Immunohistochemical detection of leptospiral antigens in cases of naturally occurring abortions in sheep

doi:10.1016/j.smallrumres.2007.04.006

Small Ruminant Research

Volume 74, Issues 1–3, January 2008, Pages 119-122

https://doi.org/10.1016/j.smallrumres.2007.04.006 Get rights and content

Abstract

This study was carried out to determine the leptospiral antigens in naturally occurring 108 abort sheep fetuses. To determine the antigen localizations in tissue sections (kidney, liver, lung and spleen) of each fetus were stained with immunoperoxidase (IP) technique and then were examined under light microscope. The results of this study showed that 19 (17%) out of 108 fetuses were positive for the presence of leptospiral antigens. In the 19 positive cases, leptospiral antigens were found in lung (n = 10; 9%), liver (n = 7; 6%), kidney (n = 12; 11%) and spleen (n = 2; 2%) samples. Microscopic studies demonstrated that leptospiral antigens were located in the cytoplasm of macrophages in interalveolar and interlobular septum of the lung; in the cytoplasm of macrophages in the portal regions and hepatocytes of the liver; in the cytoplasm of epithelial cells of renal pelvis, in the cytoplasm of epithelial cells of cortical and medullar tubules, and macrophages of intertubular region in the kidney. In the spleen, antigens were detected in the cytoplasm of macrophages throughout the parenchymal tissue. In conclusion, the results of this study demonstrate that leptospirosis could be a major disease causing abortions in sheep.

Introduction

Leptospirosis is an important infectious disease of livestock animals and humans caused by serovars of Leptospira interrogans. It is particularly important due to causing abortion and stillbirth in farm animals. Leptospira pomana, L. hardjo and L. grippotyphosa have been the most common serovars isolated from sheep (Ellis et al., 1983, Bulu et al., 1990, Maxie, 1993). Because the leptospires die readily in tissues or body fluids, diagnosis of leptospirosis in individual animals is often difficult (Ellis et al., 1983, Maxie, 1993). Leptospirosis may be diagnosed by the presence of leptospiral antigens in tissue sections. A variety of silver staining, immunofluorescence, immunoperoxidase (IP) and immunogold silver techniques are common laboratory techniques for diagnosis (Ellis et al., 1983, Scanziani et al., 1989, Scanziani et al., 1991). An IP staining procedure to detect leptospiral antigens in tissues has been shown to be an accurate and reproducible technique and be able to be used on formalin-fixed and paraffin-embedded tissues. Also, there is a positive correlation between results obtained from culture studies and results obtained from the IP staining methods on infected tissues (Ellis et al., 1983).

The IP method is demonstrably more sensitive than silver staining tissue samples (Scanziani et al., 1989, Scanziani et al., 1991, Szeredi and Haake, 2006) and more specific than serology performed using the microscopic agglutination test. Compared with culture, the sensitivity of the IP was 78% and its specificity was 100% (Scanziani et al., 1989).

Objective of this study was to determine the presence of leptospiral antigens in tissue sections in naturally occurring abort sheep fetuses by using IP technique.

Section snippets

Materials and methods

Totally, 108 ovine aborted fetuses were necropsied. All the samples were collected from Erzurum and Van provinces (of eastern Turkey). Abortions had been come from the different flocks and regions, and the number of abortions in the flocks had not been known. Tissue samples from liver, lung, spleen and kidney of each fetus were taken for histological and IP examinations. These samples were routinely processed staining for haematoxylin and eosin, and Levaditi-Manovelian as well as IP (Ellis et

Results

In IP staining, the presence of leptospiral antigens were detected in 19 (17%) out of 108 ovine abort fetuses. Leptospiral antigens were found in lung (n = 10; 9%), liver (n = 7; 6%), kidney (n = 12; 11%) and spleen (n = 2; 2%) tissue samples (Table 1). These IP positive abortive fetuses had come from the different flock.

Microscopic studies demonstrated that the leptospiral antigens were located in the cytoplasm of macrophages in interalveolar and interlobular septum of the lung; in the cytoplasm of

Discussion

Diagnosis of leptospirosis in animals usually depends on serological examination in the live animal or a combination of histopathology and culture in the dead animal (Ellis et al., 1983). The frequencies of leptospirosis were reported in sheep as 3% (Bulu et al., 1990) and 4% (Ozkan et al., 1993) with serological tests in Erzurum region. Recently, some local studies in eastern Turkey show that the seroprevalences of leptospirosis in cattle were found to be 36% (Sahin et al., 2002), 38% (Genc et

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The presence of antibodies in the fetal fluid of five cattle fetuses (5.2 %) aborted at an average gestational age of greater than 7 months has also been reported (Moore et al., 2003). With use of immuno-peroxidase staining, there were 19 (17 %) of 108 aborted sheep fetuses in which there were leptospiral antigens in the lung, liver, kidney and spleen (Saglam et al., 2008). In none of the fetuses, however, was there a positive diagnosis with use of serology procedures.
Leptospirosis is a globally distributed disease associated with reproductive failures in livestock; however, its pathogenesis has not been fully elucidated. Results from the present study indicate there is a presence of Leptospira sp. in organs and fluids of fetuses from ewes slaughtered in the semiarid region of Brazil. Twenty-nine fetuses from 23 ewes determined to be Leptospira sp.-positive using PCR were sampled (14 and 15 in dry and rainy seasons, respectively). Fetal samples of blood, central nervous system (CNS), lung, liver, spleen, stomach contents, peritoneal fluid, kidney, bladder, urine and reproductive system were collected. Diagnostic methods included the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Of the 29 fetuses, 24 (82.8 %) had at least one Leptospira sp.-positive organ or fluid, as determined using PCR, and of a total of 209 samples, 62 (29.7 %) contained leptospiral DNA. Of the 99 samples collected during the dry season, 42 (42.4 %) were positive, and of 110 samples collected during the rainy season, 20 (18.2 %) were positive (P = 0.0001). There was deoxyribonucleic acid (DNA) sequencing of three samples of kidney, CNS and liver, and in all of these, there was 99.3 % similarity with Leptospira interrogans. Leptospires were present in cultures of pooled samples from fetuses with deformities. Results indicate there is vertical (maternal-to-fetus) transmission which would represent an alternative transmission route for the spread of Leptospira sp. in ewes, suggesting molecular detection is essential in the investigation of leptospirosis in fetuses to identify animals that have been infected with this bacterium.
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Immunohistochemical detection of leptospiral antigens in cases of naturally occurring abortions in sheep

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Res. Vet. Sci.

Determination of the seroprevalence of leptospirosis in cattle by MAT and ELISA in Hatay, Turkey

Turk. J. Vet. Anim. Sci.

The studies on the spreading and serotype of leptospirosis occurrences in cattle and sheep in some cities of the East Anatolia (Kars, Artvin, Gümüşhane, Erzurum)

J. Etlik Vet. Microbiol.

Leptospira interrogans serovar hardjo in the kidneys and genital tracts of naturally infected sheep

New Microbiol.

Detection of leptospires in tissue using an immunoperoxidase staining procedure

Aust. Vet. J.

New developments in immunoperoxidase techniques and their application

Arch. Pathol. Lab. Med.

Seroprevalence of brucellosis and leptospirosis in aborted dairy cows

Turk. J. Vet. Anim. Sci.

The urinary system