Effect of Female Sex Hormones on the Immune Response against Chlamydia abortus and on Protection Conferred by an Inactivated Experimental Vaccine in a Mouse Model.

Mice are valuable models extensively used to test vaccine candidates against Chlamydia abortus and to clarify immunopathological mechanisms of the bacteria. As this pathogen has the ability to reactivate during pregnancy, it is important to deepen the knowledge and understanding of some of the effects of female hormones on immunity and vaccination. This study is aimed at describing the role of sex hormones in the pathology of OEA during chlamydial clearance using ovariectomised mice and also gaining an understanding of how 17ß-oestradiol or progesterone may impact the effectiveness of vaccination. Animals were treated with sex hormones and infected with C. abortus, and the kinetics of infection and immune response were analysed by means of bacterial isolation, histopathology, and immunohistochemistry. In a second phase of the study, protection conferred by an experimental vaccine after hormone treatment was assessed. Oestradiol showed a stimulatory effect on the immune response during infection, with a more efficient recruitment of macrophages and T-cells at the infection site. Furthermore, after vaccination, oestradiol-treated animals showed a stronger protection against infection, indicating that this hormone has a positive effect, stimulating a specific memory response to the pathogen.

Regeneration of lingual musculature in rats using myoblasts over porcine bladder acellular matrix.

OBJECTIVES: To use tissue engineering muscle repair (TEMR) for regenerating the lingual musculature of hemiglossectomized rats using neonatal myoblasts (NM) on porcine acellular urinary bladder matrix (AUBM). MATERIAL AND METHODS: The study used 80 male rats. A volumetric muscle loss (VML) injury was created on the left side of the tongue. The rats were randomized into four groups: Group 1 (AUBM + myoblasts); Group 2 (AUBM); Group 3 (myoblasts); and Group 4 (control). NM were obtained from neonatal rats. The animals were weighed on day 0 and just before euthanasia. Five rats in each group were euthanized at days 2, 14, 28, and 42; the tongues were prepared for morphometric analysis, postoperative left hemitongue weight, and immunohistochemical analysis (desmin, CD-31, and anti-neurofilament). RESULTS: The weight gain from greatest to least was as follows: AUBM + myoblasts > myoblasts > AUBM > control. The tongue dorsum occupied by VML, and difference in mg between control side and intervened side from least to great was as follows: AUBM + myoblasts < myoblasts < AUBM < control. The order from highest to lowest antibody positivity was as follows: AUBM + myoblasts > myoblasts > AUBM > control. CONCLUSION: The use of porcine AUBM and NM for the regeneration of lingual musculature was found to be an effective TEMR treatment for repairing tongue VML injury.

The inhibitory effect of flavonoids and their gut-derived metabolites on the replication of Chlamydia abortus in the AH-1 ovine trophoblast cell line.

The obligate intracellular bacterium Chlamydia abortus causes abortion and constitutes a worldwide threat for livestock. Plant-derived flavonoids have antimicrobial effects against veterinary and human pathogens and may be of help in the fight against C. abortus. The anti-infective efficacy against C. abortus of the flavonoids apigenin and naringenin, and of four of their derived metabolites was explored using an in vitro model of ovine trophoblast cells. Overall, the anti-infective effectiveness was apigenin > naringenin > benzoic acid > 3-(4-hydroxyphenyl) propionic acid ~ 4-hydroxyphenyl acetic acid ~ 4-hydroxybenzoic. At the lowest concentration tested (10 µM), apigenin, naringenin and benzoic acid inhibited the formation of C. abortus inclusions by 80%, 67%, and 39%, respectively. The cytotoxicity and anti-proliferative effects on the trophoblast host cells also differed greatly between the tested compounds. Our findings suggest that flavonoids may be of therapeutic value against C. abortus infection but metabolic conversion has a substantial and variable effect on their anti-chlamydial activity. Our results also support the notion that the mechanisms of anti-infective action may involve combined effects of the compounds against the host cells and the bacteria.

Cholangiocarcinoma and hepatic myelolipoma incarcerated in a peritoneopericardial diaphragmatic hernia with pulmonary metastasis and carcinomatosis in a cat.

CASE SUMMARY: A 14-year-old female neutered Persian-cross cat was presented with a 1 week history of anorexia and lethargy. On physical examination, marked tachypnoea and dyspnoea were evident. Radiographs of the thorax revealed a globoid-shaped cardiac silhouette with heterogeneous opacity consistent with a peritoneopericardial diaphragmatic hernia (PPDH), pulmonary nodules compatible with metastasis, seven sternal segments and a small liver in the cranial abdomen with loss of serosal detail. On echocardiography, there was no evidence of cardiac tamponade. Triple-phase CT angiography demonstrated a mixed soft tissue-, mineral- and fat-attenuated liver mass arising from the left hepatic lobes that showed a pronounced heterogeneous contrast-enhancement pattern within the pericardial sac, which was producing a marked mass effect on the adjacent structures. Additionally, there was an increase in attenuation of the mesenteric fat and peritoneal effusion. The pulmonary nodules were confirmed. Imaging findings were compatible with a malignant hepatic neoplasia incarcerated in a PPDH, lung metastasis and carcinomatosis. Owing to the poor prognosis, the cat was humanely euthanased. Histopathological diagnosis was cholangiocellular carcinoma and hepatic myelolipoma, pulmonary metastasis and carcinomatosis. RELEVANCE AND NOVEL INFORMATION: Hepatic cholangiocarcinoma incarcerated in a PPDH with pulmonary metastasis and carcinomatosis has not been previously described. Suspicion of a hepatic neoplasia should be raised in cases of PPDH and pulmonary nodules.

Radiographic, ultrasonographic, and computed tomographic characteristics of an accessory liver lobe in a cat.

A 5-year-old male Norwegian Forest cat presented with increased hepatic serum biochemical parameters. Abdominal radiography showed an oval cranioventral mass and ultrasound revealed a mobile mass attached to one hepatic lobe. Computed tomography (CT) confirmed that the mass was attached to the right medial liver lobe. Differential diagnoses were an accessory liver lobe, benign neoplasia, and focal nodular hyperplasia. The mass was removed and histopathology confirmed the mass to be normal liver tissue. Accessory liver lobe should be included in the differential diagnosis of a mobile cranial abdominal mass with a similar ultrasonographic or CT appearance to the liver.

Development of an in vivo model of Chlamydia abortus chronic infection in mice overexpressing IL-10.

Chlamydia abortus, like other members of the family Chlamydiaceae, have a unique intracellular developmental cycle that is characterized by its chronic nature. Infection of a flock can remain undetected for months, until abortion occurs the following reproductive season but, to date, neither the location nor the mechanisms that maintain this latent phase are fully understood. Studies have shown that IL-10 produced as a response to certain micro-organisms sustains the intracellular survival of pathogens and increases host susceptibility to chlamydial infections. In order to induce a sustained infection C. abortus, transgenic mice that constitutively express IL-10 were infected and the immunological mechanisms that maintain infection in these mice were compared with the mechanisms of a resistant wild-type mouse strain. Viable bacteria could be detected in different tissues of transgenic mice up to 28 days after infection, as analysed by bacterial isolation and immunohistochemistry. Chronic infection in these mice was associated with an impaired recruitment of macrophages, decreased iNOS activity at the site of infection and a more diffuse distribution of inflammatory cells in the liver. This murine model can be of great help for understanding the immunological and bacterial mechanisms that lead to chronic chlamydial infections.

IMAGING DIAGNOSIS-SPONTANEOUS PNEUMOMEDIASTINUM SECONDARY TO PRIMARY PULMONARY PATHOLOGY IN A DALMATIAN DOG.

A 1.5-year-old, 23 kg intact male Dalmatian dog was evaluated for acute respiratory insufficiency without a previous history of trauma or toxic exposition. Imaging revealed pneumomediastinum, pneumothorax, diffuse unstructured interstitial pulmonary pattern, pulmonary interstitial emphysema, and pneumoretroperitoneum. Histopathological evaluation of the lungs revealed perivascular and peribronchial emphysema, mild lymphocytic interstitial pneumonia with atypical proliferation of type II pneumocytes in bronchioles and alveoli. A lung disease resembling fibrosing interstitial pneumonia in man and cats has been previously reported in Dalmatians and should be included as a differential diagnosis for Dalmatians with this combination of clinical and imaging characteristics.

Isolation of Chlamydia abortus from a laboratory worker diagnosed with atypical pneumonia.

BACKGROUND: Identifying the aetiological agent of atypical pneumonia in human can sometimes be a tedious process, especially in cases where Mycoplasma pneumoniae, Legionella species and Chlamydia pneumoniae are ruled out. In such cases, a correct anamnesis of the patient is basic to clarify which pathogens might have produced the infection. For this reason, health professionals including veterinarians and laboratory personnel working with zoonotic pathogens should keep their doctors informed. CASE PRESENTATION: A human case of atypical pneumonia linked to Chlamydia abortus is reported. A 47-year-old male, a veterinarian researcher into chlamydiae, developed respiratory symptoms, breathing problems and high fever. Serological analyses ruled out the involvement of several respiratory pathogens, such as M. pneumoniae, Legionella pneumophila, Rickettsia conorii and C. pneumoniae, and Chlamydia abortus was identified as the possible aetiological agent of the infection. The isolation of C. abortus from the patient's sputum and subsequent molecular analysis confirmed the presence of this microorganism. CONCLUSION: As far as we know, although C. abortus has not been previously described as capable of causing pneumonia in humans, this is the first reported case of atypical pneumonia in which C. abortus is thought to have played an aetiological role.

Ante-mortem diagnosis of caprine tuberculosis in persistently infected herds: influence of lesion type on the sensitivity of diagnostic tests.

Caprine tuberculosis is a major health problem for goats, and an important zoonosis. Eradication programmes using the comparative skin test are being developed to aid in decreasing the prevalence of infection. However, persistent tuberculosis infections are found in herds subjected to eradication programmes. In the present study a commercial IFN-γ release assay and an experimental ELISA based on MPB70, were evaluated as potential ancillary tests to detect infection. The relationship between the three techniques (skin test, IFN-γ release and ELISA) and histopathological lesions was analyzed in 162 goats from herds with persistent tuberculosis infection. The presence of related pathogens (paratuberculosis and pseudotuberculosis) was also studied. The IFN-γ release assay and the ELISA had a higher sensitivity than the comparative skin test (65.3% and 66.3% vs 44.5%) using as a gold standard a combination of histopathological analysis and isolation. Used in combination, ELISA and the skin test detected 89.1% of goats with tuberculosis while a combination of IFN-γ assay and skin test detected 78.2%. The types of macroscopic and microscopic lesions reflected the results of diagnostic tests.

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers.

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.

A case of lymphocytic-plasmacytic jejunitis diagnosed by double-balloon enteroscopy in a dog.

A 3 yr old male English setter dog was presented for evaluation of a 6-wk history of intermittent diarrhea. After standard gastroduodenoscopy and colonoscopy showed normal mucosa, double-balloon endoscopy (DBE) was used via both oral and anal approaches. Gross changes consistent with inflammation in the jejunum were seen, and biopsy specimens were obtained. Histologic analysis confirmed a diagnosis of lymphocytic-plasmacytic jejunitis. Clinical remission of the disease occurred after 3 mo of therapy with prednisone, metronidazole, and a novel protein diet. Use of DBE has not been previously reported in dogs with inflammatory bowel disease, and isolated lymphocytic-plasmacytic jejunitis has not been described. The described cases of intestinal inflammatory disease diagnosed by conventional endoscopy were related to pathologic changes in the duodenum, ileum or colon, but not the jejunum. The main advantage of the DBE technique allowed examination of portions of the small intestine (jejunum) that were not commonly accessible by standard endoscopic techniques, and permitted a minimally invasive collection of biopsy samples compared with surgical biopsy. This case highlights the need to consider using DBE in animals with gastrointestinal disorders, whose symptoms are not readily explained by routine tests, conventional endoscopy, and dietary or therapeutic trials.

B cells are essential for moderating the inflammatory response and controlling bacterial multiplication in a mouse model of vaccination against Chlamydophila abortus infection.

The use of inactivated vaccines associated with suitable adjuvants has been demonstrated to confer a good level of protection against Chlamydophila abortus. However, the basis of the immune protective response induced by these vaccines has been poorly studied. B cells act as an immune regulatory population during primary infection by C. abortus. Thus, it was considered of interest to study the role of B cells in an infection after immunization with a killed vaccine. For this, C57BL/6 and B-cell-deficient mice were immunized with a killed vaccine against C. abortus using QS-21 as the adjuvant. After challenge, the course of infection was established by analysis of morbidity, C. abortus burden in the liver, and histopathological changes. The immune response induced was studied by real-time PCR techniques. Experiments involving transfer of immune serum from vaccinated or previously infected mice were also carried out. The lack of B cells reduced the protection conferred by the QS-21 adjuvant vaccine. Vaccinated B-cell-deficient mice showed a 1,000-fold-greater bacterial burden in the liver than their wild-type counterparts. Obvious differences existed in the liver, where a severe neutrophilic reaction and extended areas of necrosis were observed with vaccinated B-cell-deficient mice. An analysis of the immune response pointed to a significant increase in inflammatory cytokines and chemokines and the deficient production of transforming growth factor beta. The transfer of antibodies restored the level of protection. This study demonstrates that B cells play a crucial role in controlling C. abortus multiplication and prevent an exacerbated inflammatory response.

Postanesthetic brachial triceps myonecrosis in a Spanish-bred horse.

This report describes a case of postanesthetic brachial triceps myonecrosis affecting only the left forelimb of a horse. A fatal unilateral postanesthetic myonecrosis has not been previously reported in the horse. This article describes the factors in the horse's history, the anesthetic protocol, and the treatment that may have led to this condition.

Broncholithiasis in a cat: clinical findings, long-term evolution and histopathological features.

A 14-year-old neutered male Persian cat was evaluated because of an acute exacerbation of a chronic cough of 2-3 years of duration. Physical examination was normal except for the auscultation of accentuated breath sounds and wheezes cranially on both sides of the chest. Complete blood count, biochemical parameters and urinalysis were normal. Thoracic radiographs showed a generalised nodular pattern with multiple mineral opacities. Oral prednisone and doxycycline were prescribed. Two weeks later, the frequency of the cough was significantly reduced. Terbutaline was recommended for relief of acute exacerbations. Three years later the cat was evaluated again due to a non-related disease that led to the euthanasia of the cat. Concerning its respiratory disease, the cat had experienced nearly asymptomatic periods of 3-6 weeks of duration punctuated by acute exacerbation periods of 7-10 days, during which terbutaline was useful to relieve the cough. Thoracic radiographs showed a mild increase in the size and extent of the pulmonary mineralisation. Histopathologically, mild bronchitis and bronchiectasis were evident, accompanied by calcified bronchial plugs and marked hyperplasia and hypertrophy of the seromucinous glands. Based on clinical and pathoanatomical findings, a final diagnosis of miliary broncholithiasis and bronchiectasis was made. Broncholithiasis should be considered in differential diagnosis of pulmonary mineralisation in cats. When no concomitant diseases are present, this rare disease appears to have a slowly progressive evolution that does not appear to carry a bad prognosis and may be satisfactorily managed with combinations of bronchodilators and corticosteroids.

Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.

Genetic and pharmacological inhibition of poly(ADP-ribose) polymerase-1 interferes in the chlamydial life cycle.

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1-/- and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1-/- than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1-/- fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.

Relationship between the immune response and protection conferred by new designed inactivated vaccines against ovine enzootic abortion in a mouse model.

Chlamydophila abortus is a gram-negative obligate intracellular bacterium and the etiological agent of ovine enzootic abortion (OEA), an economically important disease in many countries. Inactivated vaccines have been reported to induce immunity in ewes and they have been used for many years. However, some outbreaks have been reported in correctly vaccinated flocks, so it is clear that new vaccines are necessary to address adequate protection and to avoid the shedding of the microorganism. This idea lead us to design inactivated vaccines, in a previously established mouse model, evaluating different inactivation procedures and new adjuvants. To assess the protection conferred, the results were analyzed on the basis of clinical signs and the isolation of C. abortus from spleen. These findings were correlated with the immune response induced by the vaccines, as determined by the production of C. abortus-specific IFN-gamma and IL-4 from splenocyte cultures and the detection of IgG isotypes in serum. BEI was found to be the best C. abortus-inactivation procedure. The inactivated vaccines adjuvated with QS-21 (QS) or Montanide 773 (M7) induced the best protection both against homologous and heterologous challenge, with an adequate (Th1-like) immune response. Finally, these selected vaccines were evaluated in a pregnant mouse model, in which they were seen to confer good protection and to avoid the C. abortus persistence in uterus after delivery. With these results, this mouse model could be considered as an adequate tool for selecting and optimizing effective vaccines against OEA.

B-cell-deficient mice show an exacerbated inflammatory response in a model of Chlamydophila abortus infection.

The resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection is dependent on gamma interferon and CD8(+) T cells, and classically, B cells have been considered to play a minimal role in host defense. The role of B cells in the immune response was studied by using a model of infection in mice with genetically modified immunoglobulin M transmembrane domains ( micro MT). In the absence of B cells, infection with C. abortus leads to an acute severe fatal disease that involves a disseminated intravascular coagulation syndrome. micro MT mice displayed an increased level of proinflammatory cytokines in serum, and an increased number of neutrophils was observed in the lesions. The possible deleterious role of neutrophils in the pathogenesis of disease in micro MT mice was determined by depletion of the neutrophils with the monoclonal antibody RB6-8C5. This led to an enhancement of the bacterial burden and early mortality in both micro MT and wild-type mice, while necrotic lesions remained. Analysis of the presence of immunoregulatory cytokines showed significantly lower levels of transforming growth factor beta in the sera of micro MT mice. However, mice lacking mature B cells were able to establish a specific immune response that protected them from a secondary challenge. Taken together, these data suggest an immunomodulatory role for B cells in the early events of C. abortus primary infection that can protect mice against an exaggerated inflammatory response.

Previous infection with the nematode Nippostrongylus brasiliensis alters the immune specific response against Chlamydophila abortus infection.

An experimental mouse model to analyze the interaction between the immune responses elicited following infection with Nippostrongylus brasiliensis and Chlamydophila abortus has been established. Mice infected with C. abortus 7 days after N. brasiliensis showed an increased bacterial multiplication in spleen and liver compared to bacteria-alone infected mice. However the morbidity of these mice, expressed as weight loss, was significantly lower. Analysis of the immune responses elicited showed that spleen from co-infected mice had reduced IFN-gamma production in response to C. abortus antigen. The bias towards a type 2 response in co-infected mice was confirmed by an increase in the production of IL-4 and in the lower ratio IgG2a/IgG1. In pregnant mice co-infection caused a delay in the time of abortion and an increased systemic susceptibility to C. abortus infection.