Two Cases of Natural Infection of Dengue-2 Virus in Bats in the Colombian Caribbean

Abstract

Dengue, a mosquito-borne zoonotic disease, is the most common vector-borne disease in tropical and subtropical areas. In this study, we aim to demonstrate biological evidence of dengue virus infection in bats. A cross-sectional study was carried out in the departments of Cordoba and Sucre, Colombia. A total of 286 bats were captured following the ethical protocols of animal experimentation. The specimens were identified and euthanized using a pharmacological treatment with atropine, acepromazine and sodium pentobarbital. Duplicate samples of brain, heart, lung, spleen, liver, and kidney were collected with one set stored in Trizol and the other stored in 10% buffered formalin for histopathological and immunohistochemical analysis using polyclonal antibodies. Brain samples from lactating mice with an intracranial inoculation of DENV-2 were used as a positive control. As a negative control, lactating mouse brains without inoculation and bats brains negative for RT-PCR were included. Tissue sections from each specimen of bat without conjugate were used as staining control. In a specimen of Carollia perspicillata captured in Ayapel (Cordoba) and Phylostomus discolor captured in San Carlos (Cordoba), dengue virus was detected, and sequences were matched to DENV serotype 2. In bats RT-PCR positive for dengue, lesions compatible with viral infections, and the presence of antigens in tissues were observed. Molecular findings, pathological lesions, and detection of antigens in tissues could demonstrate viral DENV-2 replication and may correspond to natural infection in bats. Additional studies are needed to elucidate the exact role of these species in dengue epidemics.

Keywords: antibodies; arbovirus; flavivirus; immunohistochemistry; pathology; zoonoses.

Figure 1

143 bp amplicons of C. perspicillata in brain, (lane 2), heart (lane 13), lung (lane 14), liver (lane 15), spleen (lane 16); YFV positive control (lane 19) and negative control (lane 20). Molecular weight marker 100 pb Invitrogen (lane 10).

Figure 2

Phylogenetic tree showing 4 clades that correspond to the 4 serotypes of the DENV. Sequences MG011655 and MG011656 (marked with an asterisk) were detected in bats from Cordoba, Colombia, the sequences are in the clade, these sequences correspond to serotype 2 of DENV. DENV-2.

Figure 3

(A): Brain of C. perspicillata with the presence of gliosis (G), perineural edema (E), vasculitis (V), non-suppurative meningitis (NSM) of lymphoid type B. (H&E stain 400×). (B): Liver of C. perspicillata with lymphoid infiltrate (LI) around portal triad (C) (H&E stain 400×). (C): Lung of C. perspicillata with hyperplasia of lymphoid tissue associated with bronchi (HLT), thickening of alveolar septa, interstitial pneumonia (H&E stain 100×). (D): Spleen of C. perspicillata with moderate lymphoid depletion (MLD) in lymph node (H&E stain 100×).

Figure 4

(A): Lung of P. discolor with presence of congestion (H&E stain 100×). (B): Liver of P. discolor with severe eosinophilic lymphocyte mixed necrotic hepatitis and (H&E stain 400×).

Figure 5

(A): Brain of C. perspicillata which shows the presence of antigens for DENV using IHC. (400×). (B): Lung of C. perspicillata which shows the presence of antigens for DENV using IHC. (400×). (C): Spleen of C. perspicillata which shows the presence of antigens for DENV using IHC. (400×).

Figure 6

(A): Brain of P. discolor which shows the presence of antigens for DENV using IHC. (400×). (B): Kidney of P. discolor which shows the presence of antigens for DENV using IHC. (400×).

Figure 7

Staining controls: Mouse brain (A) without infection (400×). (B): Bat´s kidney PCR negative to Dengue (400×).