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Forty-six golden jackals (Canis aureus) were collected between November 2020 and February 2021 in five counties of Serbia. Lung samples were screened for the presence of Pneumocystis DNA by pan-Pneumocystis PCR on the mtLSU rRNA gene obtaining PCR products of 370 bp in length. Pneumocystis DNA was detected in the lungs from 6/46 (13.04%) golden jackals. Four were females and two were males; four were classified as adults and two as subadults. Positive samples were confirmed in 4/5 investigated counties. No gross pathologic lung lesions were observed in this study. The sequences of Pneumocystis spp. from golden jackals were identical to one another and showed the highest similarity with Pneumocystis spp. sequences of dogs (98% nucleotide identity). The genetic variation was comparable to Pneumocystis spp. of raccoon dogs (95-97% nucleotide identity), red foxes (91-95% nucleotide identity), ferrets (86% nucleotide identity), and another Pneumocystis type in dogs (P. canis Ck2, 81% nucleotide identity) was higher. Golden jackals may be carriers and may play a nonnegligible role in the spread of Pneumocystis spp. Although this finding cannot be directly related to any clinical manifestation or pathologic lesions, a possible role in the exacerbation of different pulmonary disorders should be considered.

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans., Cissé, Ma et al. utilize genomic data from Pneumocystis species infecting macaques, rabbit, dogs and rats to investigate the molecular basis of host specificity in Pneumocystis. Their analyses provide insight to the specific adaptations enabling the infection of humans by P. jirovecii.

Infections of pigs with porcine circovirus type 2 (PCV2) can lead to various clinical conditions including reproductive disorders (PCV2-RD). In general, a transplacental infection of fetuses leads to mummification and stillbirth. So far, PCV2-RD has mainly been described in specific-pathogen-free (SPF) herds or farms with a high proportion of gilts. From December 2018 to February 2019, a high abundance of mummified fetuses (15.5%) was observed in two farrowing groups in an Austrian piglet-producing farm. PCV2 DNA was detected using qPCR in organs of all six investigated fetuses (2.07 × 108–1.09 × 1012 PCV2) genome equivalents/g tissue and via in situ hybridisation in organs from five fetuses, while histologic lesions were not observed in a single fetal heart. All isolates were sequenced and identified as PCV2d. After the implementation of a regular vaccination of all sows against PCV2, the abundance of mummified fetuses dropped to 3.5% in May 2019. In contrast to previous reports about PCV2-RD, this farm was neither an SPF herd nor a start-up herd with a high proportion of gilts. The implementation of regular PCV2 vaccination helped to reduce the abundance of mummified fetuses substantially.

‘Staggering disease’ is a neurological disorder considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 28 out of 29 cats with non-suppurative meningoencephalomyelitis and ‘staggering disease’-like neurological disorder from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work strongly indicates RusV as the long-sought cause of feline ‘staggering disease’. Given its broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans.

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of trained annotators and to validate a deep learning-based algorithm for the THS. Digitized, iron-stained cytological specimens were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked inter-observer variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance of the could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared to the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7% whereas the algorithm had an accuracy of 92.3% with no marked difference in correlation to chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

At a commercial farrow-to-feeder pig system with 2,100 sows in Serbia, lesions resembling porcine ear necrosis syndrome were observed in 80% of the weaned pigs at 45–50 days of age. Pathomorphological examinations were carried out on 10 pigs that had been found dead. The gross lesions ranged from mild, superficial dermatitis to severe, deep inflammation with exudation, ulceration and necrosis. Histopathological examination revealed erosive and ulcerative dermatitis of the pinna with neutrophilic and lymphocytic infiltration and bacterial colonies in the crusts. Staphylococcus aureus (MRSA strain), Staphylococcus hyicus and Streptococcus group C were cultivated from eight, S. hyicus from two ear tissue scraping samples. All 10 samples were positive for treponemes and phylogenetic analysis of two polymerase chain reaction products confirmed the relationship to Treponema (T.) medium/vincentii and Treponema pedis. Treponemes were also detected in seven oral swabs that were analysed to obtain evidence of the transmission of this bacterium by ear biting. The contribution of non-infectious factors to this misbehaviour could not be ruled out as the crude protein concentration of the feed was inappropriate and the climate of the pig house was suboptimal. The concentrations of selected mycotoxins in the feed were not elevated. However, the contribution of both infectious and non-infectious factors to the onset of disease was most probable.

Enteric disease in pigs is usually of multifactorial etiology, including infectious and non-infectious factors. In many cases of endemic diarrhea in weaner-to-finisher pigs, the combination of 2 or more microorganisms leads to aggravation of intestinal lesions and, consequently, clinical signs. We autopsied a 4-mo-old fattening pig with diarrhea and diagnosed severe fibrinonecrotizing typhlocolitis. Numerous spiral-shaped bacteria and amoeba-like PAS-positive protozoa were observed in the cecal and colonic mucosa and submucosa. Brachyspira hyodysenteriae was detected by PCR from colonic content. By in situ hybridization, large numbers of Entamoeba polecki were found within the lamina propria and submucosa; moderate numbers of Blastocystis sp. and scattered trichomonads were present in intestinal content. In addition, Entamoeba polecki, Balantidium spp., Blastocystis sp., and Trichomonas sp. were also detected by PCR. info:eu-repo/semantics/acceptedVersion

Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species, {"references":["Ma L, Chen Z, Huang DW, Cissé OH, Rothenburger JL, Latinne A, Bishop L, Blair R, Brenchley JM, Chabé M, Deng X, Hirsch V, Keesler R, Kutty G, Liu Y, Margolis D, Morand S, Pahar B, Peng L, Van Rompay KKA, Song X, Song J, Sukura A, Thapar S, Wang H, Weissenbacher-Lang C, Xu J, Lee C-H, Jardine C, Lempicki RA, Cushion MT, Cuomo CA, Kovacs JA. 2020. Diversity and complexity of the large surface protein family in the compacted genomes of multiple Pneumocystis species. mBio 11:e02878-19. https://doi.org/10 .1128/mBio.02878-19."]}

Hippocampal necrosis and hippocampal sclerosis in cats is a neuropathological entity which is a major concern in feline epilepsy. The aim of our study was to identify associated pathologic brain lesions possibly serving as aetiological triggers in this condition. Therefore, the formalin-fixed and paraffin waxembedded brain tissue of 35 cats diagnosed with hippocampal necrosis or sclerosis was examined retrospectively. In 26 cats inflammatory infiltrates could be found in the hippocampus or adjacent brain regions. Fifteen out of these animals demonstrated mild to moderate infiltrations by lymphocytes and complement deposition in the hippocampus similar to human limbic encephalitis, seven showed unspecific, predominantly non-suppurative inflammation, and two demonstrated suppurative inflammation of the hippocampus or adjacent brain regions. Additionally, one cat was diagnosed with central nervous manifestation of feline infectious peritonitis virus and another one with cerebral Toxoplasma gondii infection. Intracranial neoplasia was present in five cases altogether. Three of them comprised meningioma which was present additionally to lesions resembling limbic encephalitis in two cases, and a dentate gyrus alteration in one case. The other two tumour-associated cases comprised oligodendroglioma. Structural alterations of the dentate gyrus together with hippocampal sclerosis were encountered in three cases in total. Besides the case associated with a meningioma, one case demonstrated lesions resembling limbic encephalitis. A vascular infarct in the temporal lobe was encountered in one cat. In four cases no lesions other than hippocampal necrosis or sclerosis were found. The involvement of feline immunodeficiency virus infections, which may be able to produce hippocampal lesions, was not encountered in the cats examined.

We evaluated 43 published cases of dogs with confirmed Pneumocystis infection regarding the value of clinical parameters indicating the presence of the disease as well as tools for the detection of the pathogen. The assessed parameters included clinical signs, laboratory findings, results of thoracic radiography, autopsy, histopathology, methods for the detection of Pneumocystis, as well as medical therapy. Pneumocystosis was diagnosed most often in certain breeds (Cavalier King Charles Spaniel, Miniature Dachshund) with a predisposition for impaired immunity. The median age of the dogs was 1 y. Chronic therapy-resistant respiratory signs, such as tachypnea, dyspnea, and cough, along with leukocytosis, neutrophilia, and hypogammaglobulinemia, were the most frequently described clinical and clinicopathologic abnormalities. Pneumocystosis can be masked by coinfections with other respiratory pathogens, and the successful detection of Pneumocystis organisms is of major relevance. Several detection methods have been used in the past, but only a few provide reliable results. In 2017, the cytologic evaluation of Giemsa-stained bronchoalveolar lavage samples is generally used, even if sensitivity is only moderate. More reliable results can be achieved using special stains or sensitive molecular techniques. Fast and reliable detection of Pneumocystis is the essential basis for appropriate treatment and higher survival chances for dogs.

The causative role of some infectious agents found in cases of feline pneumonia is under debate, because they are also part of the physiological microbiota of the respiratory tract of healthy animals. In this retrospective study, archived formalin-fixed and paraffin-wax-embedded lung samples of 69 severe and lethal cases of pneumonia in cats were examined by immunohistochemistry (IHC) for the detection of nine selected infectious agents: Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma felis, M. gateae, Chlamydia felis, feline herpesvirus type 1, feline coronavirus, canine distemper virus, and Toxoplasma gondii. The intention was to elucidate their immediate involvement in pneumonia formation. Due to the cross-reactivity of the applied antibodies, a species-specific polymerase chain reaction (PCR) for both targeted Mycoplasma species was applied additionally. In the 42 cases (60.9%) positive for at least one pathogen, several agents were present in a high proportion of the samples (P. multocida – 34.8%, B. bronchiseptica – 29.0%), while others were present in a moderate (feline herpesvirus type 1 – 18.8%, M. gateae – 13.0%, M. felis – 10.1%) or low percentage (T. gondii – 1.4%). All samples were negative for C. felis, feline coronavirus and canine distemper virus. Mixed infections of up to four pathogens were more frequent than single infections. Mycoplasma preferably colonised lung tissue damaged by other pathogens because they never occurred as single infections. Pasteurella multocida, B. bronchiseptica, M. felis, feline herpesvirus type 1 and T. gondii showed abundant replication within lung lesions, thus suggesting a prominent role in pneumonia formation.

Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies., Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

Porcine circovirus type 2 (PCV2) causes a variety of clinical conditions including PCV2-associated reproductive disease (PCV2-RD) characterized by late term abortions and mummifications. The generally accepted diagnostic triad includes the presence of reproductive disorders, the histopathological finding of myocarditis, and detection of moderate to high viral loads within the heart tissue. A new threshold of 109 PCV2 genome equivalents (GE)/g heart tissue is suggested to fulfil the third criterion using the diagnostic settings of quantitative real time PCR and in situ hybridization of 30 fetal heart tissues. The need to identify histopathological lesions in fetal heart tissue appears to be invalid or overestimated in confirming a diagnosis of PCV2-RD, at least at the individual fetus level. The highest viral loads (1012 GE/g tissue) were detected in autolyzed and mummified piglets and were identified as PCV2d, although concurrent detection of PCV2d + a and PCV2d + b also occurred.

Respiratory diseases in pigs are mostly polymicrobial, and the involved pathogens can vary from farm to farm. The impact of Pneumocystis carinii ( P. c.) f. sp. suis on respiratory disorders has not been comprehensively appraised because tests were limited to lung tissue samples and, for this reason, it was not possible to detect the fungus in living animals. In the present study, bronchoalveolar lavage fluid (BALF) from 12 pigs and oral fluid samples from 9 pigs were tested for the presence of Pneumocystis by quantitative real-time PCR. The results from these 2 clinical specimens were compared with Pneumocystis quantities in lung tissue samples. Pneumocystis quantities in BALF correlated significantly to those in lung tissue. BALF has proved to be an adequate specimen for detection of various respiratory pathogens in pigs, and the collection procedure directly on farms is also well established. In contrast to the BALF results, all oral fluid samples were negative. Thus, specimens from the lower respiratory tract should generally be preferred for the detection of Pneumocystis. Additionally, under farm conditions, oral fluid is mainly collected in the form of collective samples per pen. In the present study, oral swab sampling of individual pigs was intended but failed in 3 of 12 pigs because they did not salivate sufficiently. As a conclusion, only BALF can be recommended as a useful tool for Pneumocystis herd monitoring in vivo.

A 3.5-y-old Whippet mixed-breed dog was presented with a history of respiratory distress, exercise intolerance, and generalized demodicosis. Hematologic alterations included marked leukocytosis and neutrophilia. Radiographic examination showed a diffuse interstitial and mild peripheral alveolar lung pattern and pneumomediastinum. Because the cytologic examination of the bronchoalveolar aspirate was not diagnostic and a persistent perforation of the upper respiratory tract could not be ruled out, the dog was submitted to thoracoscopy, and subsequently the left cranial lung lobe as well as mediastinal and sternal lymph nodes were resected. Pulmonary pneumocystosis with spread to the thoracic lymph nodes was suspected after histologic investigation of lung and lymph nodes, which was confirmed by in situ hybridization, PCR, and subsequent Sanger sequencing. We document a rare, simultaneous occurrence of severe pulmonary and thoracic lymph node pneumocystosis with spontaneous pneumomediastinum in a dog. Definitive diagnosis was achieved through the use of Grocott methenamine silver staining, in situ hybridization, and PCR.

Pneumocystis sequences can be downloaded from GenBank for purposes as primer/probe design or phylogenetic studies. Due to changes in nomenclature and assignment, available sequences are presented with a variety of inhomogeneous information, which renders practical utilization difficult. The aim of this study was the descriptive evaluation of different parameters of 532 Pneumocystis sequences of mitochondrial and ribosomal origin downloaded from GenBank with regard to completeness and information content. Pneumocystis sequences were characterized by up to four different names. Official changes in nomenclature have only been partly implemented and the usage of the "forma specialis", a special feature of Pneumocystis, has only been established fragmentary in the database. Hints for a mitochondrial or ribosomal genomic origin could be found, but can easily be overlooked, which renders the download of wrong reference material possible. The specification of the host was either not available or variable regarding the used language and the localization of this information in the title or several subtitles, which limits their applicability in phylogenetic studies. Declaration of products and geographic origin was incomplete. The print version of this manuscript is completed by an online database which contains detailed information to every accession number included in the meta-analysis.

Pneumocystis carinii f. sp. suis (PCS) nucleic acid and antibody profiles on two Austrian-farrow-to-finish farms were investigated. Furthermore, associations with other respiratory pathogens were evaluated. Respiratory specimen and sera from pigs of five age classes between the 1st week and the 3rd month of life as well as samples from sows were analyzed. On Farm A, PCS infection occurred early in life. The suckling piglets were already infected in the 1st week of life and the pigs remained positive until the 3rd month of life. On Farm B, pigs were infected later, between 3 and 4 months of age. The maximum PCS nucleic acid load on Farm A was 8.3 log10 genome copies/mL BALF, whereas on Farm B the PCS burden was significantly lower, with 4.0 log10 genome copies/mL BALF. Anti-PCS antibodies were detected in sows, as maternal antibodies in suckling piglets and as an immunological reaction to infection. On both farms, PCS infection was accompanied by several co-infections. On Farm A, there were concurrent infections with PRRSV, a virulent strain of Haemophilus parasuis, and Mycoplasma hyopneumoniae. On Farm B, PCS was accompanied by infections with swine influenza virus, Mycoplasma hyopneumoniae, and a non-virulent strain of Haemophilus parasuis. The results clearly show that the PCS profiles can vary between farms. Younger pigs may be more susceptible as they had higher PCS burdens. It is possible that PCS may contribute to a respiratory disease in pigs and further investigation of its potential role is warranted.

Zusammenfassung Ziel war zu prüfen, ob die an einer oder zwei Injektionsstellen durchgeführte simultane Applikation zweier Vakzinen gegen das porzine Circovirus Typ 2 (PCV2) bzw. Mycoplasma hyopneumoniae (M. hyo.) bei Saugferkeln ebenso wirksam ist wie die Verabreichung der einzelnen Impfstoffe. Material und Methoden: 598 Ferkel bildeten bei gemischter Aufstallung fünf Versuchsgruppen. Das Plazebo Porcilis® Diluvac forte sowie die Vakzinen Porcilis® M HYO („M HYO“) und Porcilis® PCV („PCV“) wurden jeweils in der 1. und 3. Lebenswoche (LW) wie folgt appliziert: Gruppe A: Plazebo/PCV, Gruppe B: M HYO/M HYO, Gruppe C: Plazebo/Plazebo, Gruppe D: M HYO/M HYO + PCV (kombinierte Einzeldosis), Gruppe E: Plazebo/M HYO + PCV (getrennte Injektionsstellen). Die Wirkung der simultanen Impfung wurde anhand von Tageszunahmen, pathomorphologischer Lungenveränderungen, Morbidität, Mortaliät und der Immunantwort gegen die verwendeten Antigene bestimmt. Die Verträglichkeit der simultanen Vakzination wurde anhand lokaler und systemischer Impfreaktionen bewertet. Ergebnisse: Die phasenweise schlechte Bestandsgesundheit, bedingt durch Saugferkeldurchfälle (n = 111) in der 1.–3. LW, ließ sich nicht eindeutig mit PCV2 in Zusammenhang bringen. Dennoch zeigten alle geimpften Tiere tendenziell höhere Tageszunahmen als die Kontrolltiere. Unterschiede zwischen den Gruppen hinsichtlich Lungenveränderungen, Morbidität und Mortalität waren nicht signifikant. M.-hyo.- und PCV2-spezifische Antikörpertiter lagen bei geimpften Tieren signifikant höher als bei nicht geimpften. Bei je einem Ferkel der Gruppen A (PCV2) und C (Plazebo) kam es zu lokalen Impfreaktionen. Schlussfolgerung und klinische Relevanz: In Betrieben mit suboptimaler Bestandsgesundheit lässt sich ein positiver Effekt beider Impfungen nachweisen. Die simultane Impfung gegen PCV2 und M. hyo. hatte keine negativen Auswirkungen. In Bezug auf die Steigerung der durchschnittlichen Tageszunahmen war der Effekt der simultanen Impfungen dem der Einzelimpfungen gleichwertig. Der Einsatz der Simultanimpfung gegen M. hyo. und PCV2, der Vorteile hinsichtlich Arbeitsaufwand und Tierschutz bringt, kann daher in der Praxis befürwortet werden.

The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72 pigs, 5.5-10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for mycotoxin analysis. The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44 and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15 samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the syndrome they are not the exclusive triggering factor.

Native European passerine birds are frequently clinically inapparent carriers of haemosporidian parasites of the genus Plasmodium. Clinical disease and death are only exceptionally reported. In the present study, tissue samples of 233 wild passerine birds found dead in Eastern Austria were examined by in situ hybridization (ISH) and partial cytochrome B gene sequence analysis for the presence, abundance and taxonomic assignment of Plasmodium spp. In 34 cases (14.6%), ISH yielded a positive result with large numbers of developmental stages in different cell types of the spleen, liver, brain and lung. The abundance of the tissue stages, which was comparable to fatal cases of avian malaria in penguins, suggested a major contribution to the cause of death. Genetic analysis revealed infections with representatives of three different valid species of Plasmodium, Plasmodium elongatum, Plasmodium lutzi and Plasmodium vaughani. Genetically identical parasite lineages had been found in a previous study in penguins kept in the Vienna zoo, providing evidence for the role of wild birds as reservoir hosts. Further, this study provides evidence that several species of Plasmodium are able to abundantly proliferate in endemic wild birds ultimately resulting in mortalities.

In this retrospective study, 218 pig lung tissue samples were analyzed to examine a possible association between Pneumocystis spp. using in situ hybridization, Bordetella bronchiseptica (B.b.) using immunohistochemistry (IHC), Mycoplasma hyopneumoniae (M.h.) by quantitative PCR, and Pasteurella multocida (P.m.; IHC). Compared to the bacterial agents (B.b., 5%; M.h., 30%; P.m., 23%), Pneumocystis occurred with a higher prevalence (51%). Co-infections with two or three pathogens were present in 28% of the examined cases. Those of Pneumocystis and M.h. were most commonly seen, followed by Pneumocystis and P.m. and M.h. and P.m. Histologically, interstitial pneumonia was found in both the Pneumocystis positive lungs and lungs with a mild M.h. infection. The B.b. and P.m. positive lungs were mainly associated with suppurative bronchopneumonia and severe M.h. cases with fibrinous or fibrino-haemorrhagic pneumonia. In suckling piglets, the number of samples positive for Pneumocystis predominated, whereas samples from fattening pigs were mainly positive for bacteria or Pneumocystis and bacteria.

During the last years two studies for the investigation of the etiology of porcine ear necrosis were carried out at the Clinic for Swine of the University of Veterinary Medicine Vienna. In study 1, parameters, which are discussed in this context, were collected by veterinary practitioners by completing specially designed questionnaires in farms with symptoms of the porcine ear necrosis syndrome. In study 2, samples of piglets and feed were collected for laboratory analysis of the most important infectious agents as well as mycotoxins. In the present manuscript, the results of both projects were compared. Even if the selection criteria of both studies differed, the affected age class was comparable (5.5 to ten weeks of life in study 1 and six to ten weeks of life in study 2). The herd-specific prevalence of the porcine ear necrosis syndrome varied considerably with percentages between 2 and 10, respectively, to 100%. The evaluation of questionnaires in study 1 showed that 51% of the farms had problems with cannibalism. Particles of plant material, which were frequently seen on the histologic slides of study 2, could have got into the tissue by chewing the ears of the pen mates or cannibalism. Whereas in study 1 the negative effect of parameters as high pig density, suboptimal climate, missing enrichment material and bad quality of feed and water were considered, in study 2 all these factors were checked at sample collection and ruled out as precursor for cannibalism. In both studies bacterial agents proved to be a crucial co-factor for the expansion of the necroses to deeper tissue layers, whereas viral pathogens were classified less important. In both projects it was not possible to estimate the direct impact of infectious agents and mycotoxins as direct trigger of the necroses as well as their participation as co-factors or precursor in the sense of an immunosuppression or previous damage of blood vessels or tissue.

Two field studies were conducted to investigate the influence of age on the efficacy of vaccination against Porcine Circovirus Diseases (PCVD) in animals with high levels of maternally derived antibodies (MDA). A total of 416 piglets (Study 1) and 600 piglets (Study 2) were randomly allocated to one of three groups. Two groups in each study received a single dose of a PCV2 subunit vaccine, one group at 1 week old and the other at 3 weeks of age. The third group was left untreated. Animals vaccinated at 3 weeks of age showed a significantly higher average daily weight gain and significantly reduced viraemia following PCV2 infection than the respective control groups. This difference was not observed in pigs vaccinated at 1 week of age. Furthermore, only animals vaccinated at 3 weeks of age showed an increased serological response and a higher frequency of IgM-positive animals compared with controls. The data indicated that PCV2 vaccination in the presence of high MDA levels is efficacious when used in 3-week old but not in 1-week old pigs. As the range of MDA titres of pigs vaccinated at both 1 and 3 weeks of age were comparable, the data suggest that PCV2 vaccine efficacy was independent of the level of MDA. It appears that other age-related factors affecting the active and passive transfer of immunity may perhaps have interfered with the efficacy of the vaccine in 1-week old piglets. These findings have implications for future PCV2 vaccine testing and administration strategies.

Pneumocystis carinii f. sp. suis is a fungus multiplying in the respiratory tract of pigs which occasionally is associated with interstitial pneumonia. Identification of Pneumocystis in tissue samples is considered difficult and there are only scarce data on its occurrence in European pigs. This investigation presents an in situ hybridization (ISH) procedure for identification of Pneumocystis spp. in paraffin wax embedded tissue samples and its application for labeling the agent in lung samples of pigs with interstitial pneumonia. Thirty-two out of 100 lung samples from pigs on Austrian farms were identified as positive, five of them with multiple, 12 with moderate and 15 with few organisms but Grocott’s methenamine silver staining demonstrated that only 20 cases were unequivocally positive for Pneumocystis carinii. In addition to interstitial pneumonia Pneumocystis-positive pigs were more frequently affected with granulomatous pneumonia than Pneumocystis-negative pigs. Frequently concurrent infections with different viral or bacterial lung pathogens were noted but there was no positive correlation between Pneumocystis- and PCV-2-infections. With other infections, no clear-cut differences between Pneumocystis-positive and Pneumocystis-negative animals were found. This study shows that Pneumocystis infections occur frequently in Austrian pigs with interstitial pneumonia. It remains to be shown which are the factors triggering severe multiplication and whether infection with Pneumocystis alone is able to induce lung disease in pigs.

Vaccines against M. hyopneumoniae and PRRSV are often administered at the same time. The administration of the two vaccines mixed in one syringe (simultaneous use) would save labour and increase animal welfare by reducing the number of injections. The aim of this study was to verify under field conditions, if the simultaneous administration of the two vaccines, would be comparable to the administration of the individual vaccines. 1000 piglets were divided into five groups and were either non vaccinated or vaccinated against M. hyopneumoniae and/or PRRSV-EU-type. Body weight, local reactions, morbidity and mortality were measured and clinical examinations were carried out. Blood samples were analysed serologically for the presence of antibodies against PRRSV and M. hyopneumoniae by ELISA. M. hyopneumoniae-induced lung lesion scoring (with a maximum score of 55 points) was performed at the slaughterhouse. In the 1000 pigs included in this study, no clinically relevant local or systemic reactions were observed following vaccination. No significant differences in average daily weight gain (ADWG), mortality, clinical parameters or in the prevalence of pneumonia and pleuritis at the slaughterhouse were detected between the different study groups. Significant differences were observed for the serological results, with M. hyopneumoniae antibody positive pigs in the vaccinated groups being significantly higher than in the non-vaccinated groups. Regarding the severity code of the lung lesions of the affected lungs, the lung scores from the simultaneously vaccinated pigs were significantly lower (0.92) than those from non-vaccinated pigs (1.77). Due to the low infectious pressure of both PRRSV and M. hyopneumoniae on this farm, we have to conclude that efficacy of the vaccines could not adequately be tested.

In this controlled, blinded and randomised study, Circovac®, an inactivated PCV2 vaccine was used for active immunisation of 3-week-old piglets (0.5 ml, i.m., one-shot). In a PCVD affected farm, a total of 1105 piglets from three consecutive farrowing batches were included. The vaccine efficacy was assessed by clinical parameters such as mortality, underweight pigs and improvement of average daily weight gain (ADWG) during the fattening period. Additionally, the effect of the vaccination over time on viremia and seroconversion was investigated in animals from the 1st and the 3rd batch. Mortality rate from start of fattening until slaughter was reduced by 7.40% in the vaccinated group (p < 0.0001). ADWG from 13th to 21st week was improved in the vaccinated group by 64.83 g/day (p < 0.0001) and by 41.43 g/day during the whole fattening stage. Furthermore, vaccination reduced the rate of underweight animals, especially for the obtained weight data in the 21st week of life with 5.15% difference (p = 0.044). The percentage of recently infected animals, characterised by being positive for IgM antibodies, as well as the detection of specific PCV2 DNA, was reduced in the vaccinated animals and it was found concomitant with higher mortality and underweight animals in the non-vaccinated group. From the study results it can be concluded that the vaccination of piglets with Circovac® reduced the viral shedding and protected the animals successfully as shown by the improvement of clinical parameters, and that it can be utilised as a valuable instrument against PCVD.

Background: Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds. Methods: One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods. Results: All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs. Conclusion: Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.