Role of Immunohistochemistry in the Diagnosis of Leptospirosis in Neotropical Primates

Abstract

Background: Leptospirosis is considered the most widespread zoonosis worldwide, occurring more frequently in tropical and developing regions. The aim of the present study was to detect the presence of Leptospira spp. in different primate tissues, using immunohistochemical (IHC) assays, taking advantage of the considerable number of necropsies compatible with a diagnosis of leptospirosis in neotropical primates at the Animal Pathology Laboratory (APL) of the University of Passo Fundo (UPF) in the northern region of Rio Grande do Sul. Materials, Methods & Results: Paraffin-embedded primate tissue samples were selected from necropsy examinations and subjected to IHC. The streptavidin-biotin-peroxidase method was used with diaminobenzidine chromogen (DAB) to verify immunostaining. Of the101 primates tested for Leptospira spp., 51.48% were positive; taining was distributed between lung (76.92%), liver (44.23%), and kidney (32.69%) tissue. Analysis of the combined anatomopathological verification data of the studied organs revealed a high frequency of lesions commonly observed in the tissues of animals exposed to the pathogen. For complementary diagnosis, an anti-Leptospira spp. antibody test was performed in primates at the UPF-Zoo, from which a population of the necropsied animals originated. The microscopic agglutination test (MAT) was utilized, which demonstrated 90.47% positivity in 21 individuals; sejroe and panama were the most frequent serovars. Discussion: Different intensities of tissue immunostaining were observed. Areas of fragmented or diffuse staining were considered to indicate equal positivity to that indicated by areas of staining with preserved morphology. Of 52 Leptospirapositive primates, most presented some degree of staining in lung samples, which shows a high level of involvement for this organ in primate leptospirosis. Conventional pathological diagnostic methods do not allow fort issue antigen recognition, thus making the IHC technique important to facilitate conclusive antigen sample verification. In the liver, leptospires were detected mostly between the sinusoids, hepatocytes, and Kupffer cells. In kidney tissues, staining indicated small agglomerates in the tubular lumen, interstitium, and glomeruli. All these forms of presentation have been previously reported. Considering that we detected the highest number of positive samples in lung tissue, followed by those from liver and kidney tissue, we argue that the IHC technique, when applied to samples of these three tissues, decreases the chance of false negatives. Anatomopathological studies of primate leptospirosis are scarce. In dogs, renal lesions are characterized by the necrosis and degeneration of tubular epithelium, cellular debris, and hyaline cylinders. In the liver, hepatocyte cord dissociation and biliary pigment accumulation within the canaliculi and hepatocellular necrosis are observed. These findings are similar to those from our study. In the lung, diffuse alveolar lesions are reported, with hemorrhage and edema, in add...

Serological survey and DNA screening of Leptospira spp. in free‐living adult tufted capuchin monkeys (Cebus apella nigritus) in a forest reserve Southeast São Paulo State, Brazil

“…Microscopic agglutination test results showed that the Icterohemorrhagiae serogroup was the most frequent serovar found in the free‐living capuchin monkeys ( Cebus apella nigritus ), with 30.76% of sera reactive animals. Non‐human primates reactive to this serogroup have been demonstrated in several regions of Brazil: Acre, Amazon Region, 10 Rio Grande do Sul 8 South Region, Sergipe, 11 Northeast Region and Tocantins, 15 Central Region. However, in the Southeast Region, the serovars Autumnalis and Hardjo were more frequent 14 .…”

“…In Brazil every year, there are more than 3000 cases in human beings and 280 to 300 deaths 7 . However, the occurrence in non‐human primates is still poorly investigated and could be an important issue, since these animals could act as sources of infection to staff in zoos, personnel involved in diagnostic laboratories, professionals, other species of primates, or other animals that share the same habitat 8,9 …”

“…The microscopic agglutination technique (MAT) is the serological gold standard used for leptospirosis antibodies diagnosis. However, serum reactivity of non‐human primates infected with leptospires could be of short duration 8 . Some animal species, including Cebus apella , eliminated the agent in the urine despite having MAT titers of 100 or less 10 .…”

“…Serological surveys carried out in non‐human primates at different Brazilian regions such as Rio Grande do Sul, Acre, Sergipe, Bahia, São Paulo, and Tocantins states 8,10–14 examined free‐living and captive zoo animals. All of them were sera reactive against different Leptospira spp.…”

“…serogroups and serovars, without any special predominance. However, positivity to Icterohemorrhagiae serogroup, the most frequent cause of human leptospirosis in Brazil, was found in almost all investigations 8,10,11,13,15 . Leptospiral DNA was not detected via Polymerase Chain Reaction (PCR) in blood and urine samples collected from 58 non‐human primates 16 …”