Detection of rabies antibodies in wild boars in north-east Romania by a rabies ELISA test.

BACKGROUND: In the last few decades, Romania has been considered one of the European countries most affected by animal rabies, but a combination of oral rabies vaccination (ORV) campaigns in foxes alongside mandatory vaccination of pets has substantially decreased the number of rabies cases in recent years. The objective of this study was to detect rabies antibodies in wild boar serum and thoracic fluid samples collected during the hunting season after ORV campaigns in north-eastern Romania in order to identify if wild boars are substantial competitors to foxes for ORV baits. RESULTS: When the 312 wild boar samples were tested by ELISA (BioPro ELISA, Czech Republic), 42.31% (132/312) demonstrated rabies antibodies. In order to compare these wild boar results in terms of the percentage of immunisation, fox samples were also included in the study, and in this case only 28.40% (98/345) demonstrated rabies antibodies by ELISA. To check the diagnostic sensitivity and specificity of this ELISA, those samples with a sufficient volume from both species that had tested either negative or positive with an initial ELISA were then tested with the Fluorescent Antibody Virus Neutralisation (FAVN) assay. The overall concordance between the BioPro ELISA and FAVN test was 74.26% (75/101) in wild boar samples and 65.66% (65/99) in fox samples, 140 out of 200 samples being correlated with the two methods, although no significant statistical difference (p = 0.218) between the two species was registered. We found a good agreement by both tests for the ELISA-positive samples (91.30%), however the situation was different for the ELISA-negative samples, where a low agreement was demonstrated (41.18%). CONCLUSIONS: This study reports for the first time the presence of rabies antibodies in wild boar samples collected during the hunting season in Romania after ORV campaigns in rabies endemic areas. It is also the first study to demonstrate that ELISA BioPro can be used on wild boar samples with satisfactory results compared to the FAVN test for this species.

Disseminated Infection with Aspergillus fumigatus in a Scarlet Macaw Parrot (Ara macao)-A Case Report.

A 3-year-old male scarlet macaw parrot (Ara macao) was presented to the Exotic Animal Clinic at the Faculty of Veterinary Medicine, Iași University of Life Sciences (Iași, Romania) for its postmortem examination. According to the owner, the parrot had been raised only in captivity and after 5 days of inappetence, lethargy, and mild respiratory clinical signs, the parrot died. The post mortem examination revealed various-sized granulomas and caseous plaques in the lungs, air sacs, spleen, intestinal serosa, and liver. Microscopically, the granulomas were characterized by a necrotic center and the infiltration of numerous multinucleated giant cells and epithelioid-like cells and by the presence of hyphae typical of Aspergillus spp. Moreover, in the liver tissue, a diffuse inflammation, with numerous fungal hyphae, was noted. The fungal culture and the PCR assay allowed for the isolation and identification of Aspergillus fumigatus from the lung and liver samples. The macroscopical lesions and the histopathological findings, with the fungal isolation and molecular confirmation of Aspergillus fumigatus by nested PCR, provided the basis for the diagnosis of disseminated aspergillosis. To the authors' best knowledge, this is the first report of disseminated infection caused by Aspergillus fumigatus in a scarlet macaw parrot (Ara macao).

Whole genome sequencing and phylogenetic characterisation of rabies virus strains from Moldova and north-eastern Romania.

BACKGROUND: Rabies is the oldest fatal zoonotic disease recognised as a neglected tropical disease and is caused by an RNA virus belonging to the genus Lyssavirus, family Rhabdoviridae. METHODOLOGY/PRINCIPAL FINDINGS: A deep molecular analysis was conducted on full-length nucleoprotein (N) gene and whole genome sequences of rabies virus from 37 animal brain samples collected between 2012 and 2017 to study the circulation of rabies virus (RABV) variants. The overall aim was to better understand their distribution in Moldova and north-eastern Romania. Both Sanger and high throughput sequencing on Ion Torrent and Illumina platforms were performed. Phylogenetic analysis of the RABV sequences from both Moldova and Romania revealed that all the samples (irrespective of the year of isolation and the species) belonged to a single phylogenetic group: north-eastern Europe (NEE), clustering into three assigned lineages: RO#5, RO#6 and RO#7. CONCLUSIONS/SIGNIFICANCE: High throughput sequencing of RABV samples from domestic and wild animals was performed for the first time for both countries, providing new insights into virus evolution and epidemiology in this less studied region, expanding our understanding of the disease.

An Overview on Leishmaniasis in Romania: Diagnosis and Therapeutics.

Leishmaniasis, a vector-borne disease considered to be one of the twenty neglected diseases by the World Health Organization, represents one of the public health concerns in endemic countries. In humans, as well as in animal counterparts, the infection can evolve with different clinical localizations, such as those that are cutaneous, mucocutaneous and visceral. Romania has been traditionally considered a nonendemic country for Leishmania species infection and has had sporadic positive human cases; however, the climate change recorded in recent decades has created potentially optimal conditions for the preponderant vectors of Phlebotomus spp., which has lately been identified in various parts of country. Moreover, with people and dogs (the prevailing hosts) traveling in endemic countries, the disease was imported and diagnosed in both species, and became a medical concern. In this review, we focused on the: (1) epidemiological data of leishmaniasis cases, both in humans and animals, reported by Romania; (2) diagnostic tools available for confirmation since there is a lack of gold-standard laboratory methods for human and dog patients; and (3) conventional antileishmanial therapy.

Expression of collagenases (matrix metalloproteinase-1, -8, -13) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in naturally occurring bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are among the most common skin tumors in cattle; their etiology is associated with infection by bovine papillomavirus (BPV) types-1/-2 which are considered oncogenic. Degradation of the extracellular matrix (ECM), especially collagenolysis, is a key event during a series of relevant physiological processes, including tissue remodeling and repair. Various types of proteins are implicated in the regulation of ECM degradation: among these, matrix metalloproteinases (MMPs), a group of zinc-dependent endoenzymes, and tissue inhibitors of matrix metalloproteinases (TIMPs) are known to play a major role. Previous studies reported that aberrant expression of collagenolytic MMPs (MMP-1/-8/-13) and unbalancing between MMPs and TIMPs represent a critical step in tumor growth and invasion; however, studies regarding this topic in bovine cutaneous fibropapillomas are lacking. The aim of this study was to investigate the expression of the collagenases MMP-1/-8/-13 and TIMP-3 in naturally occurring fibropapillomas harboring BPV-2 DNA and normal skin samples. Here, by immunohistochemistry and western blotting analysis, we demonstrated overexpression of MMP-8/-13 along with a down-regulation of MMP-1, associated with a decrease in TIMP-3 levels in tumor compared with normal skin samples. This is the first study describing MMP-1/-8/-13 and TIMP-3 expression in bovine cutaneous fibropapillomas and our results suggest that an impaired expression of collagenases along with an imbalance between MMPs/TIMPs may contribute to an increased collagenolytic activity, which in turn could be important in ECM changes and tumors development.

Expression of matrix metalloproteinases (MMPs)-2/-7/-9/-14 and tissue inhibitors of MMPs (TIMPs)-1/-2 in bovine cutaneous fibropapillomas associated with BPV-2 infection.

Introduction: Bovine papillomaviruses -1/-2 (BPVs) are small non-enveloped double-stranded DNA viruses able to infect the skin of bovids and equids, causing development of neoplastic lesions such as bovine cutaneous fibropapillomas and equine sarcoid. Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases that degrade basal membrane and extracellular matrix, whose function is essential in physiological processes such as tissue remodeling and wound healing. MMPs activity is finely regulated by a balancing with expression of tissue inhibitors of MMPs (TIMPs), a process that is impaired during tumour development. BPV infection is associated with upregulation of MMPs and /or their unbalancing with TIMPs, contributing to local invasion and impairment of extracellular matrix remodeling in equine sarcoid; however, studies regarding this topic in bovine fibropapillomas are lacking. Methods: The aim of this study was to perform an immunohistochemical and biochemical analysis on a panel of MMPs and TIMPs in BPV-2 positive bovine cutaneous fibropapillomas vs. normal skin samples. Results: Immunohistochemistry revealed a cytoplasmic expression of MMP-2 (15/19), a cytoplasmic and perinuclear immunoreactivity of MMP-7 (19/19) and MMP-9 (19/19), along with a cytoplasmic and nuclear pattern of MMP-14 (16/19), accompanied by a cytoplasmic expression of TIMP-1 (14/19) and TIMP-2 (18/19) in tumour samples; western blotting revealed an overexpression of MMP-2 (8/9), MMP-7 (9/9) and MMP-9 (9/9), and a decreased level of MMP-14 (9/9), TIMP-1 (9/9) and TIMP-2 (9/9) in tumour versus normal skin samples. Moreover, gelatine zymography confirmed the expression of pro-active MMP-2 (9/9) and MMP-9 (9/9) and, most importantly, indicated the presence and increased activity of their active forms (82 and 62 kDa, respectively) in tumour samples. Discussion: This is the first study describing MMPs and TIMPs in bovine cutaneous fibropapillomas and our results suggest that their unbalanced expression in presence of BPV-2 may play a significant role in tumour development. A further analysis of supplementary MMPs and TIMPs could bring new important insights into the papillomavirus induced tumours.