Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica

A group of Caesarian-derived, colostrum-deprived lambs was inoculated intranasally and intratracheally with a virulent Mycoplasma ovipneumoniae isolate selected from ovine mammary studies and propagated in an ovine mammary gland. Other groups of lambs were inoculated with M. ovipneumoniae in combination with Pasteurella haemolytica type Al or P. haemolytica alone. The M. ovipneumoniae isolate alone did not induce any specific pneumonic lesions in the lambs and when combined with P. haemolytica type Al did not increase the severity of the P. haemolytica-type lesions. Fifty percent of lambs inoculated with P. haemolytica developed a purulent and exudative bronchopneumonia with pleurisy and high titres of P. haemolytica were recovered from these lesions.     

Experimental staphylococcal mastitis in bitches: clinical, bacteriological, cytological, haematological and pathological features

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.     

Epididymal and testicular lesions in rams following experimental infection with Actinobacillus seminis

AIM: To investigate and assess the epididymal and testicular lesions in rams up to 44 days after inoculation with Actinobacillus seminis via various routes. METHODS: Forty-four young (18-24 months old) rams were randomly divided into nine test and two control groups (n=4 per group). The test rams were infected by installation, drenching or injection of A. seminis organisms cultured for 24 h in a brain-heart infusion (BHI) broth containing 2.3 x 10(9) cells/ml, via the following nine routes: intra-epididymal (1 ml), intravenous (3 ml), intra-urethral (3 ml), intra-preputial (3 ml), vas deferens (1 ml), intramuscular (3 ml), oral (10 ml), intranasal (3 ml), and intra-conjunctival (3 drops). All test rams were necropsied 9-44 days post-inoculation (p.i.). Control rams were subdivided into in-contact and non-contact groups and necropsied at 45 and 46 days p.i., respectively. Thin tissue sections were examined for histopathology. RESULTS: Gross lesions were evident only in rams inoculated intra-epididymally. Epididymides on the inoculated side were two to three times larger than those on the un-inoculated side, and the testes attached to the inoculated epididymides were also enlarged. Fibrinopurulent periorchitis and tunica vaginalitis were seen in three rams and atrophy in one. Microscopically, epididymitis was present in 17 (47%) rams, the highest incidence being in the cauda, followed by the caput and the corpus epididymis. Seminiferous tubular degeneration with areas of lymphocytic infiltration were seen in four rams: three inoculated via the cauda epididymis and one via the urethra. No epididymal and/or testicular lesions were seen in rams inoculated via the nasal and conjunctival routes. CONCLUSIONS: Injection of A. seminis in young rams by all routes except intra-conjunctival and intranasal resulted in epididymitis, predominantly in the cauda epididymis. Development of lesions in the reproductive tract following non-genital routes of inoculation supports earlier suggestions that non-venereal transmission of genital actinobacillosis occurs. This study confirmed the predilection of A. seminis for the epididymis, especially the cauda.     

The experimental production of mastitis in ewes as a determinant of the virulence of ovine ureaplasma strains

Seven out of eight ovine ureaplasma strains inoculated into the mammary gland of suckling ewes produced a mastitis. The pattern of infection was single phase in 5 ewes, persisting for 12-41 days, and biphasic in 3 ewes, persisting in 2 of them until weaning at 60 days and 3 months post-infection. Sucking lambs did not become infected in the eye or nasal areas, and did not transfer infection to the control contralateral glands.     

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis.     

Urogenital infection and seminal excretion after inoculation of bulls and rams with chlamydiae.

Five mature rams and 4 bulls were inoculated parenterally with bovine or ovine chlamydial strains of type 1 and 2. One to 3 days later, all animals developed a chlamydemia lasting 4 to 8 days. Chlamydial agents were isolated from the semen near the end of the chlamydemic phase. All rams and 3 of 4 inoculated bulls excreted chlamydiae in the semen for 22 to 29 days. From 8 to 39 days after inoculation, selected rams or bulls were killed to test for chlamydial infection in the urogenital tract and other organs. Chlamydiae were isolated in developing chicken embryos from testis, epididymis, and accessory sex glands. Bulls examined 29 and 39 days after inoculation did not harbor chlamydiae. Chlamydiae were also not isolated from 3 control bulls which were from the same herd as the principal bulls. All inoculated bulls and rams had a group-specific chlamydial antibody response within 7 days. The titers reached maximal levels of 128 to 512 at 14 days after inoculation. Subsequently, the antibody titers decreased gradually. Seminal plasma collected at different times after animals were inoculated did not fix complement in the presence of chlamydial group antigen. The number of polymorphonuclear leukocytes in the semen increased during the experiment. The semen was grossly purulent in 2 rams inoculated with the type 2 chlamydial strain of polyarthritis.     

The potential of the ovine udder as a model to determine the pathogenicity of bovine ureaplasmas

Various methods of inducing mastitis in the ovine mammary gland with two bovine ureaplasma strains were investigated. The most successful method was by inoculation of fresh broth cultures on two successive days, 24 h apart. Eight more bovine strains were inoculated by this means and three successfully infected the glands.     

Apoptotic Cell Death, bax and bcl-2 Expression During Sheep Mammary Gland Involution

Involution in ovine mammary tissue was studied by light and electron microscopy, and bax and bcl-2 protein distribution was examined by immunohistochemistry from the last day of lactation until the 8th day of drying off. The mammary gland alveoli were examined and the area of glandular epithelium was evaluated morphometrically. Regression of mammary gland epithelium by apoptosis was first identified 2 days after the end of lactation, and increased until day 8. Bax protein was detected throughout this period and was highest on the eighth day. A weak positive reaction for bcl-2 was observed only on days 1 and 8 after cessation of lactation. It is concluded that sheep mammary gland involution involves cell death by apoptosis and that bcl-2 gene family members are involved in the process.     

Surgical treatment of a mammary gland comedocarcinoma in an Arabian mare: Post-operative management,and histopathological and immunohistochemical features

A lactating 20-year-old, brown, Arabian mare, weighing about 300 kg, presented for bleeding from one teat and severe swelling of the entire mammary gland. The mare had untreated mastitis 10 months before. Consequently, a gangrenous teat developed after chronic bloody and purulent discharges. The teat was removed surgically by the field veterinarian. At that time, the mammary gland increased in size. Bloody and purulent discharges restarted 10 days previously. Under general anaesthesia, the entire mammary gland was removed. Comedocarcinoma was diagnosed by histopathological assessment. Immunohistochemical staining was performed for pan-cytokeratin and vimentin. Microscopic examination of immunohistochemical stained slides revealed expression of pan-cytokeratin. In conclusion, this report describes clinical, macroscopic, histopathological and immunohistochemical characteristics of comedocarcinoma that did not metastasise to regional lymph nodes. Reports in the field of equine oncology contribute to improved general knowledge in equine medicine, contributing to better diagnosis and treatment.     

Concurrent pathological and bacteriological findings in the urogenital organs and mammary glands of sows culled because of chronic vulvovaginal discharge and swine urogenital disease (SUGD): a case study

The urogenital organs and mammary glands of sows, culled because of excessive vulval discharge, milking problems, and urogenital infections (swine urogenital disease, SUGD) in their history (n=1070 sows) were examined. The culled sows were assigned to three groups according to parity: parity 1 (n=356); 2-6 (n=354); and >6 (n=360). Necropsy findings associated with these groups were analysed separately. Bacteriological examination of vulval discharges was performed. Escherichia coli and a large number of Gram-positive and Gram-negative organisms were found in all samples of vulval discharge. Except ovarian degeneration and oedematous endometrium, older sows had more (P<0.05) pathological changes in the oviduct, ovaries, and uteri than younger (parity 1) sows. More (P<0.05) parity 1 sows had hyperaemic and congested vaginal walls and haemorrhages into the vaginal lumen than sows of higher parity, which suffered more (P<0.05) from accumulation of purulent material in the vaginal lumen, fibrinopurulent exudate adherent to the wall of the vagina, multifocal vaginal erosions and ulcerations, and purulent, mucopurulent or purulohaemorrhagic exudate in the vagina or on the cervix. Except acute pyelonephritis, mucosal hyperaemia, and congestion of the urinary bladder, more (P<0.05) sows of higher parity had pathological changes in their urinary organs. More (P<0.05) parity 1 sows had acute or chronic purulent exudative mastitis than sows of higher parity, which had more (P<0.05) mammary gland abscessation, mammary gland cysts, and fibrous mastitis. All parity 2-6 and >6 sows had mammary gland and bladder changes, parity >6 sows had changes in the kidney, and renal pelvis, and parity 1 sows had mammary gland changes. Most parity 1 sows had bladder, kidney, and renal pelvis alterations and most parity 2-6 animals had pathological kidney and renal pelvis changes.     

Pathology of Campylobacter jejuni abortion in sheep

Campylobacter jejuni was inoculated intravenously into pregnant ewes on gestation days 114 and 123 to reproduce ovine abortion. All ewes aborted 7-12 days post-inoculation. High numbers of C. jejuni were isolated from ewe tissues (caruncle, bile, cecal feces), fetal tissues, and placenta. C. jejuni colonies were identified in caruncles and placenta by light microscopy and immunoperoxidase techniques. Histologically, inoculated ewes had a severe purulent endometritis with vasculitis. Placentas from inoculated ewes and field cases showed necrosis and purulent inflammation; however, placentas from inoculated ewes had large numbers of bacterial colonies compared to few bacteria found in field cases. Histologically, only one fetus from the inoculated ewes showed lesions (purulent bronchopneumonia), whereas all fetuses from field cases had a distinct bronchopneumonia, and one fetus showed multifocal hepatic necrosis. These results suggest that C. jejuni (serotypes Penner 1 and Lior 2) is an important abortifacient organism for sheep.     

The pathogenicity and pathogenesis of Mycoplasma capricolum subsp. capripneumoniae (F38) in the caprine mammary gland

The right mammary gland of 12 lactating goats was inoculated intracisternally with 1 ml of Mycoplasma capricolum subsp. capripneumoniae (Mcc) containing 10>⁶ colony-forming units (CFU), while their left mammary halves received 1 ml of sterile PPLO broth only. Two goats served as uninfected controls. The clinical mastitis that developed in the infected mammary halves within 24 h was initially acute but became increasingly chronic by the end of the experiment at 24 days post inoculation (DPI). The disease was characterized by atrophy of the infected mammary halves, leading to marked agalactia and an increase in somatic cell counts, with a preponderance of neutrophils initially and lymphocytes later. The Mycoplasma was re-isolated from infected mammary secretions up to 16 DPI but not from blood. Histopathology revealed that the mastitis was acute and purulent initially, followed by infiltration of lymphonuclear cells and fibroplasia in the lymphomononuclear cells and fibroplasia in the interacinar tissue, and later by massive fibrosis. Immunohistology demonstrated the presence of Mycoplasma-like bodies localized mainly on the surface of acinar/duct epithelial cells. The studies showed that Mcc was highly pathogenic in the caprine mammary gland.     

Experimental intrauterine inoculation of pregnant ewes with ureaplasmas

The pregnancies of 13 ewes which were inoculated intrauterine with one of two strains of ovine ureaplasmas resulted in 9 normal and 3 abnormal births, and one ewe was found to be no longer pregnant on postmortem examination. Vaginal ureaplasma infection was detected in the majority of ewes only after lambing. Of the 12 ewes examined at postmortem, ureaplasmas were isolated from the uterus of 5 out of the 6 necropsied up to 21 days post-partum. The vulvar/preputial areas of the majority of lambs that survived were infected with ureaplasmas for the duration of the experiment, but infections of the nasal cavity and eye areas, detected at birth in 4 lambs, were resolved within 8 days post-partum.The only pathological effects detected that could possibly be attributed to ureaplasma infection were a placentitis in an ewe that aborted, and the resorption of the foetus in another.     

Sows intramammarily inoculated with Escherichia coli influence of time of infection, hormone concentrations and leucocyte numbers on development of disease

The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland. Ten cross-bred primiparous sows were intramammarily inoculated with living E. coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition. Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made. All seven sows inoculated close to parturition developed a rectal temperature of >39.5 degrees C during the first 48 h post-partum and two of them also showed other signs of clinical disease. In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5 degrees C during the first 48 h post-partum and none of them showed any other sign of clinical discase. There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition. There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol-17beta and 15-ketodihydro-PGF2alpha before inoculation. Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition. In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group. The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of chinical coliform mastitis in the sow.     

脂多糖对小鼠乳腺组织中Toll-like Receptor4表达的影响

将40只雌性ICR小鼠,受孕后随机分为试验组和对照组。雌鼠产后10-11d,试验组经第4对乳头灌注LPS,对照组灌注生理盐水,分别于灌注后不同时间采集样本,组织学分析乳腺病理变化;分析对各组乳腺组织中TLR4和TNF-α mRNA表达变化。组织学结果显示,灌注1．5h后乳腺组织中炎性细胞增多,6、12h乳腺腺泡内有大量的炎性细胞浸润,腺泡结构崩解;6h TLR4 mRNA表达极显著高于对照组（P〈0．01）;4个试验组中TNF-αmRNA表达极显著高于对照组（P〈0．01）。试验结果表明LPS能够增强TLR4和TNF-α mRNA表达。     

The course of mastitides in cows infected into the mammary gland with strains of Mycoplasma bovigenitalium and M. bovis]

Six cows were inoculated into the mammary gland with eight mycoplasma strains isolated from the genital tract of bulls and two type strains. The milk of cows infected with Mycoplasma bovigenitalium strains isolated from the genital tracts of bulls showed a change in the appearance and contained large quantities of mycoplasmas and specific antibodies. The mastitis was most intense in about 9 days and began to subside in 17 days infection. The type strain of M. bovigenitalium PG11 failed to produce mastitis. On the other hand, the type strain of M. bovis PG45 produced severe mastitis after a 14-day latency period, with the infection spreading to the uninoculated quarters, causing atrophy of the mammary gland, and persisting till slaughter. The sera of all cows that developed mastitis after experimental infection contained high titres of specific antibodies. The two infecting mycoplasma species were recovered from the inner organs and mammary glands of these cows after slaughter.     

Effect of suckling intensity on human growth hormone binding, biochemical composition and histological characteristics of ovine mammary glands

Suckling both, or only one contralateral mammary gland during 15 days postpartum was utilized to study lactogenic hormone binding to mammary microsomal membranes and quantitative mammary morphology in ewes. Binding of radiolabeled human growth hormone was specific for lactogenic hormones. Non-radiolabeled human growth hormone, ovine and bovine prolactin and human placental lactogen effectively competed with radiolabeled human growth hormone for binding sites but ovine and bovine growth hormone were completely ineffective. Specific binding of radiolabeled human growth hormone to 600 μg of membrane protein averaged 23 ± 3% in all lactating glands. Neither days postpartum nor treatment of contralateral mammary glands substantially altered hormone binding in lactating glands. Specific human growth hormone binding (6 ± 0.5%) in non-suckled glands (15 days postpartum both udder halves) was significantly lower (P<0.01) than in lactating tissue but only a moderate and variable reduction in specific binding was measured in membranes from glands non-suckled for 15 days but contralateral to a suckled gland (14 ± 4%). Specific binding was approximately doubled in assays with 600 compared with 300 μg of membrane protein and the pattern of binding among variously suckled glands was not changed by treatment of membranes with 4 M MgCl₂ prior to assay. Most secretory cells from all lactating glands had rounded, basally displaced nuclei, apical fat globules, secretory vesicles and abundant densely stained basal cytoplasm (ergastoplasm). Alveolar lumenal area was maximal (50% of tissue area) and stromal tissue area was minimal. After 15 days of non-suckling (both udder halves) mammary cells were engorged with lipid, ergastoplasm was reduced and nuclei were irregularly shaped and randomly displaced compared with lactating tissue. In addition, lumenal area was reduced and stromal tissue more evident. Lack of suckling for 5 days had little apparent effect on mammary cytology. Like lactogenic hormone binding, mammary tissue morphology was only moderately altered by 15 days of non-suckling when the remaining gland was suckled. RNA concentration was lowest (2.1 ± 0.3 mg/g) in mammary tissue from ewes in which neither gland was suckled for 15 days postpartum but non-suckling interval had no significant effect when contralateral glands were suckled. DNA concentration was not significantly influenced by suckling treatments. Relative lactogenic hormone binding closely corresponded to changes in cytological and biochemical indices of secretory cell function.     

LPS对小鼠乳腺组织中NF—KB、ACCa和β-CaseinmRNA表达的影晌

为探讨灌注不同质量浓度LPS对小鼠乳腺组织中NF-xB、ACCa和pCaseinmRNA表达的影响，选择30只雌性ICR小鼠为试验动物，受孕后随机分为试验组和对照组，试验组经第4对乳头灌注不同质量浓度LPS（0．1，5，10，50，100mg／L），对照组灌注生理盐水，于灌注后6h采集乳腺组织样品，组织学方法分析乳腺组织的病理变化；采用RT—PCR方法分析乳腺组织中NF_KB、ACCa和B—CaseinmRNA表达的变化。病理切片结果显示，随着LPS灌注浓度的增加，乳腺小叶间炎性细胞数量逐步增加，乳腺小叶内腺泡结构破坏程度也变大；对乳腺组织中NF-xB、ACCa和pCasein的mRNA表达水平进行分析时，发现灌注LPS小鼠乳腺组织中NF-KBmRNA的表达水平随着LPS灌注浓度的增加而逐步增加，而ACCa和pCaseinmRNA的表达水平则随着LPS灌注浓度的增加而逐步降低。结果表明，灌注LPS能够上调N-KBmRNA的表达，下调ACCa和肛CaseinmRNA的表达。     

Evaluation of techniques to demonstrate foot-and-mouth disease virus in bovine tongue epithelium: comparison of the sensitivity of cattle, mice, primary cell cultures, cryopreserved cell cultures and established cell lines

Tongue epithelia infected with each of the 7 serotypes of foot-and-mouth disease virus (FMDV) were used to evaluate in vivo and in vitro systems for the detection of FMDV. Cattle inoculated by the intradermal route in the tongue (IDL) and suckling mice inoculated intraperitoneally were compared for susceptibility to FMDV with freshly prepared bovine thyroid cell cultures; cultures from cryopreserved bovine thyroid, bone marrow, mammary gland, myocardium, tongue, ovary and kidney cells; cultures from cryopreserved embryonic ovine kidney, newborn ovine kidney, ovine testicle, bone marrow, and chloroid plexus cells; and the continuous porcine kidney cell lines MVPK-1 and S6. The mean titers determined for each serotype in each system were statistically compared. The FMDV titers obtained in freshly prepared bovine thyroid cell cultures and by cattle IDL inoculation were the highest and were statistically indistinguishable. The titers obtained by suckling mouse inoculation were significantly lower than the titers obtained in thyroid cultures for serotypes A, C, Asia 1, and SAT 3. The cattle IDL assay was significantly more sensitive than the mouse assay for serotype A. The cell cultures from the cryopreserved newborn ovine kidney and embryonic ovine kidney were significantly less susceptible to serotype Asia 1 when compared with the fresh bovine thyroid cultures, but not significantly different when compared with the cattle assay for all serotypes. Cryopreservation of bovine thyroid cells directly after trypsinization resulted in the loss of susceptibility to FMDV serotype SAT 2. The other cryopreserved cell culture systems exhibited no or minimal susceptibility to all 7 serotypes, or exhibited considerable inconsistency. The established cell lines MVPK-1 and S6 were not susceptible to serotype A, and were less sensitive to serotype C than other culture systems. Quality control of cell cultures used to evaluate field specimens for FMDV was critical. The cell cultures of cryopreserved ovine kidney cells provided the most practical diagnostic system.