Evaluation of a commercial double-recognition ELISA for caprine tuberculosis diagnosis: Sensitivity, specificity, and correlation to macroscopic and microscopic lesions.

Ante-mortem diagnosis of caprine tuberculosis, as in other ruminant species, is a complex process. Therefore, it is sometimes necessary to apply a combination of methods that increases the sensitivity of diagnosis while maintaining adequate specificity. In the present study, we analysed the use of ELISA based on a double-recognition methodology for the diagnosis of goat tuberculosis. Serum samples from animals with tuberculosis, confirmed by histopathological studies and presence of Ziehl-Neelsen-positive bacteria in the lesions, were used to assess sensitivity. Sera from flocks without tuberculosis were used to assess diagnostic specificity. The relationship between ELISA results and the type of tuberculous lesions, both macroscopic and microscopic, was analysed. The sensitivity and specificity of ELISA test were 69.9% and 97.9%, respectively. Sensitivity was higher in animals with macroscopic lesions, indicating more advanced forms of the disease. Similarly, a higher sensitivity was found in animals with microscopic lesions associated with active tuberculosis, such as cavitary lesions.

Bone regeneration in critical-sized mandibular symphysis defects using bioceramics with or without bone marrow mesenchymal stem cells in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats.

OBJECTIVES: To compare new bone formation in mandibular critical-sized bone defects (CSBDs) in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats filled with bioceramics (BCs) with or without bone marrow mesenchymal stem cells (BMSCs). METHODS: A total of 64 adult female Sprague-Dawley rats were randomized into four groups (n = 16 per group): Group 1 healthy, Group 2 diabetic, Group 3 osteoporotic, and Group 4 diabetic-osteoporotic rats. Streptozotocin was used to induce type 1 diabetes in Group 2 and 4, while bilateral ovariectomy was used to induce osteoporosis in Group 3 and 4. The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxypatatite 60% and ß-tricalcium phosphate 40%) alone and eight with BMSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In all groups (healthy, diabetics, osteoporotics, and diabetics-osteoporotics), the CSBDs filled with BC + BMSCs showed greater radiological bone union, BMD, histological bone union, and more VEGF and BMP-2 positivity, in comparison with CSBDs treated with BC alone (at 4 and 8 weeks). CONCLUSIONS: Application of BMSCs cultured on BCs improves bone regeneration in CSBDs compared with application of BCs alone in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats.

Use of buccal fat pad-derived stem cells cultured on bioceramics for repair of critical-sized mandibular defects in healthy and osteoporotic rats.

OBJECTIVE: To compare new bone formation in mandibular symphysis critical-sized bone defects (CSBDs) in healthy and osteoporotic rats filled with bioceramics (BCs) with or without buccal fat pad mesenchymal stem cells (BFPSCs). MATERIALS AND METHODS: Thirty-two adult female Sprague-Dawley rats were randomized to two groups (n = 16 per group): group 1 healthy and group 2 osteoporotic (with bilateral ovariectomy). The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxyapatite 60% and ß-tricalcium phosphate 40%) alone and eight with BFPSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In both groups, CSBDs filled with BC + BFPSCs showed greater radiological bone union, BMD and histological bone union, and more VEGF and BMP-2 positivity, compared with CSBDs treated with BC alone at 4 and 8 weeks. CONCLUSIONS: The application of BFPSCs cultured on BCs improves bone regeneration in CSBDs compared with BCs alone in healthy and osteoporotic rats. CLINICAL RELEVANCE: Our results may aid bone regeneration of maxillofacial CSBDs of both healthy and osteoporotic patients, but further studies are necessary.

First report of a hawfinch (Coccothraustes coccothraustes) parasitized by Harpirhynchus nidulans in the Iberian Peninsula.

Some avian Harpirhynchidae mites live under the skin and develop cutaneous cysts. Despite the obvious lesions that these parasites can produce, little is currently known about the behavioural disturbances that cyst-forming mites may cause in infected wild birds. We report an infection by Harpirhynchidae mites in a hawfinch (Coccothraustes coccothraustes) in southeast Spain. The bird was easily captured due to its inability to fly. During clinical examination it was found to have multiple severe traumatic injuries, possibly due to a blow or a fall, as result of which the bird was euthanized. At necropsy, the hawfinch was found to be in good body condition. Two yellowish and friable mite-filled cysts were detected in the subalar region of both wings. Mites were morphologically identified as Harpirhynchus nidulans, and histological analysis of the cystic lesions was also performed. This is the first time that the occurrence of a hawfinch infected by H. nidulans in the Iberian Peninsula has been reported.

Effects of diabetes on oxidative stress, periodontal ligament fiber orientation, and matrix metalloproteinase 8 and 9 expressions during orthodontic tooth movement.

OBJECTIVES: To evaluate the influence of diabetes on oxidative stress, periodontal ligament (PDL) orientation, and matrix metalloproteinase (MMP) 8 and 9 expressions during orthodontic tooth movement in a rat model. MATERIALS AND METHODS: An orthodontic appliance was placed in 60 Sprague-Dawley rats divided into three groups: normoglycemics (n = 20) and two streptozotocin-induced diabetic groups, one untreated (n = 20) and one insulin-treated (n = 20). At 24, 48, and 72 h and 1 week, rats were sacrificed. At each time point, myeloperoxidase (MPO) and malondialdehyde (MDA) were quantified by spectrophotometry, tooth movement was evaluated by micro-CT analysis, and hematoxylin and eosin staining was used to evaluate PDL fiber orientation and immunohistochemistry staining with semi-quantitative H-score analysis of MMP-8 and MMP-9 was performed.. RESULTS: At 24 h, MPO activity was significantly higher in untreated-diabetics than normoglycemics. At 24 and 48 h, the MDA level in untreated-diabetic rats was significantly higher than in normoglycemics and insulin-treated animals. At 72 h and 1 week, PDL fibers were oriented significantly more irregularly in untreated-diabetics than in normoglycemics. At all time points, MMP-8 and MMP-9 expressions were significantly higher in both diabetic groups than in the normoglycemic group. After the second day, tooth movement was significantly greater in untreated-diabetics than in the insulin-treated and normoglycemic groups. CONCLUSIONS: Mechanical stress in untreated-diabetic rats produces more inflammatory response, oxidative stress, tooth movement, PDL disorganization, and MMP-8 and MMP-9 expressions than among normoglycemics. Insulin reverses these effects, favoring the reorganization of periodontal ligament. CLINICAL RELEVANCE: Our results suggest that the application of orthodontic force in diabetic patients would increase inflammation and delay periodontal restructuring. Insulin would partly reverse this situation although glycemic decompensation episodes may occur. For these reasons, the periods between fixed orthodontic appliance activations should be of sufficient duration to allow adequate tissue recovery.

Bone union formation in the rat mandibular symphysis using hydroxyapatite with or without simvastatin: effects on healthy, diabetic, and osteoporotic rats.

OBJECTIVE: The objective is to compare new bone formation in critical defects in healthy, diabetic, and osteoporotic rats filled with hydroxyapatite (HA) alone and HA combined with simvastatin (SV). MATERIALS AND METHODS: A total of 48 adult female Sprague-Dawley rats were randomized into three groups (n = 16 per group): Group, 1 healthy; Group 2, diabetics; and Group 3, osteoporotics. Streptozotocin was used to induce type 1 diabetes in Group 2, while bilateral ovariectomy was used to induce osteoporosis in Group 3. The central portion of the rat mandibular symphysis was used as a physiological critical bone defect. In each group, eight defects were filled with HA alone and eight with HA combined with SV. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In all groups (healthy, diabetics, and osteoporotics), the defects filled with HA + SV presented greater radiological bone union, BMD, histological bone union, and more VEGF and BMP-2 positivity, in comparison with bone defects treated with HA alone. CONCLUSIONS: Combined application of HA and SV improves bone regeneration in mandibular critical bone defects compared with application of HA alone in healthy, diabetic, and osteoporotic rats. CLINICAL RELEVANCE: This study might help to patients with osteoporosis or uncontrolled diabetes type 1, but future studies should be done.

Effect of progesterone on the vaccination and immune response against Chlamydia abortus in sheep.

Chlamydia abortus produces ovine enzootic abortion (OEA). Symptoms are not observed until the organism colonises the placenta, eventually causing abortion. Infected animals become carriers and will shed the organism in the following oestruses. This process suggests that sex hormones might play an important role in the physiopathology of OEA, affecting the success of chlamydial clearance and also jeopardising the effectiveness of vaccination. However, the mechanisms through which sex hormones are involved in chlamydial pathogenicity remain unclear. The aim of this study, therefore, was to determine the effect of progesterone on the immune response against C. abortus and on the protection conferred by an experimental inactivated vaccine in sheep. Eighteen sheep were ovariectomised and divided into four groups: vaccinated and progesterone-treated (V-PG), vaccinated and non-treated (V-NT), non-vaccinated and non-treated (NV-NT) and non-vaccinated and progesterone-treated sheep (NV-PG). Animals from both PG groups were treated with commercial medroxyprogesterone acetate impregnated intravaginal sponges before and during the vaccination (V-PG) or just before challenge (NV-PG). The animals from both V groups were subcutaneously immunised with an experimental inactivated vaccine, which was seen to confer high protection in previous studies. All sheep were challenged intratracheally with C. abortus strain AB7 and were sacrificed on day 8 post-infection. Morbidity was measured as the variation in rectal temperature and samples of sera were collected for antibody and cytokine (IFN-γ and IL-10) analysis by commercial ELISA. In addition, lung and lymph node samples were collected for chlamydial detection by qPCR and for histopathological and immunohistochemical analyses. Sheep from the V-PG group showed less severe or no lesions and lower morbidity than the other groups. They also had the highest abundance of regulatory T-cells. The sheep from V-NT also manifested high antibody levels against C. abortus and less severe lesions than those observed in non-vaccinated sheep, which showed high morbidity, low antibody levels and severe lesions, especially in NV-NT. These results confirm the effectiveness of the experimental vaccine employed and suggest that progesterone could enhance the effect.

Effect of female sex hormones on the developmental cycle of Chlamydia abortus compared to a penicillin-induced model of persistent infection.

BACKGROUND: Chlamydia abortus, an obligate intracellular pathogen with an affinity for placenta, causes reproductive failure. In non-pregnant animals, an initial latent infection is established until the next gestation, when the microorganism is reactivated, causing abortion. The precise mechanisms that trigger the awakening of C. abortus are still unknown. Sexual hormones such as estradiol and progesterone have been shown to affect the outcome of infection in other species of the family Chlamydiaceae, while estrogens increase chlamydial infection, progesterone has the opposite effect. To try to establish whether there is a relationship between these events and the latency/ reactivation of C. abortus in the reproductive tract of small ruminants, ovine endometrial (LE) and trophoblastic (AH-1) cells were treated with estradiol or progesterone prior to their infection with C. abortus. The results are compared with those obtained for treatment with penicillin prior to infection, which is a well-established model for studying persistent infection in other chlamydial species. Cells were examined by transmission electron microscopy, and an mRNA expression analysis of 16 genes related to the chlamydial developmental cycle was made. RESULTS: The changes observed in this study by the action of sex hormones seem to depend on the type of cell where the infection develops. In addition, while the changes are morphologically similar to those induced by treatment with penicillin, the patterns of gene expression are different. Gene expression patterns therefore, seem to depend on the persistence induced models of C. abortus used. Hormone treatments induced aberrant forms in infected endometrial cells but did not affect the chlamydial morphology in trophoblast cells. At the genetic level, hormones did not induce significant changes in the expression of the studied genes. CONCLUSIONS: The results suggest that penicillin induces a state of persistence in in vitro cultured C. abortus with characteristic morphological features and gene transcriptional patterns. However, the influence of hormones on the C. abortus developmental cycle is mediated by changes in the host cell environment. Furthermore, a persistent state in C. abortus cannot be characterised by a single profile of gene expression pattern, but may change depending on the model used to induce persistence.

Intratracheal infection as an efficient route for testing vaccines against Chlamydia abortus in sheep.

Pregnant ewes have been widely used to test vaccines against Chlamydia abortus. However, this model entails many disadvantages such as high economic costs and long periods of pregnancy. The murine model is very useful for specific studies but cannot replace the natural host for the later stages of vaccine evaluation. Therefore, a non-pregnant model of the natural host might be useful for a vaccine trial to select the best vaccine candidates prior to use of the pregnant model. With this aim, two routes of infection were assessed in young non-pregnant sheep, namely, intranasal (IN) and intratracheal (IT). In addition, groups of non-vaccinated sheep and sheep immunised with an inactivated vaccine were established to investigate the suitability of the model for testing vaccines. After the experimental infection, isolation of the microorganism in several organs, with pathological and immunohistochemical analyses, antibody production assessment and investigation by PCR of the presence of chlamydia in the vagina or rectum were carried out. Experimental IT inoculation of C. abortus induced pneumonia in sheep during the first few days post-infection, confirming the suitability of the IT route for testing vaccines in the natural host. The course of infection and the resulting pathological signs were less severe in vaccinated sheep compared with non-vaccinated animals, demonstrating the success of vaccination. IN infection did not produce evident lesions or demonstrate the presence of chlamydial antigen in the lungs and cannot be considered an appropriate model for testing vaccines.

N-cadherin expression in feline mammary tumors is associated with a reduced E-cadherin expression and the presence of regional metastasis.

The cadherin family of adhesion molecules regulates cell-cell interactions. N-cadherin is expressed by neural and fibroblast cells but not by normal epithelial cells. In human medicine, the role of N-cadherin in breast cancer remains controversial, but some studies have described the switch from E-cadherin to N-cadherin as a critical step in the malignant progression of neoplastic cells. The present study was carried out on 160 feline mammary tumors (21 adenomas and 139 carcinomas). The relationship between the immunohistochemical expression of N-cadherin in neoplastic epithelial cells and 2 established prognostic factors such as regional metastasis and tumor grade was examined. The results of the study showed a statistically significant relation between the expression of N-cadherin and the 2 prognostic factors, and also a reduced expression of E-cadherin in tumors that expressed N-cadherin.

IFN-γ expression in placenta is associated to resistance to Chlamydia abortus after intragastric infection.

Intragastric infection mimics the natural route of infection of Chlamydia abortus (etiological agent of ovine enzootic abortion). In the mouse model, intragastric experimental infection induces very mild signs of infection followed by late term abortions, as it is shown by the natural ovine host. In order to evaluate the immune mechanisms associated to the dissemination of the pathogen from the gastrointestinal tract, we have administered an intragastric dose of C. abortus to pregnant mice. Systemic and local expression of cytokines, tissue colonization and excretion of bacteria after parturition were monitored during pregnancy. Susceptible CBA/J mice showed a higher bacterial colonization of the placenta and excretion of live bacteria after parturition that were related to a higher local IL-10 expression. By contrast, resistant C57BL/6 mouse strain had higher local IFN-γ mRNA expression in the placenta just before parturition and a transient bacterial colonization of the reproductive tract, with no excretion of C. abortus after parturition. In summary, intragastric infection not only mimics the natural route of infection of C. abortus, but can also be useful in order to understand the immunopathogenesis of chlamydial abortion in the mouse.

Reduced expression of E-cadherin and ß-catenin and high expression of basal cytokeratins in feline mammary carcinomas with regional metastasis.

Feline mammary carcinomas are highly aggressive neoplasms. Several mechanisms are thought to be involved in their progression, including the loss of epithelial adhesion molecules. The present study was carried out on 21 adenomas and 139 mammary carcinomas. Of the carcinomas, 66 were not reported to have metastasized, while the remaining 73 had evidence of regional lymph node metastasis at the moment of diagnosis. The relationship was examined between the expression of the E-cadherin-ß-catenin complex and basal (CK5/6, CK14) and luminal (CK8/18) cytokeratin expression. In the medical literature, carcinomas expressing basal cytokeratins are reported as having a poor prognosis in human breast cancer. Results revealed that preservation of the expression of E-cadherin and ß-catenin is a significant feature of carcinomas without metastasis, whereas carcinomas with metastasis reveal the loss of one or both adhesion molecules. Additionally, basal cytokeratin expression was statistically associated with the presence of regional metastasis. Furthermore, the expression of E-cadherin-ß-catenin was significantly correlated with the high expression of CK18 and low expression of CK5/6.

Evaluating the efficacy of cinnamaldehyde and Echinacea purpurea plant extract in broilers against Eimeria acervulina.

Coccidiostats could be phased out as feed additives before 1 January 2013 for public health and food safety reasons, and, as a replacement, bioactive compounds found in plants are currently being investigated since they are more likely to be found acceptable by consumers. The aim of our study was to evaluate the effect of cinnamaldehyde (CIN) and Echinacea purpurea plant extract (EP) as additives by analyzing the performance traits, oocyst excretion and intestinal lesions following experimental infection with Eimeria acervulina. A total of 72 Ross male broilers were raised from 1 to 35 d and randomly assigned to four dietary treatments: control, without additives (C); 150 mg kg(-1) cinnamaldehyde (CIN); 1000 mg kg(-1)E. purpurea plant extract (EP); 150 mg kg(-1) cinnamaldehyde plus 1000 mg kg(-1)E. purpurea plant extract (CIN+EP). At 25 d, 12 chickens per treatment were orally infected with E. acervulina. Coccidia infestation led to lower performance but with no significant differences between the infected groups. Oocyst output reached its peak from 6 to 9 d post-infection in all treatments. At duodenal level, gross lesion scores were lower for cinnamaldehyde diets (P<0.05). A similar trend was observed in the microscopic lesion scores, with a non-significant reduction as a result of cinnamaldehyde addition (P>0.05). Scoring methods for macro- and microscopic lesions showed a positive linear relationship (G=+0.70). Further studies are necessary to assess the possible anticoccidian action of the cinnamaldehyde and its value as an alternative or adjunct in therapeutic or prophylactic strategies.

Cyclooxygenase-2 expression is related with localization, proliferation, and overall survival in canine melanocytic neoplasms.

A direct relationship has been firmly established between cyclooxygenase-2 (COX-2) expression and malignant behavior in human melanoma. This report examines the relationship between COX-2 expression and tumor location, mitotic and proliferative indices, degree of T CD3(+) lymphocyte infiltration, overall survival, and frequency of recurrence and metastasis of 57 melanocytic tumors (25 oral and 32 cutaneous). COX-2 was highly or moderately expressed in 88% of oral neoplasms (22 of 25), whereas for their cutaneous counterparts, COX-2 expression was low or insignificant in 75% of cases (24 of 32). High and moderate COX-2 expression levels were observed in 73% of melanocytic tumors with a mitotic index ≥ 3 per 10 high-power fields (26 of 36), whereas in 81% of tumors with a mitotic index < 3 (17 of 21), expression was mild or absent. There were 41 cases with known clinical outcomes; of those showing high, moderate, and mild COX-2 expression, 83.3% (10 of 12), 37.5% (3 of 8), and 25% (2 of 8) died, respectively, whereas 100% of animals showing no COX-2 expression (13 of 13) were still alive at the last follow-up. COX-2 expression was statistically correlated with tumor location, mitotic and percentage Ki-67 proliferative indices, and overall survival, frequency of neoplastic recurrence and metastasis. Regression analysis also showed disease-specific predictive value for COX-2 expression for subjects with melanocytic neoplasms. Additionally, only high COX-2 expression showed significant differences in overall survival, in comparison with moderate, mild, or absent expression. These results suggest that high COX-2 expression may be considered a prognostic biomarker and potentially as a target for therapeutic and preventive strategies in canine melanocytic neoplasms.

Microscopical and immunological features of tuberculoid granulomata and cavitary pulmonary tuberculosis in naturally infected goats.

Caprine tuberculosis is caused by bacteria of the Mycobacterium tuberculosis complex (Mycobacterium bovis and Mycobacterium caprae). Although typical tuberculoid granulomata are usually observed in the lungs and lymph nodes of infected goats, the presence of cavitary lesions with exuberant mycobacterial growth is also a common feature in this species. The aim of this study was to characterize the immunological mechanisms that lead to liquefaction and cavity formation by comparing granulomata and cavitary lesions. Samples from animals positive by skin testing were collected for microscopical and immunohistochemical examination. Samples were also collected for analysis of cytokine gene expression in the lesions by real time polymerase chain reaction. There were marked differences between granulomata and cavitary lesions. In cavitary lesions there was a substantial population of neutrophils and a significant decrease in the number of CD4(+) T cells, with concomitant increases in other T-cell populations (CD8(+) and cells expressing the γδ form of the T-cell receptor). The enzyme iNOS was strongly expressed by macrophages in the cavitary lesions. There was no difference in the balance of pro- and anti-inflammatory cytokine gene expression in the lesions. These findings suggest that cavitary lesions are reactivation sites, where conditions are optimal for Mycobacterium proliferation and that immunological mechanisms may underlie the severe destruction of lung tissue that characterizes the cavitary pathology.

Histopathological lesions in 15 cats with leishmaniosis.

Recent research into the prevalence of Leishmania infantum infection in the Mediterranean basin points to the involvement of cats as a reservoir host, but only sporadic cases of feline leishmaniosis have been reported. Feline leishmaniosis presents primarily as cutaneous disease and diagnosis is based on the demonstration of the parasite by skin biopsy. The present report describes the microscopical changes in tissue biopsies from 15 cats with leishmaniosis. The biopsies were derived from the skin, ocular tissue and mucocutaneous junctions. The most common histopathological feature was diffuse granulomatous inflammation with macrophages containing numerous amastigotes. Other patterns included granulomatous perifolliculitis and lichenoid interface dermatitis, where there were fewer parasitized macrophages. The presence of amastigotes was confirmed by immunohistochemistry in each case. The results of the study confirm the value of histopathological and immunohistochemical techniques for the diagnosis of feline leishmaniosis.

Diagnosis of placental pathogens in small ruminants by immunohistochemistry and PCR on paraffin-embedded samples.

A histological study was carried out on 58 formalin-fixed and paraffin-embedded samples of placenta from sheep and goats that had aborted, and the placental lesions were graded. Sequential histological sections of each cotyledon were then immunostained with specific antibodies and used for PCR detection of Chlamydophila abortus, Coxiella burnetii, Salmonella Abortusovis, Brucella melitensis, Listeria monocytogenes and Toxoplasma gondii. Most of the cotyledons showed different degrees of placentitis. The proportional agreement between the two techniques was 0.879 (kappa value 0.746). C abortus was the most prevalent pathogen. Mixed infections were common.

Chlamydophila abortus infection in the mouse: a useful model of the ovine disease.

Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.

High prevalence of antibodies against Chlamydiaceae and Chlamydophila abortus in wild ungulates using two "in house" blocking-ELISA tests.

Few data are available on the prevalence and relevance of chlamydiae in wild mammals, and even fewer studies have been conducted to determine the prevalence of Chlamydophila abortus in wildlife hosts, most probably due to the absence of suitable species-specific serological assays for testing sera from wild animals. In light of this, we have developed two in-house blocking-ELISA tests for detection of antibodies against Chlamydiaceae and C. abortus in wild ungulates, and analyzed the relationship between geographical and biological factors and the prevalence of antibodies against Chlamydiaceae and C. abortus in 434 wild ungulates from Spain, including sera from European wild boar, Red deer, Fallow deer, Roe deer, Mouflon, Barbary sheep, Southern chamois, and Iberian ibex. Serology revealed that 41.7+/-4% of the sera were positive for the b-ELISA-LPS (Chlamydiaceae-specific) and 18.9+/-3% for the b-ELISA-rPOMP (C. abortus-specific). Antibodies against Chlamydiaceae lipopolysaccharide (LPS) were detected in sera from all eight ungulate species, the prevalence ranging from 23 to 60%. Iberian ibex was the only wild ungulate not showing seropositivity to the C. abortus specific polymorphic outer membrane protein (POMP). The prevalence of anti-POMP antibodies in the other seven wild ungulate species ranged from 7 to 40%. While significant seroprevalence differences were detected among species and among sampling regions, no effect of age and sex was observed. The high prevalence levels found should be considered with regards to livestock and human health, and warrant further research.

Immunohistochemical characterization of a pulmonary large-cell carcinoma in a dog.

Primary pulmonary tumors are less common in dogs than secondary (metastatic) tumors. Most primary tumors are malignant and of epithelial origin. Pulmonary large-cell carcinoma is considered extremely rare in domestic animals, and some of the few reported cases actually may have been cases of malignant pulmonary histiocytosis. An 8-year-old female Wire Fox Terrier with diffuse alveolar pattern radiographically was euthanatized when it failed to respond to antibiotic therapy. Histologically, pulmonary alveolar spaces contained clusters of large round anaplastic cells with ample eosinophilic cytoplasm and large irregularly shaped nuclei with prominent nucleoli. Immunohistochemistry was used to distinguish large-cell carcinoma from malignant pulmonary histiocytosis. Tumor cells had strong immunoreactivity for cytokeratin, consistent with epithelial origin. However, a substantial percentage of the neoplastic cells co-expressed vimentin and MHC-II. The type II alveolar epithelial cell was considered the cell of origin of the neoplasm based on the presence of lamellar bodies in some neoplastic cells and immunoreactivity for surfactant protein A and thyroid transcription factor-1.