Gyula Balka

Naturally occurring parvoviral infection in Hungarian broiler flocks

The major enteric disease (ED) complex in broiler chickens is runting–stunting syndrome and in turkey broilers is poult enteritis mortality syndrome. Viruses from numerous families have been identified in the intestinal tracts of poultry with ED, such as Astroviridae, Coronaviridae, Reoviridae, Rotaviridae, and Parvoviridae. The objective of the present study was to directly demonstrate the presence of the scarcely known chicken parvovirus (ChPV) and turkey parvovirus (TuPV) in Hungarian flocks experiencing clinical signs of ED. ChPV and TuPV infection were demonstrated in 15 chicken flocks and two turkey flocks, in intestinal samples collected between 2008 and 2010. The histopathological investigation revealed enteritis in the duodenum and jejunum, and atrophy of the lymphoid organs. Indirect immunohistochemistry (IHC) suggested the intestinal epithelium of chickens and turkeys as a potential replication site of the virus, similarly to other parvoviruses, while in case of the turkey samples IHC positivity was also observed in the bursa of Fabricius, liver and pancreas. However, no direct connection could be established between the presence of the pathogen in the above-mentioned tissues and the histopathological changes observed in the investigated flocks. The phylogenetic analysis performed on the partial nucleic acid sequence of the NS1 gene revealed an evident clustering tendency of the ChPV and TuPV strains, but also highlighted the potential reciprocal role of these two species in the epidemiology of these viruses. The role of the ChPV and TuPV in the ED is far from understood, but the results of the present study emphasize the fact that in certain, still not fully elucidated conditions, ChPV and TuPV may participate in the emergence of ED in chicken flocks, as suggested by previous experimental infections.

Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus

Abstract A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV®). One TCID50 was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.

Nanoparticle Encapsulated Lipopeptide Conjugate of Antitubercular Drug Isoniazid: In Vitro Intracellular Activity and in Vivo Efficacy in a Guinea Pig Model of Tuberculosis

Considering that Mycobacterium tuberculosis (Mtb) can survive in host phagocytes for decades and currently applied drugs are largely ineffective in killing intracellular Mtb, novel targeted delivery approaches to improve tuberculosis chemotherapy are urgently needed. In order to enhance the efficacy of a clinically used antitubercular agent (isoniazid, INH) a novel lipopeptide carrier was designed based on the sequence of tuftsin, which has been reported as a macrophage-targeting molecule. The conjugate showed relevant in vitro activity on Mtb H37Rv culture with low cytotoxicity and hemolytic activity on human cells. The conjugate directly killed intracellular Mtb and shows much greater efficacy than free INH. To improve bioavailability, the conjugate was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles and tested in vivo in a guinea pig infection model. External clinical signs, detectable mycobacterial colonies in the organs, and the histopathological findings substantiate the potent chemotherapeutic effect of orally administered conjugate-loaded nanoparticles.

Antimycobacterial activity of peptide conjugate of pyridopyrimidine derivative against Mycobacterium tuberculosis in a series of in vitro and in vivo models

New pyridopyrimidine derivatives were defined using a novel HTS in silico docking method (FRIGATE). The target protein was a dUTPase enzyme (EC 3.6.1.23; Rv2697) which plays a key role in nucleotide biosynthesis of Mycobacterium tuberculosis (Mtb). Top hit molecules were assayed in vitro for their antimycobacterial effect on Mtb H37Rv culture. In order to enhance the cellular uptake rate, the TB820 compound was conjugated to a peptid-based carrier and a nanoparticle type delivery system (polylactide-co-glycolide, PLGA) was applied. The conjugate had relevance to in vitro antitubercular activity with low in vitro and in vivo toxicity. In a Mtb H37Rv infected guinea pig model the in vivo efficacy of orally administrated PLGA encapsulated compound was proven: animals maintained a constant weight gain and no external clinical signs of tuberculosis were observed. All tissue homogenates from lung, liver and kidney were found negative for Mtb, and diagnostic autopsy showed that no significant malformations on the tissues occurred.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV) in oral fluid of pigs

Recently oral fluid has become a novel sample type for pathogen nucleic acid and antibody detection, as it is easy to obtain with non-invasive procedures. The objective of the study was to analyze porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV) circulation in growing pigs from three Polish production farms, using Real Time PCR and ELISA testing of oral fluid and serum. Oral fluids were collected every 2weeks, in the same 3-4 pens of pigs aged between 5 and 17weeks. Additionally, blood samples were collected every 4weeks from 4 pigs corresponding to the same pens as oral fluid and tested for the presence of PRRSV nucleic acid (pooled by 4) and antibodies. In farm A no PRRSV circulation was detected and only maternal antibodies were present. In farm B and farm C antibodies to PRRSV in serum and oral fluid were detected in most samples. In farm B PRRSV Type 1 was detected in 80.9% of oral fluid samples and in 58.3% of serum pools, and in farm C in 92.8% of oral fluid samples and 75% serum pools. Striking differences were observed between different pens in PRRSV detection patterns. In farms B and C ORF5 sequence analysis showed the presence of wild type strains which were about 84-85% identical to the modified live vaccine used. In all three farms two waves of IAV shedding with oral fluid were detected, in weaners and fatteners.

Novel mastadenovirus infection and clinical disease in a pygmy marmoset (Callithrix [Cebuella] pygmaea).

We describe the detection and successful isolation of a novel mastadenovirus from a pygmy marmoset (Callithrix [Cebuella] pygmaea) that died following an episode of severe respiratory signs. Pathologic/histopathologic examination revealed hydrothorax and catarrhal bronchopneumonia with pronounced desquamation of the bronchiolar epithelial cells, while in other airways a marked hyperplasia of the epithelial lining and numerous giant cells could be observed. We obtained partial sequence data from the adenoviral DNA-dependent DNA-polymerase gene of the isolated strain and analyses of this region showed the highest level of identity to the recently described bat adenoviruses (strains PPV1 and TJM) and the type 2 canine adenovirus. Similar results were gained by phylogenetic calculations indicating that this novel marmoset adenovirus is only distantly related to reference Old and New World primate adenoviruses and formed a monophyletic group with bat and canine adenoviruses and the equine adenovirus 1. Even though the source of the infection remained unknown, our results could imply the possibility of a cross-species transmission of the virus from an anonymous host to the pygmy marmoset.

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).

Abstract Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10–50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.

Immunohistochemical Characterization of Type II Pneumocyte Proliferation after Challenge with Type I Porcine Reproductive and Respiratory Syndrome Virus

The aim of this study was to characterize histologically and immunohistochemically the lung lesions developing in growing pigs, 10 and 21 days after experimental challenge with a field strain of porcine reproductive and respiratory syndrome virus (PRRSV). Lung lesions were scored for (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intra-alveolar necrotic debris, (4) intra-alveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation. Immunohistochemistry was performed using antibodies specific for cytokeratin, Ki67, thyroid transcription factor (TTF)-1, the myelomonocytic marker MAC387 and PRRSV. Anti-TTF-1 identified type II pneumocytes and there was marked proliferation of these cells compared with control lung (P <0.05). Anti-cytokeratin labelled type I and II pneumocytes as well as bronchial epithelial cells; however, this labelling was not suitable for cell counting purposes. There was a correlation between lesion severity and the number of cells expressing Ki67 (P <0.05).

DISTRIBUTION OF PORCINE CIRCOVIRUS 2 CAP ANTIGEN IN THE LYMPHOID TISSUE OF PIGS AFFECTED BY POSTWEANING MULTISYSTEMIC WASTING SYNDROME

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Bačka County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Bačka County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyers patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.

First report of a malignant collision skin tumour with malignant melanoma and anaplastic sarcoma components in a dog.

This paper describes the occurrence of a rare skin tumour that has been removed surgically from the upper lip of a 13-year-old Tibetan spaniel. The tumour was 0.5 cm in diameter and macroscopically appeared as a single dermal mass, but histopathological analysis identified it as a biphasic collision mixed tumour. In the anatomically uniform tumour, 70% (4 mm in diameter) of the total parenchyma was formed by a high-grade sarcoma (with the presence of giant cells), and about 30% of it (1 mm in diameter) was a malignant melanoma (again with the presence of giant cells). The histologically distinct, but anatomically uniform tumour parts were separated by a macroscopically invisible, non-neoplastic epithelial process originating from the overlying hyperplastic epidermis. The two malignant components did not infiltrate the peritumoural vessels and each others substance. In the sarcoma part, the mitotic and apoptotic indexes were 32 and 8, respectively, whereas in the melanoma part the same parameters were 10 and 6, respectively. During the immunohistochemical investigations anti-α-SMA, anticytokeratin AE1-AE3, anti-Melan-A, anti-Ki-67 and anti-claudin-5 antibodies were applied. In conclusion, this is the first report of a primary cutaneous malignant biphasic collision mixed tumour formed by an anaplastic sarcoma with giant cells and a malignant melanoma.