Comparative examination of a rapid immunocytochemical test for the detection of highly pathogenic avian influenza virus in domestic birds in field outbreaks.

The quantitative real-time reverse polymerase chain reaction (RRT-PCR) is the preferred test method for the diagnosis of avian influenza (AI), but can be performed only in specialized laboratories. Different antigen detection methods for the diagnosis of AI were previously reported to be specific and sensitive in field outbreaks. These tests can be performed in basic countryside labs. Brain smears of domestic birds (n = 105) collected during AI field outbreaks were examined with immunocytochemistry (IC). The results were statistically analysed by comparing IC to brain histology (BH), and immunohistochemistry (IHC), to gross pathological examination (GP) (n = 105), and RRT-PCR (n = 91). AI was diagnosed with RRT-PCR in 66 cases. IC and IHC were positive in 59/66 (90%) and 60/66 (91%) cases, respectively. Lesions suspicious for AI were detected with GP and HP in 66/66 (100%) and 61/66 (92%) cases, respectively. An almost perfect agreement was found between RRT-PCR, IC, IHC, and HP. Substantial agreement was found between IC and GP, between IHC and GP, between HP and GP, and between RRT-PCR and GP. The chromogen-based IC test presented in this study produces durable staining, which can be evaluated using a simple brightfield microscope. The test is rapid (can be completed in 2 h), sensitive (90%), specific (100%), and cost-effective, which makes the method suitable for routine diagnostic tests in AI epidemics.RESEARCH HIGHLIGHTSAvian influenza virus (AIV) antigen detection was examined in field outbreaks.Bird brain smears were tested using immunocytochemistry (IC).IC results strongly correlated with real-time RT-PCR results.The IC method was rapid, specific, sensitive, and cost-effective in AIV field outbreaks.

Detection of Frederiksenia sp. isolated from a cat with nephritis - Short communication.

In this paper we report the phenotypic and partial genetic characterisation of a novel bacterium strain isolated from a cat with severe nephritis. Multilocus sequence analysis was performed on the 16S rRNA and three housekeeping (recN, rpoB, infB) gene sequences obtained by PCR. In accordance with the results of phenotypic tests, the phylogenetic analyses confirmed the relatedness of the new strain (6036) to the family Pasteurellaceae. On the phylogenetic trees, strain 6036 appeared in a separate branch, closest to that of the type species (Frederiksenia canicola) of the genus Frederiksenia. These two bacteria shared 95.14 and 76.88% identity in their partial 16S rRNA and recN gene sequences, respectively. The rpoB- and infB-based phylogenetic analyses indicated that strain 6036 is most closely related to Bibersteinia trehalosi (with 90.58% identity) and [Haemophilus] felis ATCC 49733 (89.50% identity), respectively. The predicted genome identity values, based on the recN gene sequences, suggested that strain 6036 can be classified into the genus Frederiksenia as a novel species. A PCR method, specific to strain 6036, was developed to allow its rapid and accurate identification and differentiation from F. canicola and other species of Pasteurellaceae. The minimal inhibitory concentrations of 18 antimicrobial agents for strain 6036 were also determined.

Low incidence of Schmallenberg virus infection in natural cases of abortion in domestic ruminants in Hungary.

An epizootic caused by a new orthobunyavirus called Schmallenberg virus (SBV) was recognised in European ruminants in 2011 and 2012. The re-emergence of the infection was reported in several countries in the subsequent years. Although the main clinical sign of SBV infection is abortion, the impact of SBV in natural cases of abortion in domestic ruminants had not been systematically examined before this study. The aim of the study was to investigate the role of SBV infection and to compare it to the importance of other causes of abortion by examining 537 natural cases of abortion that had occurred between 2011 and 2017 in Hungary. The cause of abortion was determined in 165 (31%) cases. An infectious cause was proved in 88 (16%) cases. SBV infection was found only in a total of four cases (0.8%) using real-time polymerase chain reaction. Three of them proved to be inapparent SBV infection, and one case was attributed to SBV-induced abortion by detecting non-purulent encephalitis and SBV nucleoprotein by immunohistochemistry in a brain tissue sample. According to the results, SBV played a minor role in natural cases of domestic ruminant abortion in Hungary during the 7-year period following the first SBV outbreak in 2011.

Isolation and complete genome sequence analysis of a novel ovine adenovirus type representing a possible new mastadenovirus species.

Pathological examination of a suckling male lamb showed severe viral pneumonia with suspected bacterial superinfection. Adenovirus was detected by immunohistochemical examination of the affected lung samples. Detection of the suspected adenovirus by PCR and subsequent isolation of the virus were successful. Using next-generation sequencing, the full genome of this ovine adenovirus was sequenced and analysed. A genome sequence comparison showed that it was a novel mastadenovirus type (named "ovine adenovirus 8") that did not belong to any of the established adenovirus species. The genome is 36,206 bp long, containing 93-bp inverted terminal repeats and 29 predicted genes, including the two genus-specific genes (encoding proteins V and IX). Ovine adenovirus 8 shows the closest relationship to ovine adenovirus 6. These two viruses seem to merit the establishment of a novel ovine mastadenovirus species for them, for which we proposed the name "Ovine mastadenovirus C".

High mortality caused by Bibersteinia trehalosi septicaemia in adult sheep - A case report.

The disease induced by Bibersteinia trehalosi usually occurs in lambs. It is triggered by certain stress factors and often emerges in the form of severe outbreaks. In adult sheep, only sporadic cases have been reported so far. This paper reports a B. trehalosi-induced high-mortality case occurring only in adult sheep. Seventy out of 628 adult sheep (11%) died in the affected pen during the six days of the outbreak. None of the 146 lambs kept in the neighbouring pen showed any clinical signs during that period. Several preceding events (shearing, vaccination and antiparasitic treatment) can be regarded as factors predisposing to the disease. Five adult sheep (4 females and 1 male) were sent for laboratory examination. Clinical, gross pathological, histological and bacteriological examinations revealed results corresponding to those reported previously in lambs that had died of a B. trehalosi-induced septicaemia.

Re-emergence of bovine haemorrhagic septicaemia in Hungary.

This paper reports an outbreak of haemorrhagic septicaemia caused by Pasteurella multocida B:2 in beef calves, a disease that has not been described in the Hungarian literature since 1943, and has not been reported to the World Organisation For Animal Health (OIE) since 1970. Acute haemorrhagic septicaemia was confirmed in beef calves on one small farm, and was suspected on two further nearby holdings with concomitant unexplained losses. The source of the infection could not be determined. Apart from a short duration of depression and loss of appetite, the affected calves developed characteristic distal limb oedema. Gross findings in two calves submitted for laboratory examinations included subcutaneous oedema and haemorrhages on serous membranes, and in one case severe pharyngeal lymph node enlargement was observed. Histological examinations revealed lesions characteristic of septicaemia. Moderate to large amounts of Pasteurella antigens were detected in all organs tested by immunohistochemistry. Two isolates of P. multocida (Pm240, Pm241) were cultured from these cases and examined in detail. These were identified as P. multocida ssp. multocida biovar 3. Both were toxA negative and belonged to serotype B:2. Multilocus sequence typing was used to assign these to a new sequence type (ST64) that is closely related to other haemorrhagic septicaemia causing strains of P. multocida regardless of the host.

Subclinical Tick-Borne Encephalitis Virus in Experimentally Infected Apodemus agrarius.

OBJECTIVES: In this study, we investigated the dose dependence of tick-borne encephalitis virus (TBEV) infection in one of the reservoirs, i.e. Apodemus agrarius, a small rodent species. METHODS: The animals were challenged with TBEV per os and intramuscularly with infectious doses ranging from 1 to 1,500 plaque-forming units (pfu). Clinical signs were recorded and clinical and pathological features were evaluated by histological, immunohistochemical, and serological methods. RESULTS: High perorally administered infectious doses resulted in virus replication in the brain, which is the first sign of subclinical viral encephalitis in the Apodemus genus. The animals seroconverted at infectious doses greater than 100 pfu, and all animals remained asymptomatic. CONCLUSION: Our work shows the first evidence that subclinical TBEV encephalitis may occur in Apodemus species, depending on the virus load of the inoculum. The antiviral response of the local innate immune system may influence the resistance of Apodemus individuals to lower infectious doses. Per oral/nasal infection seems to be more dangerous for the host than other routes of infection.

Full-length genome sequence analysis of a Hungarian porcine reproductive and respiratory syndrome virus isolated from a pig with severe respiratory disease.

Here, we report the isolation of a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) strain from a clinical outbreak of severe respiratory problems and high fever. Next-generation sequencing was used to determine the complete genome sequence of the isolate (9625/2012). The virus belongs to a new branch within subtype 1, clade D, and shows the highest similarity to PRRSV Olot/1991 and to the Amervac vaccine strain. Mutation analysis of 9625/2012 revealed no evidence of recombination but did show a high proportion of amino acid substitutions in the putative neutralizing epitopes, suggesting an important role of selective immune pressure in the evolution of PRRSV 9625/2012.

First isolation and characterization of Brucella microti from wild boar.

BACKGROUND: Brucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary. RESULTS: The B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 µg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile. CONCLUSIONS: Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity in this Brucella species.

Vascular lesions and pneumonia in a pig fetus infected by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) associated reproductive disease was diagnosed in a herd containing only gilts. A single case of abortion occurred and no other disorder was evident in the herd. PCV2 antigen and/or DNA were detected in two aborted fetuses. One of the fetuses, revealing both PCV2 DNA and antigen, presented multinucleated giant cells, severe vascular lesions (intramural oedema, fibrinoid necrosis, mild lympho-histiocytic vasculitis, fibrin thrombi) and mild non-suppurative inflammation in the lungs. Other abortifacient infections were not found. This is the only report of PCV2-induced abortion in Hungary since 1999, when PCV2-associated disease was first discovered in the country.

First detection of Pasteurella multocida type B:2 in Hungary associated with systemic pasteurellosis in backyard pigs.

This is the first report of Pasteurella multocida type B in Hungarian pigs. This disease was observed in backyard-raised pigs in three households within a small area. Neither the source of the infection nor the epidemiological connection between any of the premises could be determined. The most consistent lesion was dark red discolouration of the skin of the ventral neck and brisket, with accompanying oedema and haemorrhages. The morbidity was low and lethality relatively high, with three dead (50%) and two euthanised (33%) out of six affected animals. A total of three isolates of P. multocida (P55, P56 and P57) were cultured from these cases and examined in detail. These were identified as P. multocida ssp. multocida biovar 3. All were toxA negative and belonged to serotype B:2. Multilocus sequence typing was used to assign these to a new sequence type (ST61) that is closely related to other haemorrhagic septicaemia causing strains of P. multocida regardless of the host. M13 polymerase chain reaction and virulence-associated gene typing also show that type B strains form a highly homogeneous, distinct phylogenic group within P. multocida.

Establishment of a Mycoplasma hyorhinis challenge model in 5-week-old piglets.

Introduction: Mycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate. Methods: Five-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals. Results: Pericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain. Discussion: Our results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection.

Microbiological and pathological examination of fatal calf pneumonia cases induced by bacterial and viral respiratory pathogens.

The infectious origin of fatal cases of calf pneumonia was studied in 48 calves from 27 different herds on postmortem examination. Lung tissue samples were examined by pathological, histological, bacterial culture, virus isolation and immunohistochemical methods for the detection of viral and bacterial infections. Pneumonia was diagnosed in 47/48 cases and infectious agents were found in 40/47 (85%) of those cases. The presence of multiple respiratory pathogens in 23/40 (57.5%) cases indicated the complex origin of fatal calf pneumonia. The most important respiratory pathogens were Mannheimia-Pasteurella in 36/40 (90%) cases, followed by Arcanobacterium pyogenes in 16/40 (40%) cases, Mycoplasma bovis in 12/40 (30%) cases, and bovine respiratory syncytial virus in 4/40 (10%) cases. Histophilus somni was detected in 2/40 (5%) cases, while bovine herpesvirus-1, bovine viral diarrhoea virus and parainfluenza virus-3 were each found in 1/40 (2.5%) case. Mastadenovirus, bovine coronavirus, influenza A virus or Chlamydiaceae were not detected.

Detection of porcine circovirus in rodents - short communication.

Porcine circoviruses (PCV) are present worldwide, infecting domestic pigs and wild boars alike. Studies under laboratory conditions indicated that PCV can be taken up by mice and the virus can replicate in these animals. The possible role of rodents in maintaining and transmitting PCV2 infection in the field has not been investigated yet. The present study reports the detection of PCV2, the pathogenic form of the virus, in mice and rats. A number of rodents, such as mice, rats and voles, were collected at PCV2-infected farms and also outside pig herds and tested for the presence of the virus by polymerase chain reaction (PCR). The results indicated that PCV2 can be present both in mice and rats (65.0% and 23.8% positivity, respectively) on the infected premises, but those rodents that were collected outside pig farms remained negative for PCV2.

Tissue tropism of highly pathogenic avian influenza virus subtype H5N1 in naturally infected mute swans (Cygnus Olor ), domestic geese (Aser Anser var. domestica), pekin ducks (Anas platyrhynchos) and mulard ducks ( Cairina moschata x anas platyrhynchos).

The 2006 epidemic due to highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Hungary caused the most severe losses in waterfowl which were, according to the literature at the time, supposed to be the most resistant to this pathogen. The presence of pathological lesions and the amount of viral antigen were quantified by gross pathology, histopathology and immunohistochemistry (IHC) in the organs of four waterfowl species [mute swans (n = 10), domestic geese (n = 6), mulard ducks (n = 6) and Pekin ducks (n = 5)] collected during the epidemic. H5N1 subtype HPAIV was isolated from all birds examined. Quantitative real-time reverse transcriptase-polymerase chain reaction (qRRT-PCR) was also applied on a subset of samples [domestic geese (n = 3), mulard (n = 4) and Pekin duck (n = 4)] in order to compare its sensitivity with IHC. Viral antigen was detected by IHC in all cases. However, the overall presence of viral antigen in tissue samples was quite variable: virus antigen was present in 56/81 (69%) swan, 22/38 (58%) goose, 28/46 (61%) mulard duck and 5/43 (12%) Pekin duck tissue samples. HPAIV subtype H5N1 was detected by qRRT-PCR in all birds examined, in 19/19 (100%) goose, 7/28 (25%) mulard duck and 12/28 (43%) Pekin duck tissue samples. As compared to qRRTPCR, the IHC was less sensitive in geese and Pekin ducks but more sensitive in mulard ducks. The IHC was consistently positive above 4.31 log10 copies/reaction but it gave very variable results below that level. Neurotropism of the isolated virus strains was demonstrated by finding the largest amount of viral antigen and the highest average RNA load in the brain in all four waterfowl species examined.

Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody.

Following the introduction of African swine fever virus (ASFV) into Europe in 2007, ASFV infection has spread continuously over the past years and it became a high level disease threat in Europe and also Asia. Examination of suspect clinical cases for ASF with rapid and sensitive laboratory methods can substantially contribute to the detection and characterization of new outbreaks. In this study two sensitive tests were developed for the detection of the p72 major capsid protein of ASFV both in cell culture with an immunocytochemical (IC) and in tissue samples with an immunohistochemical (IHC) method using a commercially available mouse monoclonal antibody (clone 1BC11). The IC test was able to detect the virus at high virus dilutions in cell culture and the IHC test indicated the presence of ASFV in all formalin-fixed and paraffin-embedded tissue samples collected from two wild boars. The reported IC and IHC methods were found to be useful ancillary laboratory tests for research purposes and for the diagnosis of acute ASF.

Rapid spread and emergence of heartworm resulting from climate and climate-driven ecological changes in Hungary.

In Europe, Dirofilaria immitis persists mainly in the southern countries with a Mediterranean climate. Because spreading of heartworms from these countries towards the northern ones could be observed in the past decades, necropsy records of 4076 Hungarian dogs were reviewed for heartworm infections. The first autochthonous canine D. immitis case was detected on the Great Hungarian Plain in 2007. Until 2011, the number of heartworm infection cases was low, and these cases were restricted to a small part of the Great Hungarian Plain. Since 2012, the number of cases has increased considerably, and the rapid expansion of the parasite's geographic range could also be observed. Our retrospective study has revealed that most of the Hungarian territory became a heartworm endemic region, and the prevalence of infection greatly multiplied over the past 12 years. The establishment, rapid spread, and emergence of D. immitis may be mainly explained by the warming climate in Hungary. However, the partly climate-driven spread of the most important reservoir host in wildlife, the golden jackal (Canis aureus) from the Mediterranean Balkan Peninsula might have also played a significant role. This study is an example of the rapid spread and emergence of pathogens resulting from climate and climate-driven ecological changes. Because a continuous increase in the temperature and further dispersal of golden jackals in Europe are projected, further spread and emergence of heartworm can be expected. Similar spread and emergence of D. immitis could be observed in North America. It cannot be excluded that similar reasons (global warming and rapid dispersal and population growth of the most important wild canine reservoir host) are in the background on both continents.

Papillomavirus-associated fibropapillomas of red deer ( Cervus elaphus ).

Oval, firm, cutaneous tumours with a rough, hairless, pigmented surface, exhibiting a moderately pronounced papillary structure were detected on the abdominal skin of two young red deer ( Cervus elaphus ). One animal was shot in Lower Austria in 2004, the other at a deer farm in Hungary in 2007. Histological examination of both samples classified the tumours as fibropapillomas, showing marked proliferation of fibroblasts and connective tissue, accompanied by hyperkeratosis, parakeratosis and acanthosis of the overlaying epidermis, and occasional foci of inflammation. The distribution of cytokeratin and vimentin was characterised in the lesion. The presence of papillomavirus (PV) antigen was demonstrated by immunohistochemistry in both cases. Papillomavirus-specific DNA was successfully amplified by PCR from one sample. The obtained partial nucleotide sequence of the L2 ORF exhibited the highest critical identity values with the homologous regions of Delta-papillomaviruses, especially the Roe deer papillomavirus (93%). Phylogenetic analysis of the partial L2 ORF sequence alignment of 10 papillomaviruses by both neighbour-joining and maximum parsimony method confirmed that the Red deer PV is very closely related to the Western roe deer papillomavirus (CcPV1).

Evidence of CPV2c introgression into Croatia and novel insights into phylogeny and cell tropism.

Canine parvovirus type 2 (CPV2) emerged for the first time in 1978 and evolved into two antigenic variants CPV2a and CPV2b and the third new antigenic variant CPV2c reported in 2000 in Italy. During 2014 unexplained outbreaks of gastroenteritis were observed in kennels where an extensive vaccination program was ongoing and where vaccinated animals showed pathologic lesions consistent with typical parvovirosis. The aim of this study was to investigate whether CPV2 could have played a role in the emergence of these cases and to evaluate genetic or pathological specificities of the virus and the disease. Using PCR and phylogenetic analysis we showed that the CPV2c variant is circulating in Croatia and is in close relationships with isolates from North and South America. Histopathological lesions and cell tropism that are known for CPV2 we are reporting the identification of the virus in glial cells and ovaries. It seems that evolution of CPV and CPV2a-c and adaptation to dogs are two independent events. Croatian isolates had specific and some unique amino acid mutations under positive selection. The effect of the alterations on the immunoglobulin binding cannot be excluded.

Unusual transmission route of Lymphogranuloma venereum; following sexual contact with a female donkey.

Here, we present a 20-year-old man who presented with painful inguinal and femoral masses. He gave a history of sexual contact with a mare 14 days before his recent illness. He was diagnosed with lymphogranuloma venereum based on the histopathological findings and a high titre of IgG (1:1400).