Histological and histochemical characterisation of mammary gland tissue of cows infected with foot-and-mouth disease by contact exposure

Foot-and-mouth disease virus was observed to replicate in secretory epithelial cells of bovine mammary gland alveoli as a result of systemic infection initiated by exposure to infected animals. Viral antigens were demonstrated using fluorescent antibody and immunoperoxidase labelling techniques before the development of signs of clinical disease. In addition, labelled antigens were observed associated with cytoplasmic-like fragments in luminal membrane limited structures. Histologically, lesions of the alveolar secretory epithelium consisted of focal necrosis of these cells which eventually sloughed into the lumen.     

Immune cell differentiation in mammary gland tissues and milk of cows chronically infected with Staphylococcus aureus

This study identifies and compares the distribution of mononuclear cells in the mammary gland tissues and milk of healthy and chronically infected with Staphylococcus aureus cows. Somatic cell counts (SCCs) during the 3 months before the study were > 1 x 10(6) cell/ml in the infected quarters and < 1 x 10(5) cell/ml in the infection-free quarters. Immediately after slaughter, samples from the tissues above the gland cistern and supra-mammary lymph node were collected. No histological differences were found between the supra-mammary lymph nodes of the healthy and infected udders, and both appeared normal. In the milk of the healthy infection-free mammary glands, SCC was < 50,000 cells/ml) while epithelial cells were the predominant type. The percentage of CD18+ was low than 45%, of which over three-quarters were polymorphonuclear (PMN), and less than one- quarter were mononuclear cells. The later comprised CD4+ or CD8+ T-lymphocytes, macrophages (Mo) but not B-cells. In the tissues, there were few CD18+ leukocytes, and most of the cells were T-lymphocytes. The number of B-lymphocytes bearing CD21+ was similar to that of CD8+ and were localized in the connective tissue as clusters of 2-5 cells, mainly in areas with no alveoli, or as single cell having a dendritic like form. The number of Mos was negligible. In the milk of the infected glands, SCC exceeded 700,000 cells/ml, of which > 95% were CD18+ positive. The distribution of the leukocytes had two patterns: one presented (> 80%) of PMN cells and a small number of mononuclear cells; the second had less than 50% PMN and many mononuclear cells. The CD8+ cells in these infected sections were observed throughout the mammary epithelial cells (MEc) around the alveoli and in the alveolar lumen (AL). The numbers and the location of CD21+ B-lymphocytes were similar to those in the infection-free mammary glands. The number of CD5+ positive cells was lower than T and B- cells combined and were located throughout the mammary epithelial cells, around the alveoli and within the connective tissue. Mo numbers were high in most of those infected quarters, and were localized around the connective tissue and within the AL.     

Coagulase-negative staphylococci and mammary gland infections in cows

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria from bovine mammary gland milk samples. The objective of this study was to determine the type of inflammation evoked by CNS in the mammary gland of cows during their first lactation. Twenty-four Israeli-Holstein heifers in their first lactation were tested for bacteriological status, somatic cell count (SCC) and differential leucocyte count in milk 60-120 days postparturition and every 50-60 days after until drying off. Following the first testing, the 96 quarters of the 24 heifers were classified as follows: 69.8% as no bacterial growth (NBG), 27.1% infected with CNS and 3.1% infected with Staphylococcus aureus. During lactation, 84.5% quarters had no change in their classification, 6.2% were newly infected with other pathogens, 3.1% were classified as self-cured and in 6.2% sporadic bacteria were isolated. Among the CNS, S. intermedius, S. chromogenes and S. haemolyticus were the most frequently isolated. Milk from CNS-infected quarters had significantly higher SCC than milk from NBG quarters. An analysis of the leucocyte pattern in milk from CNS vs. NBG quarters revealed a significant increase in polymorphonuclears and a significant decrease in the percentage of total lymphocytes and lymphocytes bearing CD4+ or CD8+. The high percentage of CNS-infected quarters that remained unchanged in their bacterial status during the first lactation, indicates that those CNS have the ability to elude the immune system and persist in the mammary gland for a long time. The persisting infection, resulting to some extent from an increase of SCC by some CNS strains, suggests that in the near future control steps will have to be taken into consideration, in order to enhance the improvement of milk quality.     

Effect of pasteurization and evaporation on foot-and-mouth disease virus in whole milk from infected cows.

The effects of pasteurization and evaporation on foot-and-mouth disease virus in whole milk from infected cows obtained one day postinoculation were studied. Virus survived the heating of milk at high temperature-short time pasteurization at 75 degrees C for 15-17 seconds. In addition, virus from infected milk survived heating at 80 degrees C for the same time. Infective virus also survived in the pasteurized milk after evaporation at 65 degrees C to 50% of the original volume. The bovine udder was found to be highly susceptible to foot-and-mouth disease virus replication. Seven log10 plaque-forming units/ml of virus were recovered in whole milk 24 hours postinoculation, and decreasing titers were recovered for as long as seven days postinoculation.     

Gram-negative bacterial infections of the mammary gland in cows

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were less than 28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P less than 0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.     

腺病毒介导O型口蹄疫病毒VP1基因在关中奶山羊乳腺上皮细胞中的表达

构建O型口蹄疫病毒抗原表位VP1的重组腺病毒载体,并在体外和体内培养试验中检测腺病毒介导VP1的表达。利用PCR技术,从原始质粒中扩增VP1基因片段,双酶切后连接载体构建重组穿梭质粒pHBAd-MCMVVP1-GFP,并经磷酸钙沉淀法携带骨架质粒按比例转染HEK293A细胞,获得P1代腺病毒颗粒,经多轮扩增后进行浓缩纯化,获得复制缺陷型腺病毒颗粒,测定病毒滴度。进行体外感染乳腺上皮细胞,通过GFP的荧光表达量确定最佳感染复数。然后用RT-PCR和Western blotting检测感染的乳腺上皮细胞中抗原表位VP1的表达。进一步用纯化的腺病毒通过乳头滴定法感染羊乳腺组织,检测其乳汁中VP1的表达。结果显示,PCR、双酶切和基因测序等结果表明基因扩增和腺病毒载体构建正确,以其感染关中奶山羊乳腺上皮细胞和乳腺组织时,均可检测到VP1蛋白表达。结果表明,成功构建了含VP1基因的重组腺病毒载体,且能够介导VP1基因在体外和体内培养试验中有效表达,为进一步研究纯化分离的VP1蛋白的抗原性并提高抗原优化技术提供基础研究。     

Localization of the sheep FcRn in the mammary gland

Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells.     

Utilization of intestinal triglyceride-rich lipoproteins in mammary gland of cows

Elution profiles of total lipoproteins, apolipoprotein B (apoB) concentrations in lipoproteins, and plasma triglyceride (TG) levels were examined in early-, late-, and non-lactating cows. Additionally, arteriovenous (A-V) differences were also measured to elucidate the uptake of TG and apoB-containing lipoproteins in mammary gland. Non-lactating cows showed three major peaks corresponding to triglyceride-rich lipoprotein (TRL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fraction, whereas both early- and late-lactating cows revealed two peaks corresponding to TRL and HDL. The peak area of TRL in early- and late-lactating cows were significantly (p < 0.05) smaller than that in non-lactating cows. The plasma TG levels and apoB-48 concentrations of TRL in early- and late-lactating cows were also significantly (p < 0.01) lower. Furthermore, early lactating cows showed significantly (p < 0.05) larger A-V differences in both plasma TG and apoB-48 concentration of TRL than those in late- and non-lactating cows. These results suggested that TG in exogenous (intestinal) TRL was utilized for milk fat synthesis in lactating mammary gland of cows by the receptor-mediated uptake.     

Epidemiology of mammary gland disorders in multiparous Finnish Ayrshire cows

Logistic regression was used to investigate the effects of host characteristics, milk production and 23 veterinary diagnoses on the odds of contracting acute and chronic mastitis, and teat injury among 41 989 multiparous Finnish Ayrshire cows that calved during 1983.

Cows which had higher previous-lactation milk yields were at increased risk of acute and chronic mastitis, and teat injury. All of the mammary gland disorders were directly interrelated. Abomasal disorder, indoor hypomagnesemia, prolapsed uterus and vagina, and abortion were not risk factors for any of the three mammary gland disorders; however, retained placenta, udder edema, ketosis, non-parturient paresis, rumen acidosis, traumatic reticuloperitonitis, silent heat, ovarian cyst and other types of infertility were all direct risk factors for at least two mammary gland disorders.     

Linear hoof defects in sheep infected with foot-and-mouth disease

During the epidemic of foot-and-mouth disease (FMD) in The Netherlands in 2001, a sheep farm was identified that had been subclinically infected with the disease. The FMD virus genome was detected in 12 of 16 probang samples collected from the sheep and the virus was isolated from four of these samples. Linear defects were observed, 1 to 3 cm from the coronary band, in the hooves of several of the sheep. The defects were thought to have been caused by the FMD infection. It was thought that the distance of the defects from the coronary band might be an indication of the time since the animals had been infected. To determine the growth rate of the claws of sheep, the growth of the hoof horn of uninfected lambs and ewes was measured; in the lambs the growth rate was 0.44 mm per day and in the ewes it was 0.29 mm per day.     

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.     

Localization of culture-derived soluble Babesia bovis antigens in the infected erythrocyte

Immunoprecipitates derived from crossed immunoelectrophoresis of Babesia bovis culture supernatant fluid against a polyspecific anti-B. bovis serum were used to produce monospecific rabbit antibodies to individual B. bovis antigens. These antibodies were utilized in an immunofluorescence test to identify the location of the respective antigens within the infected erythrocyte. Two antigens were found on or near the erythrocyte membrane, while a third antigen was directly associated with the parasite itself.     

Detection of foot-and-mouth disease virus infected cattle using infrared thermography

In this study, infrared thermography (IRT) was assessed as a means of detecting foot-and-mouth disease virus (FMDV)-infected cattle before and after the development of clinical signs. Preliminary IRT imaging demonstrated that foot temperatures increased in FMDV-infected animals. The maximum foot temperatures of healthy (n = 53), directly inoculated (DI) (n = 12), contact (CT) (n = 6), and vaccine trial (VT) (n = 21) cattle were measured over the course of FMD infection. A cut-off value was established at 34.4 °C (sensitivity = 61.1%, specificity = 87.7%) with the aim of detecting FMDV-infected animals both before and after clinical signs were observed. Seven of 12 (58%) DI and 3/6 (50%) CT animals showed maximum foot temperatures exceeding the 34.4 °C cut-off before the development of foot vesicles. In contrast, only 5/21 (24%) VT animals displayed pre-clinical foot temperatures above this cut-off possibly indicating partial vaccine protection of this group. These results show IRT as a promising screening technology to quickly identify potentially infected animals for confirmatory diagnostic testing during FMD outbreaks. Further evaluation of this technology is needed to determine the value of IRT in detecting animals with mild clinical signs or sub-clinical infections.     

Secretory antibody responses in cattle infected with foot-and-mouth disease virus.

Antibody responses in serum, saliva, nasal secretions, or esophageal-pharyngeal fluid of foot-and-mouth disease virus-infected steers were examined by single radial immunodiffusion and mouse-neutralization tests. In steers infected with type O foot-and-mouth disease virus, high serum antibody titers were detected within 10 days after infection. Antibody was first detected in saliva at 30 days and gradually increased to a plateau at about 90 days. Small amounts of antibody continued to be secreted in saliva and in nasal secretions for at least 6 months. Antibody was not detected in esophageal-pharyngeal fluid. The major antibody activity in secretions was due to secretory immunoglobulin A as revealed by radioimmunoelectrophoresis.