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.

A study on latency in calves by five vaccines against bovine herpesvirus-1 infection.

Four bovine herpesvirus-1 (BHV-1) commercial vaccines, three of which (vaccines B, D, E) were modified live vaccines (MLV) and one (vaccine A) identified as a live strain of BHV-1 gE negative, were used for vaccination of calves, using three calves for each vaccine. Three months after vaccination calves were subjected to dexamethasone (DMS) treatment following which virus was recovered from calves inoculated with vaccine B and from those given vaccine D. No virus reactivation was obtained in calves, which received vaccines A or E. The DNA extracted from the two reactivated viruses was subjected to restriction endonuclease analysis. The restriction pattern of the isolate obtained from calves vaccinated with vaccine D differs significantly from that of the original vaccine, whereas the reactivated virus from calves given vaccine B conserved the general pattern of the original vaccine strain. For each reactivated virus in this experiment (B and D) as well as for the isolate obtained from calves vaccinated with a further MLV (vaccine C) in a previous trial, three calves were inoculated. No clinical signs of disease were detected in any of the inoculated calves during the observation period. When the nine calves were exposed 40 days later to challenge infection with virulent BHV-1, they remained healthy and no virus was isolated from their nasal swabbings. These results indicate that some BHV-1 vaccines considered in the project can establish latency in the vaccinated calves, however, the latency does not appear to interfere with the original properties of the vaccines in terms of safety and efficacy.

Vaccination of calves against bovine herpesvirus-1: assessment of the protective value of eight vaccines.

Eight separate, but related experiments, were carried out in which groups of six calves were vaccinated with one of eight commercial vaccines. In each experiment the vaccinated calves were subsequently exposed to three calves infected with virulent bovine herpesvirus-1 (BHV-1). In each experiment, all infected donor calves developed a typical severe infectious bovine rhinotracheitis (IBR) infection and excreted virus in their nasal secretions of up to 10(8.00) TCID50/0.1 ml. One live BHV-1 gE-negative vaccine (A) and three modified live vaccines (B, C, D), administered intranasally, all protected against clinical disease. The calves vaccinated with one vaccine (C) also did not excrete virus in the nasal secretions, whereas the calves protected by vaccines A, B and D excreted virus in their nasal secretions but at low titres (10(0.66)-10(1.24) TCID50/0.1 ml). A fourth modified live vaccine (E), given intramuscularly, failed to prevent mild clinical disease in the calves which also excreted virus in the nasal secretions at titre of 10(1.00) TCID50/0.1 ml. An analogous result was given by the calves vaccinated with either of the two inactivated vaccines (F and G) or with a BHV-1 subunit vaccine (H). All calves developed mild clinical signs and excreted virus at titres of 10(2.20)-10(3.12) TCID50/0.1 ml. Calves vaccinated with C vaccine were subsequently given dexamethasone, following which virus was recovered from their nasal secretions. The virus isolates did not cause disease when calves were infected and appeared to be closely related to the vaccine strain.

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.

A sensitive and specific PCR/Southern blot assay for detection of bovine herpesvirus 4 in calves infected experimentally.

A PCR/Southern blot assay for detection of bovine herpesvirus 4 (BHV-4) in the background of bovine cellular DNA was developed. A BHV-4 specific sequence within the gene coding for the glycoprotein B (gB) was selected for primer sequences to guarantee the specificity of the assay. With a detection limit of six molecules BHV-4 DNA in the background of 1 microg of cellular DNA (equals about 150,000 bovine cells) this PCR/Southern blot assay represents a highly sensitive method for detection of BHV-4 DNA. At low concentrations of BHV-4 genomes, this assay also allows to estimate the copy number of BHV-4: a distinction between fewer than 6, 6-59 and more than 60 BHV-4 genomes/100 microl DNA suspension was possible. Tissue and blood samples of two calves, infected experimentally with BHV-4 were examined for the prevalence of BHV-4 DNA 130 days post infection. Ten days before taking samples, one of the calves was immuno-suppressed with dexamethasone. In both calves, BHV-4 DNA was detected in the leucocyte fraction of the blood, and beyond that in lower quantities in the spleen and the kidney of the immuno-suppressed calf. It is assumed that a latent BHV-4 infection was activated after application of dexamethasone and that the leucocyte fraction of the blood represents one site of latency of BHV-4 in cattle.

Tailored surgery for esophageal body diverticula.

OBJECTIVE: The aim of our study was to identify the presence of associated functional disorders (dysmotility or gastro-esophageal reflux, GER), to select patients who need surgery and to plan a tailored surgical treatment in patients affected by esophageal body diverticula. METHODS: We report on 51 consecutive patients with esophageal body diverticula, observed at our department, who underwent a thorough functional evaluation by means of radiology, endoscopy and manometry; 24 h pH-monitoring was performed in 11 patients who complained of symptoms of GER. The treatment of choice was planned in each patient on the basis of the following elements: the need of diverticulum excision and correction of esophageal body dismotility, LES dysfunction or GER. RESULTS: An esophageal motor dysfunction was detected in 73% (37 patients) of our total cases with an impaired LES function in 53% (27 patients); GER was identified in nine out the 11 patients submitted for 24 h pH-monitoring. On the overall series, we observed dysmotility or GER in 49/51 patients (96%). Sixteen patients did not require surgical treatment and eight patients refused it; 27 patients underwent tailored surgery. The overall complication rate was 11% (two esophageal fistulae, one acute coronary disease) with 7% mortality rate (one septic shock from esophageal leakage and one myocardial infarction). At follow up (average 47 months; range 6-103 months) 92% satisfactory results (Visick I and II) and only 8% of poor results were observed in our series. None of 13 patients who underwent conservative management had major complications at mean follow-up of 64 months. CONCLUSIONS: Based upon our experience, we believe that any case of diverticulum of the esophageal body deserves a complete physiopathological evaluation because an underlying functional disorder is associated in most cases. The evidence that the diverticulum per se can be considered as the ultimate phenomenon of an underlying functional disease determined the need for a tailored surgery, planning treatment of the functional disorder as the primary goal, not necessarily associated with a diverticulectomy. In our experience a tailored surgical treatment provided best results.

Further investigations on the efficacy of a non-specific defence inducer evaluated in calves exposed to infectious bovine rhinotracheitis virus.

Six calves were given the immunomodulator Baypamun and housed together with another six calves of which, three were experimentally infected with bovine herpesvirus-1 (BHV-1), whereas the remaining three served as untreated controls. The three experimentally infected calves as well as the three controls developed clinical signs of the typical acute form of infectious bovine rhinotracheitis (IBR). Of the calves treated with Baypamun, those that had only one injection of the immunomodulator, either at the start of the experiment (time 0) or 2 days later, underwent a much milder form of IBR and recovered in a shorter time than the experimentally infected calves or the controls. The calves that received four injections of the immunomodulator, i.e. at time 0 and subsequently for the next 3 days, remained healthy throughout the 30 days of observation. Moreover, the virus shedding by the Baypamun treated calves was significantly reduced. It was speculated that the use of an immunomodulator, eventually associated with a vaccination programme, would be a feasible approach to reduce significantly the onset of outbreaks of BHV-1, one of the main infectious agent initiating the respiratory disease in cattle.

A serological survey of bovine herpesvirus-1 infection in selected dairy herds in northern and central Italy.

Serum samples from a total of 6979 dairy cattle from 55 herds in northern Italy (51 herds) and central Italy (4 herds), were examined by the serum neutralization test for the presence of antibody to bovine herpesvirus-1 (BHV-1). It was found that 84.31% of the farms selected in northern Italy and all the farms from central Italy had seropositive animals at titers of 1:4 or higher. The prevalence of infection was essentially the same among the cattle populations of the two selected areas of the country, being of 34.99% in the north and of 38.65% in central regions. A comparison of the data from the present study with those obtained in a serological survey conducted in Italy in 1966, shows that the rate of seropositive cattle to BHV-1 has increased by about 5.0% in the last 30 years.

A non-specific defence inducer in preventing clinical signs of infectious bovine rhinotracheitis in calves.

One calf was infected with bovine herpesvirus-1 (BHV-1) and mixed with five other calves, of which one had been vaccinated with a BHV-1 modified live vaccine one month earlier. The other four calves were vaccinated at the time the experimentally infected calf developed the first signs of the disease (fever, depression, nasal discharge), i.e. on post infection day (PID) 2. In addition to the vaccine, two of the four PID 2 vaccinated calves also received a non-specific defence (NSD) inducer (Baypamun, Bayer AG) at the same time as the vaccine. The calf that was vaccinated 1 month before the start of the experiment, as expected, did not show any signs of the disease. Of the remaining four, the two vaccine-only calves experienced a classical form of infectious bovine rhinotracheitis. However, the two calves that had also received the NSD inducer remained generally healthy during the entire observation period of 30 days. It was speculated that the use of a NSD inducer once an outbreak of a respiratory disease has started on a farm could be of significant help in an emergency in reducing the clinical manifestations in those animals that may subsequently be infected.

The use of a non-specific defence mechanism inducer in calves exposed to bovine herpesvirus-1 infection: preliminary trials.

The efficacy of an immunomodulator, the Baypamun (Bayer AG), was tested in calves which were subsequently exposed to bovine herpesvirus-1 (BHV-1) infection. Two groups of calves, of 8 animals each, were used for two trials. In one trial, 4 calves were treated with the immunomodulator and the remaining 4 were left as untreated controls. Two hours after the first injection of Baypamun, all calves, i.e. the treated and the untreated ones, were challenged with BHV-1. Comparing them to the untreated controls, the calves which were given the immunomodulator developed milder signs of the disease and shed a significantly lower concentration of virus. In the second experiment, 4 calves were given Baypamun and 4 calves were not, as above, but they were not inoculated with BHV-1; however, they were housed together with the calves of the former group (experimentally infected with BHV-1). The 8 calves all became infected but those that had been treated with Baypamun did not show any clinical signs of the disease, whereas the untreated calves underwent a clinical response which was considered to be typical of BHV-1 respiratory infection, as usually seen under natural conditions. In this case the shedding of virus by the calves treated with the immunomodulator also underwent a significant reduction.

A study on neonatal calf diarrhea induced by rotavirus.

This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea. The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits. When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.

Immunosuppression as a factor in allowing mucosal disease to occur.

In order to verify the role of immunosuppression in the pathogenesis of Bovine Viral Diarrhea Virus (BVDV) infection, two experiments have been carried out. In one experiment, calves previously infected with cytopathogenic (CP) or non-cytopathogenic (NCP) BVDV were treated with dexamethasone (DMS) 30 days later. In the other experiment, calves were simultaneously exposed to BVDV infection and to DMS treatment. In both experiments the DMS treated calves developed a more serious disease which in one calf was fatal. It was speculated that immunosuppression represents one of the most significant "key factors" in the occurrence of the bovine viral diarrhea/mucosal disease in cattle.

Field evaluation of the efficacy of Romovac 50, a new inactivated, adjuvanted bovine rotavirus vaccine.

An inactivated bovine rotavirus vaccine, prepared with an adjuvant which gives a water-in-oil emulsion (Montanide ISA 50, adjuvant) was used in field trials for prevention of calf neonatal diarrhoea. The vaccine, which was designated as Romovac 50, when injected into cows in their last month of pregnancy, proved to be as effective as the traditional vaccine prepared with Freund's incomplete adjuvant. Thus, the incidence of the naturally occurring neonatal diarrhoea was significantly reduced in calves which were fed mammary secretion from their vaccinated dams, compared to the calves delivered from the unvaccinated cows. Romovac 50 also has the advantages over the traditional Freund's adjuvanted vaccine in that it is less viscous and thus more readily injected and less irritant to tissues.

The nightly spontaneous alkalinization of the stomach.

Authors evaluated eighty-four 24-hour intragastric pH recordings of 73 subjects in different pathophysiologic conditions. Among them there were 12 healthy subjects, 27 duodenal ulcer patients, 10 of whom were examined both before and after treatment with H2 receptor antagonists, 18 gastro-esophageal refluxers and 16 other patients who complained of dyspepsia of various origins. Authors observed a spontaneous nocturnal alkalinization phenomenon of the stomach (SNA) which began in the latter part of the night; its onset was strongly related with sleeping and interrupted by waking up. This phenomenon was absent in duodenal ulcer patients but could be restored by treatment with anti-H2 drugs. The authors, moreover, gave evidences of a relation between SNA and the vagal integrity and modulation of the gastric secretions.

Surgical treatment of achalasia: a retrospective comparative study.

A retrospective study carried out on 74 patients among 101 consecutive cases of achalasia of the esophagus operated from 1967 to 1989 is reported. On 21 patients observed between 1967 and 1975, a standard transabdominal Heller cardiomyotomy was performed (group A). From 1976 to 1989, the treatment of choice was a Heller myotomy associated with a modified Dor's fundoplication. In 80 consecutive cases (group B) the extension of myotomy was regulated by intraoperative monitoring of lower esophageal sphincter pressure. A 5-year follow-up with questionnaires, physical examination, and barium swallows was carried out on 16 patients in group A and on 58 patients in group B. In 75.6% of the cases (56 patients) follow-up examinations included esophageal manometry and 24-hour esophageal pH monitoring. Recurrence of dysphagia was recognized in 3 cases in group A (18.7%) and in 2 cases in group B (3.4%) (P = 0.053); postoperative gastroesophageal reflux, measured as a percentage of total reflux time, showed a significantly lower mean value in group B than in group A (1.8% vs. 4.1%, P < 0.01). This study suggests that an anti-reflux procedure lowers postoperative gastroesophageal reflux after Heller myotomy. Due to the low incidence of postoperative reflux and the negligible recurrence of dysphagia, Heller myotomy associated with a modified Dor's fundoplication may represent the surgical treatment of choice for achalasia of the esophagus.

Effects in calves of mixed infections with bovine viral diarrhea virus and several other bovine viruses.

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced. Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus. From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.

Twenty-four-hour intragastric pH-metry: evidence of nightly gastric alkalinization in subjects with and without active duodenal ulcer.

Continuous 24-hour gastric pH monitoring was performed in 68 subjects to evaluate the spontaneous nightly alkalinization phenomenon (SNA). Two groups of patients were compared: the first (group A) was composed of 27 subjects with active duodenal ulcer disease; group B included 12 healthy volunteers and 29 patients with gastroesophageal reflux. Patients with duodenal disease had an alkalinization of 196.2 +/- 97.9 seconds in duration, while healthy subjects and even gastroesophageal refluxers exhibit a SNA lasting 5269 +/- 748.3 seconds (t = 6.72; p < 0.001). The absence of SNA was used as a predictive test of duodenal ulcer disease and its discriminating ability was evaluated. The evidence of this phenomenon was able to exclude the disease in 90% of group B subjects (negative predictive value) while its absence suggested correctly the presence of duodenal disease in 82% of patients (positive predictive value). The lack of SNA in duodenal ulcer patients is so frequent that its absence might be a diagnostic sign of the peptic disease.

An experimental contribution to the study of the pathogenesis of bovine viral diarrhea virus infection.

This presentation summarizes the results of a study on the pathogenesis of bovine viral diarrhea (BVDV) infection. The cytopathic (CP) strain TVM-2 of BVDV induced in calves an overt clinical disease which is usually recorded as the acute primary BVDV infection observed under natural conditions. In contrast the non-cytopathic (NCP) strain New York-1 of BVDV did not cause any significant signs of disease. However, when the calves were immunosuppressed by treatment with dexamethasone (DMS) the biotype of BVDV involved did not seem to be as important as it appeared to be in an immunologically normal animal. This was shown in this study by the NCP BVDV which caused a fatal disease in calves treated with DMS. A mixed infection given to calves by injecting them with both CP and NCP BVDV, did not result in any particularly serious disease. So, the potential immunosuppressive activity of BVDV itself for the host has not been proven under the experimental procedures used in this experiment. Finally, a modified-live CP BVDV vaccine was unable to cause clinical disease when injected into calves that had been infected previously with strain New York-1 of BVDV.

Diaplacental infections with ruminant pestiviruses.

Pestiviruses are capable of causing diaplacental infections. Maternal viremias are important for localizing virus in the ruminant placentome. Placental lesions occur with cytopathic BVDV and noncytopathic BDV. The ruminant fetus is very susceptible to pestivirus infections once the virus crosses the placenta because the fetus is 1) agammaglobulinemic, 2) immunologically immature, and 3) it has many immature organ systems with undifferentiated cells. Cytopathic BVDV (NADL) in calves and noncytopathic BDV (BD-31) in lambs cause a variety of clinical syndromes including early embryonic death, abortion, stillbirth, malformed fetuses, and/or low birth weight with viral persistence and immunological tolerance. The cytopathic BVDV (NADL) reviewed herein caused pulmonary, placental and dermal lesions when infection occurred at 80-90 days gestation. In contrast, infection at 140-150 days resulted in retinal dysplasia and cerebellar hypoplasia. The lesions were attributed to direct viral cytopathology. Noncytopathic BDV (BD-31) in lambs caused weak lambs, with hairy fleece and tonic-clonic tremors. The lambs were of low birth weight, persistently viremic and immunologically tolerant. The lambs are hypothyroid and had severe hypomyelination. It is hypothesized that the central lesion leading to many of the neural, skeletal and dermal lesions was the endocrine dysfunction leading to hypothyroidism.