Vet Pathol 29:495-502 (1992)
Rift Valley Fever Virus-induced Encephalomyelitis and Hepatitis in Calves M.
K.
RIPPY, M.
J. TOPPER, C. A.
MEBUS, AND
J. C.
MORRILL
Pathology and Disease Assessment Divisions, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD (MKR, MJT, JCM); and US Department of Agriculture, Plum Island Animal Research Center, Greenport, NY (CAM) Abstract. Three calves (Nos. 1,2 = 7 days old; No.3 = 21 days old) were inoculated subcutaneously with virulent Rift Valley fever (RVF) virus. All calves became viremic and clinically ill, but the two 7-day-old calves were moribund and were euthanatized subsequently on post-inoculation day (PID) 3. Highest viral titers were measured in the serum, with lesser concentrations in the brain, heart, spleen, and liver of these animals. Viral antigens were detected by immunohistochemical analysis only in the livers, where positive staining was localized in coalescing foci of hepatocellular necrosis. The 21-day-old calf appeared to recover after viremia and pyrexia but became lethargic and ataxic and was euthanatized on PID 9. The calf was no longer viremic, and R VF virus was isolated only from the brain. Microscopic examination of the central nervous system revealed diffuse perivascular infiltrates of lymphocytes and macrophages, multifocal meningitis, and focal areas of neuronal necrosis and aggregates of macrophages, lymphocytes, and neutrophils throughout all regions of the brain and cervical spinal cord. There was positive immunohistochemical staining for viral antigens within the cytoplasm of neurons and glial cells throughout the central nervous system. Thus, RVF virus can cause encephalomyelitis in calves, and the specific virologic diagnosis can be made by immunohistochemical localization of viral antigens in formalin-fixed tissues. Key words: Calves; encephalomyelitis; hepatitis; Rift Valley fever.
Rift Valley fever (RVF) virus, a Phlebovirus of the family Bunyaviridae, is enzootic throughout most of sub-Saharan Africa. The virus is probably maintained in nature through a cycle involving mosquito vectors and domestic livestock or through transovarial transmission in certain floodwater Aedes mosquitoes. 10 Occasional epidemics occur in enzootic areas when alterations in environmental conditions promote expansion ofvector populations in the presence oflarge numbers of susceptible hosts. The importation ofRVF virus into Egypt, a nonenzootic area where suitable vector and susceptible host populations coexist, resulted in a devastating epidemic in 1977 .11 The virus affects primarily domestic ruminants and human beings. Viral infection of sheep, cattle, and goats results in severe economic losses due to decreased milk production, abortion, and death. Fulminant neonatal disease may be the first indication of RVF in regions where increased incidences of abortion are attributable to other abortogenic agents. Fatality rates in lambs, calves, and kids less than 1 week old may approach 100%, and virtually all pregnant sheep that are infected will abort.t-" Infected neonates succumb to severe neerotizing hepatitis.v' The disease in human beings is
usually a transient illness, although complications of hemorrhagic fever, retinal disease, or encephalitis occur in a small percentage of infections. IS RVF virus-induced encephalitis has been reported in natural infections in human beings and in experimentally infected gerbils and certain strains of rats, but the pathologic characterization of the central nervous system (eNS) lesions has not been described in RVF virus-infected ruminants. Weiss reported viral encephalitis in two lambs born to ewes vaccinated with the Smithburn neurotropic strain ofRVF vaccine while pregnant." Those lambs died within 6 days of birth, but a description of the lesions and clinical data were not provided. The present report describes the lesions and virologic events leading to necrotizing encephalomyelitis in a calf infected experimentally with virulent RVF virus and contrasts findings with those in calves that succumbed to necrotizing hepatitis. In addition, immunohistochemical examination was used to localize RVF viral antigens in tissues from infected calves. This report is the first study of RVF virusinduced encephalomyelitis in a calf and immunohistochemicallocalization ofRVF viral antigens in tissues from infected ruminants.
495 Downloaded from vet.sagepub.com by guest on March 5, 2015
496
Rippy et al.
Materials and Methods
Vet PathoI29:6, 1992
Immunohistochemistry
Experimental design Three crossbred Hereford calves (Nos. I, 2, 7 days old; No.3, 21 days old) were inoculated subcutaneously with 5 x 10' plaque-forming units of virulent ZH-501 strain of Rift Valley fever (RVF) virus and served as unvaccinated, infected, control animals during safety and efficacy testing of a mutagen-attenuated RVF vaccine. The animals were commingled with their dams and housed under BSL-3 + containment. The calves were examined daily, and rectal temperatures were recorded. Whole blood for virologic, immunologic, and clinicopathologic assays was obtained by jugular venipuncture immediately prior to virus inoculation and daily through post-inoculation day (PID) 5 and on PID 7 and 9. Complete necropsies were done immediately after death and tissues were obtained for viral quantification and light microscopic examination. Only the cervical spinal cord was examined histologically. Tissue preparation Tissue samples were taken for viral quantification and light microscopic examination. Tissues were homogenized (10% w/v) using sterile sand, mortar, and pestle in Hanks' balanced salt solution (HBSS) with 0.5% human serum albumin and antibiotics (100 U penicillin and 100 J.Lg streptomycin sulfate/ ml), clarified by centrifugation, and stored at 70 C. For microscopic examination, tissue samples were fixed by immersion in 10% neutral buffered formalin, and paraffin sections were prepared and stained with Mayer's hematoxylin and eosin. Specimens representative of the different regions of the brain were taken from each calf according to a standard neurovirulence protocol." Sections of liver and brain were stained with Brown and Brenn Gram's stain Giemsa and Gomori's methamine silver stain for possibl~ identific~tion of bacteria or fungi. Additional sections were processed for immunohistochemical analysis. Virologic, immunologic, and clinicopathologic assays Serum was separated from peripheral blood by centrifugation and stored at -70 C. Homogenates (10% w/v) of selected tissues were prepared for virus quantification. RVF virus was quantified in serial lO-fold dilutions of each serum specimen or tissue homogenate. Viral plaques were counted under agarose in 16-mm Vero cell monolayers, and viral titers were calculated as previously described. 16 Serum plaque reduction neutralization tests were done using serial fourfold dilutions of sera in HBSS.14 The plaque-reduction neutralization titer was expressed as the highest dilution of serum that caused an 80% reduction in RVF viral plaques. Sera were tested for RVF virus-specific IgM and IgG antibodies by enzyme-linked immunosorbent assay as previously described.8.12 Total and differential white blood cell, hematocrit, and platelet counts were performed manually on ethylenediaminetetraacetic acid anticoagulated blood. Serum aspartate aminotransferase, gamma glutamyltransferase, and lactic dehydrogenase were measured with a Multistat III Plus (Instrumentation Laboratories, Lexington, MA) centrifugal chemical analyzer.
RVF viral antigens were demonstrated in paraffin-embedded fixed tissue specimens by immunoperoxidase or immunoalkaline phosphatase using a modified avidin biotin peroxidase complex method as previously described." Briefly, 5-J.Lm sections of tissue were mounted on 3-aminopropyltriethoxysiane-coated slides, deparaffinized, and hydrated. The sections were immersed in a 0.05% solution of protease VIII (subtilysin, Sigma Chemical Co., St. Louis, MO), pH 7.8, at 37 C for 3 minutes and washed in phosphate-buffered saline (PBS), pH 7.4. Polyclonal mouse antiserum to RVF virus was used as the primary antibody at a dilution of I : 1,000. The antiserum was prepared against the T-I and ZH-501 strains of RVF virus. This antibody does not react with normal bovine tissues. After incubation with the primary antibody at room temperature for I hour, slides were rinsed in PBS and incubated at room temperature with biotinylated horse anti-mouse IgG, then by the avidin biotin peroxidase complex method (Vectastain, Vector Labs, Burlingame, CA). Diaminobenzadine was the substrate for peroxidase and 6-bromo-2-hydroxy-3-naphthoic acid was the substrate for alkaline phosphatase. Sections were counterstained with Mayer's hematoxylin. Polyclonal mouse antiserum to yellow fever virus and normal mouse serum were substituted for the primary antibody and served as assay controls.
Results Clinical observations
By post-inoculation day (PID) 2, all three calves were febrile (Fig. 1), noticeably lethargic, and did not suckle actively. All calves had marked conjunctival and scleral mucous membrane congestion, and calf No. 2 showed signs of approximately 10-15% dehydration, i.e., slightly sunken eyes and a loss in skin elasticity. On PID 3, the youngest calves (Nos. 1,2) were moribund and were euthanatized. The eldest calf(No. 3) appeared to recover and became more active on PID 4 through 7. But on PID 8, this calf developed a subnormal rectal temperature and congested conjunctiva and sclera and was lethargic. On PID 9, the calf had a rectal temperature of 36.4 C, refused to suckle, and was reluctant to move. Although the calf could stand unassisted, it was unsteady and continually moved its head from side to side in an unnatural manner. When manipulated, the animal would fall down but could rise again, though with difficulty. The animal was euthanatized on PID 9. Laboratory findings
All three calves were viremic on PID 1 through 3 and viremia was detected in calf No. 3 through PID 5 (Fig. 1). Viremia in calf No. 3 peaked at 5.110g 1o plaqueforming units (PFU)/ml on PID 2 and became undetectable by plaque assay on PID 7. Viral titers oftissue homogenates from these calves are presented in Table
Downloaded from vet.sagepub.com by guest on March 5, 2015
Rift Valley Fever in Calves
Vet Pathol 29:6, 1992
497
2000 1800
42 41
AST
1400
40 1000 39
0
600
38
0
20~ ~iiiii:r:-r----,--~=~=:::;:::==~==::;=~
37 36
500
GGT
35 0
2
3
4
5
6
7
8
400
9 -l
:J
7
E
6
::::>
5
LL
n,
200 100
~
CJ 0
t======1~""""-"""'-~--"Ir----..----...
o-t---,--...,--.---,....-........,-........,----,..-----,-----,
4 0
-l
300
3
10000
2
8000
LDH
-l
:J 0
0
2
3
4
5
6
6000 4000
DAYS POST - INOCULATION
2000
Fig. 1. Rectal temperatures and serum viremia titers of calf Nos. 1-3 inoculated with RVF virus on day O... = calf No.3;. = calf No. 2;. = calf No. I.
O+--r--r----r---,-.,---,--,.--.------=T
1. Only a single plaque, observed on two separate repeatable assays, was detected in the spleen homogenate from calfNo. 3, and virus was not detected in the liver. However, a viral titer of 3.9 log., PFU/g was found in the brain. No RVF virus-specific IgM, IgG, or neutralizing antibodies were found in the sera of those calves that died on PID 3; however, specific IgM and IgG were first detected on PID 4 and 5, respectively, in calf No.3. A plaque-reduction neutralization titer of 1: 160 was detected in calf No. 3 on PID 7 and rose to 1: 640 by PID 9. Serum hepatic enzymes (aspartate aminotransferase [AST], gamma glutamyltransferase [GGT], and lactic dehydrogenase [LDH]) were markedly elevated in the calves (Nos. 1, 2) that died on PID 3 (Fig. 2). Serum AST values were noticeably increased in calf No. 3 on PID 2 but had returned to near baseline levels by PID 4. Only slight elevations in GGT and LDH levels were seen in calf NO.3. There were dramatic changes in hematologic values during the first 3 days after infection, and trends were similar in all three calves. Hematocrit values were elevated from 26-38% to 4652%, platelet counts were reduced, and severe leukopenia was seen. The mean white blood cell (WBC) count for all three calves decreased from 8,900 cells/ J,ll on PID 0 to a mean nadir of 2,500 cells/J,ll on PID 2. The WBC count ofcalf No. 3 decreased from 10,400
Fig. 2. Serum AST, GGT, and LDH values ofRVFvirusinoculated calf Nos. 1-3 ... = calf No.3; • = calf No.2; • = calf No. I.
o
2
3
4
5
6
7
8
9
DAYS POST - INOCULATION
cells/J,ll on PID 0 to 3,600 cells/J,ll on PID 2. A persistent leukopenia was seen throughout the course of illness in this animal. In calf No.3, neutrophil counts decreased from 5,600 cells/ul to 2,700 cells/ul, and lymphocyte counts decreased from 3,100 cells/J,ll to 600 cells/J,ll on PID 2.
Table 1. Rift Valley fever viral titers* in cerebrospinal fluid (CSF) serum, and organ homogenates of virus-infected calves. Organ
Calf No.
It
2t
3:1:
5.1 CSF 3.0
Rift Valley Fever Virus-induced Encephalomyelitis and Hepatitis in Calves M.
K.
RIPPY, M.
J. TOPPER, C. A.
MEBUS, AND
J. C.
MORRILL
Pathology and Disease Assessment Divisions, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD (MKR, MJT, JCM); and US Department of Agriculture, Plum Island Animal Research Center, Greenport, NY (CAM) Abstract. Three calves (Nos. 1,2 = 7 days old; No.3 = 21 days old) were inoculated subcutaneously with virulent Rift Valley fever (RVF) virus. All calves became viremic and clinically ill, but the two 7-day-old calves were moribund and were euthanatized subsequently on post-inoculation day (PID) 3. Highest viral titers were measured in the serum, with lesser concentrations in the brain, heart, spleen, and liver of these animals. Viral antigens were detected by immunohistochemical analysis only in the livers, where positive staining was localized in coalescing foci of hepatocellular necrosis. The 21-day-old calf appeared to recover after viremia and pyrexia but became lethargic and ataxic and was euthanatized on PID 9. The calf was no longer viremic, and R VF virus was isolated only from the brain. Microscopic examination of the central nervous system revealed diffuse perivascular infiltrates of lymphocytes and macrophages, multifocal meningitis, and focal areas of neuronal necrosis and aggregates of macrophages, lymphocytes, and neutrophils throughout all regions of the brain and cervical spinal cord. There was positive immunohistochemical staining for viral antigens within the cytoplasm of neurons and glial cells throughout the central nervous system. Thus, RVF virus can cause encephalomyelitis in calves, and the specific virologic diagnosis can be made by immunohistochemical localization of viral antigens in formalin-fixed tissues. Key words: Calves; encephalomyelitis; hepatitis; Rift Valley fever.
Rift Valley fever (RVF) virus, a Phlebovirus of the family Bunyaviridae, is enzootic throughout most of sub-Saharan Africa. The virus is probably maintained in nature through a cycle involving mosquito vectors and domestic livestock or through transovarial transmission in certain floodwater Aedes mosquitoes. 10 Occasional epidemics occur in enzootic areas when alterations in environmental conditions promote expansion ofvector populations in the presence oflarge numbers of susceptible hosts. The importation ofRVF virus into Egypt, a nonenzootic area where suitable vector and susceptible host populations coexist, resulted in a devastating epidemic in 1977 .11 The virus affects primarily domestic ruminants and human beings. Viral infection of sheep, cattle, and goats results in severe economic losses due to decreased milk production, abortion, and death. Fulminant neonatal disease may be the first indication of RVF in regions where increased incidences of abortion are attributable to other abortogenic agents. Fatality rates in lambs, calves, and kids less than 1 week old may approach 100%, and virtually all pregnant sheep that are infected will abort.t-" Infected neonates succumb to severe neerotizing hepatitis.v' The disease in human beings is
usually a transient illness, although complications of hemorrhagic fever, retinal disease, or encephalitis occur in a small percentage of infections. IS RVF virus-induced encephalitis has been reported in natural infections in human beings and in experimentally infected gerbils and certain strains of rats, but the pathologic characterization of the central nervous system (eNS) lesions has not been described in RVF virus-infected ruminants. Weiss reported viral encephalitis in two lambs born to ewes vaccinated with the Smithburn neurotropic strain ofRVF vaccine while pregnant." Those lambs died within 6 days of birth, but a description of the lesions and clinical data were not provided. The present report describes the lesions and virologic events leading to necrotizing encephalomyelitis in a calf infected experimentally with virulent RVF virus and contrasts findings with those in calves that succumbed to necrotizing hepatitis. In addition, immunohistochemical examination was used to localize RVF viral antigens in tissues from infected calves. This report is the first study of RVF virusinduced encephalomyelitis in a calf and immunohistochemicallocalization ofRVF viral antigens in tissues from infected ruminants.
495 Downloaded from vet.sagepub.com by guest on March 5, 2015
496
Rippy et al.
Materials and Methods
Vet PathoI29:6, 1992
Immunohistochemistry
Experimental design Three crossbred Hereford calves (Nos. I, 2, 7 days old; No.3, 21 days old) were inoculated subcutaneously with 5 x 10' plaque-forming units of virulent ZH-501 strain of Rift Valley fever (RVF) virus and served as unvaccinated, infected, control animals during safety and efficacy testing of a mutagen-attenuated RVF vaccine. The animals were commingled with their dams and housed under BSL-3 + containment. The calves were examined daily, and rectal temperatures were recorded. Whole blood for virologic, immunologic, and clinicopathologic assays was obtained by jugular venipuncture immediately prior to virus inoculation and daily through post-inoculation day (PID) 5 and on PID 7 and 9. Complete necropsies were done immediately after death and tissues were obtained for viral quantification and light microscopic examination. Only the cervical spinal cord was examined histologically. Tissue preparation Tissue samples were taken for viral quantification and light microscopic examination. Tissues were homogenized (10% w/v) using sterile sand, mortar, and pestle in Hanks' balanced salt solution (HBSS) with 0.5% human serum albumin and antibiotics (100 U penicillin and 100 J.Lg streptomycin sulfate/ ml), clarified by centrifugation, and stored at 70 C. For microscopic examination, tissue samples were fixed by immersion in 10% neutral buffered formalin, and paraffin sections were prepared and stained with Mayer's hematoxylin and eosin. Specimens representative of the different regions of the brain were taken from each calf according to a standard neurovirulence protocol." Sections of liver and brain were stained with Brown and Brenn Gram's stain Giemsa and Gomori's methamine silver stain for possibl~ identific~tion of bacteria or fungi. Additional sections were processed for immunohistochemical analysis. Virologic, immunologic, and clinicopathologic assays Serum was separated from peripheral blood by centrifugation and stored at -70 C. Homogenates (10% w/v) of selected tissues were prepared for virus quantification. RVF virus was quantified in serial lO-fold dilutions of each serum specimen or tissue homogenate. Viral plaques were counted under agarose in 16-mm Vero cell monolayers, and viral titers were calculated as previously described. 16 Serum plaque reduction neutralization tests were done using serial fourfold dilutions of sera in HBSS.14 The plaque-reduction neutralization titer was expressed as the highest dilution of serum that caused an 80% reduction in RVF viral plaques. Sera were tested for RVF virus-specific IgM and IgG antibodies by enzyme-linked immunosorbent assay as previously described.8.12 Total and differential white blood cell, hematocrit, and platelet counts were performed manually on ethylenediaminetetraacetic acid anticoagulated blood. Serum aspartate aminotransferase, gamma glutamyltransferase, and lactic dehydrogenase were measured with a Multistat III Plus (Instrumentation Laboratories, Lexington, MA) centrifugal chemical analyzer.
RVF viral antigens were demonstrated in paraffin-embedded fixed tissue specimens by immunoperoxidase or immunoalkaline phosphatase using a modified avidin biotin peroxidase complex method as previously described." Briefly, 5-J.Lm sections of tissue were mounted on 3-aminopropyltriethoxysiane-coated slides, deparaffinized, and hydrated. The sections were immersed in a 0.05% solution of protease VIII (subtilysin, Sigma Chemical Co., St. Louis, MO), pH 7.8, at 37 C for 3 minutes and washed in phosphate-buffered saline (PBS), pH 7.4. Polyclonal mouse antiserum to RVF virus was used as the primary antibody at a dilution of I : 1,000. The antiserum was prepared against the T-I and ZH-501 strains of RVF virus. This antibody does not react with normal bovine tissues. After incubation with the primary antibody at room temperature for I hour, slides were rinsed in PBS and incubated at room temperature with biotinylated horse anti-mouse IgG, then by the avidin biotin peroxidase complex method (Vectastain, Vector Labs, Burlingame, CA). Diaminobenzadine was the substrate for peroxidase and 6-bromo-2-hydroxy-3-naphthoic acid was the substrate for alkaline phosphatase. Sections were counterstained with Mayer's hematoxylin. Polyclonal mouse antiserum to yellow fever virus and normal mouse serum were substituted for the primary antibody and served as assay controls.
Results Clinical observations
By post-inoculation day (PID) 2, all three calves were febrile (Fig. 1), noticeably lethargic, and did not suckle actively. All calves had marked conjunctival and scleral mucous membrane congestion, and calf No. 2 showed signs of approximately 10-15% dehydration, i.e., slightly sunken eyes and a loss in skin elasticity. On PID 3, the youngest calves (Nos. 1,2) were moribund and were euthanatized. The eldest calf(No. 3) appeared to recover and became more active on PID 4 through 7. But on PID 8, this calf developed a subnormal rectal temperature and congested conjunctiva and sclera and was lethargic. On PID 9, the calf had a rectal temperature of 36.4 C, refused to suckle, and was reluctant to move. Although the calf could stand unassisted, it was unsteady and continually moved its head from side to side in an unnatural manner. When manipulated, the animal would fall down but could rise again, though with difficulty. The animal was euthanatized on PID 9. Laboratory findings
All three calves were viremic on PID 1 through 3 and viremia was detected in calf No. 3 through PID 5 (Fig. 1). Viremia in calf No. 3 peaked at 5.110g 1o plaqueforming units (PFU)/ml on PID 2 and became undetectable by plaque assay on PID 7. Viral titers oftissue homogenates from these calves are presented in Table
Downloaded from vet.sagepub.com by guest on March 5, 2015
Rift Valley Fever in Calves
Vet Pathol 29:6, 1992
497
2000 1800
42 41
AST
1400
40 1000 39
0
600
38
0
20~ ~iiiii:r:-r----,--~=~=:::;:::==~==::;=~
37 36
500
GGT
35 0
2
3
4
5
6
7
8
400
9 -l
:J
7
E
6
::::>
5
LL
n,
200 100
~
CJ 0
t======1~""""-"""'-~--"Ir----..----...
o-t---,--...,--.---,....-........,-........,----,..-----,-----,
4 0
-l
300
3
10000
2
8000
LDH
-l
:J 0
0
2
3
4
5
6
6000 4000
DAYS POST - INOCULATION
2000
Fig. 1. Rectal temperatures and serum viremia titers of calf Nos. 1-3 inoculated with RVF virus on day O... = calf No.3;. = calf No. 2;. = calf No. I.
O+--r--r----r---,-.,---,--,.--.------=T
1. Only a single plaque, observed on two separate repeatable assays, was detected in the spleen homogenate from calfNo. 3, and virus was not detected in the liver. However, a viral titer of 3.9 log., PFU/g was found in the brain. No RVF virus-specific IgM, IgG, or neutralizing antibodies were found in the sera of those calves that died on PID 3; however, specific IgM and IgG were first detected on PID 4 and 5, respectively, in calf No.3. A plaque-reduction neutralization titer of 1: 160 was detected in calf No. 3 on PID 7 and rose to 1: 640 by PID 9. Serum hepatic enzymes (aspartate aminotransferase [AST], gamma glutamyltransferase [GGT], and lactic dehydrogenase [LDH]) were markedly elevated in the calves (Nos. 1, 2) that died on PID 3 (Fig. 2). Serum AST values were noticeably increased in calf No. 3 on PID 2 but had returned to near baseline levels by PID 4. Only slight elevations in GGT and LDH levels were seen in calf NO.3. There were dramatic changes in hematologic values during the first 3 days after infection, and trends were similar in all three calves. Hematocrit values were elevated from 26-38% to 4652%, platelet counts were reduced, and severe leukopenia was seen. The mean white blood cell (WBC) count for all three calves decreased from 8,900 cells/ J,ll on PID 0 to a mean nadir of 2,500 cells/J,ll on PID 2. The WBC count ofcalf No. 3 decreased from 10,400
Fig. 2. Serum AST, GGT, and LDH values ofRVFvirusinoculated calf Nos. 1-3 ... = calf No.3; • = calf No.2; • = calf No. I.
o
2
3
4
5
6
7
8
9
DAYS POST - INOCULATION
cells/J,ll on PID 0 to 3,600 cells/J,ll on PID 2. A persistent leukopenia was seen throughout the course of illness in this animal. In calf No.3, neutrophil counts decreased from 5,600 cells/ul to 2,700 cells/ul, and lymphocyte counts decreased from 3,100 cells/J,ll to 600 cells/J,ll on PID 2.
Table 1. Rift Valley fever viral titers* in cerebrospinal fluid (CSF) serum, and organ homogenates of virus-infected calves. Organ
Calf No.
It
2t
3:1:
5.1 CSF 3.0
