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Incidence of leptospiral abortion in Brazilian dairy cattle
Preventive Veterinary Medicine 40 (1999) 271±275
Incidence of leptospiral abortion in Brazilian dairy cattle Helio Langoni*, Luiz Carlos de Souza, Aristeu Vieira da Silva, Maria CecõÂlia R. Luvizotto, Antonio Carlos Paes, Simone Baldini Lucheis Departamento de Higiene VeterinaÂria e SauÂde PuÂblica, Faculdade de Medicina VeterinaÂria e Zootecnia, UNESP, Campus de Botucatu, Distrito de RubiaÄo JuÂnior, s/n-Botucatu-Sao Paulo, CEP 18618.000, Brazil Accepted 11 February 1999
Abstract We examined dams' paired serum samples and foetal kidneys (histopathologically and attempting leptospiral cultures) from 120 Brazilian Holstein abortions from 10 herds near Sao Paulo, Brazil, in 1996. Leptospiras was isolated from 15 foetuses. The Leptospira interrogans serovar hardjo was obtained from four, pomona from three and wolffi from eight foetuses. Ten of these 15 foetuses showed a four-fold rise in titres between the day of abortion and the second samples taken 15 days later. Fifty-seven other foetuses had histologic evidence of leptospires; 27 of their dams (47%) had four-fold rises in titres. In total, 72 of 120 aborted foetuses had evidence of leptospiral infection. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Abortion; Leptospira interrogans; Cattle-microbiological disease
1. Introduction Bovine leptospirosis causes serious financial loss due to abortion, stillbirths, death, decrease in milk production and infertility, mainly in tropical and sub-tropical countries (Ellis, 1984; Ellis et al., 1982; Elder et al., 1985; Prescott et al., 1988). Different serovars have been found in South America, including those from the hebdomadis sub-group (Oliveira et al., 1980; Riedemann et al., 1986; ), pomona (Dragui et al., 1986; Riedemann et al., 1986) and wolffi (Dragui et al., 1986). However, in these reports isolation was * Corresponding author. Tel.: +55-14-82-135-19; fax: +55-14-82-123-43; e-mail: [email protected] 0167-5877/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 9 9 ) 0 0 0 2 0 - 3
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H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
performed from samples collected at a single time for each abortion. There are no reports of `rising titres' in the dams which confirms the causal role for the agent. We studied the presence of anti-leptospiral agglutinin in bovine paired samples (from the time of abortion and 15 days later), using several leptospiral serovars. We also report the association between serologic results in the dams and both leptospiral isolation from their aborted foetus kidneys and the presence of leptospires in the histopathological examination of foetal kidneys. 2. Material and method One hundred and twenty Holstein foetuses and 240 serum samples from their mothers (sampled on the day of abortion and 15 days later) were studied. The criteria for selecting the 10 herds were a history of abortion on the farm and farm location in the region of Botucatu, SaÄo Paulo, Brazil. None of the herds were vaccinated for leptospirosis. All recognized abortions between April 1996 and October 1996 were included. The aborted foetuses were sent to the Laboratory of Diagnosis of Zoonosis at the Faculty of Veterinary Medicine and Animal Science. 2.1. Microscopic seroagglutination Serum samples were assayed for 21 leptospiral serovars: andamana, australis, autumnalis, brasiliensis, bratislava, butembo, castellonis, copenhageni, canicola, cynoptery, djasiman, grippotyphosa, hardjo, icterohaemorraghiae, javanica, panama, pomona, pyrogenes, tarassovi, shermani and wolffi. The samples were maintained in Ellinghausen medium with weekly cleaving, using antigens that had been cultured from 4 to 14 days and that were free from contamination and self-agglutination (Faine, 1982). 2.2. Microbiological culture The foetal kidneys were isolated under aseptic conditions. The kidneys were crumbled (rising two sterile surgical blades) and re-suspended in 10% buffered sterile saline (w/v) with 25 mg/ml of neomycin and furazolidone. The suspension was transferred to sterile tubes under room temperature for at least 1 h for further inoculation. After that, three serial dilutions from 0.5 ml from the supernatant, on logarithmical scale (10ÿ1±10ÿ3) were performed in buffered saline; 0.5 ml of each dilution was inoculated in tubes with Fletcher semi-solid medium and then incubated at 308C to observe bacterial growth every week. A sterile platinum inoculating loop was used to place a drop of culture on a slide for microscopic examination, and then it was covered with an 18 18 mm2 cover glass. The material was observed with a dark-field microscope (40 objective and 10 or 15 eyepiece). In cases where leptospires were identified, the material was transferred to two new tubes, making a new passage for isolated leptospires with a daily tube observation for the presence of an opalescent ring.
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
273
2.3. Histopathology Pieces of the kidneys were collected in 10% formalin for leptospiral silver staining according to Kerr (1938). 3. Results The most prevalent seropositivity in the dams on the day of abortion was to bratislava, hardjo, pomona and wolffi (Table 1). Twenty-seven (22.5%) of the 120 aborting dams had at least a four-fold increase in titres between the first and second samples of serum of the serovars hardjo (12.5%), pomona (1.7%) and wolffi (8.3%), without simultaneous isolation of Leptospire (however, with positive results in the histopathological examination of the kidneys of their foetuses). The serological response was lower in 30 additional cases however, for all 57 dams in Table 2, the histopathological examination of their foetus was positive. Culture attempts from the foetal kidneys, gave positive results in 15 of 120 (12.5%) cases; there was an opalescent ring development and spirochets were visualized under dark-field observation. Specific reference antiserum showed the presence of Leptospira interrogans serovar hardjo in four (26%), pomona in three (20%) and wolffi in eight (53%) of these 15 cases. Table 3 shows the distribution of rising titres observed in these 15 dams. 4. Discussion Several studies reported a great variation of serological profiles of different leptospiral serovars in cattle (Ellis, 1984; Ellis et al., 1982; Elder et al., 1985), as we also found. If we consider only four-fold increases of the serovars hardjo, pomona and wolffi, in the 15 cases with positive isolation of the microrganisms of kidneys, we have a Table 1 Distribution of microscopic seroaggultination-test titres to serovars of Leptospira sp. in 120 Brazilian Holsteins sampled on the day of abortion in 1996 (row percents) Serovara bratislava canicola castellonis hardjo icterohaemorraghiae pomona tarassovi wolffi a
Titre 100
200
400
800
1600
seronegative
18 2 5 24 6.2 13 4.4 40
10 1 2 16 0.0 8.5 0.5 23
2.0 0.0 0.0 13 0.0 0.0 0.0 15
0.0 0.0 0.0 5.4 0.0 0.0 0.0 2.5
0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.5
70 97 93 41 94 79 95 19
Only serovars found in 4 dams are shown.
274
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
Table 2 Distributions of paired titres for Brazilian Holsteins dams whose aborted foetuses had histopathology consistent with leptospirosis but negative cultures (1996) Titre of dams Leptospira serovar
N dams
Initial (day of abortion) *
Final (15d after abortion)
hardjo
12 10 3 3
NR 100 200 200
400 200 400 800
pomona
4 3 2
NR 100 NR
200 200 400
wolffi
8 8 2 2
NR 100 200 200
200 400 400 800
*
NR: no reaction.
seroconversion in only 10 dams (67; 95% CI: 38, 88%). On the other hand, if we analyse the 57 cases with negative culture results but in which leptospires nevertheless were visible in histopathological sections of kidneys, we observed that for the same serovars, 47% (95% CI: 34, 61%) of the dams seroconverted. In total, 72 out of 120 aborted foetuses (60%; 95% CI: 51, 69%) had evidence of leptospiral infection, although only 37 of 120 dams (31%; 95% CI: 23, 40%) had evidence of acute seroconversion. A serological study carried out by Dragui et al. (1986) showed a higher serovar wolffi prevalence, followed by serovar pomona in cases of abortion. As in the study, serovar pomona's importance was also highlighted by Elder et al. (1985) who obtained serological titres significantly related to bovine abortion.
Table 3 Distribution of paired titres for Brazilian Holsteins dams whose aborted foetuses had both positive cultures and positive histopathology indicating leptospirosis Titer of dams Serovars isolated
n dams
Initial (day of abortion)
Final(15d later)
hardjo n4
2 2
100 400
400 800
pomona n3
2 1
100 400
800 800
wolffi n8
4 2 2
200 100 400
800 400 800
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
275
These results reassure the importance of Leptospire on bovine abortion etiology. We stress our finding that many dams with infected foetuses failed to show four-fold rising titres even though their titres were low at the time of abortion. Acknowledgements This work was supported by grants from FAPESP ± Fundac,aÄo de Amparo a Pesquisa do Estado de SaÄo Paulo, Brazil. References Dragui, M.G., Zurbriggem, M.A., Vanzini, 1986. Serological survey of bovine leptospiroses in Corrientes province, Argentina, Vet. Arg. 3, 357±358. Elder, J.K., Pepper, P.M., Hill, M.W.M., 1985. The significance of leptospiral titres associated with bovine abortion. Austr. Vet. J. 62, 258±262. Ellis, W.A., 1984. Bovine leptospirosis in the tropics: Prevalence, pathogenis and control. Prev. Vet. Med. 2, 411±421. Ellis, W.A., O'Brien, J.J., Neill, S.D., 1982. Bovine leptospirosis: Serological findings in aborting cows. Vet. Rec. 110, 178±180. Faine, S. (Ed), 1982. Guidelines for the Control of Leptospirosis, World Health Organization, Geneva, 171 pp.. Kerr, D.A., 1938. Improved Warthin ± Starry method of staining spirochetes in tissue section. An. J. Clin. Pathol. 2, 63±67. Oliveira, S.J., Pianta, C., Gomes, M., 1980. Abortos em bovinos no Rio Grande do Sul, Brasil. Isolamento de leptospiras do sorogrupo hebdomadis. Bol. Inst. Pesq. Vet. Desiderio Finamor 1, 51±56. Prescott, J.F., Miller, R.B., Nicholson, V.M., 1988. Seroprevalence and association with abortion of leptospirosis in cattle in Ontario. Can. J. Vet. Res. 52, 1988. Riedemann, S., Leal, H., Zaamora, J., 1986. The first isolation of Leptospira interrogans serovar castellonis in Chile. J. Vet. 33, 393±394.
Incidence of leptospiral abortion in Brazilian dairy cattle Helio Langoni*, Luiz Carlos de Souza, Aristeu Vieira da Silva, Maria CecõÂlia R. Luvizotto, Antonio Carlos Paes, Simone Baldini Lucheis Departamento de Higiene VeterinaÂria e SauÂde PuÂblica, Faculdade de Medicina VeterinaÂria e Zootecnia, UNESP, Campus de Botucatu, Distrito de RubiaÄo JuÂnior, s/n-Botucatu-Sao Paulo, CEP 18618.000, Brazil Accepted 11 February 1999
Abstract We examined dams' paired serum samples and foetal kidneys (histopathologically and attempting leptospiral cultures) from 120 Brazilian Holstein abortions from 10 herds near Sao Paulo, Brazil, in 1996. Leptospiras was isolated from 15 foetuses. The Leptospira interrogans serovar hardjo was obtained from four, pomona from three and wolffi from eight foetuses. Ten of these 15 foetuses showed a four-fold rise in titres between the day of abortion and the second samples taken 15 days later. Fifty-seven other foetuses had histologic evidence of leptospires; 27 of their dams (47%) had four-fold rises in titres. In total, 72 of 120 aborted foetuses had evidence of leptospiral infection. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Abortion; Leptospira interrogans; Cattle-microbiological disease
1. Introduction Bovine leptospirosis causes serious financial loss due to abortion, stillbirths, death, decrease in milk production and infertility, mainly in tropical and sub-tropical countries (Ellis, 1984; Ellis et al., 1982; Elder et al., 1985; Prescott et al., 1988). Different serovars have been found in South America, including those from the hebdomadis sub-group (Oliveira et al., 1980; Riedemann et al., 1986; ), pomona (Dragui et al., 1986; Riedemann et al., 1986) and wolffi (Dragui et al., 1986). However, in these reports isolation was * Corresponding author. Tel.: +55-14-82-135-19; fax: +55-14-82-123-43; e-mail: [email protected] 0167-5877/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 9 9 ) 0 0 0 2 0 - 3
272
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
performed from samples collected at a single time for each abortion. There are no reports of `rising titres' in the dams which confirms the causal role for the agent. We studied the presence of anti-leptospiral agglutinin in bovine paired samples (from the time of abortion and 15 days later), using several leptospiral serovars. We also report the association between serologic results in the dams and both leptospiral isolation from their aborted foetus kidneys and the presence of leptospires in the histopathological examination of foetal kidneys. 2. Material and method One hundred and twenty Holstein foetuses and 240 serum samples from their mothers (sampled on the day of abortion and 15 days later) were studied. The criteria for selecting the 10 herds were a history of abortion on the farm and farm location in the region of Botucatu, SaÄo Paulo, Brazil. None of the herds were vaccinated for leptospirosis. All recognized abortions between April 1996 and October 1996 were included. The aborted foetuses were sent to the Laboratory of Diagnosis of Zoonosis at the Faculty of Veterinary Medicine and Animal Science. 2.1. Microscopic seroagglutination Serum samples were assayed for 21 leptospiral serovars: andamana, australis, autumnalis, brasiliensis, bratislava, butembo, castellonis, copenhageni, canicola, cynoptery, djasiman, grippotyphosa, hardjo, icterohaemorraghiae, javanica, panama, pomona, pyrogenes, tarassovi, shermani and wolffi. The samples were maintained in Ellinghausen medium with weekly cleaving, using antigens that had been cultured from 4 to 14 days and that were free from contamination and self-agglutination (Faine, 1982). 2.2. Microbiological culture The foetal kidneys were isolated under aseptic conditions. The kidneys were crumbled (rising two sterile surgical blades) and re-suspended in 10% buffered sterile saline (w/v) with 25 mg/ml of neomycin and furazolidone. The suspension was transferred to sterile tubes under room temperature for at least 1 h for further inoculation. After that, three serial dilutions from 0.5 ml from the supernatant, on logarithmical scale (10ÿ1±10ÿ3) were performed in buffered saline; 0.5 ml of each dilution was inoculated in tubes with Fletcher semi-solid medium and then incubated at 308C to observe bacterial growth every week. A sterile platinum inoculating loop was used to place a drop of culture on a slide for microscopic examination, and then it was covered with an 18 18 mm2 cover glass. The material was observed with a dark-field microscope (40 objective and 10 or 15 eyepiece). In cases where leptospires were identified, the material was transferred to two new tubes, making a new passage for isolated leptospires with a daily tube observation for the presence of an opalescent ring.
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
273
2.3. Histopathology Pieces of the kidneys were collected in 10% formalin for leptospiral silver staining according to Kerr (1938). 3. Results The most prevalent seropositivity in the dams on the day of abortion was to bratislava, hardjo, pomona and wolffi (Table 1). Twenty-seven (22.5%) of the 120 aborting dams had at least a four-fold increase in titres between the first and second samples of serum of the serovars hardjo (12.5%), pomona (1.7%) and wolffi (8.3%), without simultaneous isolation of Leptospire (however, with positive results in the histopathological examination of the kidneys of their foetuses). The serological response was lower in 30 additional cases however, for all 57 dams in Table 2, the histopathological examination of their foetus was positive. Culture attempts from the foetal kidneys, gave positive results in 15 of 120 (12.5%) cases; there was an opalescent ring development and spirochets were visualized under dark-field observation. Specific reference antiserum showed the presence of Leptospira interrogans serovar hardjo in four (26%), pomona in three (20%) and wolffi in eight (53%) of these 15 cases. Table 3 shows the distribution of rising titres observed in these 15 dams. 4. Discussion Several studies reported a great variation of serological profiles of different leptospiral serovars in cattle (Ellis, 1984; Ellis et al., 1982; Elder et al., 1985), as we also found. If we consider only four-fold increases of the serovars hardjo, pomona and wolffi, in the 15 cases with positive isolation of the microrganisms of kidneys, we have a Table 1 Distribution of microscopic seroaggultination-test titres to serovars of Leptospira sp. in 120 Brazilian Holsteins sampled on the day of abortion in 1996 (row percents) Serovara bratislava canicola castellonis hardjo icterohaemorraghiae pomona tarassovi wolffi a
Titre 100
200
400
800
1600
seronegative
18 2 5 24 6.2 13 4.4 40
10 1 2 16 0.0 8.5 0.5 23
2.0 0.0 0.0 13 0.0 0.0 0.0 15
0.0 0.0 0.0 5.4 0.0 0.0 0.0 2.5
0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.5
70 97 93 41 94 79 95 19
Only serovars found in 4 dams are shown.
274
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
Table 2 Distributions of paired titres for Brazilian Holsteins dams whose aborted foetuses had histopathology consistent with leptospirosis but negative cultures (1996) Titre of dams Leptospira serovar
N dams
Initial (day of abortion) *
Final (15d after abortion)
hardjo
12 10 3 3
NR 100 200 200
400 200 400 800
pomona
4 3 2
NR 100 NR
200 200 400
wolffi
8 8 2 2
NR 100 200 200
200 400 400 800
*
NR: no reaction.
seroconversion in only 10 dams (67; 95% CI: 38, 88%). On the other hand, if we analyse the 57 cases with negative culture results but in which leptospires nevertheless were visible in histopathological sections of kidneys, we observed that for the same serovars, 47% (95% CI: 34, 61%) of the dams seroconverted. In total, 72 out of 120 aborted foetuses (60%; 95% CI: 51, 69%) had evidence of leptospiral infection, although only 37 of 120 dams (31%; 95% CI: 23, 40%) had evidence of acute seroconversion. A serological study carried out by Dragui et al. (1986) showed a higher serovar wolffi prevalence, followed by serovar pomona in cases of abortion. As in the study, serovar pomona's importance was also highlighted by Elder et al. (1985) who obtained serological titres significantly related to bovine abortion.
Table 3 Distribution of paired titres for Brazilian Holsteins dams whose aborted foetuses had both positive cultures and positive histopathology indicating leptospirosis Titer of dams Serovars isolated
n dams
Initial (day of abortion)
Final(15d later)
hardjo n4
2 2
100 400
400 800
pomona n3
2 1
100 400
800 800
wolffi n8
4 2 2
200 100 400
800 400 800
H. Langoni et al. / Preventive Veterinary Medicine 40 (1999) 271±275
275
These results reassure the importance of Leptospire on bovine abortion etiology. We stress our finding that many dams with infected foetuses failed to show four-fold rising titres even though their titres were low at the time of abortion. Acknowledgements This work was supported by grants from FAPESP ± Fundac,aÄo de Amparo a Pesquisa do Estado de SaÄo Paulo, Brazil. References Dragui, M.G., Zurbriggem, M.A., Vanzini, 1986. Serological survey of bovine leptospiroses in Corrientes province, Argentina, Vet. Arg. 3, 357±358. Elder, J.K., Pepper, P.M., Hill, M.W.M., 1985. The significance of leptospiral titres associated with bovine abortion. Austr. Vet. J. 62, 258±262. Ellis, W.A., 1984. Bovine leptospirosis in the tropics: Prevalence, pathogenis and control. Prev. Vet. Med. 2, 411±421. Ellis, W.A., O'Brien, J.J., Neill, S.D., 1982. Bovine leptospirosis: Serological findings in aborting cows. Vet. Rec. 110, 178±180. Faine, S. (Ed), 1982. Guidelines for the Control of Leptospirosis, World Health Organization, Geneva, 171 pp.. Kerr, D.A., 1938. Improved Warthin ± Starry method of staining spirochetes in tissue section. An. J. Clin. Pathol. 2, 63±67. Oliveira, S.J., Pianta, C., Gomes, M., 1980. Abortos em bovinos no Rio Grande do Sul, Brasil. Isolamento de leptospiras do sorogrupo hebdomadis. Bol. Inst. Pesq. Vet. Desiderio Finamor 1, 51±56. Prescott, J.F., Miller, R.B., Nicholson, V.M., 1988. Seroprevalence and association with abortion of leptospirosis in cattle in Ontario. Can. J. Vet. Res. 52, 1988. Riedemann, S., Leal, H., Zaamora, J., 1986. The first isolation of Leptospira interrogans serovar castellonis in Chile. J. Vet. 33, 393±394.