Detection of retinoid receptors in non-neoplastic canine lymph nodes and in lymphoma

This study evaluated the difference in retinoid receptor expression between non-neoplastic lymph nodes and nodal lymphoma in dogs. Retinoid receptor expression was evaluated by immunohistochemistry in 32 canine lymph nodes. The lymph nodes had been previously diagnosed as non-neoplastic (6 normal and 7 hyperplastic lymph nodes) and B- and T-cell lymphoma (19 cases). Immunohistochemistry for retinoic acid receptors and retinoid-X receptors (and their subtypes α, β, and γ) was performed in all cases. In addition, immunohistochemistry for CD3 and CD79a was performed in all lymphoma cases. Non-neoplastic lymphocytes were negative for all retinoid receptors. Retinoic acid receptor-γ was detected in 100% of B-cell lymphoma and 78% of T-cell lymphoma, while retinoid X receptor-γ was positive in 78% of T-cell lymphoma cases. When normal lymph node architecture was still present, a contrast between retinoid-negative benign cells and retinoid-positive malignant cells was clear. Retinoid receptors were expressed in neoplastic, but not in benign lymphocytes, suggesting their value for both diagnosis and treatment of canine lymphoma.     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

An Inhibitor of Cholesterol Biosynthesis and the Alveolar Macrophages

An accumulation of lipid material was observed in the alveolar macrophages of animals treated per os with an inhibitor of cholesterol biosynthesis (trans-1, 4-bis) (2-dichlorobenzyl-aminomethyl) cyclohexane dihydrochloride: AY-9944). This lipid is probably 7-dehydro-cholesterol which is a precursor of cholesterol and accumulates in various organs, especially in the lungs. In the rats and pigs the reaction is limited to the alveolar macrophages which are expectorated or degenerate. In dogs some of the alveolar macrophages are also expectorated while others continue to collect a cholesterol-like lipid until large crystals form, which provoke the formation of giant cells and later fibrocytes, giving rise to “cholesterol granulomata”.     

HP-PRRSV抑制猪肺eNOS表达与猪肺微血栓形成的相关性研究

为了解高致病性猪繁殖与呼吸综合征病毒（HP-PRRSV）与肺微血栓病变的关系及内皮型一氧化氮合酶（eNOS）的表达对微血栓的影响,选取50头70日龄的鄂通两头乌猪,4头作为正常对照,46头感染HP-PRRSV。采取50头猪的肺组织,应用HE染色观察组织病理变化并对微血栓病变程度进行等级评分,将46头感染猪分为轻度微血栓组、中度微血栓组、重度微血栓组,每组选取4头猪进行后续研究。应用免疫组化、荧光定量PCR和Western blot技术,明确HP-PRRSV和eNOS在微血栓猪肺中的表达、分布及变化。组织病理学研究结果显示感染猪肺组织肺泡间隔明显增宽,毛细血管有不同程度的淤血、出血,肺泡腔内有巨噬细胞、脱落的肺泡上皮细胞及淋巴细胞,肺泡壁有大量微血栓。HP-PRRSV主要分布于肺泡巨噬细胞及少量的淋巴细胞的细胞质。随着HP-PRRSV病毒含量的增加,微血栓病变越严重。微血栓病变越严重,死亡率越高。eNOS主要分布于肺泡Ⅱ型细胞、巨噬细胞、血管内皮细胞及支气管平滑肌细胞的细胞质,感染猪肺组织的eNOS mRNA和蛋白表达显著低于未感染对照组。研究结果表明HP-PRRSV使eNOS的表达下调,并参与感染猪肺组织微血栓的形成,增加感染猪的死亡率。     

Immunohistochemical Detection of Retinoid Receptors in Tumors from 30 Dogs Diagnosed with Cutaneous Lymphoma

Background: Retinoids exert their effects by binding to retinoid receptors. Two types of retinoid receptors have been described: retinoic acid receptor (RAR) and retinoid X receptor (RXR), and their subtypes α, β, and γ. The expression of subtypes varies depending on the disease process. This study intended to detect the pattern of retinoid receptor expression in cutaneous lymphomas in dogs. Hypothesis: Cutaneous lymphomas in dogs have variable expression of retinoid and retinoid X receptors. Animals: Biopsy specimens from 30 dogs with cutaneous lymphoma. Methods: Tissues of dogs with cutaneous lymphoma were evaluated by immunohistochemistry for expression of retinoid receptors. The tissues were tested for the presence of 3 RAR and RXR subtypes (α, β, and γ). Lymphoma immunophenotype was determined by the use of the immunohistochemical markers CD79a (B‐cell) and CD3 (T‐cell) in all cases. Results: Twenty‐nine of 30 dogs were CD3 positive. The retinoid receptors expressed with the greatest frequency were RARβ (87% of cases), and RXRα and RXRγ (77% of cases). The expression of RARγ was not observed. Conclusions and Clinical Relevance: Retinoid and rexinoid receptor binding drugs may have an impact on the treatment of dogs with cutaneous lymphoma.     

Porcine mononuclear phagocyte subpopulations in the lung,blood and bone marrow: dynamics during inflammation induced by Actinobacillus pleuropneumoniae

Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14⁺ CD163⁺ CD203α^+/− MHCII^+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14⁺ CD163⁺ CD203α⁻ MHC II⁻ population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14⁻ CD163⁻ cells maturing directly into CD14⁺ CD163⁻ that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14⁻ CD163⁻ CD203α⁻ MHCII⁻ MP directly switching into CD14⁺ CD163⁺ CD203α⁻ MHCII⁻ MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions.     

Effect of dietary crude protein level on growth performance,blood characteristics,and indicators of intestinal health in weanling pigs

An experiment was conducted to test the hypothesis that reducing crude protein (CP) in starter diets for pigs reduces post-weaning diarrhea and improves intestinal health. In total, 180 weanling pigs were allotted to 3 diets containing 22, 19, or 16% CP. Fecal scores were visually assessed every other day. Blood samples were collected from 1 pig per pen on days 1, 6, 13, 20, and 27, and 1 pig per pen was euthanized on day 12. Results indicated that reducing dietary CP reduced (P < 0.01) overall average daily gain, gain to feed ratio, final body weight, and fecal scores of pigs. Pigs fed the 16% CP diet had reduced (P < 0.01) serum albumin compared with pigs fed other diets. Blood urea nitrogen, haptoglobin, interleukin-1β, and interleukin-6 concentrations in serum were greatest (P < 0.01) on day 13, whereas tumor necrosis factor-α and interleukin-10 concentrations were greatest (P < 0.01) on day 6. Villus height in the jejunum increased (P < 0.05) and crypt depth in the ileum was reduced (P < 0.01) if the 19% CP diet was fed to pigs compared with the 22% CP diet. A reduction (P < 0.05) in mRNA abundance of interferon-γ, chemokine ligand 10, occludin, trefoil factor-2, trefoil factor-3, and mucin 2 was observed when pigs were fed diets with 16% CP. In conclusion, reducing CP in diets for weanling pigs reduces fecal score and expression of genes associated with inflammation.     

Comparison of beta-ligands used in cattle production: structures,safety, and biological effects

Technologies that increase the efficiency and sustainability of food animal production to provide meat for a growing population are necessary and must be used in a manner consistent with good veterinary practices, approved labeled use, and environmental stewardship. Compounds that bind to beta-adrenergic receptors (β-AR), termed beta-adrenergic receptor ligands (β-ligands), are one such technology and have been in use globally for many years. Though all β-ligands share some similarities in structure and function, the significance of their structural and pharmacological differences is sometimes overlooked. Structural variations in these molecules can affect absorption, distribution, metabolism, and excretion as well as cause substantial differences in biological and metabolic effects. Several β-ligands are available for use specifically in cattle production. Ractopamine and zilpaterol are beta-adrenergic agonists approved to increase weight gain, feed efficiency, and carcass leanness in cattle. They both bind to and activate β1- and β2-AR. Lubabegron is a newly developed selective beta-adrenergic modulator with unique structural and functional features. Lubabegron displays antagonistic behavior at the β1- and β2-AR but agonistic behavior at the β3-AR. Lubabegron is approved for use in cattle to reduce ammonia emissions per unit of live or carcass weight. Additionally, lubabegron can withstand prolonged use as the β3-AR lacks structural features needed for desensitization. Due to these unique features of lubabegron, this new β-ligand provides an additional option in cattle production. The individual properties of each β-ligand should be considered when making risk management decisions, as unique properties result in varying human food safety profiles that can determine appropriate safe β-ligand use.     

Novel H5 clade 2.3.4.6 viruses with both α-2,3 and α-2,6 receptor binding properties may pose a pandemic threat

The emerging H5 clade 2.3.4.6 viruses of different NA subtypes have been detected in different domestic poultry in China. We evaluated the receptor binding property and transmissibility of four novel H5 clade 2.3.4.6 subtype highly pathogenic avian influenza viruses. The results show that these viruses bound to both avian-type (α-2,3) and human-type (α-2,6) receptors. Furthermore, we found that one of these viruses, GS/EC/1112/11, not only replicated but also transmitted efficiently in guinea pigs. Therefore, such novel H5 subtype viruses have the potential of a pandemic threat.     

Pathogenic mechanisms implicated in the intravascular coagulation in the lungs of BVDV-infected calves challenged with BHV-1

Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (MΦs) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of MΦs in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNFα, IL-1α, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary MΦs. In this regard, the co-infected calves, in spite of presenting a greater number of IMΦs than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNFα and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.     

Distribution of cells immunopositive for AM-3K, a novel monoclonal antibody recognizing human macrophages, in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits

The monoclonal antibody AM-3K, which was developed using human pulmonary macrophages as the immunogen, immunocytochemically labels most human macrophages except for blood monocytes and dendritic cell populations. AM-3K also shows cross-reactivity in some animal species. To evaluate the usefulness of AM-3K, the present study investigated the detailed distribution of AM-3K-immunopositive macrophages in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits. Zamboni's solution-fixed, paraffin-embedded sections were the most available for the immunocytochemistry with AM-3K. In all animal species examined, AM-3K labeled most macrophages in splenic red pulp, lymph node sinuses and thymus, and tissue macrophages in the interstitium of various organs and sites such as the kidneys, lungs, heart, pancreas, intestines, and skin. Alveolar macrophages and perivascular microglial cells were also immunoreactive for AM-3K. Interestingly, Kupffer cells of dogs, cats, and horses were labeled for AM-3K, but those of cattle, pigs, and rabbits were not. Furthermore, in tumor tissues and inflammatory lesions such as liver fibrosis and encephalomalacia that were obtained from dogs, infiltrating macrophages were stained with AM-3K, but not all infiltrating macrophages reacted to AM-3K. In addition, only 30-50% of pulmonary and peritoneal macrophages obtained from cats and dogs were reactive for AM-3K. AM-3K did not react with blood monocytes, dendritic cell populations, and osteoclasts. These observations indicate that AM-3K specifically labels most exudate and tissue macrophages in the animal species examined. However, the expression of antigens recognized by AM-3K on macrophages may be dependent on differential maturation stages or different functions evoked by some conditions. AM-3K immunoreaction products were seen on the cytoplasmic membrane of macrophages by immunoelectron microscopy. AM-3K would be useful for detection of macrophage populations in the animal species examined here.     

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3⁺, CD4⁺ and γδ T-cells; whereas CD8⁺ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.     

Inhibition of transient receptor potential vanilloid type 1 through α2 adrenergic receptors at peripheral nerve terminals relieves pain

The activation of α₂ adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α₂ receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α₂ receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α₂ receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α₂ receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α_2C receptor mRNA quantified by real-time PCR was highest followed by those of α_2A and α_2B receptors in DRGs. α_2A and α_2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α₂ receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia.     

The Concentrations of Copper,Zinc and Molyrdenum in Swine Liver and the Relationship to the Distrirution of Solurle Copper- and Zinc-Rinding Proteins

The concentrations of copper, zinc and molybdenum were measured in liver samples from 21 normal slaughter pigs (average age about 6 months) and in 36 sows (average age about 2 years). The following mean values were found: Slaughter pigs: 15 ± 8 µg Cu/g, 45 ± 7 μg Zm/g and 1.0 ± 0.2 μg Mo/g wet weight; sows: 46 ± 70 μg Cu/g, 70 ± 26 μg Zn/g and 1.3 ± 0.3 μg Mo/g wet weight. The concentrations of all 3 elements were significantly higher in the sows than in the young pigs. There was no correlation between the concentrations of copper, zinc or molybdenum. The recorded copper levels in the slaughter pigs were in accordance with the levels of non-supplemented pigs given in the literature. The soluble hepatic copper- and zinc-binding proteins were separated into 3 different fractions by gel filtration. With increasing copper and zinc levels in the liver, a higher relative amount of these elements were found in the low molecular weight fraction.     

Pathological,ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: Porcine epidemic abortion and respiratory syndrome (PEARS))

Summary

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6‐day‐old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re‐isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum.

Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

In vivo evaluation of vaccine efficacy against challenge with a contemporary field isolate from the α cluster of H1N1 swine influenza virus

Influenza A virus vaccines currently contain a mixture of isolates that reflect the genetic and antigenic characteristics of the currently circulating strains. This study was conducted to evaluate the efficacy of a trivalent inactivated swine influenza virus vaccine (Flusure XP) in pigs challenged with a contemporary α-cluster H1N1 field isolate of Canadian swine origin. Pigs were allocated to vaccinated, placebo, and negative-control groups and monitored for respiratory disease for 5 d after challenge. On the challenge day and 5 d after challenge the serum of the vaccinated pigs had reciprocal hemagglutination inhibition antibody titers 40 for all the vaccine viruses but ≤ 20 for the challenge virus. Gross lesions were present in the lungs of all pigs that had been inoculated with the challenge virus, but the proportion of lung tissue consolidated did not differ significantly between the placebo and vaccinated pigs. However, the amount of virus was significantly reduced in the nasal secretions, lungs, and bronchoalveolar lavage fluid in the vaccinated pigs compared with the placebo pigs. These results indicate that swine vaccinated with Flusure XP were partially protected against experimental challenge with a swine α-cluster H1N1 virus that is genetically similar to viruses currently circulating in Canadian swine.