PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Almeida S.R., Diel D.G., Rissi D.R., Weiblen R. & Flores E.F. 2008. [Clinic and pathological characterization of acute mammillitis in lactating ewes inoculated with bovine herpesvirus 2.] Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes inoculadas experimentalmente com o herpesvírus bovino 2. Pesquisa Veterinária Brasileira 28(1):87-94. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.

• Bovine herpesvirus-2 BoHV-2 mammillitis sheep experimental infection

Abstract in Portuguese:

ABSTRACT.- Almeida S.R., Diel D.G., Rissi D.R., Weiblen R. & Flores E.F. 2008. [Clinic and pathological characterization of acute mammillitis in lactating ewes inoculated with bovine herpesvirus 2.] Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes inoculadas experimentalmente com o herpesvírus bovino 2. Pesquisa Veterinária Brasileira 28(1):87-94. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.

Abstract in English:

ABSTRACT.- Silva M.S., Brum M.C.S., Weiblen R. & Flores E.F. 2007. [Identification and differentiation of herpesvirus types 1 and 5 isolated from clinical samples in central-southern Brazil, Argentina and Uruguay (1987-2006).] Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006). Pesquisa Veterinária Brasileira 27(10):403-408. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesviruses types 1 and 5 (BoHV-1; BoHV-5) are genetically and antigenically closely related such they can not be distinguished by routine diagnostic tests. As BoHV-1 has been historically associated with respiratory and genital disease, herpesviruses isolated from these clinical syndromes have been tentatively – and sometimes definitively - diagnosed as BoHV-1. Likewise, cases of herpetic neurological infection in cattle have been generally attributed to BoHV-5. This study reports the identification of 40 herpesvirus isolates from different clinical specimens and syndromes in central-southern Brazil, Argentina and Uruguay (1987-2006) by the use of a PCR able to differentiate between BoHV-1 and BoHV-5. BoHV-1 isolates (n=16) were identified in cases of respiratory disease (n=3), vulvovaginitis and/or balanoposthitis (n=3), in semen of healthy bulls (n=5) and in cases of neurological disease (n=5). Viruses identified as BoHV-5 (n=24) were isolated predominantly from cases of neurological disease (n=21), but also from semen of healthy bulls (n=2) and from a spleen of a calf with systemic disease (n=1). These results show that both BoHV-1 and BoHV-5 are not strictly associated with their respective diseases; yet are frequently involved in clinical conditions otherwise attributed to the other virus. These findings also reinforce the need of correctly identifying the herpesvirus isolates as to better understand their pathogenesis and epidemiology.

• Bovine herpesvirus BoHV-1 BoHV-5 differential diagnosis epidemiology

Abstract in Portuguese:

ABSTRACT.- Silva M.S., Brum M.C.S., Weiblen R. & Flores E.F. 2007. [Identification and differentiation of herpesvirus types 1 and 5 isolated from clinical samples in central-southern Brazil, Argentina and Uruguay (1987-2006).] Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006). Pesquisa Veterinária Brasileira 27(10):403-408. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesviruses types 1 and 5 (BoHV-1; BoHV-5) are genetically and antigenically closely related such they can not be distinguished by routine diagnostic tests. As BoHV-1 has been historically associated with respiratory and genital disease, herpesviruses isolated from these clinical syndromes have been tentatively – and sometimes definitively - diagnosed as BoHV-1. Likewise, cases of herpetic neurological infection in cattle have been generally attributed to BoHV-5. This study reports the identification of 40 herpesvirus isolates from different clinical specimens and syndromes in central-southern Brazil, Argentina and Uruguay (1987-2006) by the use of a PCR able to differentiate between BoHV-1 and BoHV-5. BoHV-1 isolates (n=16) were identified in cases of respiratory disease (n=3), vulvovaginitis and/or balanoposthitis (n=3), in semen of healthy bulls (n=5) and in cases of neurological disease (n=5). Viruses identified as BoHV-5 (n=24) were isolated predominantly from cases of neurological disease (n=21), but also from semen of healthy bulls (n=2) and from a spleen of a calf with systemic disease (n=1). These results show that both BoHV-1 and BoHV-5 are not strictly associated with their respective diseases; yet are frequently involved in clinical conditions otherwise attributed to the other virus. These findings also reinforce the need of correctly identifying the herpesvirus isolates as to better understand their pathogenesis and epidemiology.

Abstract in English:

Diel D.G., Fonseca E.T., Souza S.F., Mazzanti A., Bauermann F., Weiblen R. & Flores E.F. 2005. [Bovine herpesvirus 5 may use the olfactory and trigeminal pathways to invade the central nervous system of rabbits, depending upon the route of inoculation.] O Herpesvírus bovino tipo 5 (BoHV-5) pode utilizar as rotas olfatória ou trigeminal para invadir o sistema nervoso central de coelhos, dependendo da via de inoculação. Pesquisa Veterinária Brasileira 25(3):164-170. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesvirus type 5 (BoHV-5) is a major etiological agent of meningoencephalitis in cattle. Following replication in the nasal mucosa, viral invasion of the brain is thought to occur mainly by the olfactory pathway. To address the role of this pathway in the pathogenesis of neurological infection in a laboratory model, 30 days old rabbits had the main olfactory bulbs (MOBs) surgically removed and were subsequently inoculated intranasally (IN) or conjunctivally (IC) with a highly neurovirulent BoHV-5 strain (SV-507). Following IN inoculation, 10 out of 10 (100 %) control rabbits developed neurological disease. The clinical onset ranged from day 5 to 10 post-inoculation (pi, average 7.5 days); nine being euthanized in extremis and one recovering after a mild clinical course. In contrast, only one rabbit (9.1 %) of the group lacking the MOBs (n=11) developed neurological disease (onset at day 17 pi). Dexamethasone administration to the survivors (n=10) at day 50pi was followed by virus shedding in nasal and/or ocular secretions by 8 animals, demonstrating that the virus was able to reach the trigeminal ganglia (TG) during acute infection. These results demonstrate that the olfactory route provides the main, yet not the sole access to the brain of rabbits following IN inoculation. To address the role of a second pathway, groups of control (n=12) or MOB-lacking rabbits (n=12) were inoculated into the conjunctival sac (IC), following which the virus would be expected to use the ophtalmic branch of the trigeminal nerve to reach the brain. Ten control rabbits (83.3 %) developed neurological disease upon IC inoculation (onset 15.3 days [11 to 20]). Previous ablation of the MOBs did not affect the frequency and course of neurological disease: ten out of 12 rabbits (83.3 %) lacking the MOBs developed neurological disease (onset 9 to 15 dpi, average: 12.7 days) upon IC inoculation. These results demonstrate that both IN and IC routes may operate in the transport of BoHV-5 to the brain of experimentally infected rabbits, depending on the route of inoculation. IN inoculation results in a fast and efficient transport by the olfactory pathway, the trigeminal route providing an alternative, much slower and less efficient transport; IC inoculation results in efficient viral transport by the trigeminal route, yet with a delayed kinetics comparing to the transport provided by the olfactory pathway.

• Bovine herpesvirus type 5 BoHV-5 neurological infection rabbits.

Abstract in Portuguese:

Diel D.G., Fonseca E.T., Souza S.F., Mazzanti A., Bauermann F., Weiblen R. & Flores E.F. 2005. [Bovine herpesvirus 5 may use the olfactory and trigeminal pathways to invade the central nervous system of rabbits, depending upon the route of inoculation.] O Herpesvírus bovino tipo 5 (BoHV-5) pode utilizar as rotas olfatória ou trigeminal para invadir o sistema nervoso central de coelhos, dependendo da via de inoculação. Pesquisa Veterinária Brasileira 25(3):164-170. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine herpesvirus type 5 (BoHV-5) is a major etiological agent of meningoencephalitis in cattle. Following replication in the nasal mucosa, viral invasion of the brain is thought to occur mainly by the olfactory pathway. To address the role of this pathway in the pathogenesis of neurological infection in a laboratory model, 30 days old rabbits had the main olfactory bulbs (MOBs) surgically removed and were subsequently inoculated intranasally (IN) or conjunctivally (IC) with a highly neurovirulent BoHV-5 strain (SV-507). Following IN inoculation, 10 out of 10 (100 %) control rabbits developed neurological disease. The clinical onset ranged from day 5 to 10 post-inoculation (pi, average 7.5 days); nine being euthanized in extremis and one recovering after a mild clinical course. In contrast, only one rabbit (9.1 %) of the group lacking the MOBs (n=11) developed neurological disease (onset at day 17 pi). Dexamethasone administration to the survivors (n=10) at day 50pi was followed by virus shedding in nasal and/or ocular secretions by 8 animals, demonstrating that the virus was able to reach the trigeminal ganglia (TG) during acute infection. These results demonstrate that the olfactory route provides the main, yet not the sole access to the brain of rabbits following IN inoculation. To address the role of a second pathway, groups of control (n=12) or MOB-lacking rabbits (n=12) were inoculated into the conjunctival sac (IC), following which the virus would be expected to use the ophtalmic branch of the trigeminal nerve to reach the brain. Ten control rabbits (83.3 %) developed neurological disease upon IC inoculation (onset 15.3 days [11 to 20]). Previous ablation of the MOBs did not affect the frequency and course of neurological disease: ten out of 12 rabbits (83.3 %) lacking the MOBs developed neurological disease (onset 9 to 15 dpi, average: 12.7 days) upon IC inoculation. These results demonstrate that both IN and IC routes may operate in the transport of BoHV-5 to the brain of experimentally infected rabbits, depending on the route of inoculation. IN inoculation results in a fast and efficient transport by the olfactory pathway, the trigeminal route providing an alternative, much slower and less efficient transport; IC inoculation results in efficient viral transport by the trigeminal route, yet with a delayed kinetics comparing to the transport provided by the olfactory pathway.

Abstract in English:

Flores E.F., Weiblen R., Vogel F.S.F., Roehe P.M., Alfieri A.A. & Pituco E.M. 2005. [Bovine viral diarrhea virus (BVDV) infection in Brazil: history, current situation and perspectives.] A infecção pelo vírus da Diarréia Viral Bovina (BVDV) no Brasil - histórico, situação atual e perspectivas. Pesquisa Veterinária Brasileira 25(3):125-134. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV infection and associated diseases have been reported in Brazil since the late 1960ties. Several serological, virological, clinical and pathological reports demonstrate the widespread distribution of BVDV infection among Brazilian cattle. In addition to variable levels of positive serology in beef and dairy cattle, BVDV antibodies have been occasionally detected in swine, wild boars, goats, cervids and water buffaloes. BVDV infection has been diagnosed in aborted fetuses, buffy coats of persistently infected (PI) animals, clinical specimens from animals suffering from different clinical syndromes, semen of bulls of artificial insemination (AI) centers, in healthy fetuses and in commercial fetal bovine serum and/or cultured cells. About 50 isolates have been genetically and/or antigenically characterized up to date, whilst roughly an equivalent number of isolates awaits characterization. Most of the characterized isolates belong to BVDV-1 genotype, non-cytopathic (NCP) biotype, yet some BVDV-2 (and some CP BVDV) have been identified as well. Brazilian BVDV isolates display a high antigenic variability and are markedly different from North American vaccine strains. A few inactivated, polyvalent vaccines are currently licensed in the country, yet vaccination is still incipient in many regions: only about 2.5 million doses were marketed in 2003. The low serological cross-reactivity between vaccine strains and field isolates has recently stimulated national industries to develop vaccines containing Brazilian BVDV-1 and BVDV-2 strains. The overall knowledge about BVDV infection in Brazil has grown considerably in the last years, due to an increasing number of laboratories performing diagnosis and research. Studies on the pathogenesis, serological and molecular epidemiology and production of reagents for diagnosis have contributed decisively for the recent growing knowledge on BVDV infections in the country.

• Bovine viral diarrhea virus BVDV epidemiology diagnostic.

Abstract in Portuguese:

Flores E.F., Weiblen R., Vogel F.S.F., Roehe P.M., Alfieri A.A. & Pituco E.M. 2005. [Bovine viral diarrhea virus (BVDV) infection in Brazil: history, current situation and perspectives.] A infecção pelo vírus da Diarréia Viral Bovina (BVDV) no Brasil - histórico, situação atual e perspectivas. Pesquisa Veterinária Brasileira 25(3):125-134. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV infection and associated diseases have been reported in Brazil since the late 1960ties. Several serological, virological, clinical and pathological reports demonstrate the widespread distribution of BVDV infection among Brazilian cattle. In addition to variable levels of positive serology in beef and dairy cattle, BVDV antibodies have been occasionally detected in swine, wild boars, goats, cervids and water buffaloes. BVDV infection has been diagnosed in aborted fetuses, buffy coats of persistently infected (PI) animals, clinical specimens from animals suffering from different clinical syndromes, semen of bulls of artificial insemination (AI) centers, in healthy fetuses and in commercial fetal bovine serum and/or cultured cells. About 50 isolates have been genetically and/or antigenically characterized up to date, whilst roughly an equivalent number of isolates awaits characterization. Most of the characterized isolates belong to BVDV-1 genotype, non-cytopathic (NCP) biotype, yet some BVDV-2 (and some CP BVDV) have been identified as well. Brazilian BVDV isolates display a high antigenic variability and are markedly different from North American vaccine strains. A few inactivated, polyvalent vaccines are currently licensed in the country, yet vaccination is still incipient in many regions: only about 2.5 million doses were marketed in 2003. The low serological cross-reactivity between vaccine strains and field isolates has recently stimulated national industries to develop vaccines containing Brazilian BVDV-1 and BVDV-2 strains. The overall knowledge about BVDV infection in Brazil has grown considerably in the last years, due to an increasing number of laboratories performing diagnosis and research. Studies on the pathogenesis, serological and molecular epidemiology and production of reagents for diagnosis have contributed decisively for the recent growing knowledge on BVDV infections in the country.

Abstract in English:

Groff F.H.S., Merlo M.A., Stoll P.A., Stepan A.L., Weiblen R. & Flores E.F. 2005. [Epidemiology and control of pseudorabies outbreaks in the state of Rio Grande do Sul, Brazil, 2003.] Epidemiologia e controle dos focos da doença de Aujeszky no Rio Grande do Sul, em 2003. Pesquisa Veterinária Brasileira 25(1):25-30. Depto Medicina Veterinária Preventiva, Universi-dade Federal de Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br Aujeszky’s disease (AD) or pseudorabies is an important viral disease of swine and has significant economic impact on the pig industry worldwide. The infection produces direct and indirect economic losses, mainly due to restrictions to international trade of swine products. Since the beginning of the 20th century, AD had been notified in several Brazilian regions, yet the state of Rio Grande do Sul (RS) remained as a “provisionally free” area under the International Organization of Epizooties (OIE) guidelines. In 2003, two outbreaks were notified in swine herds located in northern RS, boundary with Santa Catarina, a state where the infection is endemic. Control/eradication measures consisted in tracing back all swine movements, quarantine and eradication of the affected herds by sanitary slaughter under official inspection. In outbreak # 1 (Pinheirinho do Vale, January/2003) six herds were affected, one of which presented animals with clinical signs. Starting from this outbreak, 146 herds and 42.399 pigs were traced back, six herds (7.822 animals) being depopulated – the herd where the index outbreak plus five other herds with positive serology. The outbreak # 2 (Aratiba, September/2003) resulted in a wider spread of the infection, involving another three counties and 77 herds (9 with clinical signs, 68 with positive serology). From the index case, 109.316 pigs in 630 herds were traced back; 28.443 animals from positive herds were slaughtered. Total numbers reached 151.715 traced back animals in 776 herds; 71 herds were found serologically positive. The adopted measures were efficient to eliminate the outbreaks and to stop the disease spread to neighboring areas, reestablishing the sanitary status previous to the outbreaks.

• Aujeszky’s disease pseudorabies ADV PRV epidemiology.

Abstract in Portuguese:

Groff F.H.S., Merlo M.A., Stoll P.A., Stepan A.L., Weiblen R. & Flores E.F. 2005. [Epidemiology and control of pseudorabies outbreaks in the state of Rio Grande do Sul, Brazil, 2003.] Epidemiologia e controle dos focos da doença de Aujeszky no Rio Grande do Sul, em 2003. Pesquisa Veterinária Brasileira 25(1):25-30. Depto Medicina Veterinária Preventiva, Universi-dade Federal de Santa Maria (UFSM), Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br Aujeszky’s disease (AD) or pseudorabies is an important viral disease of swine and has significant economic impact on the pig industry worldwide. The infection produces direct and indirect economic losses, mainly due to restrictions to international trade of swine products. Since the beginning of the 20th century, AD had been notified in several Brazilian regions, yet the state of Rio Grande do Sul (RS) remained as a “provisionally free” area under the International Organization of Epizooties (OIE) guidelines. In 2003, two outbreaks were notified in swine herds located in northern RS, boundary with Santa Catarina, a state where the infection is endemic. Control/eradication measures consisted in tracing back all swine movements, quarantine and eradication of the affected herds by sanitary slaughter under official inspection. In outbreak # 1 (Pinheirinho do Vale, January/2003) six herds were affected, one of which presented animals with clinical signs. Starting from this outbreak, 146 herds and 42.399 pigs were traced back, six herds (7.822 animals) being depopulated – the herd where the index outbreak plus five other herds with positive serology. The outbreak # 2 (Aratiba, September/2003) resulted in a wider spread of the infection, involving another three counties and 77 herds (9 with clinical signs, 68 with positive serology). From the index case, 109.316 pigs in 630 herds were traced back; 28.443 animals from positive herds were slaughtered. Total numbers reached 151.715 traced back animals in 776 herds; 71 herds were found serologically positive. The adopted measures were efficient to eliminate the outbreaks and to stop the disease spread to neighboring areas, reestablishing the sanitary status previous to the outbreaks.

Abstract in English:

Lima M., Flores E.F., Weiblen R., Flores F.S.F. & Arenhart S. 2004. [Characterization of bovine viral diarrhea virus (BVDV) types 1 and 2 isolates for use in vaccines.] Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas. Pesquisa Veterinária Brasileira 24(1):35-42. Depto Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to ³1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates. At day 240 post-vaccination, the animals received a booster administration (IBSP-2: 107.3 TCID50 and SV-253: 106.8 TCID50). Revaccination resulted in a strong anamnestic response in most animals, with increasing antibody titers mainly to BVDV-2. These are promising results towards the future use of these strains in modified-live vaccines for the control of BVDV infection in Brazil.

• Bovine viral diarrhea virus (BVDV) modified-live vaccine attenuation.

Abstract in Portuguese:

Lima M., Flores E.F., Weiblen R., Flores F.S.F. & Arenhart S. 2004. [Characterization of bovine viral diarrhea virus (BVDV) types 1 and 2 isolates for use in vaccines.] Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas. Pesquisa Veterinária Brasileira 24(1):35-42. Depto Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to ³1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates. At day 240 post-vaccination, the animals received a booster administration (IBSP-2: 107.3 TCID50 and SV-253: 106.8 TCID50). Revaccination resulted in a strong anamnestic response in most animals, with increasing antibody titers mainly to BVDV-2. These are promising results towards the future use of these strains in modified-live vaccines for the control of BVDV infection in Brazil.

Abstract in English:

Spilki F.R, Esteves P.A., Lima M., Franco A.C., Chiminazzo C., Flores E.F., Weiblen R., Driemeier D. & Roehe P.M. 2004. Comparative pathogenicity of bovine herpesviruses type 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a). Pesquisa Veterinária Brasileira 24(1):43-49. Centro de Pesquisas Desidério Finamor, Fepagro Saúde Animal, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@ufrgs.br The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

• Infectious bovine rhinotracheitis IBR BHV-1.1 BHV-1.2a subtypes pathogenicity.

Abstract in Portuguese:

Spilki F.R, Esteves P.A., Lima M., Franco A.C., Chiminazzo C., Flores E.F., Weiblen R., Driemeier D. & Roehe P.M. 2004. Comparative pathogenicity of bovine herpesviruses type 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a). Pesquisa Veterinária Brasileira 24(1):43-49. Centro de Pesquisas Desidério Finamor, Fepagro Saúde Animal, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@ufrgs.br The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

Abstract in English:

ABSTRACT.- Franco A.C., Spilki F.R., Esteves P.A., Lima M., Weiblen R., Flores E.F., Rijsewijk F.A.M. & Roehe P.M. 2002. A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge. Pesquisa Veterinária Brasileira 22(4):135-140. [Um mutante gE-negativo de herpesvírus bovino tipo 1.2a é atenuado para bovinos e induz proteção frente ao desafio com vírus de campo.] Centro de Pesquisas Veterinárias Desidério Finamor, Fepagro-Saúde Animal, Cx. Postal 2076, Porto Alegre, RS 90001-970, Brazil. E-mail: proehe@ufrgs.br The authors previously reported the construction of a glycoprotein E-deleted (gE·) mutante of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE·, was designed as a vacinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE·. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas nonvaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE· could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental vírus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE· was shown to be suitable for use as a BHV-1 differential vaccine vírus.

• BHV-1 differential vaccine gE deletion IBR vacina diferencial gE.

Abstract in Portuguese:

RESUMO.- Franco A.C., Spilki F.R., Esteves P.A., Lima M., Weiblen R., Flores E.F., Rijsewijk F.A.M. & Roehe P.M. 2002. A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge. Pesquisa Veterinária Brasileira 22(4):135-140. [Um mutante gE-negativo de herpesvírus bovino tipo 1.2a é atenuado para bovinos e induz proteção frente ao desafio com vírus de campo.] Centro de Pesquisas Veterinárias Desidério Finamor, Fepagro-Saúde Animal, Cx. Postal 2076, Porto Alegre, RS 90001-970, Brazil. E-mail: proehe@ufrgs.br Em estudo prévio os autores reportaram a construção de um mutante do Vírus da Rinotraqueíte Infecciosa Bovina (IBR) ou Herpesvírus Bovino tipo 1.2a (BHV-1.2a), do qual foi deletado o gene que codifica a glicoproteina E. Esse mutante (265gE-) foi construído a partir de uma amostra autóctone do vírus, tendo como objetivo seu uso como amostra vacinai em vacinas diferenciais, capazes de permitir a diferenciação entre animais vacinados e infectados com vírus de campo. Para determinar a atenuação e eficácia do 265gE· como imunógeno, bezerros foram inoculados por via intranasal com 106,9 DICC50 do mesmo. O vírus foi detectado em secreções dos animais inoculados em títulos mais baixos e por um período mais curto do que a amostra virulenta parental, inoculada em animais controle. Vinte e um dias após, os animais inoculados com o vírus mutante foram desafiados com a amostra parental, apresentando somente sinais leves de infecção. Os animais controle apresentaram intensa rinotraqueíte e excretaram vírus em títulos mais elevados e por mais tempo do que os vacinados. Seis meses após a vacinação, foi examinada a capacidade de reativação da infecção nos bezerros, através da administração de corticosteróides. O mutante 265gE- não foi reativado dos animais vàcinados. Os sinais clínicos consequentes à reativação do vírus parental foram muito atenuados nos animais vacinados, em comparação com os não vacinados. Além disso, a excreção de vírus de campo foi consideravelmente reduzida nestes últimos. Em vista de sua atenuação, imunogenicidade e efeito protetivo frente ao desafio com uma amostra virulenta de BHV-1 e subseqüente reativação, o mutante 265gE- demonstrou apresentar grande potencial para ser utilizado como vírus vacinai em vacinas diferenciais contra o BHV-1.

Abstract in English:

ABSTRACT.- Scherer C.F.C., Flores E.F., Weiblen R., Kreutz L.C., Dürr J.W., Brum L.P., Quadros V.L. & Lima M. 2000. [A rapid virus-neutralization test for detection of antibodies against bovine viral diarrhea virus (BVDV) in milk.] Técnica rápida de neutralização viral para pesquisa de anticorpos contra o vírus da Diarréia Viral Bovina (BVDV) no leite. Pesquisa Veterinária Brasileira 22(2):45-50. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. The identification of bovine viral diarrhea virus (BVDV) positive herds through detection of antibodies in milk may viabilize large scale control/eradication programs. With this objective, the virus neutralization test (VN) was adapted to detect BVDV antibodies in milk. The adaptation consisted of a reduction in the time of incubation followed by detection of viral antigens in the indicator cells by immunofluorescence (IFA) and allowed readings at 24 hours. The rapid virus neutralization test (RVN) was initially tested in 1,335 serum samples, showing a 93. 7% sensitivity and 91.1 % agreement with the traditional VN. The RVN was also used to test 423 bovine sera that were toxic for cell culture in the traditional VN test, detecting 316 (74.7%) positive samples. Testing of matched serum and milk samples from BVDV seropositive cows showed that the VNR can detect antibodies in the milk of cows with serum neutralizing titers as low as 10. Anti-BVDV neutralizing activity was detected in milk of 97.4% (191/196) of cows with serum titers 3320; in 92.9% (79/85) of cows with titers of 160; in 88% (59/67) of cows with serum titers of 80. The frequency of BVDV antibodies in milk was 76.9% (40/52) for cows with serum titers of 40; 61.3% (19/31) for cows with titers of 20 and 33.3% (10/30) for cows with serum titers of 20. These results demonstrate that the RVN test is adequate for detecting BVDV antibodies in milk, mainly in cows having moderate to high serum titers, and therefore may be used for testing bulk milk samples to identify herds with viral activity. The use of this test may viabilize large scale programs for control/eradication of BVDV infection. It allows to assay a large number of samples and identify positive herds through testing milk routinely submitted for somatic cell counts (SCC), reducing costs with individual sample collection, shipping and testing.

• Bovine viral diarrhea virus BVDV diagnosis antibodies milk Vírus da Diarréia Viral Bovina diagnóstico leite anticorpos.

Abstract in Portuguese:

RESUMO.- Scherer C.F.C., Flores E.F., Weiblen R., Kreutz L.C., Dürr J.W., Brum L.P., Quadros V.L. & Lima M. 2000. [A rapid virus-neutralization test for detection of antibodies against bovine viral diarrhea virus (BVDV) in milk.] Técnica rápida de neutralização viral para pesquisa de anticorpos contra o vírus da Diarréia Viral Bovina (BVDV) no leite. Pesquisa Veterinária Brasileira 22(2):45-50. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. A identificação de rebanhos positivos para o vírus da Diarréia Viral Bovina (BVDV) através de detecção de anticorpos no leite pode viabilizar programas de controle em larga escala. Com esse objetivo, a técnica de soroneutralização (SN) foi adaptada para a pesquisa de anticorpos em amostras de leite. A adaptação consistiu na redução do tempo de incubação do teste, seguida da detecção de antígenos virais por imunofluorescência. A redução do tempo de incubação minimizou os efeitos tóxicos do leite sobre as células de cultivo, além de permitir a obtenção dos resultados em 24 horas. A técnica rápida (SNR) foi inicialmente testada em 1.335 amostras de soro bovino, apresentando sensibilidade de 93,7% e concordância de 91, 1% em relação à SN tradicional. A SNR foi também utilizada para testar 423 amostras de soro bovino que apresentaram toxicidade para as células na SN tradicional, detectando 316 (74,7%) amostras positivas. O teste de amostras de soro e leite de 520 vacas em lactação demonstrou que a SNR pode detectar anticorpos no leite de vacas com títulos séricos a partir de 10. Atividade neutralizante anti-BVDV no leite foi detectada em 97,4% (191/196) de vacas com títulos séricos 3 320; em 92,9% (79/85) de vacas com títulos de 160; em 88% (59/67) de vacas títulos de 80. A freqüência de animais positivos na SNR foi de 76,9% (40/52) para animais com títulos séricos de 40; 61,3% (19/31) com títulos de 20 e de 33,3% (10/30) para vacas com títulos de 10. Esses resultados demonstram que a técnica de SNR é adequada para a pesquisa de anticorpos anti-BVDV no leite, principalmente em animais com títulos moderados e altos de anticorpos. Essa técnica pode ser utilizada para testar amostras coletivas de leite e identificar rebanhos com atividade viral. A utilização dessa técnica pode viabilizar programas regionais de combate à infecção, pois permite testar um grande número de amostras e identificar rebanhos positivos através do leite enviado rotineiramente para contagem de células somáticas (CCS), reduzindo significativamente os custos com a coleta individual, transporte e teste de amostras.

Abstract in English:

ABSTRACT.- Spilki F.R., Esteves P.A., Franco A.C., Lima M., Holz C.L., Batista H.B.C.R., Driemeier D., Flores E.F., Weiblen R. & Roehe P.M. 2002. [Neurovirulence and neuroinvasiveness of bovine herpesvirus type 1 and 5 in rabbits.] Neurovirulência e neuroinvasividade de Herpesvírus bovinos tipos 1 e 5 em coelhos. Pesquisa Veterinária Brasileira 22(2):58-63. Centro de Pesquisas Veterinárias Desidério Finamor, Estrada do Conde 6000, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@orion.ufrgs.br In order to determine the capacity of bovine herpesvirus type 1 and 5 (BHV-1 and BHV-5) to invade, multiply and spread along the central nervous system (CNS) (neuroinvasiveness), as well as their potential to induce neurological illness (neurovirulence), 30 to 35 days old rabbits were inoculated with the BHV-5 strain EVI 88 / 95 and Los Angeles and Cooper BHV-1 strains, by the intrathecal (IT) and intranasal (IN) routes. The BHV-5 strain induced severe neurological clinical signs in 100% (12/12) of the rabbits inoculated by both routes. Histopathological examination revealed multifocal non-suppurative meningoencephalitis, characterized by multifocal gliosis and perivascular cuffing. Virus was recovered from many parts of the brain. Both BHV-1 strains, when inoculated via 1T route, were not neurovirulent. The strain Los Angeles, after IN inoculation, induced signs of severe respiratory disease (7/7), as well as signs of neurological impairment, indistinguishable from those induced by BHV-5, in 57% (4/7) of the infected rabbits. However, the rabbits with nervous signs revealed at histopathology vasculitis and thrombosis in lungs and brain, the latter with foci of neuronal necrosis, but no lesions indicative of encephalitis, suggesting that neural damage was probably consequent to tissue anoxia. The BHV-1 strain Cooper, after IN inoculation, induced only mild signs of respiratory disease. These findings indicate that the BHV-5 strain was both neuroinvasive and neurovirulent, since it was capable of invading, spreading and multiplying in the rabbits brains by both routes of inoculation, yet causing neurological disease, apparently consequent to vírus induced neural damage. The BHV-1 Los Angeles strain was not neuroinvasive, whereas its neurovirulence was probably consequent to tissue anoxia, which histologically seemed not to be related to direct viral pathogenic effect. The BHV-1 strain Cooper was neither neurovirulent nor neuroinvasive for rabbits. It is possible that these observations bear relationship with the frequent association of BHV-5 with encephalitis in cattle, as opposed to BHV-1 encephalitis, which is a rare event in nature.

• Infectious bovine rhinotracheitis IBR BHV-1 bovine encephalitis herpesvirus BHV-5 Rinotraqueíte infecciosa bovina herpesvírus da encefalite bovina.

Abstract in Portuguese:

RESUMO.- Spilki F.R., Esteves P.A., Franco A.C., Lima M., Holz C.L., Batista H.B.C.R., Driemeier D., Flores E.F., Weiblen R. & Roehe P.M. 2002. [Neurovirulence and neuroinvasiveness of bovine herpesvirus type 1 and 5 in rabbits.] Neurovirulência e neuroinvasividade de Herpesvírus bovinos tipos 1 e 5 em coelhos. Pesquisa Veterinária Brasileira 22(2):58-63. Centro de Pesquisas Veterinárias Desidério Finamor, Estrada do Conde 6000, Cx. Postal 47, Eldorado do Sul, RS 92990-000, Brazil. E-mail: proehe@orion.ufrgs.br Com o objetivo de avaliar a capacidade dos herpesvírus bovinos tipos 1 e 5 (BHV-1 e BHV-5) de invadir e replicar no sistema nervoso central (SNC) (neuroinvasividade), bem como sua capacidade de induzir doença neurológica (neurovirulência), coelhos com 30 a 35 dias de idade foram inoculados com uma amostra do Herpesvírus da Encefalite Bovina (BHV-5; amostra EVI 88/95) ou com amostras de BHV-1 (Los Angeles ou Cooper), pelas vias intratecal (IT) e intranasal (IN). A inoculação da amostra de BHV-5, tanto pela via 1T como IN, induziu sinais clínicos neurológicos em 100% (12/12) dos coelhos inoculados. Os exames histopatológicos revelaram um quadro de meningoencefalite não-purulenta multifocal, caracterizada por gliose multifocal e infiltrados perivasculares. O vírus foi isolado de várias áreas do SNC desses animais. As amostras de BHV-1, quando inoculadas pela via IT, não foram neurovirulentas. A amostra Los Angeles de BHV-1, quando administrada pela via IN, induziu sinais respiratórios severos, além de sinais neurológicos em 57% (4/7) dos animais inoculados. Entretanto, o exame histopatológico destes quatro animais revelou vasculite e trombose no pulmão e cérebro, este último apresentando focos de necrose neuronal, porém sem lesões indicativas de encefalite. Isso sugere que os sinais neurológicos foram, provavelmente, consequentes a prejuízos no fluxo sangüíneo encefálico, e não a danos neuronais provocados pela inoculação desse vírus. A amostra Cooper de BHV-1, quando inoculada pela via IN, induziu apenas sinais leves de infecção respiratória. Estes resultados indicam que apenas a amostra de BHV-5 foi capaz de invadir e replicar no encéfalo dos coelhos quando inoculada tanto por via IN como IT, apresentando neuroinvasividade e neurovirulência. É possível que estas observações tenham relação com o fato de amostras de BHV-5 freqüentemente causarem encefalites, em contraposição a infecções pelo BHV-1, onde encefalites são raramente observadas.