Ole Lerberg Nielsen

The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

A serotype 1- and serotype 3-specific detection of Mareks disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Mareks disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.

Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcriptionpolymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment of the N-gene and could detect various strains of IBV in allantoic fluid from inoculated chicken embryos, and in tracheal tissue preparations from experimentally infected chickens. Four pairs of oligonucleotides selectively primed the amplification of the S1 gene of Massachusetts/Connecticut, D1466, D274/D3896 and 793B strains of IBV, respectively. Groups of specific pathogen free chickens were experimentally inoculated with the Massachusetts (H120, M41), the D1466 and the 793B strains of IBV, and tracheal tissue preparations were made from each bird for RT-PCR and for immunohistochemistry (IHC) up to 3 days post-inoculation. The N-gene RT-PCR detected IBV in 82% of the chickens, while IHC only detected IBV in 60%. This difference was significant (P<0.02). The detection rate by N-gene RT-PCR varied from 67 to 100% for the various strains of IBV inoculated. The S1 gene oligonucleotide pairs were applied to the same tissue preparations and they detected specifically the Massachusetts (M41 and H120), the D1466 and the 793B strains of IBV at rates varying between 58 and 92%. When the mixtures of the primers were applied, the detection rate in tissue preparations was reduced to the level of 50 to 67%. It is concluded that the direct detection of IBV in tracheal tissues by RT-PCR is more sensitive than IHC and that the RT-PCR technique is able to distinguish between types of IBV.

Some new aspects of the pathology, pathogenesis, and aetiology of disseminated lung lesions in slaughter pigs.

From 40 pigs rejected for human consumption at slaughter due to an apparent presence of pyemic lung lesions (defined as disseminated processes containing pus and/or necrotic material), the lungs, spleen, liver, and kidneys were subjected to an extended macroscopic examination. Several lung lesions were sampled from each animal for histological and bacteriological examination. Samples from the kidneys and spleens were also subjected to bacteriological examination. At gross level, four groups of lung lesions were identified: 1) disseminated foci with contents of pus and/or necrotic material (n=26); 2) disseminated or multifocally located ecchymoses with a central area of fibroplasia (n=9); 3) non‐pneumonic lesions, i.e., disseminated areas of atelectasis (n=1) or haemorrhagic areas developing due to the process of slaughter (n=1); and 4) suppurative lesions without a disseminated distribution pattern (n=3). Histologically, the disseminated suppurative/necrotic foci were identified as: A) abscesses (n=10); B) necrotic lesions (n=6); and C) ectatic or ectatic‐like bronchioles with contents of pus and necrotic material (n=10). The macroscopic observation of disseminated centres of fibroplasia with peripheral ecchymoses (n=9) was confirmed histopathologically. The livers of five pigs contained multiple areas of chronic interstitial fibrosis related to migration of Ascaris suum larvae (“milk spotted liver”). Such hepatic lesions were significantly (p<0.01) related to the simultaneous occurrence of disseminated pulmonary ecchymoses with a central area of fibroplasia. Generally, all lung lesions of each individual animal contained identical monocultures of bacteria following this pattern: Staphylococcus aureus (abscesses); Actinomyces hyovaginalis (necroses); S. aureus, A. hyovaginalis, and Arcanobacterium pyogenes (ectatic and ectatic‐like bronchioles). Areas with fibrosis were sterile or contained bacteria considered to be a result of contamination. Apart from one kidney, from which S. aureus was cultured, all other organs were sterile. It is concluded that difficulties exist in differentiating pulmonary pyemic lesions from non‐pyemic lesions at the gross level. Thus, it was not possible to distinguish between abscesses/necroses and ectatic bronchioles, the pathogenesis of the latter being uncertain. However, the chronic non‐pyemic lesions related to the migration of A. suum larvae should be identified by the absence of pus/necrosis. S. aureus was predominantly isolated from abscesses, whereas, and most surprisingly, A. hyovaginalis was the dominant bacterium isolated from the pulmonary necroses.

Pulmonary infections in swine induce altered porcine surfactant protein D expression and localization to dendritic cells in bronchial-associated lymphoid tissue

Surfactant protein D (SP‐D) is a pattern‐recognition molecule of the innate immune system that recognizes various microbial surface‐specific carbohydrate and lipid patterns. In vitro data has suggested that this binding may lead to increased microbial association with macrophages and dendritic cells. The aim of the present in vivo study was to study the expression of porcine SP‐D (pSP‐D) in the lung during different pulmonary bacterial infections, and the effect of the routes of infection on this expression was elucidated. Furthermore, the aim was to study the in vivo spatial relationship among pSP‐D, pathogens, phagocytic cells and dendritic cells. Lung tissue was collected from experimental and natural bronchopneumonias caused by Actinobacillus pleuropneumoniae or Staphylococcus aureus, and from embolic and diffuse interstitial pneumonia, caused by Staph. aureus or Arcanobacterium pyogenes and Streptococcus suis serotype 2, respectively. By comparing normal and diseased lung tissue from the same lungs, increased diffuse pSP‐D immunoreactivity was seen in the surfactant in both acute and chronic bronchopneumonias, while such increased expression of pSP‐D was generally not present in the interstitial pneumonias. Co‐localization of pSP‐D, alveolar macrophages and bacteria was demonstrated, and pSP‐D showed a patchy distribution on the membranes of alveolar macrophages. SP‐D immunoreactivity was intracellular in dendritic cells. The dendritic cells were identified by their morphology, the absence of macrophage marker immunoreactivity and the presence of dendritic cell marker immunoreactivity. Increased expression of pSP‐D in the surfactant coincided with presence of pSP‐D‐positive dendritic cells in bronchus‐associated lymphoid tissue (BALT), indicating a possible transport of pSP‐D through the specialized M cells overlying (BALT). In conclusion, we have shown that pSP‐D expression in the lung surfactant is induced by bacterial infection by an aerogenous route rather than by a haematogenous route, and that the protein interacts specifically with alveolar macrophages and with dendritic cells in microbial‐induced BALT. The function of the interaction between pSP‐D and dendritic cells in BALT remain unclear, but pSP‐D could represent a link between the innate and adaptive immune system, facilitating the bacterial antigen presentation by dendritic cells in BALT.

Purification, characterization and immunolocalization of porcine surfactant protein D

Surfactant protein D (SP‐D) is a collectin believed to play an important role in innate immunity. SP‐D is characterized by having a collagen‐like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+‐dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro‐organisms. This paper describes the tissue immunolocalization of porcine SP‐D (pSP‐D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP‐D. Porcine SP‐D was purified from porcine bronchoalveolar lavage (BAL) by maltose‐agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate–polyacrylamide gel electrophoresis as a band of ∼53 000 MW in the reduced state and ∼138 000 MW in the unreduced state. Porcine SP‐D was sensitive to collagenase digestion and N‐deglycosylation, which reduced the molecular mass to ∼24 000 MW and ∼48 000 MW respectively, in the reduced state. N‐deglycosylation of the collagen‐resistant fragment, reduced the molecular mass to ∼21 000 MW showing the presence of an N‐glycosylation site located in the CRD. Porcine SP‐D bound to solid‐phase mannan in a dose and Ca2+‐dependent manner with a saccharide specificity similar to rat and human SP‐D. The purified protein was used for the production of a monoclonal anti‐pSP‐D antibody. The antibody reacted specifically with pSP‐D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP‐D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.

A pig model of acute Staphylococcus aureus induced pyemia

BackgroundSepsis caused by Staphylococcus aureus constitutes an important cause of morbidity and mortality in humans, and the incidence of this disease-entity is increasing. In this paper we describe the initial microbial dynamics and lesions in pigs experimentally infected with S. aureus, with the aim of mimicking human sepsis and pyemia.MethodsThe study was conducted in anaesthetized and intravenously inoculated pigs, and was based on bacteriological examination of blood and testing of blood for IL-6 and C-reactive protein. Following killing of the animals and necropsy bacteriological and histological examinations of different organs were performed 4, 5 or 6 h after inoculation.ResultsClearance of bacteria from the blood was completed within the first 2 h in some of the pigs and the highest bacterial load was recorded in the lungs as compared to the spleen, liver and bones. This probably was a consequence of both the intravenous route of inoculation and the presence of pulmonary intravascular macrophages. Inoculation of bacteria induced formation of acute microabscesses in the lungs, spleen and liver, but not in the kidneys or bones. No generalized inflammatory response was recorded, i.e. IL-6 was not detected in the blood and C-reactive protein did not increase, probably because of the short time course of the study.ConclusionThis study demonstrates the successful induction of acute pyemia (microabscesses), and forms a basis for future experiments that should include inoculation with strains of S. aureus isolated from man and an extension of the timeframe aiming at inducing sepsis, severe sepsis and septic shock.

Intravenous inoculation of Staphylococcus aureus in pigs induces severe sepsis as indicated by increased hypercoagulability and hepatic dysfunction.

Nine pigs were inoculated intravenously once or twice with 10(8) Staphylococcus aureus per kilogram body weight and sacrificed 12, 24 and 48 h after inoculation. Three sham-infected pigs served as controls. Blood samples were taken for bacteriology, haematology and clinical chemistry. A necropsy was carried out and tissue samples were collected for bacteriology and histology. The onset of clinical disease was seen at 7-8 h after inoculation. The blood bacterial counts remained low throughout the study. All infected pigs developed sepsis characterized by fever, neutrophilia, increased levels of C-reactive protein (CRP) and IL-6, and decreased levels of serum iron. The CRP and IL-6 levels peaked at 36 h, whereas IL-1beta and tumour necrosis factor-alpha showed no obvious changes. Thromboelastography showed increasing hypercoagulability from 12 h and onwards, whereas the platelet numbers declined slightly throughout the experiment. The levels of serum aspartate aminotransferase and bilirubin were elevated at 24 and 36 h. In conclusion, sepsis and severe sepsis were induced as evidenced by dysfunction of the blood clotting system and the liver.

A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study

Johansen LK, Frees D, Aalbæk B, Koch J, Iburg T, Nielsen OL, Leifsson PS, Jensen HE. A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study, APMIS 2010; 119: 111–8.

Polysynovitis after Oligofructose Overload in Dairy Cattle

Acute bovine laminitis is a systemic disease with local manifestations primarily affecting the claws. However, distension of the tarsocrural joints has been observed after experimental oligofructose overload in dairy heifers as a part of the complex interpreted as acute, clinical laminitis. Therefore, the aim of the present study was to study bovine synovial joints and tendon sheaths after oligofructose overload. Ten dairy heifers received oral oligofructose overload (17 g/kg body weight); four were killed 24h after overload and six after 72 h. Six control heifers received tap water and were killed after 72 or 96 h. Clinical examination included locomotion scoring and palpation of the tarsocrural joints. Ruminal fluid and blood was collected for measurements of pH and hydration status. Total protein concentrations and white blood cell (WBC) counts were determined in synovial fluid collected from tarsocrural joints after death. Synovial joints and tendon sheaths were examined and synovial membranes were studied microscopically. Swabs taken from the synovial cavities were subject to bacteriological culture. Heifers with oligofructose overload developed signs of ruminal and systemic acidosis. Lameness was observed in three of ten heifers 24h after overload and in all remaining heifers after 72 h. Distension of tarsocrural joints was observed from 18 h after overload and peaked at 30 h when all examined joints were moderately or severely distended. The synovial fluid was turbid and protein content and WBC counts were increased at both 24 and 72 h compared with controls. Bacterial culture was negative. Synovial membranes 24 and 72 h after overload had a fibrinous and neutrophil inflammatory reaction that regressed in severity between 24 and 72 h after overload. Heifers subjected to oligofructose overload therefore developed generalized sterile neutrophilic polysynovitis. Focus on this aspect of bovine laminitis may shed new light on the pathogenesis of this complex disease.

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization

ABSTRACT Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, “Actinobacillus salpingitidis,” and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens.