Balakrishnan Govindan

Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai - 600 051, India.

DOI : https://dx.doi.org/10.13005/bpj/461

Abstract

Leptospirosis is a worldwide zoonosis affecting domestic animals, pet animals, wild animals and human beings. It causes abortion, repeat breeding, jaundice and haemorrhagic mastitis in bovines. The present study was undertaken to understand the involvement of different leptospiral serovars with different clinical conditions of leptospirosis among bovines. A total of 1122 serum samples collected from cattle with different clinical conditions which included abortion (247), repeat breeding (429), jaundice (215), haemorrhagic mastitis (231) were subjected to Microscopic agglutination test. The study revealed the association of hardjo with abortion (21.98 per cent), repeat breeding (47.25 per cent), jaundice (15.38 per cent) and haemorrhagic mastitis (15.38 per cent). The serovar pomona was associated with abortion (19.67 per cent), repeat breeding (39.34 per cent), jaundice (21.31 per cent) and haemorrhagic mastitis (19.67 per cent). Serovar hebdomadis was associated with abortion (29.48 per cent), repeat breeding (37.17 per cent), jaundice (16.67 per cent) and haemorrhagic mastitis (16.67 per cent). Serovar ballum was found to associate with abortion (18.75 per cent), repeat breeding (46.87 per cent), jaundice (15.62 per cent) and haemorrhagic mastitis (18.75 per cent). In addition, the serovar australis was found to associate with abortion (19.29 per cent), repeat breeding (42.10 per cent), jaundice (19.29 per cent) and haemorrhagic mastitis (19.29 per cent). However from this study, serovar specificity with regard to clinical conditions in cattle could not be established as all the five serovars were involved with different clinical conditions.

Keywords

Leptospira serovars; Clinical conditions; Leptospirosis; Cattle

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Introduction

Leptospirosis is a worldwide zoonosis affecting domestic animals, pet animals, wild animals and human beings. It causes abortion, repeat breeding, jaundice and haemorrhagic mastitis in bovines. The involvement of different leptospiral serovars with different clinical conditions of leptospirosis among bovines has been reported earlier by many workers (Venkataraman and Jaganathan, 1961; Rao and Surendran, 1970; Thierman, 1982; Slee, et al., 1983; Venugopal, et al., 1986 and Ramakrishna and Venkataraman, 1994). All these studies recorded only few serovars namely hardjo, hebdomadis, pomona, autumnalis, javanica, grippotyphosa and icterohaemorrhagiae in association with different clinical conditions of leptospirosis among bovines. Whereas the association of serovars australis, ballum etc., has not been available in the perusal of the literature. But the serological evidences of leptospirosis due to australis and ballum have been documented in the literature (Srivastava and Kumar, 2003; Selvaraj, et al., 2005;  Koteeswaran, 2006; Thiyageeswaran, 2007 and  Sekar, et al., 2008). Hence a detailed study on the distrbution of different leptospiral serovars from clinically suspected cattle was undertaken in the present study.

Materials and methods

Collection of serum samples

A total of 1122 serum samples were collected from cattle with different clinical conditions and suspected for leptospirosis which included abortion (247), repeat breeding (429), jaundice (215), haemorrhagic mastitis (231). The serum samples were subjected to Microscopic agglutination test.

Microscopic agglutination test (MAT)

Preparation of antigens for MAT

A 5-8 day old liquid culture of live leptospires incubated at 29 ± 1˚ C, containing density of 2×10⁸ leptospires per ml was used (OIE, 2004). The leptospiral cultures without clumps were used as antigens in MAT. The panel of antigens used in MAT is presented in Table – 1.

Table 1: Reference strains of Leptospires* used in the study

S.No	Serogroup	Serovar	Strain
1	Australis	australis	Ballico
2	Autumnalis	rachmati	Rachmati
3	Ballum	ballum	Mus127
4	Canicola	canicola	HondUtrecht IV
5	Grippotyphosa	grippotyphosa	Moskva V
6	Hebdomadis	hebdomadis	Hebdomadis
7	Icterohaemorrhagiae	icterohaemorrhagiae	RGA
8	Javanica	Poi	Poi
9	Pomona	pomona	Pomona
10	Pyrogenes	pyrogenes	Salinem
11	Sejroe	hardjo	Hardjoprajitno
12	Tarassovi	tarassovi	Peripellicin

*Obtained from National Reference laboratory, Indian Council of Medical Research, Andaman and  Nicobar Islands, India.

 

Microscopic agglutination test

This test was conducted as per OIE (2004) in 96 well ‘U’ bottom titration plates (M/s. Laxbro, India). Serum dilutions were made in deep well (96 well) dilution plates (M/s. Laxbro, India). To 980 µl of PBS, 20 µl (1:50) of serum samples were added in individual wells. Serum dilutions (25 µl of 1:50) were added to each of the 12 wells in the A to G rows of ‘U’ bottom microplates. In the last row, only PBS 25 µl was added to all the wells which served as antigen control. Thus each row is corresponding to each sample. Twelve antigens (25 µl) were added in all the wells of respective columns (antigen 1 in column 1, antigen 2 in column 2 and so on) including in the respective antigen control wells, so that the final serum dilution was in 1 in 100. The plates were closed with lids and incubated at 37˚C for 2 h. A drop (5 µl) of mixture (final dilution of 1:100) was placed on grease-free slide and the wet preparation without cover slip was screened using 20X objective of the dark field microscope (M/s. Nikon, 200E Japan) for the presence of agglutination and/or reduction in number of organisms in comparison with the respective antigen control. A 50 per cent reduction in the number of free leptospires in the test sample comparable with the respective antigen control was considered positive with or without agglutination.

Table 2: Seroprevalence of leptospirosis in clinically suspected cattle

Clinical signs	Total screened	Total positive (MAT)		Serovars reacted
		No.	Percent	Australis	ballum	hardjo	hebdomadis	pomona	Total
Cattle                   x2 = (5.82) NS
Abortion	247	78	31.58	11(19.29%)	12 (18.75%)	20 (21.98%)	23(29.48%)	12(19.67%)	78
Repeat breeding	429	150	34.97	24 (42.10%)	30 (46.87%)	43 (47.25%)	29(37.17%)	24(39.34%)	150
Jaundice	215	61	28.37	11(19.29%)	10 (15.62%)	14 (15.38%)	13(16.67%)	13(21.31%)	61
Haemorrhagic Mastitis	231	62	26.84	11(19.29%)	12 (18.75%)	14 (15.38%)	13(16.67%)	12(19.67%)	62
Total	1122	351	31.28	57	64	91	78	61	351

NS – Statistically non- significant at 5 per cent level (P<0.05)

 

Results and discussion

A total of 1122 serum samples were collected from cattle with the history of abortion, repeat breeding, jaundice and haemorrhagic mastitis (Table – 2). Out of 1122 serum samples, 351 were found positive (31.28 per cent). The five serovars namely hardjo, hebdomadis, ballum, australis and pomona were found to be involved. The prevalence rate of hardjo (25.93 per cent) was more, followed by hebdomadis (22.22 per cent), ballum (18.23 per cent), pomona (17.38 per cent) and australis (16.24 per cent). Slee et al. (1983) proved that serovar hardjo was associating with bovine abortions in Australia, whereas Thiermann (1982) reported that hardjo could produce mastitis and abortions. Sullivan and Callan (1970), Hoare and Claxton (1972), Gordon (1977) and Higgins et al. (1980) reported association of clinical mastitis with hardjo in cattle. The association of hardjo with abortion and infertility in cattle was also reported earlier (Ellis et al., 1983 and Ramakrishna and Venkataraman, 1994). In the present study, hardjo was associated with abortion (21.98 per cent), repeat breeding (47.25 per cent), jaundice (15.38 per cent) and haemorrhagic mastitis (15.38 per cent). Knott and Dadsmell (1970) recorded abortion amongst cattle in Australia following pomona infection. The association of pomona with abortion was recorded earlier by several workers in India (Rao and Surendran, 1970; Venugopal et al., 1986). In addition to abortion, the serovar pomona also caused repeat breeding (Venugopal et al., 1986) and jaundice (John et al., 1980). In the present study serovar pomona was associated with abortion (19.67 per cent), repeat breeding (39.34 per cent), jaundice (21.31 per cent) and haemorrhagic mastitis (19.67 per cent). The serovar hebdomadis was earlier observed in aborted cows (Rao and Surendran, 1970). In the present study, hebdomadis was associated with abortion (29.48 per cent), repeat breeding (37.17 per cent), jaundice (16.67 per cent) and haemorrhagic mastitis (16.67 per cent). Serovar ballum was found to associate with abortion (18.75 per cent), repeat breeding (46.87 per cent), jaundice (15.62 per cent) and haemorrhagic mastitis (18.75 per cent). In addition, the serovar australis was found to associate with abortion (19.29 per cent), repeat breeding (42.10 per cent), jaundice (19.29 per cent) and haemorrhagic mastitis (19.29 per cent). The involvement of serovars ballum and australis with different clinical conditions of leptospirosis in cattle has not been reported earlier in the available literature.  However from this study, serovar specificity with regard to clinical conditions could not be established as all the five serovars were involved with different clinical conditions.

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