseased calves from each farm were chosen for examination. Acute and convalescent serum samples were taken from calves with signs of respiratory disease. Sera were analyzed for antibodies to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus type 3 (PI-3V), and bovine adenovirus-3 (BAV-3) by indirect ELISA kits. Among 42 serum samples collected at sample 1, seroprevalence values for viruses BHV-1, BVDV, BRSV, PI-3V, and BAV-3 were 61.9% (26), 57.1% (24), 64.2% (27), 90% (38), and 61.9% (26), respectively. Seroconversion to BVDV, BRSV, PI-3V, and BAV-3 occurred in 11.9% (5), 16.6% (7), 26.1% (11), and 21.4% (9) of animals, and 52.3% (22) had generated antibodies against one or more viral infections at sample 2. In addition, no significant relationship between seroprevalence of BHV-1, BVDV, BRSV, PI-3V, and BAV-3 and dairy herd size was observed (P > 0.05). According to serological findings, BHV-1, BVDV, BRSV, PI-3V, and BAV-3 are common pathogens of the dairy calf pneumonia in dairy herds in Mashhad area of Khorasan Razavi province, Iran.     

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.     

Detection of bovine herpesvirus-1-specific IgM using a capture enzyme immune assay with isotype-specific monoclonal antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.     

Effect of lymphocytes and broncho-alveolar washing cells on the replication of bovine herpesvirus type 1 in tracheal organ cultures

The daily addition of lymphocytes collected from a calf between 7 and 11 days after experimental infection with bovine herpesvirus type 1 (BHV-1) to bovine fetal tracheal organ cultures after infection with BHV-1 did not inhibit virus replication. The daily addition of normal lymphocytes, together with a low concentration of serum antibody against BHV-1, had a slight viral inhibitory effect which was believed to be due to antibody-dependent cell-mediated cytotoxicity. The addition of broncho-alveolar washing (BAW) cells, collected before infection or 30 days after infection of a calf with BHV-1, together with lymphocyte culture supernatant, to tracheal organ cultures immediately after infection with BHV-1 produced some inhibition of virus replication. Virus replication was markedly inhibited when BAW cells collected from the calf 18 days after infection were used in a similar manner.     

Pcr studies on the potential sites for latency of BHV-4 in calves

The aim of the study was to examine various tissues of experimentally infected calves for the BHV-4 genome so as to detect in which cells the virus persists during the latent phase of the infection. The presence of the bovine herpesvirus type 4 genome was detected by a nested PCR in a variety of tissues collected from two susceptible calves experimentally infected 62 days earlier. Mild clinical signs of bronchitis, an elevated body temperature for 2–3 days, and a slightly increased number of blood leukocytes were observed in both inoculated calves. BHV-4 was demonstrated in seven samples from the 12 different parts of the nervous system tested from each calf (29.1%), from the cornea, from lymph nodes near to the inoculation site, from the gallbladder and from the bone marrow. Thus a member of the predominantly lymphotropic Gammaherpesvirinae subfamily was detected in neural tissue and other organs that have never been associated with persistence.     

Association of bovine viral diarrhea virus with multiple viral infections in bovine respiratory disease outbreaks

We investigated eleven outbreaks of naturally occurring bovine respiratory diseases in calves and adult animals in the St-Hyacinthe area of Quebec. Specific antibodies to bovine herpesvirus-1, bovine viral diarrhea virus, respiratory syncytial virus, parainfluenza type 3 virus, reovirus type 3, and serotypes 1 to 7 of bovine adenovirus were found in paired sera from diseased animals. Several bovine viruses with respiratory tropism were involved concomitantly in herds during an outbreak of bovine respiratory disease. In addition, concomitant fourfold rises of antibody titers were frequently observed to two or more viral agents in seroconverted calves (61%) or adult animals (38%). Bovine viral diarrhea virus was found to be the most frequent viral agent associated with multiple viral infection in calves only (92%).     

Dynamics of Moraxella bovis infection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves

Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.     

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks.In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.     

The use of immunomodulators in the control of infectious bovine rhinotracheitis

Three experiments have been carried out to verify the effectiveness of an immunomodulator, Baypamun (Bayer AG) in limiting the spread of Bovine herpesvirus-1 (BHV-1), the causal agent of infectious bovine rhinotracheitis (IBR). In the first experiment, four calves infected with BHV-1 developed severe disease whereas four calves given Baypamun simultaneously with the virus had less severe disease. Four other calves in contact with the infected calves became severely ill but another four given Baypamun were only mildly affected. In the second experiment three calves infected with BHV-1, which reacted with typical disease, were allowed to remain in contact with six calves. All six calves were given Baypamun at various times following the exposure to BHV-1 infection and all showed a much reduced reaction with two treated for 4 days developing no clinical disease. Finally, in the third experiment one calf vaccinated one month before the start of the experiment did not develop any signs of disease when housed together with a calf experimentally infected with BHV-1. Of four other calves, vaccinated when the infected calf showed the first signs of disease, only the two given Baypamun in addition to the vaccine, were protected from clinical disease whereas the two given vaccine only developed classical signs of IBR. In the three experiments the virus shedding by the Baypamun-treated calves resulted to be significantly reduced.     

Recrudescence of bovine herpesvirus-5 in experimentally infected calves

A latent infection of bovine herpesvirus-5 (BHV-5) was established in 4 calves. These calves, plus 2 controls, were given dexamethasone (DM) to reactivate the latent virus. The 4 principal calves developed antibodies to BHV-5 by postinoculation day (PID) 21. Antibody titers increased until PID 42 before decreasing to low levels of PID 75. After the first DM treatment (started on PID 76), an anamnestic antibody response was demonstrated in the 4 principal calves. Calves, 2, 3, and 4 were euthanatized and necropsied at PID 121, and their antibody titers were again decreasing. The virus BHV-5 was not isolated from the tissues by conventional techniques of viral isolation but was isolated from the trigeminal ganglion and spinal cord of calf 3 by explantation techniques. The BHV-5 was isolated, using conventional viral isolation techniques, from a nasal swab sample of calf 1 on PID 91 (15 days after the first DM treatment) and from the thoracic lymph node 6 days after the start of a 2nd DM treatment. Seemingly, BHV-5 may be latently harbored in the nerve tissues or calves and this virus may be reactivated from the upper respiratory tract following subsequent DM treatment.     

Humoral immune response of the bovine fetus to in utero vaccination with attenuated bovine coronavirus

A fetal response to in utero vaccination with attenuated bovine coronavirus (9 to 49 days before parturition) was determined in 8 calves, 5 vaccinated and 3 controls. Calves were derived by hysterotomy before parturition and were maintained in a closed gnotobiotic environment. The IgA, IgM, and IgG values and coronavirus-neutralizing antibody titers were higher in the sera and intestinal loop fluid from vaccinated calves than in those from control calves. Sections of ileum and ileal lymph nodes from 1-day-old vaccinated calves, when stained with monospecific anti-bovine IgG, IgM, and IgA had numerous positively stained plasma cells. Positive fluorescence was not detected in comparable tissues from controls. When the 8 calves were given virulent coronavirus orally at 6 days of age, vaccinated calves did not become ill, whereas control calves had diarrhea in 19 to 22 hours. All calves were killed at 10 days of age. Control calves had lesions characteristic of coronavirus infection, and intestinal epithelial cells were positive by fluorescent antibody tests. In vaccinated calves, lesions of coronavirus infection were absent, and results of fluorescent antibody tests were negative. Although in utero vaccination with a coronavirus vaccine stimulated immunity in the newborn calf, the frequency of abortions (2 of 14 cows inoculated intra-amniotically) and premature births (4 of 14) precluded practical application.     

DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves

Twelve calves infected with bovine herpesvirus type 1 (BHV-1) were killed when in a latent state of infection. Latency was verified 30 days after virus inoculation of the calves by seroconversion, absence of virus shedding, and in 2 calves, by recrudescence of the infection after they were treated with dexamethasone. By in situ hybridization techniques and autoradiography, DNA of BHV-1 was detected in 13 of 23 trigeminal ganglia of latently infected calves. Viral DNA was restricted to the nucleus of nerve cells. Single neurons harboring BHV-1 DNA were observed in 4.9% of the sections (n = 325) of the trigeminal ganglia. The results obtained correspond to those known from herpes simplex virus infections in mice. The implications for the virus-host relationship are discussed.     

Effect of repeated intradermal injections of bovine herpesvirus type 1 antigen on seronegative cattle.

Forty-three cattle seronegative to bovine herpesvirus-1 (BHV-1) were given from one to five intradermal injections of BHV-1 inactivated antigen at four-week intervals. This delayed hypersensitivity test was assessed by the increase in skin thickness. The activity of the antigen was assessed in five animals which had a previous natural BHV-1 infection with clinical signs and seroconversion. Anti-BHV-1 antibodies were detected by seroneutralisation and an enzyme-linked immunoassay. Only one animal showed a significant but slight increase in skin thickness after the first test, but it was negative after a second test. The animals remained seronegative after the first test. Seroconversion was identified in 11 of the 43 animals (25 per cent) submitted to repeated delayed hypersensitivity tests. Five of 37 animals seroconverted after only two tests. The serological response was transient in seven of 11 seroconverted calves. Repeated hypersensitivity tests were therefore able to induce a serological response in seronegative calves but the response was weak and often transient. The test must therefore be applied cautiously to seronegative animals.     

Immunization against bovine herpesvirus-1 infection. Preliminary tests in calves with a DNA vaccine

A bovine herpesvirus-1 (BHV-1) vaccine expressing glycoprotein D, the form with the transmembrane anchor removed, was evaluated for inducing immunity in calves. The plasmid encoding gD of BHV-1 was injected three times to nine calves, using three animals for each of the following routes: intramuscularly (i.m.), intradermally (i.d.), or intranasally (i.n.). Three additional calves were given the plasmid vector only and served as unvaccinated controls. When calves were subjected to challenge infection with BHV-1, all vaccinated calves as well as the controls developed a typical severe form of infectious bovine rhinotracheitis. However, compared to the controls, the vaccinated calves showed earlier clearance of challenge virus. Moreover, the calves given the vaccine i.m. developed neutralizing antibody to BHV-1 between 21 and 42 days following the first injection of vaccine, whereas in calves vaccinated either i.d. or i.n., as well as the controls, antibody first appeared in their sera 14 days post-challenge infection.     

Analysis of the kinetics, isotype and specificity of serum and coproantibody in lambs infected with Cryptosporidium parvum

Enteric cryptosporidiosis was studied in colostrum-deprived lambs each infected at five days old with 10(6) oocysts. The prepatent period was three to five days and faecal oocyst concentration fell below detectable levels by day 16 after infection. Specific IgA, the only isotype detected by immunofluorescent assay in faecal extracts from infected lambs, was first evident on day 10 and titres continued to rise until day 16 of infection in association with declining oocyst output. Specific IgM and IgG antibodies were first detected in serum seven days after infection. No specific antibody was detected in uninfected control lambs. Immunoblotting methods showed that serum antibody and faecal IgA had similar profiles of antigen recognition. Antigens with approximate molecular weights of 180,000, 23,000 and 15,000 were consistent features on immunoblots performed with convalescent sera and faecal extracts. The results suggest that specific IgA in intestinal secretions has an important role in immunity to cryptosporidiosis.