In vitro growth inhibition of major mastitis pathogens by Staphylococcus chromogenes originating from teat apices of dairy heifers

Earlier field observations suggest that teat apex colonization by Staphylococcus chromogenes pre-partum in dairy heifers protects udder quarters against elevated somatic cell counts early post-partum. To explain these findings, the in vitro inhibitory capability of S. chromogenes from teat apices of heifers towards some major mastitis pathogens was tested using a modified cross-streaking method. Two out of 10 S. chromogenes isolates, both originating from two different teats from the same heifer, consistently inhibited growth of all Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis strains, but none of the Escherichia coli strains. The present study, therefore, supports the protective effect of teat apex colonization by S. chromogenes by in vitro production of inhibitory substances.     

Relationship between morphological characteristics of the teat duct and prevalence of intramammary infections with Streptococcus agalactiae in dairy cows

The objective of the study was to determine the relationship between morphological findings of the surface of teat duct particularly the level of ceratosis and the prevalence of intramammary infections (IMI). The study was conducted on a commercial dairy herd housing about 3000 lactating dairy cows. We examined 891 quarters in the middle of lactation. Duplicate samples of quarter foremilk were collected monthly. The bacteriological status of quarters was determined according to the recommendations of IDF. At the same time teats were evaluated by clinical examinations. The appearance of teat skin lesions and the status of the teat duct especially the existence of hyperceratosis (HC) was documented. Four classes of teat duct hyperceratosis were defined: without, slight, medium and severe HC. The rate of IMI in different classes of hyperceratosis of teat duct was compared by Chi-square analysis. Prevalences of intramammary infections were determined three times (P1, P2 and P3) during the study period. Prevalence of infection was high for S. aureus (P1: 5.6% vs. P2: 4.5% vs. P3: 4.3%), Sc. agalactiae (P1: 2.7% vs. P2: 2.6% vs. P3: 2.8%) and CNS (P1: 10.7% vs. P2: 8.8% vs. P3: 9.6%). Furthermore we detected IMI caused by other streptococci, yeast, E. coli and mixed infections. A positive correlation between status of HC and prevalence of IMI for Sc. agalactiae was found. At the second and third sampling time the rate of intramammary infection with Sc. agalactiae in quarters with medium HC (P2: 9.21% and P3: 13.73%) differed significantly (p < 0.05) compared to groups without (P2: 1.56% and P3: 1.91%) and slight hyperceratosis (P2: 2.33% and P3: 2.56%). The results of our study indicate a correlation between morphology of teat duct surface, especially regarding to Sc. agalactiae. On one hand HC can cause high intramammary infection rate with Sc. agalactiae. On the other hand it is possible that HC is the consequence of a quarter infection with Sc. agalactiae. Further research is required.     

The bactericidal activity of a teat dip containing chlorhexidine and cetrimide.

Using an in vivo test on teat skin the disinfectant activity of a teat dip containing chlorhexidine and cetrimide was compared with two iodophor solutions, one containing the recommended concentration of 0.5 per cent available iodine and the other a 10-fold dilution of this (0.05 per cent iodine). The test organisms used were Staphylococcus aureus and Escherichia coli and for both species the 0.5 per cent iodophor was significantly more bactericidal than either the diluted iodophor or the chlorhexidine/cetrimide teat dip (P less than 0.01). In the test against S aureus, chlorhexidine/cetrimide and the 0.05 per cent iodophor showed similar bactericidal activity, but the iodophor was significantly more bactericidal against E coli (P less than 0.01). It is argued that due to its low bactericidal activity this formulation of chlorhexidine/cetrimide is likely to be inferior to 0.5 per cent iodophor solution as a disinfectant teat dip.     

Scanning electron microscopy-aided observations on and therapy of teat canal infections

An examination of teat canal swabs established that 51 teat canals out of 68 quarters of machine-milked cows were colonized by Staphylococcus aureus. Only 31 of these quarters yielded milk from which S. aureus could be cultured, and 6 out of the 31 produced milk containing somatic cell counts in excess of 500 000/ml. No inhibitory substances could be detected in milk samples 12 h after 10 mg of sodium cloxacillin had been deposited in the test canal on 1-4 successive occasions. Teat canal swabs and milk sample cultures of the same quarters became and remained bacteriologically negative for at least a week after the last treatment. Six quarters, which according to the International Dairy Federation criteria were suffering from subclinical mastitis, became negative after local teat canal therapy. Scanning electron micrographs of one infected teat canal revealed the presence of cocci in depressions and crevices on the epithelial surface, suggesting that such cocci are not always flushed out into milk samples. Teat canal therapy should make a marked contribution to the control of bovine mastitis.     

Microbiological colonisation of bovine teat canal--significance and influencing factors

The teat canal of lactating dairy cattle is of particular importance for the defence of facultative pathogenic and pathogenic microorganisms (e.g., staphylococci) invading the bovine udder. Furthermore the canal is usually colonized with microorganisms, too. In addition to microorganisms inhibiting mastitis pathogens the teat canal is colonized by staphylococci. The microbial colonization can be influenced by the environment of the animals, the care and disinfection of the teat skin and indirectly by the effects of forces being associated with machine milking. Because of vacuum fluctuations occurring under the teat tip microorganisms, which colonize the teat canal, can invade the bovine mammary gland and cause infections there. This paper gives a review of the microbial colonization of the bovine teat duct and of influencing factors on the microbial populations as well as of the significance of the teat canal colonization for the development of mastitis.     

Cytologic observations of the bovine teat end

Cells infiltrating from the vasculature and histologic components of internal tissues of teats (mammary papilla) from noninfected udder quarters were studied, using light and electron microscopy. Morphometric analysis demonstrated a progressive increase in number of infiltrating cells from the distal teat cistern (sinus papillaris) to the junction of the Furstenberg's rosette (distal termination and convergence of mucosal folds lining the teat cistern) and the streak canal (ductus papillaris). Plasma cells contributed to cellular increases in subepithelial connective tissue and were the most prevalent infiltrating cell type. Plasma cells also penetrated the basal epithelial lining of the rosette area and occasionally migrated to the luminal surface near the squamocolumnar junction. Neutrophils and monocytes contributed to the increase in cells infiltrating the epithelial lining. Few infiltrating cells were observed in epithelium and underlying stroma of the streak canal. Cytologic comparison demonstrated a reduction in all cell types from lactating to involuting phases of lactation. Greater numbers of plasma cells, lymphocytes, and monocytes were observed in teat end tissues from quarters previously infected with Staphylococcus aureus.     

Effects of postmilking teat treatment on the colonization of Staphylococcus aureus on chapped teat skin

Sixteen Holstein cows were used to test the effect of postmilking teat treatment on colonization and intramammary infection by Staphylococcus aureus on chapped teats. Treatments were (1) chapping the teat and using 1% I2/10% glycerin postdip solution, (2) 1% I2/10% glycerin postdip solution on nonchapped teats, (3) chapping the teat and using 10% glycerin postdip solution, (4) chapping the teat and not using a postdip solution. All mammary glands were free of S aureus teat skin colonization and intramammary infection at the start of the study. Teats selected for chapping were dipped in 1N NaOH prior to 3 applications of S aureus broth culture; cultures were applied at 12-hour intervals on all teats. Treatments were applied after each milking for 30 days and were initiated after the second broth dip. Teat skin swab specimens and milk samples were collected before treatment application. Teat skin condition was scored daily. Nonchapped teats (treatment 2) did not support skin or orifice colonization by S aureus. Treatment-1 teats healed most rapidly and supported less colonization in skin and orifice than did treatment-3 and -4 teats. Teat skin scores and skin colonization were lower for treatment-3 than treatment-4 teats. A correlation between teat skin colonization and teat skin conditions was found. Two intramammary infections were found in treatment-4 quarters and 1 in a treatment-3 quarter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparatus for pasteurising teat cup liners between cows in a herringbone parlour.

In a herringbone milking parlour, teat cup liners were deliberately contaminated in turn with Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Sterp uberis. Contamination was achieved by filling the liners with milk that contained 10(6) test organisms per ml. After the clusters had been back-flushed with water at 85 degrees C for five seconds, normal swabbing methods failed to recover any contaminating organisms from the teat liners in 56 tests out of 64. After 10 seconds back-flushing no recoveries were made in the same number of tests. The apparatus developed to effect this back-flushing for a particular herringbone parlour is described, with details of its routine use during milking. For a 100-cow herd, the running cost of such equipment using a five-second back-flush is estimated at no more than 4 pounds per week and, in its present form, would not add more than 10 seconds to the total milking time for each cow. Improvements in design of the apparatus, and in milking techniques arising from the routine use of the device, are also considered.     

Resistance of mastitis pathogens in northern Germany

The data of 1692 susceptibility tests acquired from April 2003 through March 2004 in the mastitis laboratory of the Institute for Food Quality and Safety were summarized in order to help veterinarians confronted with acute mastitis in choosing the appropriate antibiotic. Two thirds of the milk samples were infected with gram-positive cocci. One third of these were identified as Streptococcus (S.) uberis, one fourth as Staphylococcus (S.) aureus. All isolates (100%) of S. uberis, S. dysgalactiae, S. agalactiae and Arcanobacterium pyogenes were susceptible to Penicillin and Ampicillin. Concering S. aureus, nearly 100% of the isolates were susceptible to Oxacillin, Cephalothin, Cefacetril, Cefquinom and Neomycin, but only 88% of the isolates were sensitive to Penicillin, Ampicillin and Cefoperazon. The gram-negative rods (Escherichia (E.) coli, Klebsiella spp. and Pseudomonas spp.) displayed an irregular resistance pattern. More than 93% of all examined isolates including Pseudomonas spp. were susceptible to Colistin. The sensitivity of E. coli and Klebsiella spp.to Marbofloxacin, Enrofloxacin and Cefquinom exceeded 96%. Thus, the susceptibility of gram-positive mastitis pathogens to common antibiotics is favourable. Because the highly effective Colistin is no longer approved for local therapy, the situation for gram-negative bacteria is more difficult.     

Effects of quarter individual and conventional milking systems on teat condition

An on-farm experiment was performed to investigate the effects of two milking systems on teat condition. An auto-tandem milking parlor made by GEA^® with a conventional milking cluster (CON) and a single tube milking system called MultiLactor^® (MULTI) from Siliconform GmbH were tested. For both milking system, in total 73 animals were milked over a period of seven months. To assess the effect of milking on teat condition, each teat was scored according to teat-condition evaluation systems. To evaluate teat condition, the teat color, ring formation at the teat base and formation of teat hyperkeratosis were assessed. The evaluation was conducted once a month after milking. The collected data were then statistically analyzed with generalized linear mixed models. The only differences found between the two milking systems occurred with respect to their effect on teat color. MULTI showed significantly better scores compared to CON. There were no significant differences between the milking systems with regard to teat-end hyperkeratosis and ring formation at the teat base.     

The prophylactic effect of a teat sealer on bovine mastitis during the dry period and the following lactation

AIM: To determine the prophylactic efficacy of a teat sealer, administered at drying off, in reducing new intramammary infections in the dry period and the following lactation. METHODS: A total of 528 cows with late lactation somatic cell counts <2 00,000 cells/ml was identified in three commercial herds. Of these, bacteriological examination showed 482 cows were uninfected in all four quarters and 46 were infected in only one quarter. At drying off, uninfected quarters were randomly allocated to the following treatments: no infusion (negative controls), infusion with a bismuth subnitrate based teat sealer, infusion with teat sealer + antibiotic, or infusion with a cephalonium-based dry cow antibiotic (positive control). New infections were identified during the dry period by periodic udder palpations and at calving by bacteriological culture. RESULTS: All three infused treatments reduced the incidence of new intramammary infections due to Streptococcus uberis, both during the dry period and at calving, by about 90% (p <0.01). The majority of the infections were due to Streptococcus uberis. For all treatments, a 50% lower incidence of clinical mastitis over the first 5 months of the ensuing lactation was reported by farmers. X-ray imaging of 19 teats showed that the teat sealer material was retained, at least in part, in the lower teat sinus over about 100 days of the dry period. CONCLUSIONS: Closure of the teat canal from day one of the dry period as achieved by the teat sealer was as effective in reducing new dry period infections as the infusion of a long-acting dry cow antibiotic formulation. The lower incidence of new infections in the ensuing lactation among the infused quarters implies that fewer subclinical infections persisted from the dry period. Use of teat sealers at drying off appears to offer the same prophylactic efficacy as the dry cow antibiotic approach.     

Influence of milking technique and lactation on the bovine teat by means of ultrasonographic examination

The teats of Brown Swiss and Austrian Simmental cows, divided into two groups, one milked by means of an automatic milking system, the other using a conventional milking parlour, were examined monthly by ultrasonography. Aim of the study was to compare the effects of two different milking machines upon the structures of the bovine teat canal and wall by ultrasonography and thereby evaluate ultrasonography as a research tool for visualisation of different influences on the bovine teat. Length and thickness of the teat canal and teat wall thickness were measured and analysed. During lactation, teat canal length and thickness increased in both groups, teat canal length decreased in conventional milked cows at the end of lactation. Shorter and narrower teat canals were observed in automatic milked cows. Differences between the groups in teat canal length and thickness were determined in early lactation. During lactation teat wall thickness showed a slight increase. Automatic milked cows displayed thinner teat walls than cows milked in the milking parlour. Teat morphology was influenced by the number and duration of lactations, milk yield, quarter of the udder and time and date of examination. It was concluded that the effect of the two different milking machines caused significant differences in bovine teat morphology and that ultrasonography proved to be an appropriate method for visualising influences of the milking technique on the bovine teat.     

In vitro and in vivo evaluation of a 0.5% chlorhexidine gluconate teat dip

The activity of a 0.5% chlorhexidine gluconate postmilking teat germicide in reducing the numbers of Staphylococcus aureus and Streptococcus agalactiae on the skin of excised teats from cows was determined. The product yielded logarithmic reductions of 4.09 and 4.10 against S aureus and Str agalactiae, respectively, compared with 3.80 and 3.81 reductions, using a 1% iodophor dip. Germicide tolerance to an organic load containing Serratia marcescens or Pseudomonas spp was also determined. Organisms were not recovered from the product 8 hours after introduction of a simulated organic load containing either species of bacteria. The germicide was further evaluated against S aureus and Str agalactiae, using experimental challenge-exposure procedures in a research dairy herd. Efficacy was 73.4% (P less than 0.001) against S aureus and 68.1% (P less than 0.005) against Str agalactiae.     

Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Radiographic method for anatomic study of the teat canal: characteristics related to resistance to new intramammary infection during lactation and the early dry period.

An in vivo radiographic method to determine teat canal anatomy has shown that resistant quarters had teat canals with a smaller diameter than that of susceptible quarters. The length of the teat canal did not appear to be related to resistance to new intramammary infections. These findings relate to new infection caused by Aerobacter aerogenes, Streptococcus agalactiae, and Staphylococcus aureus. Quarters guarded by teat canals with larger diameter were less resistent to new infection during both lactation and the early dry period compared with quarters guarded by teat canals with small diameter.     

奶山羊实验性乳房炎的病理组织学观察及促炎性细胞因子的检测

选健康泌乳期关中奶山羊8只,分成金黄色葡萄球菌感染组和大肠埃希菌感染组,每只羊分别经右乳头灌注病原性大肠埃希菌(3×103cfu)和金黄色葡萄球菌(3×102cfu),对照乳区灌注等量无菌PBS.于灌注细菌前后不同时间采集血样和乳样,测定血清和乳样中的促炎性细胞因子水平,并于72 h后取山羊乳腺组织制备病理切片观察组织...     

Radiographic method for anatomic study of the teat canal: changes between milking periods.

Radiographs of the teat canal (papillary duct) of 14 Holstein-Friesian cows were made immediately after removal of the milking machine and at 2, 4, 6, and 8 hours. The distal, middle, and proximal diameters of the canal were dilated when the machine was removed. The distal and the middle parts of the teat canal were more dilated than the proximal part of the canal. Two hours later, the canal was constricted in the 3 areas. During the next 6 hours, the canal dilated-the greatest kilation being at the proximal paet of the teat canal. The average lenght of the teat canal remained unchanged. Apparently, microorganisms can easily pass through the teat canal when it is dilated at the end of machine milking and for up to 2 hours after milking.     

Host-response patterns of intramammary infections in dairy cows

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.     

通用基因芯片对奶牛乳房炎主要致病茵的检测

应用生物信息学手段和查阅文献资料设计了金黄色葡萄球菌、大肠埃希菌、无乳链球菌、绿脓杆菌、停乳链球菌、乳房链球菌6种奶牛乳房炎主要致病菌的通用引物和金黄色葡萄球菌、大肠埃希菌、绿脓杆菌的寡核苷酸探针及无乳链球菌、停乳链球菌、乳房链球菌的特异引物,并用这3种特异引物扩增片段的纯化产物作为这3种链球菌的检测探针。在引物对样品中细菌的相应基因片段扩增的同时进行靶基因的生物素标记,扩增的产物与硝酸纤维素膜上的探针进行杂交,酶联、显色后根据芯片扫描仪的判读结果来确定奶牛乳房炎致病菌感染的种类。结果表明,建立的以16S rDNA为对象的基因芯片技术可以快速的检测出以上6种细菌,整个检测过程需要6h~7h,灵敏度高,特异性好,能快速的对奶牛乳房炎的主要致病菌做出诊断。     

The bacterial flora in the teat duct of ewes can protect against and can cause mastitis

We studied the possible effects of bacterial populations within the teat duct, in the pathogenesis of ovine mastitis. In experiment I, 32 ewes were allocated into group A (ewes from which we isolated (+++ growth) coagulase-negative staphylococci), B (ewes from whose duct we isolated (+ growth) coagulase-negative staphylococci) or C (ewes from which we isolated Bacillus spp.) and subdivided into A1, B1, C1 (n=4; challenged by deposition of 1.250 cfu of Mannheimia haemolytica into the teat duct) or A2, B2, C2 (n=4; used as uninoculated controls); group D (n=8) contained ewes with no bacteria in their teat ducts and were challenged as above. There were less bacteriological isolations of flora (P = 0.018) and challenge (P<0.05) organisms from A1 than from A2 and D ewes; the severity of pathological findings in A1 (summed up score: 27) ewes was smaller than in D (summed up score: 36) ewes (P = 0.038). No such findings were evident with B1 or C1 ewes (P>0.4). In experiment II, ewes (groups E and F, n=6) from whose duct we isolated coagulase-negative staphylococci (+ growth) were used; in group G (n=6) ewes with no bacteria in their teat ducts were included. Teat chapping was applied in E and G ewes. All E ewes developed acute clinical mastitis within 24 h after teat chapping, although we had carried out no challenge; there were more bacteriological isolations of flora organisms from E than from F and G ewes (P < 0.001); the severity of pathological findings in E (score: 28) was greater than in F (score: 3) or G (score: 14) ewes. In experiment III, eight ewes with no bacteria in their teat ducts were allocated into group H or I (n=4) and challenged into the teat (group H) or into the gland (group I) with 10(6) cfu of a Staphylococcus simulans recovered from the teat duct of a group E ewe. Group H ewes developed transiently clinical followed by subclinical mastitis (based on bacteriological and cytological evidence), whilst group I ewes developed severe clinical disease. We conclude that staphylococcal flora present in high numbers within the teat duct of ewes can afford some protection against invading microorganisms. However with impeded defence mechanisms of the teat, the same flora may invade the mammary parenchyma and cause clinical mastitis.