Herpes Virus Infection Associated with Interstitial Nephritis in a Beaked
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Arbelo et al. BMC Veterinary Research 2012, 8:243 http://www.biomedcentral.com/1746-6148/8/243 RESEARCH ARTICLE Open Access Herpes virus infection associated with interstitial nephritis in a beaked whale (Mesoplodon densirostris) Manuel Arbelo1, Edwige N Bellière2, Eva Sierra1, Simona Sacchinni1, Fernando Esperón2, Marisa Andrada1, Miguel Rivero1, Josue Diaz-Delgado1 and Antonio Fernández1* Abstract Background: The capacity for herpesvirus to cause disease in cetaceans is unclear and may be varied depending on the different conditions of individuals and between different species. Kidney pathology and intralesional virus-associated infection have been rarely reported in cetaceans. Result: On April 2004, an old adult male Blainville’s beaked whale (Mesoplodon densirostris) 420 cm long with a poor body condition was stranded on Tenerife Island. During necropsy, no gross lesions were observed in the kidneys. However, membranous glomerulonephritis, multifocal interstitial lymphoplasmacytic nephritis and acute multifocal necrotizing tubulointerstitial nephritis with intranuclear inclusion bodies was diagnosed by histological analysis. Tissue samples were submitted for bacteriological analysis and molecular viral screening. Conclusion: A novel alpha herpesvirus associated with interstitial nephritis was identified in an old adult male Blainville's beaked whale (M. densirostris) with a poor body condition stranded in the Canary Islands. This report suggests that identification of herpesvirus infection could be used as a differential diagnosis for interstitial nephritis in cetaceans. Keywords: Beaked whale, Interstitial nephritis, Alpha herpes virus, Diagnosis Background [8]. Gamma-HV have been identified in mucosal lesions in The presence of herpesviruses (HV) in cetaceans was bottlenose dolphins, Risso’sdolphins(Grampus griseus), shown in the late 1980s by electron microscopy (EM) ana- dwarf sperm whales (Kogia sima) and Blainville’s beaked lysis demonstrating HV-like particles in skin biopsies from whales (Mesoplodon densirostris) [8-10]. beluga whales (Delphinapterus leucas) [1,2]. EM analysis The capacity of HV to cause disease in cetaceans is un- also demonstrated that HV was associated with encephal- clear and may show variations depending on the different itis in a harbor porpoise (Phocoena phocoena) [3] and with conditions between individuals or within species [7-10]. skin lesions of dusky dolphins (Lagenorhynchus obscures) Recently, systemic herpes viral lesions have been reported [4]. In addition to EM, immunoperoxidase staining [3], in a striped dolphin, likely secondary to the immunosup- serum neutralization and enzyme-labeled immunosorbent pression caused by morbillivirus co-infection [11]. assays have been used as indicators for the presence of Tubulointerstitial nephritis is characterized by inflam- HV in cetaceans [5]. Alpha-HV have been associated with matory and degenerative changes in tubular epithelium fatal systemic infections in bottlenose dolphins (Tursiops associated with interstitial lymphoplasmacytic cell infil- truncatus) [6], a Cuvier’s beaked whale (Ziphius caviros- tration [12]. Interstitial lymphoplasmacytic nephritis is a tris) [7], and with cutaneous lesions in bottlenose dolphins frequent histopathological finding in cetaceans. However, intralesional etiologies or pathogenic virus or bacteria * Correspondence: [email protected] have rarely been associated with this type of lesion. Here 1 Unit of Veterinary Histology and Pathology, Institute for Animal Health we report the presence of tubulointerstitial nephritis in a (IUSA), Veterinary School, University of Las Palmas de Gran Canaria, Canary Islands, Spain Full list of author information is available at the end of the article © 2012 Arbelo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Arbelo et al. BMC Veterinary Research 2012, 8:243 Page 2 of 7 http://www.biomedcentral.com/1746-6148/8/243 beaked whale stranded in the Canary Islands caused by a To obtain additional nucleotide sequences (approxi- novel cetacean cytopathogenic alpha-HV. mately 700 bp in length), products of the external PCR from positive samples were amplified using the primers Methods DFA and KG1. Amplified DNA fragments of the predicted On April 18th, 2004, an old adult male Mesoplodon den- size were purified and cloned into the plasmid vector sirostris (420 cm long) with a poor body condition was pGEM-T (Promega Corporation) following the manufac- observed to be alive near the shore on Tenerife Island turer’s instructions and sequenced with the corresponding (Granadilla de Abona, Geographic Universal Trasverse T7 primer (forward) and SP6 primer (reverse). Mercator (UTM) coordinate system, X 348338, Y 3102515). Two clones for each sample were selected. Once the con- The whale died soon after beaching (Figure 1). Necropsy sensus nucleotide sequence was obtained and the primer was started 8 hours postmortem (code 1–2) [13]. binding sites excluded, the Clustal W option in MEGA 4.0 Routine sampling for histological, EM, immunohistologi- was used to deduce the corresponding amino acid se- cal, bacteriological and virological studies were performed quence and to construct an unrooted neighbor-joining tree. [7]. Samples fixed in 10% neutral buffered formalin were All the available cetacean HV DNA-polymerase gene routinely processed and embedded in paraffin blocks. sequences reported to date [6-11,17-20] were compared by Histological sections were stained using hematoxylin and their aminoacid (aa) sequences in the GenBank database, eosin (H&E) as well as periodic acid Schiff (PAS). Add- which is the recommended method for molecular HV clas- itionally, immunohistochemistry (IHC) for the detection of sification [21]. morbillivirus and HV antigens was performed as previously Sequences that were 100% identical were excluded (in described [14,15] Tissue samples from lung, liver and kid- those cases, only the sequence published first was conserved ney were submitted for bacteriological analysis [7,13]. in the phylogenetic tree) and other sequences from HV- For molecular viral screening, 0.5 g of frozen tissues sam- assigned species available at the International Committee ples from skin, liver, muscle, lung and kidney were macer- on Taxonomy of Viruses [22] were also included in the ated in 4.5 ml of phosphate buffered saline (PBS), using an comparison. W automatic homogenizer (Precellys 24 Dual homogenizer, Bertin Technologies, Montigny le Bretonneux, France), and Result total nucleic acid was extracted by pressure filtration Pathological findings (QuickGene DNA tissue kit S, FujiFilm Life Science, Tokyo, Gross examination revealed several Xenobalanus sp bar- Japan), following the manufacturer’s instructions. nacles attached to the fluke, multiple cutaneous scars HV DNA detection was conducted by pan nested HV and atrophy of the epaxial musculature. Intravascular PCR [16] involving DFA, KG1 and ILK primers in the ex- gas was noted within the epicardial, mesenteric and por- ternal PCR, and TGV-IYG primers in the internal PCR. tal veins as well as beneath the liver capsule. Nematodes The primer sequences have been described previously were observed within the bronchi. Fish bones, otoliths [16]. To determine positive samples, direct sequencing of and plastic foreign debris were found within the squa- the nested PCR product, approximately 220 base pairs mous stomach. Many parasitic cysts (Phyllobothrium sp.) (bp) was performed using the primers TGVseq – IYGseq. were observed in the retroperitoneal area as well as in Figure 1 Stranded specimen involved. An adult old male Blainville’s beaked whale (Mesoplodon densirostris). Mandibule and tooth features match with an old male beaked whale. Arbelo et al. BMC Veterinary Research 2012, 8:243 Page 3 of 7 http://www.biomedcentral.com/1746-6148/8/243 reniculi, which also presented with membranous glomer- ulonephritis and lymphoplasmacytic interstitial nephritis. Additional findings included intravascular non-staining vacuoles (gas-like bubbles) in the hepatic sinusoids, peri- cardial vessels, within small vessels of intestinal serosa and inter-renicular venous vessels. Mild multifocal para- sitic bronchopneumonia with intraluminal nematodes was noted. Moderate diffuse intracytoplasmic eosino- philic globules were present in the hepatocytes, as fre- quently seen in active (alive) stranded dolphins (personal observation). Cortico-adrenal steatosis and multifocal lymphoplasmacytic adrenalitis were observed in both ad- renal glands but no cell necrosis and/or viral inclusion bodies was found. Signs of testicular atrophy, likely asso- ciated with senescence, were observed with increased interstitial connective tissue and thickened basement membrane surrounding the atrophied tubules with no luminal spermatozoa. Figure 2 Histological, histochemical and ultrastructural findings. Kidney. Cortico- medular area of one reniculum. Multifocal lymphoplamacytic interstitial nephritis. H&E (100X, bar=200 μm). Up right side inset: Higher magnification of renal cortical area showing Immunohistological and ultrastructural study aggregate of lymphoplasmocytic cells and two glomeruli with (PAS HV antigen was only detected in the kidney and immuno- positive). PAS technique (400x, barr= 100 μm). Up right side