Application of fluorescent in situ hybridization for specific diagnosis of Pneumocystis carinii pneumonia in foals and pigs

Fluorescent in situ hybridization, immunohistochemistry, and Grocott's methenamine-silver nitrate staining were compared as diagnostic methods for Pneumocystis carinii pneumonia in formalin-fixed lung tissue from foals and pigs. An oligonucleotide probe targeting 18S ribosomal RNA of P. carinii was designed for in situ hybridization, and a commercially available monoclonal antibody was used for immunohistochemistry. Samples from six foals and 10 pigs with P. carinii pneumonia, as verified by Grocott's methenamine-silver nitrate staining, were examined concurrently with samples from seven animals with pneumonia caused by other pathogens. Fluorescent in situ hybridization showed distinctive positive reactions for P. carinii in all test samples. The immunohistochemical procedure, however, only revealed P. carinii in the foals. The number of P. carinii organisms observed by fluorescent in situ hybridization and immunohistochemistry far exceeded the number of organisms stained by Grocott's methenamine-silver nitrate staining. The results show that fluorescent in situ hybridization targeting ribosomal RNA can provide a specific diagnosis of P. carinii pneumonia in foals and pigs.     

Acremonium strictum pulmonary infection in a horse

A 10-year-old Thoroughbred gelding was admitted to the Veterinary Medical Teaching Hospital of the University of California-Davis with a 2-week history of intermittent fever and acute onset of lethargy, anorexia, and ataxia. Although the clinical signs were nonspecific, the results of initial hematologic and biochemical analysis were consistent with a chronic inflammatory process. Thoracic radiographs revealed an increased fine reticulonodular interstitial opacity throughout the dorsal caudal lung fields. Cytologic examination of bronchoalveolar lavage (BAL) fluid showed mixed inflammation with many mononuclear phagocytes containing single, spherical, intracytoplasmic fungal organisms. Four mold species were cultured in low numbers from the BAL fluid. One of the fungal elements observed on the culture plates was identified as Acremonium strictum by real-time polymerase chain reaction (PCR). A diagnosis of fungal pneumonia due to A strictum was made based on the results of thoracic imaging, cytologic evaluation, culture, and PCR testing. The horse made an uneventful recovery with supportive treatment and was disease-free based on normal physical, radiographic, and cytologic findings at 21 days after presentation. To our knowledge, this is the first report of isolation of A strictum from the BAL fluid of a horse with interstitial pneumonia.     

Pneumocystis carinii pneumonia in a Cavalier King Charles Spaniel

Pneumocystis carinii pneumonia was diagnosed by postmortem examination of a one-year-old Cavalier King Charles Spaniel with four-week history of dyspnea. Cytologic and histologic examination of lung tissues revealed numerous P. carinii trophozoites and cysts, and P. carinii specific DNA was detected by polymerase chain reaction. The dog showed hypogammagloblinemia and extremely low levels of serum IgG. It was considered that P. carinii pneumonia in this case was associated with an immunodeficient condition which has already been reported in Miniature Dachshunds.     

Common variable immunodeficiency in miniature dachshunds affected with Pneumonocystis carinii pneumonia.

Seven miniature dachshunds, all under the age of 1 year, were presented with polypnea, tachypnea, and exercise intolerance as a result of Pneumocystis carinii pneumonia, which was diagnosed on transtracheal aspirate cytology. In all of the dogs, historical and clinical signs were suggestive of immune incompetence. Immunological studies undertaken were leukogram parameters, serum immunoglobulin fraction quantification, lymphocyte transformation assay. CD3 and CD79a lymphocyte markers on lymphoid tissue, and anti-canine immunoglobulin G immunoperoxidase staining. The immunological studies showed hypogammaglobulinemia, deficiency of serum immunoglobulins A, G, and M, decreased lymphocyte transformation response to phytohemagglutinin and pokeweed mitogens and absence of B lymphocytes with presence of T lymphocytes in the lymphoid tissue stained with CD3 and CD79a lymphocyte markers. The preceding findings suggest that P. carinii pneumonia occurring in the miniature dachshund is a result of both a T- and B-cell deficiency. This presentation is not the classic primary severe combined immunodeficiency syndrome but rather combined variable immunodeficiency, which has been well documented in humans but never in the dog.     

RADIOGRAPHIC ASPECTS OF Pneumocystis carinii PNEUMONIA IN THE MINIATURE DACHSHUND

The thoracic radiographic changes of Pneumocystis carinii in 7 miniature Dachshunds were reviewed. The dogs were 7–12 months old and presented with polypnea, exercise intolerance and clinical signs suggestive of immune-incompetence. P. carinii pneumonia was diagnosed in all the dogs using transtracheal aspirate cytology and confirmed at postmortem in 3 dogs that died. Radiographically, diffuse pulmonary changes were present and varied from a mild interstitial and bronchial pattern to an alveolar pattern. Radiographic evidence of cor pulmonale was present in 1 dog. The most severe radiographic changes were seen in 2 of the dogs that died.     

Use of dapsone in the treatment of Pneumocystis carinii pneumonia in a foal

A 6-month-old male Quarter Horse was evaluated for chronic respiratory tract disease. Diagnostic investigations revealed pulmonary inflammation; Pneumocystis carinii was detected within macrophages. Lymphocyte subpopulation phenotyping and immunoglobulin concentration analysis were performed and results suggested immune suppression. Trimethoprim-sulfamethoxazole administration was initiated; the colt was discharged but was reexamined 8 days later because of profuse diarrhea and endotoxemia. Bacterial culture of feces recovered Salmonella spp resistant to trimethoprim-sulfamethoxazole, and a diagnosis of antimicrobial-associated colitis was made. Bilateral fibrinous hypopyon developed and was treated with topical medication and intracameral injections of human recombinant tissue plasminogen activator. Dapsone (3 mg/kg [1.4 mg/lb], PO, q 24 h; dose extrapolated from human data) was administered for treatment of P carinii pneumonia (56-day treatment period). The colt recovered from the pneumonia and diarrhea. Dapsone may be a useful adjunct to traditional treatment for P carinii pneumonia in horses or as a sole medication for horses that cannot tolerate other treatments.     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with histologically diagnosed pulmonary disease

Equine bronchoalveolar lavage (BAL) fluid collected from 70 horses and respiratory secretions (RS) obtained from 61 of these horses were evaluated cytologically and grouped according to the histological diagnosis of the lungs from which they were obtained. The histological categories included: normal lung (8 horses); pulmonary eosinophilic infiltration (9 horses); interstitial pneumonia (5 horses); pulmonary hemorrhage (5 horses); and mild (12 horses), moderate (7 horses) and severe (24 horses) chronic small airway disease. In horses with pulmonary disease, all BAL samples and all but one RS sample differed cytologically to those obtained from normal horses; however, the type and severity of the pulmonary disease could not always be determined using either BAL or RS cytology. There was a positive association between the percentage of neutrophils in BAL and the neutrophil scores in RS specimens; there was no positive association between other cell types.     

Pneumocystis carinii in large domestic animals in Denmark. A preliminary report

By microscopic examination of toluidine blue O stained autopsy lung imprints Pneumocystis carinii was shown in 6 calves out of 160 (3.8%), in 1 sheep out of 28 (3.6%) and in 8 pigs out of 119 (6.7%). This is the first report of Pneumocystis carinii in calves, sheep and pigs in Denmark.     

Animal pneumocystosis: a model for man

Pneumocystis carinii is an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. Apart from AIDS, cases of pneumocystosis have been reported in patients receiving immunosuppressive therapy associated with organ transplantation without chemoprophylaxis and in malignant blood diseases. In vitro models are only of limited interest because there is no continuous in vitro culture. The in vivo models have contributed a great deal to the understanding of human Pneumocystis carinii pneumonia. Indeed, animal models remain of prime interest for many purposes, principally comparative medicine, pathogenesis, epidemiology and immunology. Among animal models, the rabbit is a very susceptible host to P. carinii infection, and does not need glucocorticoid treatment. Moreover, antigenic and genomic data suggest that rabbit-derived Pneumocystis strains are more closely related to human Pneumocystis than those of mice or rats. We have therefore shown that the rabbit model permits the study of the pulmonary surfactant modification due to P. carinii infection. This model should be a very interesting model for pathogenesis or immune response studies in immunocompetent animals. The rabbit model could also be used for epidemiological studies. P. carinii transmission appears to be very rapid via contact of Pneumocystis-free rabbits with infected rabbits. These Pneumocystis-free animals could be helpful for characterizing the source and the reservoir and studying parasite transmission.     

Diagnosis of pneumocystis pneumonia in a 2‐year‐old King Charles Cavalier Spaniel using the polymerase chain reaction

A 2‐year‐old, female spayed, King Charles Cavalier Spaniel was presented for evaluation of dyspnea, inappetence, and lethargy. Thoracic radiographs revealed a moderate diffuse interstitial lung pattern affecting the perihilar and caudodorsal lung fields, and an echocardiogram revealed severe pulmonary hypertension. A bronchoalveolar lavage (BAL) was performed, and cytology revealed mixed inflammation with cysts and trophozoites consistent with Pneumocystis. Pneumocystis infection was later confirmed with PCR. To the author's knowledge, this report represents the first case of canine pneumocystis pneumonia diagnosed antemortem with PCR from a BAL sample. Pneumocystis represents an important, but uncommon cause of afebrile pneumonia in immunosuppressed dogs.     

Isolation and buoyant density of Pneumocystis carinii in Percoll gradients

A new gradient material, Percoll, a collodial suspension of silica particles covered with polyvinylpyrolidon, has been used to isolate Pneumocystis carinii from infected rat and human lung tissues. The specific gravity of Pneumocystis carinii was shown to be within the range of 1.018-1.062 g/ml with most pneumocysts within 1.033-1.049 g/ml.     

Pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus"

A prevalent and distinctive infectious interstitial pneumonia (IIP) of immunocompetent laboratory rats was suspected to be caused by a putative virus, termed rat respiratory virus, but this was never substantiated. To study this disease, 2 isolators were independently populated with rats from colonies with endemic disease, which was perpetuated by the regular addition of naive rats. After Pneumocystis was demonstrated by histopathology and polymerase chain reaction (PCR) in the lungs of rats from both isolators and an earlier bedding transmission study, the relationship between Pneumocystis and IIP was explored further by analyzing specimens from 3 contact transmission experiments, diagnostic submissions, and barrier room breeding colonies, including 1 with and 49 without IIP. Quantitative (q) PCR and immunofluorescence assay only detected Pneumocystis infection and serum antibodies in rats from experiments or colonies in which IIP was diagnosed by histopathology. In immunocompetent hosts, the Pneumocystis concentration in lungs corresponded to the severity and prevalence of IIP; seroconversion occurred when IIP developed and was followed by the concurrent clearance of Pneumocystis from lungs and resolution of disease. Experimentally infected immunodeficient RNU rats, by contrast, did not seroconvert to Pneumocystis or recover from infection. qPCR found Pneumocystis at significantly higher concentrations and much more often in lungs than in bronchial and nasal washes and failed to detect Pneumocystis in oral swabs. The sequences of a mitochondrial ribosomal large-subunit gene region for Pneumocystis from 11 distinct IIP sources were all identical to that of P. carinii. These data provide substantial evidence that P. carinii causes IIP in immunocompetent rats.     

Frequency of occurrence of pneumonia associated agents and their correlation with clinical and pathological-anatomical findings in pigs

The aim of this study was to investigate the incidence and influence of different causative organisms involved in the development of pneumonia and bronchopneumonia in pigs. Bronchoalveolar lavage fluid (BALF) from 339 pigs was examined molecular-biologically and culturally. The evaluation considered the following pathogens: Mycoplasma (M.) hyopneumoniae, Mycoplasma (M.) hyorhinis, PRRSV (US-Type), PRRSV (EU-Type), PCV-2, Influenzavirus Type A, alpha-haemolytic Streptococci, beta-haemolytic Streptococci, Pasteurella (P.) multocida, Bordetella (B.) bronchiseptica, Haemophilus (H.) parasuis and Actinobacillus (A.) pleuropneumoniae. This was followed by a pathological-anatomical examination in 168 pigs. Pathological-anatomical examination was evaluated for possible interstitial pneumonia, catarrhal-purulent bronchopneumonia and pleuritis. alpha-haemolytic Streptococci, PCV-2, H. parasuis, M. hyorhinis, M. hyopneumoniae, B. bronchiseptica, PRRSV (US-Type), P. multocida, PRRSV (EU-Type) and Influenzavirus Type A were evident in descending incidence in the BALF. A. pleuropneumoniae were only isolated culturally in two cases in the BALF. The frequency and semiquantitative detection rate in the bacteriological culture of alpha-haemolytic Steptococci, M. hyopneumoniae, P. multocida and B. bronchiseptica correlated significantly with the ascertained clinical evaluation score and the pathological-anatomical examination score. M. hyorhinis and Influenzavirus Type A only correlated with the severity degree of clinical appearance, while PCV-2 and PRRSV (US-Type) correlated with the frequency of pathological-anatomical changes. The higher the clinical score, the higher was the number of animals ascertained with five or more different pathogens. The more different causal agents were isolated in the BALF of one animal, the higher was the average pathological-anatomical examination score. For the diagnosis of pneumonia, especially when analysing facultative pathogens or secondary pathogens, a useful interpretation of analysis results is only possible in connection with a clinical and pathological evaluation.     

双氢青蒿素治疗大鼠卡氏肺孢子虫肺炎后的肺脏病理变化观察

为研究经双氢青蒿素治疗后卡氏肺孢子虫肺炎 (PCP)大鼠肺部病理学变化 ,以地塞米松磷酸钠皮下注射 Wistar大鼠建立卡氏肺孢子虫肺炎动物模型 ,用 60 mg/ kg双氢青蒿素治疗实验大鼠 ,杀鼠取肺 ,用光镜和电镜观察肺部病理学变化 ,同时设有感染对照组和正常对照组。结果发现肺印片中卡氏肺孢子虫 (Pc)包囊数目显著减少 ,肺组织炎症明显减轻 ,Pc滋养体表膜和核膜破裂 ,胞质中出现大量空泡和高电子密度颗粒 ,Pc包囊中也出现空泡 ,囊内小体变性坏死。研究结果表明双氢青蒿素可杀死 Pc滋养体和包囊 ,从而减轻肺组织的炎症反应     

Equine bronchoalveolar lavage cytology: survey of Thoroughbred racehorses in training

SUMMARY Sixty-two Thoroughbred horses aged between 1 and 7 years in training in Sydney had bronchoalveolar lavage (BAL) samples collected for cytological examination. All horses, except the yearlings and those with a cough, had raced at the time of the examination and the trainers reported satisfactory performance. Free erythrocytes were found In 73% of samples and haemoslderophages In 90% of the samples, Indicating Immediate or past occurrences of exercise-Induced pulmonary haemorrhage (EIPH). Bronchoalveolar fluid from the yearlings contained significantly less (P / 0.05) erythrocytes and haemosiderophages than samples from horses In other age groups. In the older horses, there was also more haemosiderln within the macrophages. No differences In BAL cytology could be attributed to gender, and there was no relationship between BAL cytology and racing performance. The main cytological findings were (mean ± sd): total nucleated cells - 832 ± 578/μL with the main cell types being: macrophages - 59 ± 10% (haemosiderophages - 20 ± 24%); neutrophlls - 9 ± 6%; lymphocytes - 31 ± 9%. The erythrocyte count was 10.3 ± 17.7% of the total cell count. Horses with chronic coughing had a higher proportion of macrophages and a lower proportion of lymphocytes in the leucocytes obtained from BAL. There was a higher occurrence of EIPH detected In BAL findings than that previously reported when endoscopic examination has been used to diagnose EIPH. The occurrence and severity of EIPH as Indicated by the BAL findings was found to be related to exercise Intensity. The cytological findings were similar to those reported in horses in the northern hemisphere. We conclude that BAL cytology may be useful In the diagnosis of various lower respiratory tract disorders and that exercise-induced pulmonary haemorrhage occurs In virtually all horses In race training.     

Pneumocystosis associated with canine distemper virus infection in a mink

An adult mink from a farm experiencing 100% mortality in affected animals was submitted for diagnostic examination. Clinical history included signs of respiratory disease, oculonasal discharge, and thickening of footpads. Canine distemper virus and Pneumocystis carinii were identified in lung tissue, suggesting immunosuppresion and secondary infection due to morbillivirus disease.     

Thoracic radiography,bronchoalveolar lavage cytopathology,and pulmonary parenchymal histopathology: a comparison of diagnostic results in 11 cats

The purpose of this study was to compare the diagnostic results and value of thoracic radiography, bronchoalveolar lavage (BAL) fluid cytopathology, and lung histopathology in 11 cats with spontaneous respiratory disease in which radiography and cytopathology were inadequate in establishing a definitive diagnosis. In these cats, radiographic patterns were characterized as bronchial (n=6), interstitial (n=3), and alveolar (n=2); other features included hyperinflation (n=3), bronchiectasis (n=2), pleural fissure lines (n=2), pulmonary nodules (n=2), atelectasis (n=1), and a tracheal mass (n=1). Bronchoalveolar lavage fluid was unremarkable in two cats. Abnormal BAL fluid showed inflammation (n=5), hemorrhage (n=2), epithelial hyperplasia (n=1), or was suspicious for neoplasia (n=1). Histopathological evaluation revealed inflammation (n=8), neoplasia (n=2), and vascular congestion (n=1). The predominant radiographic location of disease correlated with the same histopathological location in seven cats, and the cytopathological class of BAL fluid was consistent with the histopathological class of disease in seven cats. There was poor correlation between the types of cells found in the BAL fluid and the pathologist's prediction of the types of cells likely to be found in the BAL fluid based on the amount and type of airway cellularity seen on histopathological examination. The results of this study suggest that in some cats, BAL fluid cytopathology does not always correlate with the type of pulmonary disease identified on histopathology. In respiratory diseases where radiography and cytopathology fail to provide a definitive diagnosis, histopathological examination of the lung may be necessary.     

Pneumocystis Pneumonia in a Thoroughbred Racehorse

Pneumocystis pneumonia is an opportunistic respiratory infection that occurs in immunocompromised animals. In horses, pneumocystic pneumonia is observed mostly in foals and often progresses rapidly. Here, we report pneumocystic pneumonia in a Thoroughbred racehorse. A 3-year-old Thoroughbred racehorse colt had marked respiratory symptoms for 3 weeks and was unresponsive to antibiotic treatment. At necropsy, firm, tan, patchy lesions were scattered diffusely in the lungs. Microscopically, alveolar septa thickened by proliferation of collagen fibers and infiltration of inflammatory cells were observed. In the alveolar spaces, many brown-black yeast-like organisms similar to cystic forms of Pneumocystis carinii were recognized by staining with Gomori's methenamine silver. Bronchoalveolar lavage fluid (BALF) obtained before necropsy included macrophages engulfing the fungus bodies. Amplified products were obtained from BALF and lung tissue samples by Pneumocystis-specific nested PCR. Phylogenetic analysis based on the 18S rRNA gene sequence revealed that the P. carinii organism from BALF was related to the Pneumocystis spp. detected in other animals and was especially close to P. carinii derived from ferrets. This is a rare case of pneumocystic pneumonia in a colt with chronic pulmonary lesions.     

醋酸可的松和地塞米松诱发野鼠卡氏肺孢子虫肺炎的试验研究

采用16只褐家鼠、30只黄胸鼠和20只小家鼠经皮下注射醋酸可的松和地基米松12周，建立野鼠卡氏肺孢子虫肺炎模型。经剖捡，取肺组织制备涂片、印片和切片，用不同染色液染色后，进行病原体形态观察。结果表明：黄胸鼠卡氏肺孢子虫感染率最高，为100％；褐家鼠感染率次之，为81．25％；小家鼠最低，为65％；注射醋酸可的松的诱导率高于地塞米松的诱导率。支气管灌洗液涂片和肺组织印片用姬姆萨染色后，可见卡氏肺孢子虫的囊内小体和滋养体；肺组织印片和切片用哥氏银、甲苯胺蓝染色后，可见清晰的卡氏肺孢子虫的包囊壁。