SEROLOGICAL AND BIOCHEMICAL STUDIES ON BOVINE LEPTOSPIROSIS

SEROLOGICAL AND BIOCHEMICAL STUDIES ON BOVINE LEPTOSPIROSIS

ATTIA E.R.H and MOSALLAM T.E.

Leptospirosis Unite, Animal Reproduction Research Institute (ARRI)

Email: sradwanradwan@yahoo.com

INTRODUCTION

Leptospirosis is an anthropozoonosis caused by a spirochete of the genus Leptospira that lives mainly among rodents. Transmission to humans primarily occurs through contact with environments contaminated by the urine of infected animals causing serious problems including hepato-nephritis and clinical diagnosis is usually difficult because of the clinical polymorphism (Levett, 2001). Early diagnosis of leptospirosis is urgent for effective medical care, improving patient outcomes (Assez et al., 2013).

The genus Leptospirais classified serologically into two species, the pathogenic species L. Interrogans and the saprophytic species L. biflexa. There are more than 200 serovars of L. interrogans and more than 60 serovars of L. biflexa. Most of these serovars can infect different animal species, but there is a primary host reservoir for each serovar, which ensures the survival and dissemination of the organisms (Birnbaum et al., 1998). Leptospirosis causes high economic losses in live-stock due to abortion, still-birth, decreased milk production, and death of young ages. The present work was adopted to fulfill two tasks: a) diagnosis of leptospirosis in dairy herds by using two serological techniques (Microscopic Agglutination Test (MAT) and ELISA, b) estimation of some biochemical parameters to study the effect of leptospirosis on animal health.

MATERIALS and METHODS

1. Animals:-

Two hundred and eighty six (286) cows were investigated in this study. These animals were distributed in private farms in five governorates (Behera, Alexandria, Kalubia, Sharkia and Assiut) in Egypt. Some of them suffered from repeat breeders (36), some suffered from abortion (13), some had abnormal milk (100) and 137 cows were apparently healthy. All cows were non vaccinated to Leptospira.

2. Samples:

Blood sera samples of the investigated cows were collected and stored at -20°C till serological examination except sera that used for estimation of liver enzymes were immediately tested.

3. Leptospiraserovars:

Five leptospiraserovars were obtained from Animal Reproduction Research Institute, the source of these reference strains was leptospirosis reference laboratory in Center for Diseases Control (C.D.C.), Atlanta, Ga.30333, USA. Leptospiraserovarsare L.int. icterohaemorrhagiae, L.int.canicola, L.int.pomona, L.int.grippotyphosa and L.int.wolffi. These serovars were used for MAT, ELISA.  

4. Leptospiral media:

It is a serum free medium which has been used in the continuous subculture of leptospiral strains both for their maintenance and propagation(EMJH (Ellinghausen, McCullough, Johnson and Harris)base medium (Difco) USA - EMJH Enrichment (Difco) USA).

5. The Microscopic Agglutination Test (MAT):

The MAT was employed in this study to determine the presence of leptospiral antibodies and their titers in the sera of adult dairy cows against 5 leptospiralserovars (L.int.icterohaemorrhagiae, L.int. canicola, L.int.pomona, L.int.grippotyphosa and L.int. wolffi. It was carried out according to Faine et al. (1999). The MATwas performed with living reference leptospira strains cultivated for 7 days in EMJH medium at 30 ᴼC. For serological studies a serial double fold serum dilution is done using Phosphate Buffer Saline (PBS) beginning with dilution 1:100.

6. ELISA Test was carried out according to Hajkova and Jurmanova (1986)

7. Biochemical examinations:-      

1- Estimation of urea was done based on Batton and Crouch (1979).

2- Estimation of creatinine: was carried out according to the method described by Bowers and Wong (1980).

3- Estimation of aspartate aminotransferase (AST): using kinetic method which carried out adopting the method of Breuer (1996).

4- Estimation of alanine aminotransferase (ALT): using kinetic method which carried out adopting the method of Breuer (1996).

5- Estimation of γ-glutamyltransferase (GGT):- Using kinetic method which is carried out adopting the method of Kaplan (1992).

8. Statistical analysis:

Collected data were analyzed for the mean and standard error of mean. Significance of the results was evaluated by F-test, (analysis of variance) according to Petrie and Watson (1999).

RESULTS

Table 1: Leptospira seropositive cases among examined cows with reproductive disorders:

Table 2: Results of sero-diagnosis of leptospirosis using MAT among cows with reproductive disorders:

Table 3: Results of sero-diagnosis of leptospirosis using ELISA among cows with reproductive disorders:

Table 4: Distribution of positive titers against different leptospiralserovars among cows:

Table 5: Values of ALT, AST and GGT associated with leptospiral infection in cows (u/l):

* Significant at P<0.05 Means that have different subscripts were significantly different at P<0.05.

Can= L.int.canicolaIct= L.int.icterohaemorrhagiae Pom= L.int.pomona Grip= L.int.gripotyphosa Wol= L.int.wolfi

Table 6: Kidney function parameters associated with leptospiral infection in cows (mg/dl):

* Significant at P<0.05

Means that have different subscripts were significantly different at P<0.05.

Can= L.int.canicolaIct= L.int.icterohaemorrhagiae Pom= L.int.pomona Grip= L.int.gripotyphosa Wol= L.int.wolfi

DISCUSSION

In the present study, titers of 1:200 or greater were recorded as positive. According to the report of (WHO 1982) titers of 1:800 or greater were considered as indicative for an active leptospiralinfection. The results of serological tested cows sera indicated that leptospiral seropositive cases using MAT and ELISA were 45.81% and 52.10% respectively. Vanasco et al. (2001) suggested that ELISA could constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents and agreement between ELISA and MAT would be much higher if ELISA cut-off points were lowered, being 1:20. It could be concluded that MAT and ELISA could be used to screen leptospiral infection in cattle. However the ELISA was more sensitive, specific and rapid to detect antibodies in cattle with positive titer of 1:200 and more Attia and Ibrahim(2002). The obtained results revealed that agglutinins against L.int.grippotyphosa were predominant (11.89%) and (12.94%) by using MAT and ELISA respectively. The distribution of its positive titers at 1:200, 1:400, 1:800 and 1:1600 was in the following percentages 26.48%, 35.29%, 35.29% and 2.94% respectively. These results were approximately similar to that obtained byBerovich (1987), Wanyangu et al. (1987) and El-Sukhon et al. (1992). They detected agglutinins against L.int.grippotyphosa in 14%, 10.3% and 15.1% respectively in infertile cattle sera.

In Egypt, Attia (1993) detected agglutinins against L.int.grippotyphosa in 2% in cattle at Dakahlia Governorate, while 5.5% in Baladi cows sera at Giza abattoir. The author also detected antibodies in 5% in cow sera from infertile cases in private farms in Egypt. The obtained results are lower than that obtained by Eman S. Ibrahim (2007) who detected agglutinins against L.int.grippotyphosa in 17.07% in infertile cows, and higher than that obtained by Hatem (1979), Kilany (1988), Prescott et al. (1988), Truner(1988), Sebek et al. (1989) and Espi et al. (2000) who detected agglutinins against L.int.grippotyphosa in 1%, 6.5%, 2%, 7.1%, 2.9% and 2.37% respectively. The current study revealed that agglutinins against L.int.canicola were (10.49%) and (12.59%) by using MAT and ELISA respectively. The distribution of its positive titers at 1:200, 1:400, 1:800 and 1:1600 was in the following percentages 13.33%, 50.00%, 26.67% and 10.00% respectively. The obtained results were higher than results of Attia, (1993) at Giza abattoir, who detected agglutinins against L.int.canicola in 9.5% and 1% in cow sera respectively. On the other hand Ibrahim (2007) recorded agglutinins against L.int.canicola in 21.34% in cows suffering from infertility.

Concerning some biochemical studies associated with leptospiral infection in cows suffered from reproductive disorders, it was found that ALT and AST were significantly high in cows suffered from leptospirosis compared with control group. This elevation behaved rather similar in different leptospiraserovars. Moreover GGT was increased slightly in cows suffered from leptospirosis compared with control group. Blood urea and creatinine were significantly elevated compared with control group. This elevation was markedly seen in cows affected with L.int.icterohaemorrhagiae mixed with L.int.grippotyphosa, L.int.canicola, L.int.pomona and L. int. wolfi. The obtained results were similar to Levett (2001) who reported that in severe leptospirosis, the rise in  AST and ALT activities were due to release of bacterial toxins or immunological reactions. Renal function impairment is indicated by raised plasma creatinine levels. The degree of azotemia varies with severity of illness. The outer membrane of leptospires contains lipopolysaccharide (LPS) and outer membrane proteins (OMPs). The LPS is highly immunogenic and is responsible for serovar specificity. An inverse relationship between expression of transmembrane OMPs and virulence was demonstrated in serovar L.int.grippotyphosa, outer membrane components may be important in the pathogenesis of interstitial nephritis (Barocchi et al., 2002).

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