Histopathology of the Organs from Cattle with Leptospirosis

| Leptospirosis is one of the neglected diseases distributed worldwide. Leptospirosis causes severe infection in cattle. A total of four male cattle were found dead in the cowshed during July 2019 – April 2020. The necropsy procedure was conducted, and various macroscopic lesions were identified. Further, several fresh and fixed specimens were collected and transported to the laboratory for ELISA and histopathology. The qualitative ELISA showed that all kidney samples were positive against Leptospira IgG. The histopathology showed that the lung specimens were suffering from pneumonia (3/4), the liver congestion (2/4), liver fatty degeneration (1/4), and chronic nephritis and glomerulonephritis (4/4). Based on the Gram staining, all kidney samples showed the appearance of slender form bacteria that may belong to Leptospira  sp in the lumen of tubules. All dead cattle showed moderate and minimal expression of CD4+ and CD8+ in the lung and liver concomitantly. In contrast, there is no expression of CD4+, and a high expression of CD8+ was observed from the kidneys. This study demonstrated that leptospirosis in cattle impacts several organs with severe manifestation occurs on the kidneys.


Advances in Animal and Veterinary Sciences
November 2020 | Volume 8 | Issue 11 | Page 1221

sAmples collection
The lung, liver, and kidney were collected and separated into two parts. The first part was stored inside the sterile plastic bag and cooled at 4° C. The second part was stored using 10% neutral buffer formalin (NBF). The specimens were transported to the Integrated Laboratory, University of Muhammadiyah Sidoarjo, East Java, Indonesia.

enzyme-linked immunosorbent AssAys (elisA)
The fresh specimens were tested using ELISA against Leptospira IgG (LEBTOSPIRA IgG ELISA kit, Cat. No: MBS036135, MyBioSource). Before the ELISA, the organs were mashed up using the mortar and mixed with sterile distilled water. The mixture was then centrifuged at 3000 rpm for 5 minutes, and it repeated three times. The ELISA was performed following the provided procedure by the manufacturer. The optical density (OD) was read using an ELISA reader at 450 nm of wavelength. The samples were categorized as positive (OD ≥ 1.1) and negative (OD ≤ 1.0).

morphometry
The H&E slide was analyzed by a single pathologist using the scoring system. The score was calculated based on the addition of scores from several parameters include severity, duration, distribution, and types of exudation. The Gram and IHC staining slide was scored based on the distribution of the staining target (Table 2).

reSultS ANd dIScuSSIoN
The result showed that all the kidney specimens were positive against Leptospira IgG (Table 3). The kidney samples were tested against Leptospira IgG because these are the predilection of the Leptospira sp. The appearance of IgG from the kidneys indicated that all cattle infected by Leptospira sp. Further laboratory tests revealed various histopathological changes of the lung, liver, and kidney.

Advances in Animal and Veterinary Sciences
November 2020 | Volume 8 | Issue 11 | Page 1222  Severe chronic active diffuse necrotizing glomerulo-nephritis NT = not tested, + = positive, -= negative, NHC = no histopathological changes. The H&E score showed the severity of histopathology. A higher score indicated increasingly severe histopathological changes ( Table 4). The lung showed pneumonia with various manifestations that belong to acute (C2), chronic (C1), and chronic active (C4). It was suspected that tissue destruction of the lung in C1, C2, and C4 reflected grad ual pathogenesis in leptospirosis (Figures 1 and 2). However, the liver did not show significant histopathological changes ( Figure 3). Further, the kidney from C1 and C3 showed chronic interstitial nephritis with diffuse and focal distribution, respectively. Besides, the C2 and C4 showed chronic glomerulonephritis (Table 4, Figure 4, and 5).

Advances in Animal and Veterinary Sciences
November 2020 | Volume 8 | Issue 11 | Page 1223   Gram staining of lung and liver did not show the presence of slender form bacteria that may belong to Leptospi-ra sp. In contrast, the kidney showed a high presentation of slender form bacteria within the lumen (Table 4 and Figure 6). Surprisingly, it is synergistic to the immunohistochemistry score. The zero scores of Gram staining in the lung showed the appearance of CD4+ and CD8+ as normal lymphocytes within the nodular lymphoid tissue. It was different from the kidney samples. The kidney showed a high expression of CD8+ as the compensatory impacts of Leptospira sp infection (Table 4). Leptospira sp. is a pathogenic bacteria that can survive in the environment for prolonged periods. The ability of Leptospira sp. to survive in the environment enables this bacteria to cause direct and indirect infection in humans and animals. Leptospirosis commonly occurs and spreads in the unsanitary area with a high population of rats. Wild rats act as the main reservoirs of Leptospira sp. that promote freshwater contamination through its urine (Pellizzaro et al., 2019). Furthermore, it enables the waterborne disease in cattle.
Leptospira sp. is utilizing its virulence factors such as surface protein TlyC to bind the extracellular matrix of host tissue and erythrocytes (Evangelista and Coburn, 2010). In advances, this bacteria circulates through the bloodstream and releases sphingomyelinase C gene A (sphA) that induces endothelial and membrane cell damage (Narayanavari et al., 2015). In a healthy immunological response, the host body releases T-lymphocytes (CD4+ and CD8+) to eliminate the circulatory antigen. However, both CD4+ and CD8+ are suppressed during leptospirosis because leptospirosis can depress innate immune response associated with a decrease in NK cell, and adaptive immune response associated with an inhibition of CD4+ and CD8+ lymphocytes (Duarte-Neto et al., 2019).
The synergic mechanism between Leptospira virulence factors and its ability to inhibit immune response generates