Pathogenesis of feline infetious peritonitis: pathologic changes and immunofluorescence

Feline infectious peritonitis (FIP) was experimentally induced in FIP virus (FIPV) antibody-positive and antibody-negative kittens after challenge exposure to live-virus aerosol. Seropositive kittens developed antiviral immunofluorescence and lesions more rapidly after challenge exposure than did seronegative kittens. In seropositive kittens, FIPV antigen was present in macrophages and large mononuclear cells in tracheobronchial lymph nodes, lungs, and trachea on postchallenge-exposure day (PCD) 2; in liver and spleen on PCD 3; in kidneys and omentum on PCD 4; and subsequently in nasal turbinates, thoracic and abdominal lymph nodes, thymus, bone marrow, parotid salivary gland, eyes, and brain. Initial antiviral immunofluorescence on PCD 2 coincided with the onset of viremia and vascular lesions. Systemic lesions characterized by perivascular necrotizing pyogranulomatous inflammation, phlebitis and thrombosis, fibrinous serositis, and generalized lymphoid necrosis developed on PCD 3 and 4. Coronavirus-like particles were observed by electron microscopy in cytoplasmic vacuoles or smooth endoplasmic reticulum of degenerating macrophages in inflammatory lesions. In seronegative kittens, antiviral immunofluorescence in tracheobronchial lymph nodes was first detected on PCD 5, and viremia occurred on PCD 6. Systemic necrotizing lesions, comparable with those observed in seropositive kittens on PCD 3 or 4, did not occur in seronegative kittens until PCD 13 or 16. In both groups of kittens, initial viral infection in regional lymphoreticular tissue was followed by viremia and infection of macrophages in reticuloendothelial organs (liver, spleen, lymph nodes) and perivascular locations. The accelerated onset of infection and lesions indicative of an Arthus-type reaction in challenge-exposed seropositive vs seronegative kittens further supports the immune-mediated pathogenesis of FIP.     

The parotid, mandibular and lateral retropharyngeal lymph nodes of the camel (Camelus dromedarius)

The parotid, mandibular and lateral retropharyngeal lymph nodes of the dromedary camel were examined using both light and electron microscopy. All three lymph nodes were lobulated. They did not show the characteristic medulla, cortex and paracortex of typical lymph nodes. Instead, they contained lymphatic nodules, dense anodular lymphoid tissue and diffuse lymphoid tissue dispersed throughout the lymph node. Networks of sinuses were present in the diffuse lymphoid tissue. The diffuse lymphoid tissue in the periphery of all lymph nodes examined was characterized by numerous erythrocytes within and around its network of sinuses. The nodal sinuses were contiguous with the septal vessels, which are considered the possible source of erythrocytes seen in this study. The lymph nodes that were seen in this study resembled the haemolymph nodes of other mammals with regard to their content of erythrocytes but were unique in being located in sites that were typical of ordinary lymph nodes. Morphometric analysis has shown that the percentage volume densities of the stroma and the various parenchymal components were similar in the three lymph nodes.     

Intracranial,extraneural ectopic lymph node in a bovine (Bos taurus)

The brain from a field necropsied 8‐month‐old feedlot heifer presenting with an acute history of depression, lethargy, dyspnoea and anorexia was evaluated grossly and by histopathology. The meninges overlying the left cerebral hemisphere contained a 12 × 26 × 32 mm, dark red, soft, ovoid mass. Histologic examination of this tissue revealed a well‐organized lymph node with normal architecture. Organization of reactive lymphoid tissue resembling normal lymph node architecture may occur under chronic stimulation. However, there are no known aggregates of lymphoid tissue present within the cranial vault in any veterinary species. This is the first reported case of an intracranial ectopic lymph node in any species.     

Characterisation of immunosuppression in rabbits after infection with myxoma virus

Myxoma virus (MXV) causes the systemic disease myxomatosis in the European rabbit. Despite many in vitro studies on the function of MXV immunomodulatory proteins and detailed molecular knowledge of virus, little is known about the dynamics of interaction of the virus with the integrated host-immune system during infection. In this study changes in haematological profile, changes in lymphocyte subset distribution and non-specific proliferation activity of lymphocytes from different lymphoid compartments on the 2nd, 4th, 6th, 9th and 11th day after experimental infection of rabbits with MXV strain Lausanne was characterised. The relationship between alterations of immune parameters and dynamic of virus dissemination through the body was investigated. Haematological changes included moderate leucopenia with significant lymphopenia, neutrophilia, monocytosis and eosinopenia. A decrease of T cells including CD4+ and CD8+ and increase of CD79alpha+ were observed in draining popliteal lymph node 4 days after virus inoculation. From day 6, comparable changes were seen in collateral popliteal lymph node, spleen and peripheral blood. From day 9, the mentioned lymphocyte subsets tended to reach their original state in all of these lymphocyte compartments except draining popliteal lymph node. In thymus, MXV infection affected mainly CD4+CD8+ double positive thymocytes. On the other hand, proliferation activity of lymphocytes determined by the proliferation assay with plant-derived mitogens was significantly reduced from day 4 or 6 and remained reduced until the end of experiment in all observed lymphoid organs. Presence of MXV in respective lymphoid compartments preceded changes in lymphocyte subset distribution or lymphocyte activity.     

Blood vessels of lymph nodes in the pig

Pig lymph nodes have an unusual arrangement of cortical and medullary tissue, and lack a hilus. The aim of the present study was to describe the pathways by which blood reaches these lymph nodes. Natural or synthetic latex casts were made of blood vessels of superficial inguinal, popliteal and jejunal lymph nodes. Major nodal arteries approach these lymph nodes and give rise to about five branches, most of which remain on the node surface. These branches often wrap around part or all of the node in a claw-like manner and they divide to form a network on its surface. Arteries from this network penetrate the capsule directly to supply the lymphoid parenchyma. Anastomoses are common at all levels between the arteries supplying the nodes and arteriovenous anastomoses occur within the nodes. These may help to maintain and regulate blood flow within the lymph node under different conditions of stimulation.     

Histological, histochemical and immunohistochemical study of the lymph nodes of the one humped camel (Camelus dromedarius)

Prescapular, femoral, mesenteric, mediastinal and splenic lymph nodes from nine camels of one to 12 years of age were studied. There were no obvious structural differences between these different lymph nodes or between the ages. The lymph nodes were surrounded by a capsule formed of two layers, an outer thicker layer of connective tissue and an inner thinner layer mainly of smooth muscles. Trabeculae extended from the inner layer of the capsule dividing the parenchyma characteristically into incomplete lobules. Subcapsular and trabecular lymphatic sinuses were supported by a reticular fiber network. The parenchyma was uniquely different from that of other species, as it was arranged in the form of lymphoid follicles and interfollicular lymphoid tissue. The lymphoid follicles of CD22 positive lymphocytes were supported by a reticular fiber network. This fine network of α-smooth muscle actin positive cells enclosed the lymphoid follicles. The interfollicular tissue was mainly made up of diffusely distributed CD3 positive lymphocytes. MHC class II: DR was expressed by most lymphocytes of the follicles and interfollicular tissue. Lymphatic sinuses and high endothelial venules were found in the interfollicular zone. The lymphatic sinuses were lined by discontinuous endothelial cells. The wall of the high endothelial venules was infiltrated by several lymphocytes and enclosed in a layer of α-smooth muscle actin positive cells. Acid phosphatase positive cells were evenly distributed in the interfollicular zone. A few cells were localized in the lymph follicles. Alkaline phosphatase was observed in the endothelium of the lymphatic sinuses and in the lymphoid follicles.     

一例华南虎淋巴瘤的诊断与分析

通过对1例成年雌性华南虎(Panthera tigris amoyensis)发病后的临床特征、死后剖检、病理组织形态学观察,发现虎体内多种脏器均有不同程度的病变。临床特征表现为,渐进性消瘦、食欲减退至废绝,病程持续一个多月后死亡。尸体剖检可见严重黄染,淋巴结肿大坏死,肝、脾、肾、胰、肺等脏器表面均有灰白色结节。组织病理切片观察发现,淋巴滤泡结构、淋巴结结构被大片形态一致的淋巴细胞样肿瘤细胞取代。同时,在多个器官中均发现了瘤细胞呈浸润性生长,影响周围正常的组织结构,且各脏器中的肿瘤细胞与淋巴结中肿瘤细胞形态一致。最终,诊断为体表、肠系膜淋巴结B细胞恶性淋巴瘤,累及肝脾、肺、肾脏、胰腺等组织,造成机体死亡。     

FEASIBILITY OF CONTRAST‐ENHANCED ULTRASOUND‐GUIDED BIOPSY OF SENTINEL LYMPH NODES IN DOGS

Our goal was to develop and validate a technique to identify the sentinel lymph nodes of the mammary glands of healthy dogs with contrast‐enhanced ultrasound, and evaluate the feasibility of obtaining representative samples of a sentinel lymph node under ultrasound guidance using a new biopsy device. Three healthy intact female adult hounds were anesthetized and each received an injection of octafluoropropane‐filled lipid microspheres and a separate subcutaneous injection of methylene blue dye around a mammary gland. Ultrasound was then used to follow the contrast agent through the lymphatic channel to the sentinel lymph node. Lymph node biopsy was performed under ultrasound guidance, followed by an excisional biopsy of the lymph nodes and a regional mastectomy procedure. Excised tissues were submitted for histopathologic examination and evaluated as to whether they were representative of the node. The ultrasound contrast agent was easily visualized with ultrasound leading up to the sentinel lymph nodes. Eight normal lymph nodes (two inguinal, one axillary in two dogs; two inguinal in one dog) were identified and biopsied. Lymphoid tissue was obtained from all biopsy specimens. Samples from four of eight lymph nodes contained both cortical and medullary lymphoid tissue. Contrast‐enhanced ultrasound can be successfully used to image and guide minimally invasive biopsy of the normal sentinel lymph nodes draining the mammary glands in healthy dogs. Further work is needed to evaluate whether this technique may be applicable in patients with breast cancer or other conditions warranting evaluation of sentinel lymph nodes in animals.     

Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum

BACKGROUND: Lymphadenopathy in canine leishmaniosis has been reported as reactive lymphoid hyperplasia or granulomatous (histiocytic) lymphadenitis. However, we are unaware of information on the effect of latent Leishmania infection on lymph node cytology compared with clinically affected dogs. OBJECTIVES: The aim of the present study was to investigate cytologic patterns of lymphadenopathy in dogs with clinical and subclinical forms of leishmaniosis and to correlate cytologic findings with the density of Leishmania amastigotes in fine needle aspiration (FNA) smears. METHODS: FNA cytology of prescapular or popliteal lymph nodes was evaluated on 32 dogs with clinical evidence of leishmaniosis (group A), 24 subclinically infected dogs (group B), and 17 clinically healthy noninfected dogs (group C); groups were based on the results of serologic and PCR tests for Leishmania sp. Differential nucleated cell counts (based on 300 cells) and amastigote density were determined microscopically. Cytologic findings were categorized and compared among groups. RESULTS: Cytologic abnormalities were found in 19 of 32 (59.4%) dogs in group A, 1 of 24 (4.2%) dogs in group B, and 2 of 17 (11.8%) dogs in group C and were significantly more frequent in group A than group B (P <.001) or C (P = .001). In group A, 68.7% of the dogs had lymphoid hyperplasia, 12.5% had lymphoid hyperplasia and histiocytic lymphadenitis, 6.3% had histiocytic lymphadenitis, and 3.1% had lymphoid hyperplasia and neutrophilic lymphadenitis. Lymphoid hyperplasia was also noted in 1 dog in group B, and lymphoid hyperplasia and eosinophilic lymphadenitis were each found in 1 dog in group C. Lymph node smears from 31 (96.9%) dogs in group A and 6 (25%) dogs in group B were positive for Leishmania amastigotes; however, no correlation was found between the density of amastigotes and cytopathologic patterns of lymphadenopathy. CONCLUSION: Abnormal lymph node cytology is much more common in dogs with clinical leishmaniosis than in dogs with subclinical infection, and primarily involves lymphoid hyperplasia. Despite finding no association between the density of amastigotes and type of lymphadenopathy, lymph node cytology still is a valuable diagnostic tool for diagnosing canine leishmaniosis.     

The buccal lymph node (lymphonodus buccalis) in dogs: occurrence, anatomical location, histological characteristics and clinical implications

Three dogs were presented for clinical examination with bilateral buccal nodules which were identified as enlarged buccal lymph nodes. As little is known about this pathology, 150 dogs were examined by anatomical dissection for the presence of buccal lymph nodes. They were found in 13 dogs, occurring bilaterally in six dogs and unilaterally in seven dogs. Two buccal lymph nodes were bilobulated and one was double. The lymph nodes were always located dorsal to the zygomatic muscle and rostral to the masseter muscle in the region where the superior labial vein drains into the facial vein. Histology demonstrated a large amount of intranodal adipose tissue scattered throughout the lymphoid tissue. The canine buccal lymph node should not be confused with the accessory parotid or ventral buccal salivary gland and is clinically important as it can enlarge due to tumour metastasis or inflammation of the buccal region.     

Ovine nasal zygomycosis caused by Conidiobolus incongruus

About 700 sheep died on 52 farms in north-western New South Wales and south-western Queensland over a 3 mo period. Affected animals had a marked asymmetrical swelling of the face, extending from the nostril to just anterior to the eyes. They lost condition rapidly and died within 7 to 10 days. At necropsy there was a unilateral severe necrogranulomatous rhinitis with extension of inflammation into the adjacent subcutaneous tissue, nasal septum and hard palate. Metastatic lesions were present in the draining lymph nodes and in the thorax. Histological changes consisted of a granulomatous reaction with numerous eosinophilic foci of necrosis and a diffuse, heavy, mixed inflammatory cell response. Many vessels had segmental necrosis and thrombosis. Fungal hyphae were numerous, particularly within or associated with necrotic foci. Conidiobolus incongruus was isolated from nasal tissues, parotid and submandibular lymph nodes and pulmonary lesions.     

Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in Asian elephants (Elephas maximus)

Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.     

Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.     

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.     

Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats

Feline infectious peritonitis (FIP) is the most frequent lethal infectious disease in cats. However, understanding of FIP pathogenesis is still incomplete. Mutations in the ORF 3c/ORF 7b genes are proposed to play a role in the occurrence of the fatal FIPV biotype. Here, we investigated 282 tissue specimens from 28 cats that succumbed to FIP. Within one cat, viral sequences from different organs were similar or identical, whereas greater discrepancies were found comparing sequences from various cats. Eleven of the cats exhibited deletions in the 3c gene, resulting in truncated amino acid sequences. The 7b gene was affected by deletions only in one cat. In three of the FIP cats, coronavirus isolates with both intact 3c genes as well as 7b genes of full length could also be detected. Thus, deletions or stop codons in the 3c sequence seem to be a frequent but not compelling feature of FIPVs.     

Laboratory changes consistent with feline infectious peritonitis in cats from multicat environments

The present study describes the prevalence of haematological and electrophoretic changes consistent with the diagnosis of feline infectious peritonitis (FIP) in cats without FIP living in six multicat environments with different prevalence of FIP and of other diseases. The results allow designing haematological and electrophoretic profiles typical of each group, most likely depending on the management and on the health status of the group rather than on the prevalence of FIP. In fact, many cats from the colonies with open management and frequent outbreaks of infectious diseases other than FIP had one or more haematological and/or electrophoretical changes consistent with FIP, compared with the reference ranges. In the case of non-specific clinical signs such as fever or neurological signs because of diseases other than FIP, these cats would be erroneously considered as affected by FIP and euthanasized. The use of internal ranges designed on the basis of repeated samplings from non-symptomatic cats allows avoiding these misinterpretations. Results from cats with symptoms consistent with FIP living in the same colonies were also compared with both the reference ranges and the internal ones: such a comparison demonstrated that the use of internal ranges rarely affected the possibility to correctly diagnose the disease in cats with symptoms suggestive of FIP.     

Spontaneous histiocytic sarcoma of the popliteal lymph node in a young sprague-dawley rat

The present report describes a rare case of spontaneous primary histiocytic sarcoma of the popliteal lymph node in a 19-week-old female Sprague-Dawley (SD) rat. At necropsy, a 10 mm-diameter whitish nodule was found at the site of the femoral muscle in the right hindlimb. Histopathologically, the nodule comprised large pleomorphic histiocyte-like cells with abundant eosinophilic or foamy cytoplasm. Multinucleated giant cells, necrotic foci surrounded by palisading arrays of epithelioid histiocyte-like cells and phagocytosis of cell debris or erythrocytes by the neoplastic cells were occasionally observed. Invasion of the tumor cells into the surrounding adipose tissue was found focally, but there were no distal metastases. Immunohistochemically, the neoplastic cells were positive for vimentin, CD68 (ED1) and lysozyme. We concluded that this tumor occurred in the popliteal lymph node, considering the anatomical location of the lesion and the presence of the remnants of lymphoid tissue involved in the tumor.     

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting syndrome (PMWS), and to evaluate the diagnostic techniques for detecting porcine circovirus type 2 (PCV-2) and other microorganisms. Four pigs in the early stage and 11 pigs in the late stage of the disease, and eight clinically normal pigs were necropsied. Samples of lymphoid tissue and serum were also obtained from 12 slaughter pigs from the same farm. The tissues were examined histopathologically, and in situ hybridisation, serology and PCR were used to detect porcine circovirus type 1 (PCV-1) and/or PCV-2 in tissues and/or sera. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) were also investigated. Characteristic microscopical lesions of PMWS were observed in the lymphoid tissues of the pigs in all three necropsied groups; the lesions were most common and severe in the pigs in the early stage of the disease, less so in the pigs in the late stage of the disease, and least in the clinically normal pigs. PCV-2 infection was detected in all the necropsied pigs by in situ hybridisation and PCR. Only three pigs had the PCV-1 genome in serum or lymph node tissue. In contrast, the slaughter pigs had no microscopical lesions and no PCV-2 nucleic acid in their serum or tissues, and only one of them had the pCV-1 genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.