Abstract
This study demonstrated that bovine herpesvirus 5 (BoHV)-5 infected calves can develop encephalitis and remain asymptomatic. Seven calves were infected intranasally and monitored for 30 days. Cerebrospinal fluid (CSF) analysis was performed from the onset of neurological signs. Multiple sections of brain and the trigeminal ganglion were submitted to histopathology. Virus detection (PCR and isolation) was performed on CSF and tissues. Four calves developed signs of neurologic disease and died. Three calves remained asymptomatic and were euthanized 30 days post-infection. Cerebrospinal fluid mononuclear pleocytosis occurred in symptomatic and asymptomatic calves. BoHV-5 was isolated and viral DNA was detected in multiple areas of the encephalon of all calves. The viral DNA was detected in the CSF of 2 calves showing neurological signs. Histologically, inflammation was noted in the brain of all calves and confirmed that the encephalitis caused by BoHV-5 may be mild and asymptomatic.
Résumé
Encéphalite asymptomatique chez des veaux infectés expérimentalement par le virus herpétique bovin 5. Cette étude a démontré que les veaux infectés par le herpès virus bovin de type 5 (BoHV)-5 peuvent développer l’encéphalite et ne pas présenter de symptômes. Sept veaux ont été infectés par voie intranasale et surveillés pendant 30 jours. L’analyse du liquide céphalorachidien (LCR) a été réalisée dès l’apparition des signes neurologiques. De nombreuses sections du cerveau et du ganglion de Gasser ont été soumis à un examen histopathologique. La détection du virus (PCR et isolement) a été réalisée sur le LCR et les tissus. Quatre veaux ont développé des signes de maladie neurologique et sont morts. Trois veaux n’ont pas manifesté de symptômes et ont été euthanasiés 30 jours après l’infection. Une pléocytose mononucléaire du LCR s’est produite chez les veaux avec et sans symptômes. Le BoHV-5 a été isolé et l’ADN viral a été détecté dans des régions multiples de l’encéphale de tous les veaux. L’ADN viral a été détecté dans le LCR de 2 veaux manifestant des signes neurologiques. À l’histologie, l’inflammation a été remarquée dans le cerveau de tous les veaux et a confirmé que l’encéphalite causée par BoHV-5 peut être légère et asymptomatique.
(Traduit par Isabelle Vallières)
Introduction
Meningoencephalitis caused by bovine herpesvirus 5 (BoHV-5) infection is a major cause of encephalopathy in cattle in Brazil. The disease most often affects young animals and has been detected in several provinces (1–5). The infection has low morbidity and high lethality. In both natural (6,7) and experimental infections (8), some of the affected animals show only mild and transitory dysfunction with subsequent recovery.
Diverse results observed in experimental infections may be explained by the fact that the extent of neural invasion and virulence varies among different strains of BoHV-5 and according to the age of the host. The same strain is capable of inducing fatal disease in newborn calves (9) and asymptomatic disease in 5-month-old calves (10). While 1 strain causes clinical disease (11), another can cause latent infection without neurological disorders (12,13).
The establishment of latency in the host is a common characteristic of herpes viruses. Cattle may be asymptomatic carriers that will only show signs of encephalopathy if immunosuppression induces viral replication. Unlike an acute infection, there is no manifestation of neurological deficits during latent infection, even when the virus is present in several regions of the encephalon (10,12,13). Studies have suggested that encephalic inflammation may occur at the beginning of the process, even during latent infection (10,14). The objective of this study was to determine whether there was evidence to demonstrate that BoHV-5 can cause asymptomatic encephalitis in calves.
Materials and methods
This study was approved by the Animal Care Committee of the Universidade Estadual de Londrina (number 25/05, process 9671/2003).
All procedures related to the multiplication, isolation, and quantification of the BoHV-5 were done in Madin Darby bovine kidney (MDBK) cells kept in minimum essential medium (MEM) (D-MEM; Invitrogen, Carlsbad, California, USA) with 8% fetal calf serum (Invitrogen — Gibco, BRL, Carlsbad), gentamicin (55 μg/mL) and amphotericin-B (2.5 μg/mL). The virus used was the AA PAR strain isolated from the brain of a cow affected by encephalitis caused by BoHV-5 and confirmed by immunoperoxidase assay using monoclonal antibodies (15) and polymerase chain reaction (PCR) assay (16). The viral titers were measured in tissue culture infectious dose (TCID50).
Seven healthy Holstein bull calves between 20 and 58 days of age were used in this study. Calves with serum antibodies against bovine herpesvirus were excluded. The calves were kept in individual isolation pens and fed twice a day with milk substitute (Bovilac; Nutron, Toledo, Paraná, Brazil), in a total volume of 10% of their body weight (BW). Free choice water and grass hay, and a commercial pelletized ration at a maximum intake of 10% BW were available.
The calves were inoculated with BoHV-5 intranasally using 0.5 mL of suspension in each nostril with a total dose of 107.42 TCID50. Physical examinations were performed twice a day, assessing vital parameters, presence and aspect of nasal and ocular discharges, and neurological signs.
Blood samples were obtained from the jugular vein into plastic tubes without anti-coagulant, immediately before infection and every 72 h thereafter. Cerebrospinal fluid (CSF) was obtained through puncture of the atlanto-occipital space using an 18G 5.1-cm catheter stylet (BD Angiocath; Becton Dickinson, Juiz de Fora, Minas Gerais, Brazil). The CSF was sampled prior to infection and again when neurological signs developed and then every 48 h until death. In calves that remained asymptomatic, samples were obtained prior to infection and again on days 21, 23, 26, and 30 post-infection (p.i.).
Postmortem examination was performed immediately after death of sick animals or euthanasia of asymptomatic calves on the 30th day p.i. Tissues collected include trigeminal nerve ganglion and 9 different areas of the nervous system: anterior cortex (frontal), posterior cortex (occipital), dorso-lateral cortex (parietal), ventro-lateral cortex (temporal), diencephalon, mesencephalon, pons, medulla oblongata, and cerebellum. Where possible, samples were taken bilaterally. Samples were divided into 2 aliquots: 1 fixed in 10% neutral buffered formalin for histological examination and the other refrigerated at 4°C for virus isolation and PCR assay.
The presence of antibodies against bovine herpesvirus was tested by virus neutralization of the blood serum samples. Gross appearance (color and turbidity) and total and differential cell count of the CSF samples were recorded. Protein, glucose, pH, and specific gravity were also determined.
For virus isolation, each 500 μL aliquot of CSF was directly inoculated on MDBK cells and kept for 1 h at 37°C for adsorption before the medium (D-MEM) was added. For CNS tissues, samples were macerated in D-MEM (20% w/v), homogenized, and centrifuged. Antibiotic and antifungal agents were added to the supernatant, and 500 μL were inoculated into the MDBK cell culture. After 1 h at 37°C for adsorption, the fluid was collected, cells were washed with saline and medium was added for cell maintenance. The cells were then incubated at 37°C with daily observation (7 d maximum) for detection of cytopathic effect. Samples were considered negative when no cytopathic effect was observed after 3 passages. The presence of BoHV-5 DNA was detected by PCR assay (16).
All CSF samples and 500 μL of supernatant from each of the CNS macerate were tested for the presence of BoHV-5 DNA using a PCR assay designed to detect the glycoprotein C gene. For DNA extraction from fresh brain fragments, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was carried out according to Alfieri et al (17). CSF samples were used in the PCR assay without prior DNA extraction. The PCR assay was carried out using the primers designated as B5 specific for BoHV-5 (5′-CGG ACG AGA CGC CCT TGG-3′, nt 322–339) and a consensus primer designated as Bcon [5′-AGT GCA CGT ACA GCG GCT CG-3′, nt 519–538 (BoHV-1) and nt 461–480 (BoHV-5)]. The reaction was performed using 5 μL of extracted DNA and 45 μL of polymerase chain reaction (PCR)-mix consisting of 0.4 pmol of each primer (B5 and Bcon); 1.6 mM of dNTP (Invitrogen, Life Technologies, Carlsbad); 2.5 units of Platinum Taq DNA Polymerase (Invitrogen); 1 × PCR buffer (20 mM Tris–HCl pH 8.4; 50 mM KCl); 1.5 mM MgCl2; 8% dimethyl sulfoxide, and ultrapure sterile water to a final volume of 45 μL. Amplification was performed in a thermocycler (PTC 200; MJ Research, Watertown, Massachusetts, USA) with the following cycling profile: an initial step of 3 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 58°C, 1 min at 72°C, and a final extension step of 7 min at 72°C (16). Products from PCR were analyzed by electrophoresis on 2% agarose gel in TBE buffer, pH 8.4 (89 mM Tris; 89 mM boric acid; 2 mM EDTA), at constant voltage (90 V) for approximately 45 min, stained with ethidium bromide (0.5 μg/mL) and visualized under UV light.
Routine histopathological examination of CNS samples was carried out and abnormalities were classified as mild, moderate, or marked depending on the extent of the lesions identified. Perivascular cuffing was considered mild, moderate, or severe when there was up to 2, up to 4, or 5 or more layers of inflammatory cells surrounding blood vessels, respectively.
Results and discussion
The main clinical signs and progression of the disease in this study are shown in Table 1. Four of the 7 infected calves showed signs of encephalopathy starting on day 9 or 11 p.i. with most cases progressing to natural death. The period of time that affected calves showed clinical signs was variable. Calf #7 had peracute manifestation of the disease, dying 12 h after the beginning of clinical signs and after a severe episode of seizures. Calves 6 and 11 showed acute disease with progressive worsening over 3 d. Calf #1 had a longer period of disease with non-progressive clinical signs, which led to dehydration due to dysphagia and anorexia. The calf was given milk and water through an esophageal tube and was euthanized on the 10th day p.i. due to deterioration of its general condition. Previous observations in experimentally (9,11,14) and naturally infected calves (2,18) showed that either clinical presentation is possible: peracute or a more prolonged duration.
Table 1.
Clinical signs in calves with experimentally induced herpetic meningoencephalitis
| Number of symptomatic calves | Number of asymptomatic calves | ||||||
|---|---|---|---|---|---|---|---|
| 1 | 6 | 7 | 11 | 3 | 9 | 12 | |
| Age (days) | 58 | 22 | 22 | 20 | 45 | 23 | 45 |
| Signs of encephalopathy | |||||||
| Presence of signs | + | + | + | + | − | − | − |
| Onset of signs (days p.i.) | 11 | 11 | 9 | 9 | − | − | − |
| Duration of signs | 10 d | 3 d | 12 h | 3 d | − | − | − |
| Death | euthanasia | natural | natural | natural | euthanasia | euthanasia | euthanasia |
| Depression | + | + | + | + | − | − | − |
| Manias | + | + | − | + | − | − | − |
| Amaurosis | + | + | + | + | − | − | − |
| Dysphasia | + | + | + | + | − | − | − |
| Hypotonic tongue | − | + | − | + | − | − | − |
| Bruxism | + | − | − | + | − | − | − |
| Ataxia | + | + | − | + | − | − | − |
| Hypermetria | + | − | − | − | − | − | − |
| Sialorrhea | + | + | − | − | − | − | − |
| Facial myoclonus | + | + | + | − | − | − | − |
| Tonic convulsion | − | + | − | − | − | − | − |
| Clonic convulsion | − | − | + | − | − | − | − |
| Paresis and recumbence | + | + | − | + | − | − | − |
| Other manifestations | |||||||
| Anorexia (days p.i.) | 11 to 20 | 11 to 14 | 9 | 7 to 11 | − | − | − |
| Serous nasal discharge (days p.i.) | 7; 11 to 20 | 3 to 13 | − | 5 to 11 | 7; 9 and 14 | 10 to 12 | 4; 13 and 16 |
| Ocular discharge (days p.i.) | − | − | − | 5 to 11 | − | − | 16 and 17 |
| Sporadic cough (days p.i.) | − | 6 and 7 | 4 to 8 | − | − | 10 to 27 | 9 to 13 |
| Dehydration | + | − | − | − | − | − | − |
p.i. = post-infection, − = not present.
The initial clinical signs were moderate to severe depression, anorexia, amaurosis, and dysphagia followed by hypotonia of the tongue, sialorrhea, bruxism, ataxia, hypermetria, mania (compulsive acts), facial myoclonus, convulsions, paresis, and recumbence. All are consistent with signs of exaggerated and/or depressed neuronal activity. The neurological signs observed, as well as their intensity, were variable among the animals and over the study period. Similar clinical signs were observed in other studies using experimental infection (9,11,12) and in reports of natural infection (1,2,3,7,18).
All calves had serous nasal discharge at some point, except for calf #7. The nasal discharge persisted for several days in some calves, but was intermittent in other calves. The volume of the discharge was usually small. Some calves had sporadic non-productive cough that did not coincide with the nasal discharge. None of the calves developed problems of the small airways. Only calves #11 and #12 showed small amounts of ocular discharge, unlike the observations of Perez et al (14).
The experimental infection with BoHV-5 did not induce signs of encephalopathy in all the calves. Three animals did not manifest any neurological signs over the 30-day p.i. period. This result is consistent with reports of other researchers that infection with herpesvirus may not cause clinical disease when latent (9–11,19). It is believed that the lack of clinical signs is due to the absence of encephalitis in these animals. Considering studies in which high numbers of calves were inoculated experimentally, Meyer et al (11) observed a smaller incidence of calves that remained apparently healthy (1 out of 8 calves), while Vogel et al (12) could not initially induce clinical disease in any of the 12 calves used and only 3 out of 8 calves developed signs of encephalopathy after dexamethasone reactivation of the latent infection. The diversity of results possibly reflects differences in pathogenicity among the different BoHV-5 strains used in these studies.
Respiratory and cardiac rates did not differ significantly over time in calves that developed encephalopathy and in those that were asymptomatic. In calves that showed neurological signs mean body temperature had a tendency to be higher (> 40.0°C) on days 8, 11, and 12 p.i., coinciding with the onset of signs. In asymptomatic calves mean body temperature increased on days 9 and 10 p.i., and the calves remained alert and hungry. The body temperatures of calves in both groups did not exceed 40.4°C. These results reinforce the findings of Perez et al (14), but are different from those of Meyer et al (11) and Vogel et al (12) who did not find significant elevations in body temperature. Meyer et al (11) reported a discrete increase in body temperature on days 4 to 10 p.i., but it was not considered hyperthermia, as temperature values did not increase over 39.5°C. In the same study, calves infected with BoHV-1 showed hyperthermia during the initial period p.i. These results suggest that infection with the BoHV-5 is not necessarily followed by fever.
The consistent and remarkable abnormalities of the CSF cytology have diagnostic relevance as they confirm, in vivo, the brain inflammatory process (20). As shown in Figure 1, a marked pleocytosis (6 cells/mm3 before infection and 242 cells/mm3 on the 11th day p.i.) was noted in calves with signs of encephalopathy. Mononuclear cells were predominant (over 90%), which is characteristic of viral encephalitis (21). Unexpectedly, with exception of calf #9, the calves that remained asymptomatic had the same cytological abnormalities in the CSF compared with calves that had encephalopathy (9 cells/mm3 before infection and 168 cells/mm3 on the 23rd day p.i.). The total cell count decreased later. This is an indication that, even without showing neurological signs, these animals also developed meningoencephalitis, which probably decreased in intensity up to 30 days p.i. The CSF samples were clear and translucent in most cases. Eventually, a sample would be xanthochromic and turbid. Characteristic biochemical abnormalities were not observed. Although it has important diagnostic value, other researchers did not perform CSF cytology during experimental infection. The demonstration of mononuclear pleocytosis in apparently healthy calves is reported for the first time in this study, and strongly suggests that BoHV-5 causes meningoencephalitis, but the inflammation may not be accompanied by observable neurological disorders.
Figure 1.
Variation in mean mononuclear leucocyte count in the CSF of calves that developed clinical signs of encephalopathy (n = 4, continuous line) or remained asymptomatic (n = 3, dotted line) after experimental infection with BoHV-5. Unlike the protocol adopted for calves that showed clinical disease, in asymptomatic calves the CSF samples were collected starting on day 21 p.i.
Serum conversion occurred in all 7 calves; however, there was considerable individual variation in titer and the time necessary to reach maximum titer. As in previous studies (12,13), the highest titers (32 and 64) were reached by the asymptomatic calves at 21 days p.i. It is possible that the titers of serum neutralizing antibodies were not high in the calves with signs of encephalopathy because they died earlier. Perez et al (14) reported much higher titers of serum antibodies in calves with latent infection, which were detectable earlier, at 14 days p.i. However, unlike in the present study, those authors induced infection in older calves (1-year-old), which may explain the difference.
Viral DNA was confirmed by PCR assay and virus was isolated from the nervous system of all calves (Table 2). In the 3 asymptomatic calves, viral DNA was present in 3 to 5 locations in the nervous system. In all calves that manifested neurological signs, the virus was identified in a greater number of brain locations than in asymptomatic calves, indicating a broader distribution of infection. Virus was less frequently isolated from the cerebellum and medulla oblongata. The telencephalon, diencephalon and pons were positive for viral DNA in all calves. These results reaffirm that there is no preferable location by the virus in calves with apparent encephalopathy (9,11). The results from Vogel et al (12) were similar, reinforcing the concept that BoHV-5 distributes throughout the host’s encephalon, even if the infection is latent. Therefore, in contrast with an earlier statement that the rostral cortex is the best sample for BoHV-5 infection diagnosis (14), multiple sampling sites are recommended to maximize the diagnostic outcome.
Table 2.
Results of polymerase chain reaction (PCR) and virus isolation from different sections of the nervous system (NS) and cerebrospinal fluid (CSF) samples of calves experimentally infected with BoHV-5
| Nervous system area | Symptomatic calves | Asymptomatic calves | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1a | 6 | 7 | 11 | 3 | 9 | 12 | ||||||||
| PCRb | VIc | PCR | VI | PCR | VI | PCR | VI | PCR | VI | PCR | VI | PCR | VI | |
| Rostral cortex | + | CPEd* | + | − | + | CPE | + | CPE* | − | − | − | − | + | CPE* |
| Caudal cortex | + | CPE* | + | CPE* | + | CPE* | + | CPE | + | − | − | − | − | − |
| Dorso-lateral cortex | + | CPE* | + | CPE* | + | CPE* | + | CPE* | + | CPE* | + | CPE* | + | − |
| Ventro-lateral cortex | + | CPE* | + | CPE* | + | CPE* | + | CPE* | − | − | − | − | + | − |
| Diencephalon | + | − | + | CPE* | + | CPE | − | − | − | − | + | − | + | − |
| Mesencephalon | + | CPE* | − | − | + | CPE* | + | CPE* | − | − | − | − | + | CPE* |
| Pons | + | − | + | − | + | CPE* | + | CPE* | + | − | + | CPE* | − | − |
| Medulla oblongata | + | CPE* | − | − | − | − | + | CPE* | − | − | − | − | − | − |
| Cerebellum | − | − | − | − | + | − | + | CPE* | + | CPE* | − | − | − | − |
| Trigeminal ganglion | + | CPE* | + | − | + | CPE* | + | − | + | − | − | − | − | − |
| CSF | + | − | − | − | + | − | − | − | − | − | − | − | − | − |
Calf numbers 1, 6, 7, 11, 3, 9, and 12.
Viral DNA detected by PCR assay.
VI — virus isolation in cell culture.
CPE — cytopathic effect.
presence of viral DNA confirmed by PCR assay.
− = negative.
The presence of viral DNA was not always accompanied by isolation of BoHV-5, particularly in the asymptomatic calves. Vogel et al (12) did not succeed in their attempts to isolate the virus from the same category of animals. This may be explained by the fact that, in those calves, the virus should be in the latent form, and therefore without its infective potential. The presence of viral DNA in the trigeminal ganglion of 5 calves is affirmation that this is a common site for the presence of the virus (19).
Similar to recent observations in natural occurring cases (18,20), the presence of viral DNA was confirmed in the CSF from 2 calves that showed neurological dysfunction. The presence of BoHV-5 in the CSF of experimentally infected calves has only been reported once in the literature (8). Despite the presence of DNA, attempts at virus isolation were unsuccessful in both cases (Table 2). Furthermore, compared to brain tissues, the negative PCR results were more probable in CSF. Even though brain tissues are a more reliable source, the data support the antemortem use of CSF as a means to confirm the etiologic diagnosis of the disease. A negative result doesn’t rule out the disease, but a positive one can confirm the diagnosis.
Calf #6 was the only animal with macroscopic encephalic abnormalities (congestion and areas of malacia of the anterior cortex). No lesions of the brain or other organs were observed in the remaining calves. Other authors have also reported congestion and areas of malacia in experimentally infected animals (14,19) and in naturally occurring disease (3,6). The lack of lesions at postmortem is a common feature of this disease (2,11).
All calves had some degree of microscopic abnormalities of the nervous system, although at different locations and intensities. In general, the lesions were distributed in the whole brain of the calves and also in the trigeminal nerve ganglion (Tables 3 and 4). Calves 1, 6, and 7 had marked lesions in several sections of the nervous tissue, while calves 3, 9, and 11 had less severe lesions. Calf #11 showed signs of encephalopathy, but had only mild histological lesions. Although calf #12 was asymptomatic, it had moderate lesions in several portions of the nervous system. The lesions consisted of nonsuppurative necrotizing meningoencephalitis, characterized by perivascular cuffing with mononuclear cell infiltrates, cortical laminar necrosis, gliosis, and meningitis. Our results are consistent with other studies (1,3,6,7). Eosinophilic intracellular inclusion bodies, mainly located in the astrocytes, are frequently evident in animals affected by natural infection (1,3,6). However, in the present study, as well as in other experimental infections (9,14), no inclusions were seen.
Table 3.
Histological changes in brain sections and trigeminal ganglion of calves that developed encephalopathy after experimental infection with BoHV-5
| Nervous system area | Calf number
|
|||
|---|---|---|---|---|
| 1 | 6 | 7 | 11 | |
| Rostral cortex | PVCb (++)a | PVC (+++) | PVC (+++) | PVC (+) |
| Meningitis (++) | CLN (++) | CLN (++) | ||
| Necrosis (+++) | Meningitis (+++) | Meningitis (+++) | ||
| Gliosis (++) | ||||
| Necrosis (+) | ||||
| Caudal cortex | PVC (++) | PVC (++) | Meningitis (+) | Hemorrhage (+) |
| CLNc (+) | CLN (+) | |||
| Meningitis (++) | Meningitis (+) | |||
| Gliosis (+) | ||||
| Dorso-lateral cortex | PVC (+++) | PVC (++) | PVC (++) | Hemorrhage (+) |
| CLN (++) | Meningitis (+) | Meningitis (+) | ||
| Meningitis (+++) | Gliosis (+) | Gliosis (+) | ||
| Gliosis (++) | ||||
| Ventro-lateral cortex | PVC (+++) | PVC (+) | PVC (++) | Hemorrhage (+) |
| CLN (++) | Meningitis (+) | CLN (+) | ||
| Meningitis (+++) | Gliosis (+) | Meningitis (++) | ||
| Necrosis (+) | ||||
| Diencephalon | PVC (+++) | PVC (+++) | PVC (+++) | Hemorrhage (+) |
| Meningitis (+++) | Necrosis (+) | Gliosis (+) | ||
| Gliosis (++) | ||||
| Mesencephalon | PVC (+++) | PVC (+) | PVC (+++) | Meningitis (+) |
| Necrosis (++) | Gliosis (+) | Necrosis (+) | Hemorrhage (+) | |
| Meningitis (+++) | Gliosis (+) | |||
| Gliosis (++) | ||||
| Pons | PVC (+++) | PVC (+) | Gliosis (++) | (−) |
| Meningitis (+++) | ||||
| Gliosis (++) | ||||
| Medulla oblongata | PVC (+++) | PVC (++) | PVC (+) | Hemorrhage (+) |
| Gliosis (+) | Gliosis (+) | Meningitis (++) | ||
| Gliosis (+) | ||||
| Cerebellum | PVC (+++) | PVC (+) | Meningitis (++) | (−) |
| Meningitis (+) | Gliosis (++) | Hemorrhage (+) | ||
| Gliosis (+) | ||||
| Trigeminal ganglion | PVC (+) | PVC (+) | (−) | Necrosis (+) |
| Necrosis (+) | ||||
(−) absence of changes, (+) mild changes, (++) moderate changes, (+++) marked changes.
PVC — perivascular cuffing.
CLN — cortical laminar necrosis.
Table 4.
Histological changes in brain sections and trigeminal ganglion of calves that remained asymptomatic after experimental infection with BoHV-5
| Nervous system area | Calf number
|
||
|---|---|---|---|
| 3 | 9 | 12 | |
| Rostral cortex | CLN (+) | PVC (+) | PVC (++) |
| Meningitis (+) | CLN (+) | CLN (++) | |
| Gliosis (+) | Meningitis (+) | Meningitis (++) | |
| Gliosis (+) | Gliosis (++) | ||
| Caudal cortex | CLN (+) | CLN (++) | PVC (++) |
| Meningitis (+) | CLN (+) | ||
| Gliosis (++) | |||
| Dorso-lateral cortex | CLN (+) | CLN (+) | Meningitis (++) |
| Ventro-lateral cortex | (−) | Meningitis (+) | PVC (++) |
| CLN (+) | |||
| Meningitis (+) | |||
| Gliosis (+) | |||
| Diencephalon | PVC (+) | Necrosis (+) | PVC (+++) |
| Meningitis (++) | |||
| Gliosis (++) | |||
| Mesencephalon | Meningitis (+) | (−) | PVC (++) |
| Gliose (+) | |||
| Pons | PVC (+) | PVC (++) | PVC (+) |
| Hemorrhage (+) | Gliosis (++) | ||
| Medulla oblongata | (−) | (−) | PVC (+) |
| Cerebellum | PVC (+) | PVC (++) | (−) |
| Meningitis (+) | |||
| Gliosis (+) | |||
| Trigeminal ganglion | PVC (+) | (−) | Necrosis (+) |
(−) absence of changes, (+) mild changes, (++) moderate changes, (+++) marked changes.
PVC — perivascular cuffing.
CLN — cortical laminar necrosis.
The histopathological changes are uncontestable proof that calves which remained asymptomatic developed brain inflammatory processes. This explains the pleocytosis previously observed in the CSF cytology of those animals. The magnitude of the meningoencephalic inflammation, and the localization and distribution of the lesions, were likely insufficient to cause signs of the disease. This hypothesis is corroborated by the results published by Vogel et al (12,13), which demonstrated that the clinical presentation of the disease depends on how neuro-invasive and neuro-virulent the BoHV-5 strain was. Virus strains such as the one used by these authors can be highly neuro-invasive and mildly neuro-virulent, thereby causing an elevated number of asymptomatic infections. Furthermore, individual factors of resistance such as cellular immunity may contribute to the occurrence of this form of presentation. If these are correct concepts, it is reasonable to assume that the morbidity rates of this disease may be higher than the ones currently reported. On the other hand, the lethality rates may not be so elevated. These hypotheses, however, require more detailed investigation. In summary, it can be inferred that BoHV-5 infection causes encephalitis in calves, but this is not necessarily accompanied by manifestation of neurological dysfunction.
Acknowledgment
We thank Dr. Spencer Russell for reviewing and correcting the English version of this manuscript. CVJ
Footnotes
Funding for this study was provided by Brazilian Funding Agencies CNPq, FINEP and CAPES.
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (hbroughton@cvma-acmv.org) for additional copies or permission to use this material elsewhere.
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