Natural and experimental infections of lambs with Mycoplasma ovipneumoniae

Mycoplasma (M.) ovipneumoniae was isolated pure or mixed with bacteria from 47 lungs of lambs of 14 in 22 tested flocks. M. ovipneumoniae was obtained as pure culture in cases of mild bronchopneumonia. Experimental intratracheal or intranasal infection caused several days of rising body temperature above 39.7 degrees C. Nasal discharge, coughing, and dyspnea did not occur. M. ovipneumoniae was successfully re-isolated from nasal swabs, beginning 2 d from infection. Lobular catarrhal bronchopneumonia was established by postmortem examinations, 10-14 d from infection, and M. ovipneumoniae was re-isolated from the lungs. Histological patterns of lungs were characterised by interstitial cell reactions.     

The experimental infection of specific pathogen free lambs with Mycoplasma ovipneumoniae.

Six colostrum-deprived SPF lambs inoculated endobronchially with a second passage broth culture of a Scottish strain of Mycoplasma ovipneumoniae, were killed in batches of two at seven, 14 and 28 days post-inoculation. One lamb from each batch showed macroscopic and microscopic lung lesions similar to but milder than those described for respiratory mycoplasmoses in other species of animals and exhibited minor clinical symptoms. Mycoplasma were recovered from all infected but from no control animals: five infected lambs yielded mycoplasma from lung tissue. Two lambs infected with M ovipneumoniae by endobronchial intubation were placed in contact with six other SPF lambs. M ovipneumoniae was recovered from the upper respiratory tract only of all six contact lambs, but no pathological changes were noted in their lungs. Both donor lambs yielded mycoplasma from lung tissue, but microscopic lesions were detected in only one of them, and these were minimal. No seroconversion due to the infection could be demonstrated in any of the lambs by either the indirect haemagglutination or metabolic inhibition tests.     

绵羊肺炎支原体感染羊细胞因子的动态变化

为检测绵羊在感染绵羊肺炎支原体(MO)前后细胞因子的含量变化情况,将6只巴什拜羊和6只盘羊杂交羊人工感染MO,在感染前后分别采集静脉血,分离血清,用ELISA方法检测IL-1β、TNF-α、IL-8、IL-10及IFN-γ含量。结果显示:两组羊在感染后IL-1β浓度均升高,第21天巴什拜羊的IL-1β含量降低,且显著低于杂交羊(P0.05);感染后两组的TNF-α含量逐渐升高,第14~21天巴什拜羊TNF-α含量开始下降,且第14天显著低于杂交羊(P0.05),第21天极显著低于杂交羊(P0.01);感染后第14和21天,巴什拜羊的IL-8含量开始下降,而杂交羊则继续升高,且杂交羊IL-8含量均极显著高于巴什拜羊(P0.01);两组羊的IL-10含量在第5天达到最高,且杂交羊极显著高于巴什拜羊(P0.01),而第14和21天巴什拜羊显著(P0.05)和极显著(P0.01)高于杂交羊;感染后两组羊的IFN-γ含量逐渐升高,第14~21天,巴什拜羊的IFN-γ含量开始下降,而杂交羊则继续升高,第14和21天巴什拜羊显著(P0.05)和极显著(P0.01)低于杂交羊。结果提示:绵羊感染MO前后细胞因子变化明显,巴什拜羊和杂交羊细胞因子变化也有差异,这对研究支原体肺炎发病机理及免疫治疗有指导意义。     

Differences in the peripheral immune response between lambs and adult ewes experimentally infected with Mycobacterium avium subspecies paratuberculosis

The peripheral immune response, and its relationship with the outcome of the infection according to the age of the animal, has been investigated in young lambs and adult ewes experimentally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (Map). Sixteen 1.5-month-old lambs out of 24 and 23 adult ewes out of 30 were orally challenged with an ovine Map field isolate. Animals were divided into two groups: HD, infected with a higher dose of Map and LD, with a lower dose. The remaining animals were used as uninfected control groups. Animals were euthanized at 110-120 and 210-220 days post-infection (dpi). Along the experiment, the humoral response and the specific and non-specific IFN-γ production were assessed. An intradermal skin test (IDT), using avian PPD, was also performed at 90 and 195 dpi. Samples of intestine and related lymphoid tissue were taken for histological, bacteriological and PCR studies. The Ab and IFN-γ production as well as the IDT response appeared earlier and with more intensity in the adult ewes compared to the lambs. The basal non-specific IFN-γ levels increased only in the adult ewes from the HD group. Animals from the LD and HD groups were positive to PCR; however, lesions consistent with paratuberculosis were exclusively observed in the HD group, both in lambs and in adult sheep, but they only progressed to more advanced stages in the former. These results suggest that the peripheral immune response induced by Map infection in the adult ewes is more efficient to control the progression of the infection than in lambs. This could likely be due to the existence of previous contacts with Map or other mycobacteria in the adult sheep compared to the young lambs.     

Cell mediated and humoral immune responses of white-tailed deer experimentally infected with Mycobacterium bovis

The objective of this study was to improve the understanding of immune responses of whitetailed deer (Odocoileus virginianus) infected with Mycobacterium bovis. Ten mature, female, white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3)or 2 x 10(5)colony-forming units of M. bovis. Lymphocyte proliferation and humoral response to M. bovis PPD and the M. bovis protein, MPB70 were measured. Deer were tested for exposure to M. bovis by the comparative cervical skin test. Biopsy specimens of skin test sites were examined microscopically and immunohistochemically. The comparative cervical skin test correctly identified all M. bovis -inoculated deer as exposed to M. bovis. Lymphocyte proliferative responses to MPB70 were more consistent than responses to M. bovisPPD in M. bovis -inoculated deer. Antibody responses were more prominent in deer with disseminated disease than in deer with localised disease. The cellular components of delayed-type hypersensitivity reactions at skin test sites were similar to tuberculin reactions in other species. T lymphocytes of the gamma/delta phenotype were seen in increased numbers in M. bovisPPD injection sites.     

The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting

The humoral immune response over time of White Leghorn chickens experimentally infected with Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization of M. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gallisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein of M. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests.     

Heterologous antibodies to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain

An IgM capture ELISA using heterologous antibodies was developed to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain. Detection of parasite in tissues from inoculated dogs was evaluated by mouse bioassay and immunohistochemical techniques. Serum samples were obtained at regular intervals up to 62 days post-inoculation (p.i.), when the animals were necropsied and their tissues examined. Antibody levels were measured by IgM capture ELISA (McELISA), indirect hemagglutination (IHA), indirect fluorescent antibody test (IgG-IFAT) and indirect immunoenzymatic assay (IgG-ELISA). All dogs seroconverted but only one exhibited severe clinical signs of infection. IgM antibodies were detected by McELISA from the seventh day on, with decreasing IgM levels around the 27th day. Similar results were obtained from IHA, although McELISA showed earlier and longer detection of IgM antibodies. IgG antibodies were detected from the seventh day on, and throughout the period of observation. Immunohistochemical findings and mouse bioassay revealed the presence of free tachyzoites in tissues of the clinically affected dog only. These results suggest that T. gondii acute infection in dogs shows a remarkably transient IgM synthesis, and this feature may constitute an important marker of active infection. Furthermore, McELISA was shown to be a potential tool to diagnose canine toxoplasmosis.     

感染绵羊肺炎支原体前后绵羊血清中相关细胞因子的动态检测

为研究绵羊感染绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)前后体内细胞因子含量的变化,将6只盘羊杂交羊和6只巴什拜羊人工感染MO,在感染前后采集静脉血,分离血清,用ELISA方法检测IL-5、IL-9、IL-12及IL-13含量。结果显示:2组羊在感染后IL-5浓度均升高,第7(P0.05)、14(P0.01)和21天(P0.01)盘羊杂交羊IL-5含量显著和极显著高于巴什拜羊;感染后第7(P0.01)、14(P0.01)和21天(P0.05)盘羊杂交羊的IL-9浓度极显著和显著地高于巴什拜羊;在感染后的第7(P0.05)、14(P0.01)和第21天(P0.01),盘羊杂交羊的IL-12浓度显著和极显著地高于巴什拜羊;在感染后的第5(P0.05)、7(P0.05)、14(P0.01)和21天(P0.01),IL-13浓度巴什拜羊显著和极显著低于盘羊杂交羊。结果表明:绵羊感染MO前后血清中IL-5、IL-9、IL-12及IL-13的浓度有明显变化,巴什拜羊和盘羊杂交羊的上述细胞因子变化也有显著差异,这对研究支原体肺炎发病机理及临床诊断有指导意义。     

Cell-mediated and humoral immune response of chickens to Mycoplasma synoviae.

The leukocyte migration-inhibition test was employed to demonstrate the presence of cell-mediated immunity and to ascertain its relation to immunoglobulin production in Mycoplasma synoviae infection in chickens. With peripheral leukocytes and a preparation of M. synoviae used as antigen, good discrimination was obtained between naturally or experimentally infected birds and uninfected control birds. Only the infected groups showed significant inhibition. Positive migration inhibition values developed in the second week of infection, often before the appearance of hemagglutination-inhibition titers, and continued to accompany the production of immunoglobulins with some degree of correlation for at least 6 or 12 months.     

Lesions in lambs experimentally infected with bovine respiratory syncytial virus

Respiratory syncytial virus was inoculated intratracheally into five 1-week-old lambs. Three of the lambs responded clinically with fever, hyperpnea, and listlessness. Pulmonary lesions consisted of multifocal areas of consolidation, with necrosis of individual epithelial cells of the airways and accumulation of necrotic debris, macrophages, and few neutrophils in terminal airways and alveoli. Pulmonary septa in affected areas were infiltrated with numerous macrophages and lymphocytes. Viral particles were seen as buds on epithelial cells and free in bronchioles and alveoli.     

Dynamics of the humoral immune response of calves infected and re-infected with Cooperia punctata

The dynamics of the humoral immune response of calves were analysed after primary infection and re-infection with the intestinal nematode Cooperia punctata. 12 male 5 month-old Holstein-Friesian calves were randomly divided into two groups A and B. At the beginning of the experiment Group A animals were each infected experimentally with a single oral dose of 130,000 infective third stage larvae (L3) of C. punctata. The animals of Group B were kept as non-infected controls. The two calves from Group A with the highest infections died of cooperiosis at 32 and 44 days after infection (DAI), respectively. On DAI 100 the calves were treated with the recommended dose of oxfendazole. On DAI 180 the remaining four calves of Group A and three animals of Group B (B1) were infected with 260,000 L3 of C. punctata, while the other three calves of Group B (B2) served as non-infected controls. Monitoring of the humoral immune response predominantly demonstrated an IgG1 response against both adult and L3 antigen of C. punctata. Moreover, re-infections increased the levels of these immunoglobulins. IgA levels were less increased than IgG1 and no significant increase was observed in IgG2 and IgM levels. Immunoblotting analysis showed that total IgG present in the serum of the primary infected animals mainly reacted against adult proteins of 12-14 and 17-20 kDa and against L3 proteins of 33 and 43 kDa. After re-infection total IgG reacted with the same adult proteins but also with an adult 29 kDa protein.     

Cytokine expression in porcine lungs experimentally infected with Mycoplasma hyopneumoniae

To gain further insight into the pathogenesis of porcine enzootic pneumonia (PEP), cytokine expression in different pulmonary compartments was examined. Mycoplasma hyopneumoniae (Mh) and proinflammatory and immunoregulatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-alpha) were detected by immunohistochemical methods in porcine lungs experimentally infected with Mh. Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 1- to 5-week post-inoculation (wpi). Three Mh-free pigs were taken as controls. Mh-antigen was shown in paraffin-wax-embedded tissues by immunohistochemistry in the luminal surface of bronchial and bronchiolar epithelial cells of all Mh-infected pigs. Significant increase in cytokine expression was detected on snap-frozen tissues from the bronchoalveolar exudate of the airways, mononuclear cells of the alveolar septa and macrophages and lymphocytes of the peribronchial and peribronchiolar lymphoid tissue, from 1 wpi onwards, compared to expression in non-pneumonic lungs. The main cytokines in the BALT of Mh-infected animals that showed an increase were IL-2, IL-4, IL-8, IL-10 and TNF-alpha. In the alveolar septa and bronchoalveolar exudate IL-1 (alpha and beta), IL-2, IL-4, IL-8 and IL-10 expression also increased in infected animals.     

The effect of albendazole on nematode parasites in experimentally infected lambs.

Thirty-five complete oestrous cycles were studied in five nonpregnant camels (Camelus dromedarius) over a period of 15 months. The oestrous cycle did not have a luteal phase. During the cycle of 28 days the ovarian activity was strictly follicular. Follicles matured in six days, maintained their size for 13 days and regressed in eight days. Ovulation was non-spontaneous and required the stimulus of coitus. Manual stimulation of the cervix for 15 minutes did not induce ovulation. The external manifestations of the oestrous cycle as well as the changes in the ovaries and the genital tract were also studied.     

The effect of fenbendazole on nematode parasites in experimentally infected lambs.

Fenbendazole given orally to experimentally infected lambs at a dose rate of 5 mg per kg was found to be 100 per cent effective against three and 10-day larave and also against 20-day adults of H contortus, O circumcincta, N battus and T colubriformis. The same dose was also 100 per cent effective against 10-day, 17-day larvae and 27-day adult D filaria.     

Cellular and humoral local immune responses in sheep experimentally infected with Oestrus ovis (Diptera: Oestridae)

Cellular and humoral local responses were investigated following repetitive artificial Oestrus ovis infections in lambs. The presence of larvae induced a huge local recruitment of either leucocytes (T and B lymphocytes, macrophages) or granulocytes (eosinophils, mast cells and globule leucocytes). This cellular response was more pronounced in the ethmoid and sinus (development sites of second and third instar larvae) than in the septum or turbinates where first instar larvae migrate. Infected lambs produced Oestrus ovis specific IgG and IgA antibodies in their mucus. This local humoral response was mainly directed against larval salivary gland antigens and not against larval digestive tract antigens. Compared to the control animals, the sinusal mucosa of infected animals was extremely thickened and the epithelium exhibited hyperplasia, metaplasia and eosinophilic exocytosis. The possible roles of these local immune responses in the regulation of O. ovis larvae populations in sheep are discussed.