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.     

B‐Mode and Doppler Sonography of the Mammary Glands in Dairy Goats for Mastitis Diagnosis

This study aimed to evaluate the sonographic characteristics of the udder and teats and to determine the Doppler indexes of mammary artery in healthy and undergoing subclinical and clinical mastitis goats. Thirty animals among Saanen and Alpine Brown goats were arranged in three groups, healthy goats (HG), goats with subclinical mastitis (SMG) and goats with clinical mastitis (CMG). Using the B‐mode, the sonographic characteristics (echotexture and echogenicity) and biometry (diameter and area of the udder cistern, diameter and area of the teat cistern and thickness of the teat wall) were evaluated. Using Doppler ultrasonography, the vascular indexes of the mammary artery were obtained. It was observed hyperechogenicity with solid component in the gland cistern when comparing animals with clinical mastitis and healthy mammary tissue. Regarding the echotexture of the breast tissue, there was heterogeneity in the mammary parenchyma on the three groups, for the milk, it was observed homogeneity for animals on HG and SMG and heterogeneity for animals on CMG. Grey‐scale quantitative assessment revealed increase in echogenicity (mean value) for all the structures when comparing the three groups. Biometry did not reveal statistical difference between groups, for none of the evaluated structures. Doppler examination of the mammary artery showed the decrease of end diastolic velocity and raise of pulsatility index between groups. The association of B‐mode and Doppler ultrasonography is useful for the evaluation of the udder of dairy goats with mastitis. It is a sensitive and specific method for the study of this disease. Doppler mode was unable to establish reliable criteria for diagnosis of subclinical mastitis. Moreover, the quantification of echogenicity is a useful technique for the evaluation of the milk in animals with mastitis; therefore, it is suggested that it can be used as complementary technique for the diagnosis of mastitis in goats.     

Ultrasonographic assessment of the feline mammary gland

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5-9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6-9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.     

B-mode ultrasound examination of canine mammary gland neoplastic lesions of small size (diameter < 2 cm)

Ultrasonography is a valuable tool for the evaluation of neoplastic lesions in the dog and there is a growing interest in the use of this technique for the stadiation of canine mammary tumours. An accurate assessment of small sized nodules facilitates the stadiation of the mammary lesions and helps the clinician in the choice of the most indicated surgical therapy. The aim of this study was to identify those ultrasound criteria that may be useful in discriminating between benign and malignant lesions of small size (diameter smaller than 2 cm). Sixty-two nodules, < 2 cm in larger diameter, belonging to thirty-five bitches presented between January 2012 and February 2014 were evaluated. Tumours were observed by conventional ultrasound and assessed for: shape (regular-irregular), limit (defined-ill-defined), margins (regular-irregular), echogenicity (hypoechoic-isoechoic-hyperecoic), echotexture (homogeneus-heterogeneus), presence of hyperecoic halo, distal acoustic enhancement or shadowing and surrounding tissue alterations. Among the alterations in surrounding tissues, the disruption of the glandular tissue and the increase in echogenicity of the peritumoral tissues were assessed. Thereafter, bitches were subjected to mastectomy and nodules were evaluated histologically. None of the ultasound criteria considered in the current study showed a statistically significant relation with malignancy, except for the presence of alterations in the tissue surrounding the nodules. According to our results, this characteristic may indicate malignancy, however its subjectivity may affect the applicability in clinical practice. In conclusions, conventional ultrasound in bitches had a limited ability in discriminating benign and malignant mammary gland neoplastic lesions of small size (diameter?<?2 cm).     

Low expression of the antibacterial factor L‐amino acid oxidase in bovine mammary gland

In the mouse, L‐amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis.     

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.     

Role of progestin-induced mammary-derived growth hormone in the pathogenesis of cystic endometrial hyperplasia in the bitch

Endogenous progesterone and synthetic progestins may induce hypersecretion of growth hormone (GH) of mammary origin, hyperplastic ductular changes in the mammary gland, and the development of cystic endometrial hyperplasia (CEH) in dogs. It was investigated whether progestin-induced mammary GH plays a role in the pathogenesis of CEH in the bitch. During 1 year, bitches with surgically excised mammary glands and healthy control bitches received medroxyprogesterone acetate (MPA). Before and after MPA treatment, uterine and mammary tissues were collected for histological, immunohistochemical, and RT-PCR examination. After MPA administration, the mammary tissue in the control dogs had differentiated into lobulo-alveolar structures and CEH was present in all uteri of both dog groups. In the MPA-exposed mammary tissue of the control dogs, GH could only be demonstrated immunohistochemically in proliferating epithelium. After treatment with MPA the dogs of both groups had immunohistochemically demonstrable GH in the cytoplasm of hyperplastic glandular uterine epithelial cells. RT-PCR analysis of the mammary gland tissue after MPA administration demonstrated a significant higher GH gene, and lower GHR gene expression than before treatment. In the uterus, the expression of the gene encoding for GH was significantly increased in the mastectomized dogs, whereas in the control dogs the expression of the gene encoding for insulin-like growth factor-I had significantly increased with MPA administration. MPA treatment significantly down regulated PR gene expression in the uterus in both dog groups. These results indicate that progestin-induced GH of mammary origin is not an essential component in the development of CEH in the bitch.     

乳头管灌注脂多糖对兔血清酶活性的影响及病理组织学观察

探讨经乳头管灌注脂多糖(LPS)对兔血清酶活性的影响及乳腺病理组织学变化.12只泌乳兔(产后第7天)经乳头管灌注LPS(150 μg/kg体重)建立急性临床型乳房炎模型,在灌注前2 h,灌注后6、12、24、48、72 h以及5 d和7 d分别测定血清中乳酸脱氢酶(LDH)、髓过氧化物酶(MPO)、过氧化物酶(LP)及...     

环状RNA Circ0000316在大肠埃希氏菌和金黄色葡萄球菌诱导的奶牛乳腺上皮细胞炎症模型中的表达研究

奶牛乳腺炎是奶牛场的多发病和常发病,在世界各地的养殖场均有很高的发病率。环状RNA(circular RNA,circRNA)作为近年来被广泛研究的非编码RNA在奶牛乳腺炎发生过程中也发挥了一定的作用。采集患有乳腺炎的奶牛乳腺组织和健康乳腺组织检测circ0000316的表达量,同时采用大肠埃希氏菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)刺激奶牛乳腺上皮细胞,建立奶牛乳腺上皮细胞炎症模型,检测炎症模型中circ0000316的表达量。结果显示,成功建立了奶牛乳腺上皮细胞炎症模型,且circ0000316在奶牛乳腺炎组织和奶牛乳腺上皮细胞炎症模型中均呈现低表达,为研究circRNA在乳腺炎发生过程中的作用奠定了基础。     

新疆库尔勒市某奶牛场奶牛隐性乳房炎情况调查

为了检测和调查新疆库尔勒市某奶牛场泌乳奶牛乳房炎的发病率,对该奶牛场养殖的32头泌乳期奶牛用凝乳法进行了隐性乳房炎检测。结果表明,隐性乳房炎奶牛阳性率为40.63%,且乳房炎阳性率随奶牛使用年限的增加而增加;用挤奶机器挤奶的奶牛乳房炎阳性率高于手工挤奶,后乳区的乳房炎阳性率高于前乳区。     

Effects of Staphylococcus aureus mastitis on bovine mammary gland plasma cell populations and immunoglobin concentrations in milk

Bovine mammary tissue and milk samples were examined to determine effects of chronic Staphylococcus aureus mastitis on the humoral immune response. Parenchymal and teat end tissues from lactating bovine mammary glands were stained immunohistochemically to determine distribution of immunoglobulin (Ig) G1-, IgG2-, IgA-, and IgM-producing plasma cells. Numbers of all Ig-producing plasma cells tended to be higher in tissues from S. aureus infected quarters compared with controls, but most differences were not statistically different. Numbers of IgG1-producing plasma cells at the Furstenberg's rosette area of infected quarters were significantly (P less than 0.05) higher than uninfected quarters. There were no significant differences in concentrations of Ig isotypes in milk from S. aureus infected and uninfected quarters. Data suggest that the antigenic effect of chronic S. aureus infection on the humoral immune response of the bovine mammary gland is minimal. Persistency of S. aureus infection may result, in part, from suboptimal stimulation or immunosuppression of the mammary immune system.     

Mastitis in the lactating mink female (Mustela vison S.) and the development of "greasy kits"

"Greasy kits" is the result of a multifactorial disease complex with few known definitive aetiological factors. Mastitis has been hypothesized as a triggering factor although classical clinical signs of mastitis (rubor, tumor, dolor, calor) are rarely seen in lactating Danish mink females. In this study we sacrificed 2 groups of lactating mink females with a total of 78 mammary glands at day 19-30 after giving birth. The first group had raised normal mink kits while the other group had suffered severe attacks of greasy kits. We found no clinical or histopathological evidence of mastitis but isolated streptococci and staphylococci from 2 mammary glands in females raising greasy kits. These glands showed no clinical or histological signs of inflammation attributable to bacteria and we conclude that mastitis is not necessary for the generation of greasy kits.     

The Lymph Drainage of the Mammary Glands in the Bitch: a Lymphographic Study. Part I: The 1st, 2nd, 4th and 5th Mammary Glands

The objective of this paper was to study the lymph drainage of the 1st, 2nd, 4th and 5th mammary glands in the bitch using indirect lymphography. The main conclusions drawn after the study of 67 normal lactating mongrel bitches were as follows: lymph drains from the first gland, usually to the axillary nodes, and, in few cases, to the axillary and superficial cervical nodes simultaneously. The second gland drains to the axillary nodes. The fourth gland usually drains to the superficial inguinal nodes, but it may, rarely, drain to the superficial inguinal and medial iliac nodes simultaneously. The fifth gland drains to the superficial inguinal nodes. Lymphatic connection between the mammary glands could not be demonstrated. Furthermore, it was confirmed that lymph can pass from one gland to another, through their common regional lymph nodes, by retrograde flow. It was demonstrated that there is a connection between the superficial inguinal lymph nodes from either side. It is suggested that lymphatic connection between the axillary and sternal nodes and between the axillary and bronchial nodes should be possible in some cases. Lymphatics of the mammary glands that cross the midline were not demonstrated.     

Presence of subepithelial lymphoid nodules in the teat of ewes

A total of 87 clinically healthy ovine teats were examined bacteriologically (by scraping the mucosa) and histologically. Teats examined were those of lactating mammary glands with no bacteria isolated (n = 23); of mammary glands after cessation of lactation with no bacteria isolated (n = 25); of lactating mammary glands with bacteria isolated (n = 22); and of mammary glands after cessation of lactation with bacteria isolated (n = 17). The salient histological feature was subepithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Subepithelial lymphoid nodules, some with germinal centres, were detected in 43 (49%) teats. The majority of lymphoid nodules was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with the presence of leucocytic activity (P < 0.001) and with the presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced subepithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion. The findings call for further investigations into the lymphoid structures of the teat; these should elucidate the role and development of mammary mucosa-associated lymphoid tissues and may lead to strategies for enhancing non-specific defence mechanisms of the mammary gland.     

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland

The mammary gland performs a variety of immunological functions, including protecting itself from mastitis and protecting neonates from infectious agents. Several molecules that mediate lymphocyte trafficking in the immune system are also expressed in the mammary gland. This review is focused on the immunological function of these molecules, especially glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the mammary gland. GlyCAM-1 is expressed in the lactating mouse mammary gland. Endothelial cells produce this protein and secrete it into milk. The glycosylated modification of mammary gland GlyCAM-1 is different from that of the lymph nodes, and lacks the binding ability for L-selectin on lymphocytes. GlyCAM-1 in the mammary gland is not involved in lymphocyte migration, and probably has another function besides that of the lymph nodes. MAdCAM-1 is expressed on endothelial cells of small venules around mouse mammary lobules during lactation. This molecule has the ability to interact with alpha4beta7 integrin on lymphocytes and mediates lymphocyte recruitment to the mammary gland. The density of beta7+/CD3+ T-cells is correlated with the density of the MAdCAM-1-stained area, suggesting that MAdCAM-1 may mediate the migration of these cells. In contrast, there is no relationship between MAdCAM-1 expression and the number of beta7+/c-IgA+ B-cells, implying that some other factor is involved in lymphocyte migration to the mammary gland. Chemokines, such as IL-8, GRO-alpha, MCP-1, RANTES and MEC, have been detected in human and mouse mammary glands. Although little information is available, these molecules may contribute to lymphocyte migration to the mammary gland.     

Effects of two anti-inflammatory drugs on physiologic variables and milk production in cows with endotoxin-induced mastitis

OBJECTIVE: To determine the effects of 2 anti-inflammatory drugs in lactating Holstein cows with endotoxin-induced mastitis. ANIMALS: 30 multiparous Holstein cows that had been lactating for 30 to 60 days. PROCEDURE: Bacterial culture of milk samples and physical examinations established that study cows were in good health and free of mastitis. Mastitis was induced in 1 front mammary gland by intramammary administration of purified bacterial endotoxin. Cows were allocated into 1 of 3 treatment groups: untreated endotoxic mastitis (n = 9), endotoxic mastitis plus flunixin meglumine (9), and endotoxic mastitis plus isoflupredone acetate (10). Heart rate, rectal temperature, mammary surface area, and rumen motility were recorded hourly for 14 hours following endotoxin administration. Flunixin meglumine or isoflupredone acetate was administered after mammary swelling and rectal temperature > or = 40 degrees C had developed. Milk production was evaluated from 5 days before to 10 days after induction of mastitis. RESULTS: Neither drug ameliorated loss of milk production or swelling of the affected mammary gland. Both drugs reduced mean heart rate during the 14 hours following endotoxin administration, compared with untreated control cows. Cows treated with flunixin meglumine had increased rumen motility and decreased rectal temperature during the same period, compared with all other cows. CONCLUSIONS AND CLINICAL RELEVANCE: Neither drug enhanced recovery of milk production following endotoxin-induced mastitis. Flunixin meglumine decreased rectal temperature, whereas isoflupredone did not; however, it has not been established that reduction of fever is beneficial to cows with naturally occurring mastitis.     

Concentrations and specific antibodies to staphylococcal enterotoxin-C and toxic shock syndrome toxin-1 in bovine mammary gland secretions,and inflammatory response to the intramammary inoculation of these toxins

To investigate the pathological role of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) in bovine mastitis, the production of SEs and TSST-1 was investigated in staphylococci isolated from 120 mammary gland secretions (MGS, 51 from no clinical sign-mammary glands and 69 from staphylococcal mastitic-mammary glands) collected from dairy farms where staphylococcal mastitis frequently occurred in Miyagi and Yamagata prefectures from 1997 to 1998. Concentrations of these toxins and specific antibody titers in each MGS were also measured. Furthermore, SEC and TSST-1 were inoculated into lactating mammary glands and inflammatory responses were analyzed. A high percentage of staphylococci including Staphylococcus aureus and coagulase-negative staphylococci isolated from both no clinical sign- and mastitic-MGS produced both SEC and/or TSST-1. The concentration of SEC increased with the severity of the mastitis, and was significantly higher (P<0.05) in acute mastitic-than in no clinical signs-MGS. Titers of specific antibodies to TSST-1 in MGS were significantly higher (P<0.05) than those to SEC, regardless of whether or not the cows were lactating or mastitic. Specific antibodies purified from MGS neutralized each toxin in vitro. A significant increase (P < 0.05) in somatic cell counts was induced by the intramammary inoculation of SEC but not TSST-1. These findings indicated that SEC rather than TSST-1 plays an important role in the pathology of staphylococcal bovine mastitis. The inflammatory activity of TSST-1 was probably neutralized by specific antibodies in MGS.     

The Lymph Drainage of the Mammary Glands in the Bitch: a Lymphographic Study. Part II: the 3rd Mammary Gland

The objective of this investigation was to study the lymph drainage of the 3rd mammary gland in the bitch using indirect lymphography. The main conclusions drawn after the study of 62 normal lactating mongrel bitches were as follows: lymph usually drains from the third gland to the axillary and superficial inguinal nodes simultaneously, but in some cases it drains only cranially to the axillary nodes. It may, rarely, drain only caudally to the superficial inguinal nodes. A lymphatic connection between the mammary glands could not be demonstrated. It was demonstrated that there is a connection between the superficial inguinal lymph nodes on either side. It is suggested that lymphatic connection between the axillary and sternal nodes and the axillary nodes of both sides should be possible in some cases. Lymphatics of the mammary glands that cross the midline were not demonstrated. No relationship between the number of lymph nodes and the body size of the dog could be demonstrated.     

Infection following challenge of the lactating and dry udder of dairy cows with Actinomyces pyogenes and Peptostreptococcus indolicus

Challenge of 12 mammary glands of cows in mid-lactation with 10(7) colony forming units (cfu) of Peptostreptococcus indolicus on two occasions led to clinical mastitis in only four quarters. The bacteria were rarely recovered and disappeared from the secretion within 14 days. In challenges 7 days prior to drying off eight of 12 quarters became infected and at drying off all quarters challenged became infected. The infections established at drying off persisted well into the dry period. P. indolicus infection was also established in all of 12 dry glands challenged, but usually eliminated at calving or early in the next lactation. Isolation of P. indolicus was accompanied in about one-third of cases by changes in the appearance of the secretion. Intramammary challenge with Actinomyces (formally Corynebacterium) pyogenes led to clinical and subclinical infections in nine of 12 lactating glands and in all of six dry glands. Dry period infections with A. pyogenes were more severe and rarely eliminated even by antibiotic therapy. Infections during lactation were often eliminated either naturally or by antibiotic therapy. Intermittent recovery of A. pyogenes from the lactating mammary gland, without clinical signs of infection, was possible for up to 90 days after challenge. Combined infections with A. pyogenes and P. indolicus were clinically more severe with a higher frequency of systemic involvement. It was shown that in the non-lactating gland an acute mastitis, similar to 'summer mastitis' could be established either by simultaneous inoculation with A. pyogenes and P. indolicus or by subsequent inoculation of quarters excreting P. indolicus with A. pyogenes.