Mononuclear cell infiltration of the equine endometrium: immunohistochemical studies.

Endometrial sections from mares with varying degrees of mononuclear cell infiltration were examined for immunoglobulin (Ig)A-, IgM-, IgG(T)- and IgG(Fc)-containing cells, luminal and glandular epithelial cell Ig-staining and free interstitial Ig-staining, using a peroxidase anti-peroxidase technique. Mares with mild to moderate (Group 2) and mares with severe diffuse mononuclear cell infiltration, superimposed by acute endometritis (Group 3), had significantly higher numbers of Ig-containing cells than genitally-normal mares (Group 1). The differences between Groups 1 and 3 were significant for all four isotypes. In Groups 1 and 2, numbers of IgA-containing cells were significantly larger than numbers of IgM- and IgG(T)-containing cells. Generally, more glandular epithelial cells stained for IgA and IgM than for IgG(T) and IgG(Fc), and Ig-staining for all isotypes increased from Group 1 to Group 3. Free interstitial staining did not appear to differ among the three groups, but IgG(Fc)- and IgG(T)-staining generally was more intense than IgA- and IgM-staining. The efficiency of uterine defence in the mare does not seem to depend solely on humoral factors, and defects involving other components of the defence system may contribute to failure of the uterus to clear infection.

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.

Immunohistochemical localization of alpha 2-beta 1-glycoprotein in horses.

Alpha 2-beta 1-glycoprotein may be found free in horse serum or complexed with alpha-1-proteinase inhibitor to form pre-alpha 2-elastase inhibitor. There has been little information published concerning alpha 2-beta 1-glycoprotein and its possible tissue sources in horses. A peroxidase-antiperoxidase technique was used to identify alpha 2-beta 1-glycoprotein in buffy coat and bone marrow neutrophils of healthy horses. Macrophages and neutrophils in bronchoalveolar lavage samples from clinically normal horses and from horses with chronic pulmonary disease also were positive for alpha 2-beta 1-glycoprotein. Alpha 2-beta 1-glycoprotein was identified in some instances in normal equine hepatocytes of formalin-fixed liver sections. In formalin-fixed lung sections from horses with chronic, small-airway disease and chronic bronchointerstitial pneumonia, alpha 2-beta 1-glycoprotein was observed in some airway secretions and in macrophages.

Mast cells in normal and pathological specimens of the equine lung.

In lungs from 22 horses, the number and type of mast cells in histologically normal specimens were compared with those in specimens with various histological lesions. Tissues fixed in Carnoy's solution were superior to those fixed in formalin for the identification of mast cells. Fixation of lung specimens from eight horses in Carnoy's solution and formalin allowed the identification of two subpopulations of mast cells: formalin-sensitive, which predominated and formalin-resistant. Specimens with mild chronic tracheitis and mild and moderate chronic bronchitis generally had slight increases in mast cell numbers in comparison to histologically normal specimens. An increase in mast cell numbers was also observed around small and large bronchioles with mild, moderate and severe bronchiolitis and in fibrosed pulmonary pleurae in comparison to specimens with no histological lesions. The most striking increase in mast cell numbers was seen in fibrosed alveolar septa; there were approximately four times as many mast cells in these specimens as in specimens with normal histology.

Fibrin/fibrinogen in lungs and respiratory secretions of horses with chronic pulmonary disease.

The concentration of soluble fibrinogen derivatives (SFD) and protease and procoagulant activities were determined in cell-free supernatants of equine respiratory secretions obtained from horses with chronic pulmonary disease. The concentration of neutrophils was estimated from direct smears of the secretions. Lung specimens and smears of the secretions were evaluated for the presence of fibrin or fibrinogen by use of immunohistochemical methods. Thirty-five of 80 specimens tested contained SFD. Respiratory secretions from horses with moderate or severe chronic pulmonary disease contained SFD more frequently than did secretions from mildly affected horses (P less than 0.05). Respiratory secretions with vast numbers of neutrophils had significantly (P less than 0.05) higher SFD concentrations than respiratory secretions with fewer neutrophils. Protease and procoagulant activities in respiratory secretion specimens were positively correlated with neutrophil content, clinical diagnosis, and SFD concentration. Immunohistochemically, macrophages that stained for fibrin or fibrinogen were observed in direct smears of respiratory secretions from horses with moderate and severe chronic small airway disease, but not in smears from mildly affected horses. Fibrin or fibrinogen was detected in a few thickened alveolar septa from 10 horses with moderate or severe chronic small airway disease, but not in lungs from horses with mild or no evidence of chronic small airway disease. Fibrin or fibrinogen was detected in alveolar septa, granulomas, and on alveolar macrophages in lungs of all horses with chronic granulomatous and chronic bronchointerstitial pneumonia. The presence of SFD in equine respiratory secretions may be an indicator of pulmonary inflammation.

Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with clinically diagnosed chronic pulmonary disease.

Thirty-nine horses and 3 ponies underwent a thorough respiratory examination and were grouped as follows: healthy (4 horses and 1 pony); mild chronic pulmonary disease (CPD 11 horses); moderate CPD (16 horses and 1 pony); and severe CPD (8 horses and 1 pony). Bronchoalveolar lavage (BAL) fluid collected from all animals and respiratory secretions (RS) obtained from 39 of these animals were evaluated cytologically and the results were compared. It was concluded that cytological examination of either BAL fluid or RS was useful in diagnosing various equine pulmonary diseases. The only advantage that BAL offered over RS sampling was in cases in which there was no RS available in the trachea. In addition, the severity of the CPD did not always correlate with either RS or BAL cytology.

Immunohistochemical localisation of alpha 2-macroglobulin in the horse.

A peroxidase antiperoxidase technique was used to identify alpha 2-macroglobulin in formalin-fixed sections of normal equine lung and liver and in tissue sections and bronchoalveolar lavage fluid from horses with various lung diseases. Equine peripheral blood leucocytes and bronchoalveolar lavage samples from clinically normal horses were negative for alpha 2-macroglobulin. It was concluded that liver and pulmonary macrophages may be potential sources of alpha 2-macroglobulin. Although alpha 2-macroglobulin may play a role in various chronic bronchointerstitial pneumonias of the horse, it is doubtful that it is of importance in equine chronic small airway disease.

Immunohistochemical localisation of alpha-1-protease inhibitor in the horse.

Using a peroxidase anti-peroxidase technique alpha-1-protease inhibitor (alpha-1-PI) was identified in normal equine hepatocytes in formalin-fixed liver sections, and in airway secretions and macrophages in formalin-fixed lung sections of horses with chronic small airway disease and chronic bronchointerstitial pneumonia. In addition, it was identified occasionally in macrophages in bronchoalveolar lavage samples from clinically healthy horses and from horses with chronic small airway disease. Equine peripheral blood leucocytes and formalin-fixed lung sections with normal histology were negative for alpha-1-PI.

Distribution of histological lesions in the equine endometrium.

The distribution of histopathological lesions in the equine endometrium was examined to investigate the representativeness of a single biopsy specimen in terms of the whole endometrium. Five sections from each of 110 uteri obtained from slaughtered mares were evaluated microscopically and classified according to a four-category grading system used for endometrial biopsies. Depending on the extent of agreement between the categories of the homologous sections, the uteri were considered to show either good agreement (81 uteri; 73.6 per cent), moderate agreement (26 uteri; 23.6 per cent) or poor agreement (three uteri; 2.7 per cent). All the homologous sections of the group showing moderate agreement were within two adjacent categories. Disagreements were more often due to variations in the distribution of fibrotic lesions than to variations in the degree of chronic infiltrative lesions. There was no seasonal effect on the apparent degree or distribution of lesions. In most cases the examination of a single biopsy, when combined with a thorough clinical examination, should provide adequate information about the condition of a mare's endometrium.

Comparison of respiratory secretion cytology and pulmonary histology in horses.

Equine respiratory secretions (RS) collected before (33 horses) and after (76 horses) slaughter were evaluated cytologically and grouped according to the histological diagnosis of the lungs from which they were obtained. The histological categories included normal lungs (control; 17 horses), chronic small airway disease with mild (23 horses), moderate (13 horses) and severe lesions (25 horses), interstitial pneumonia (13 horses), pulmonary eosinophilic infiltration (13 horses) and pulmonary haemorrhage (5 horses). On a group basis there were significantly more neutrophils in RS of horses with mild, moderate and severe chronic small airway disease and interstitial pneumonia than in control horses. Horses with severe chronic small airway disease had significantly more lymphocytes and significantly fewer ciliated columnar epithelial cells in RS than control horses. Significantly more eosinophils were seen in RS of horses with pulmonary eosinophilic infiltration than in control horses. On an individual horse basis all horses with moderate and severe chronic small airway disease and with pulmonary haemorrhage had a RS cytology which differed from that of control horses; however, some horses with mild chronic small airway disease (11 horses), pulmonary eosinophilic infiltration (4 horses) and interstitial pneumonia (3 horses) did not have an RS cytology that differed from control horses.

Chronic small airway disease in the horse: immunohistochemical evaluation of lungs with mild, moderate and severe lesions.

The peroxidase anti-peroxidase technique was used to demonstrate free and intracellular immunoglobulin (Ig) within the lungs of 23 horses with chronic small airway disease. Histologically, all the horses had chronic bronchiolitis; however, the lesions varied in degree from mild in eight horses, to moderate in nine horses and severe in six horses. Lungs from three horses which had no gross or histopathological lesions were used as controls. In comparison with control horses, horses with mild chronic bronchiolitis had increased numbers of Ig A-containing and non-immunoglobulin staining cells around the vasculature and bronchioles. As the severity of the lesions increased so did the number of IgA-, IgG(Fc)- and in several cases non-immunoglobulin staining cells around the vasculature, bronchioles and in the alveolar septa. In severely affected horses, large amounts of free IgG(Fc) were observed interstitially and in alveoli. In areas of mucosal epithelial hyperplasia and metaplasia large amounts of free IgA and IgG(Fc) were sometimes observed interepithelially in a pattern which differed from that in control horses.

Immunohistochemical evaluation of the equine endometrium during the oestrous cycle.

Endometrial biopsies were obtained from four mares during consecutive oestrous cycles on the first day of oestrus, on the day when ovulation was detected, and four and eight days after ovulation. Cycle stages were confirmed by means of rectal palpation, ultrasonography and plasma progesterone determination. Immunohistochemical evaluation of the formalin fixed biopsy specimens was performed using a peroxidase anti-peroxidase technique. Immunoglobulin (Ig)A-, IgM-, IgG(Fc)- and IgG(T)-containing cells were detected in all biopsies; with IgA- and IgG(Fc)-containing cells generally predominating. There was no cyclical trend of Ig-containing cell numbers for any isotype. Free immunoglobulins of the four classes evaluated were frequently seen in luminal epithelium, glandular epithelium and secretions, and interstitium. This study of endometrial biopsies from a limited number of cycling mares suggests the presence in the equine endometrium of free and intracellular immunoglobulins of the classes A, M, G(Fc) and G(T) without any apparent cyclical trend.

Immunofluorescent evaluation of the lower respiratory tract of healthy horses and of horses with chronic bronchiolitis.

Pulmonary parenchymal tissues from 6 healthy horses and from 9 horses with chronic bronchiolitis were evaluated by use of an indirect immunofluorescent technique. In horses of both groups, the diffuse interstitial immunofluorescence was most intense for immunoglobulin (Ig)G, moderate for IgA, and minimal for IgM. Intensity of fluorescence was increased markedly in diseased lungs exposed to anti-IgA and anti-IgG. Around small bronchi and large bronchioles of healthy horses, IgA-containing cells generally were more numerous than were IgG-Fc fragment (Fc)-containing cells; in small bronchioles, however, IgA-containing cells and IgG (Fc)-containing cells were identified with approximately equal frequency. Immunoglobulin-containing cell types usually were located in the lamina propria and submucosa of airways, with occasional Ig-containing cells seen interepithelially and intraluminally; IgG (T)- and IgM-containing plasma cells were observed infrequently in the same regions. The number of Ig-containing cells became fewer as the diameter of the airway decreased; therefore, at the level of the terminal bronchiole, Ig-containing cells only were observed occasionally. This pattern of Ig-containing cell distribution was similar in horses with chronic bronchiolitis; however, horses with chronic bronchiolitis had more IgA-containing cells forming perivascular and peribronchiolar cuffs than did healthy horses. The number of IgG (Fc)-containing lymphocytes and non-Ig-stainable lymphocytes also were higher in horses with chronic bronchiolitis than in healthy horses, but the number did not approach the magnitude of that observed for IgA-containing cells. One horse had deposits of complement C3 and IgG (Fc) within alveolar septa.

Eimeria acervulina infections from each of the four sporocysts of a single oocyst.

Oocysts were found in all four birds each infected with one of the four sporocysts of a single oocyst of Eimeria acervulina and in three of the four birds infected with single sporocysts from two other oocysts. Possible use of these infections for genetic studies was discussed. An efficient method for collecting fecal material containing sometimes rather low levels of oocysts was also described.