Bovine serum albumin and cell counts in the diagnosis of subclinical udder infection

Summary

Puncture of the milk cisterns was performed in 120 bacteriologically positive quarters of forty‐seven lactating dairy cows on three farms. This method was used to determine whether the existing infection was an infection of the teat canal or one of the udder. The results were related to the concentration of bovine serum albumin (BSA) and the cell count in the milk. Of the bacteriologically negative quarters, both BSA levels (in 91 per cent of the quarters the BSA concentration was 0.20 mg. per ml. of milk or less) and cell counts (92 per cent contained less than 500,000 cells per ml. of milk) were low. In cases of udder infection with primary pathogenic bacteria there was a marked increase in cell count (90 per cent more than 500,000 cells per ml. of milk), whereas the increase in BSA was rather small (51 per cent still contained 0.20 mg. BSA per ml. of milk or less). While the difference in cell counts of milk from quarters with udder infections and teat canal infections with primary pathogenic bacteria was significant, the difference between the BSA levels of these two groups was not. Therefore, the cell count supplies more reliable information than does the BSA level of the milk. Of all infections, 23 per cent were found to be infections of the teat canal.     

The Use of Induced Mammary Infections for Evaluating Dry Cow Treatment Products II. Trial of a Proposed Method to Compare Three Levels of Novobiocin

Infections were induced at the end of lactation in all udder quarters of 19 cows by the infusion of 0.2 mL of a 10^-4 dilution in milk of a six hour milk culture of Staphylococcus aureus, strain 305 (A.T.C.C No. 29740). Two right or two left udder quarters were infected at 15 days and the opposite two five days before the last milking of lactation. Following the last milking all four udder quarters of eight cows were treated with 400 mg novobiocin in 10 mL of 2% aluminum monosterate in peanut oil, gelled. All udder quarters of eight other cows were treated with 50 mg novobiocin in the same vehicle and the udder quarters of three cows were treated with the vehicle only.

At calving, eight of 32 quarters treated with 400 mg novobiocin were still infected, as were 18 of 32 treated with 50 mg of novobiocin and all those quarters treated with vehicle only. Results were identical from udder quarters infected 15 and five days before drying off.

No significant differences were found between quarters in milk yield on the last day of lactation, nor the length of the dry period. An increasing number of udder quarters were infected at calving with increase in lactation age of the cow, although the small number of cows would not allow a firm conclusion. A significant difference in results was found between front and hind udder quarters, only five of 32 front quarters were infected at calving as compared to 21 to 32 hind quarters.

The method proposed was found to give essentially the same results as those from a large field trial using the same antibiotic. It should therefore be useful in evaluation trials of new antibiotic products for dry cow treatment.

     

Some Effects of the Source of Bovine Milk Leucocytes and Strain of Staphylococcus on their Interaction in Vitro

A method is described for the quantitative study in vitro of the bovine milk leucocyte: staphylococci interaction which is capable of demonstrating differences between groups of leucocytes, and, to some extent, stains of Staph. aureus.

By means of this method it has been possible to show statistically, that significant differences can occur, between quarters of the same udder, in the numbers of leucocytes competent to ingest staphylococci, and also in the numbers of staphylococci ingested.

Between 4 strains of Staph. aureus significant differences were noted in their ability to multiply in the presence of milk leucocytes; in the production of leucocidal factors; and in the reduction of Resazurin in whole, normal milk.

     

Antibiotic Treatment of Experimental Staphylococcus aureus Infections of the Bovine Mammary Gland

Experimental infections were produced in 78 quarters of 17 cows by the infusion of small numbers of a single strain of Staphylococcus aureus. In each single experiment three quarters in a cow were infected, with the fourth left as a control. At times varying from three to 60 days after the infusion of organisms, a standard intramammary antibiotic treatment was administered on a single occasion. A cure was arbitrarily defined as the absence of the organism in foremilk, from direct plating and replated incubated milk, together with return to normal somatic cell count levels as determined by an electronic counter.

With these standardized conditions the effects of a number of cow associated factors on the outcome of the therapy were determined.

Forty-three of the 78 quarters (55%) were cured by the standard treatment. Significant differences in percentages of quarters cured were found to be associated with the duration of infection before therapy, the lactation age of the cow, the length of time in lactation, somatic cell count in milk at time of treatment, the location of the quarter in the udder and individual cows. No significant effects on the outcome of the standard treatment were found associated with the number of bacteria in the secretion at the time of treatment, previous infection and cure in a quarter nor the season of the year in which treatment was given. Of the 35 quarters in which infection recurred following treatment, organisms were reisolated from 12 within four days, 18 between five and nine days, four between ten and 17 days and one after 28 days.

From these data it is apparent that if, as has been suggested, models such as described are to be used for efficacy trials, standardization of some parameters is essential.

     

A Mycoplasma Isolated from Cattle with Infectious Bovine Keratoconjunctivitis

A mycoplasma has been recovered from the eyes of calves in two naturally-occurring outbreaks of infectious bovine keratoconjunctivitis; also from a third group of calves accidentally exposed to an animal which had ocular exudates from one of the outbreaks instilled into its eyes.

The severity of the ocular lesions in infectious bovine keratoconjunctivis outbreaks may be related to a mixed infection with the mycoplasma and Moraxella bovis.

Preliminary typing studies indicate the mycoplasma is not serologically related to any known bovine mycoplasma.

     

Prototheca zopfii mastitis in a herd of dairy cows

Mastitis caused by the colourless alga Prototheca zopfii was diagnosed in 17 of 120 cows in a dairy herd. Infection occurred in animals varying from 3–14 years old and was present in one to four quarters of each cow.

Nine cases were associated with clinical mastitis characterised by the presence in milk of flakes or small clots. Somatic cell counts consistent with subclinical mastitis (>500 × 10³ cells/ml) were recorded in five of the eight remaining cows.

Histological examination of udder tissue showed the presence of granulomatous lesions associated with the presence of Prototheca.

The problem was identified and controlled by repeated microbiological examination of milk samples from all lactating cows and immediate culling of infected animals.

P. zopfii was also recovered from environmental water samples on this farm. It is suggested that infection may have occurred as a result of teat sores caused by trauma from a milking machine, and the tendency for cows to lay down on a race, the surface of which was sometimes flooded by drain water in which Prototheca were present.     

Infectious Center Assay of Intracellular Virus and Infective Virus Titer for Equine Mononuclear Cells Infected in vivo and in vitro with Equine Herpesviruses

A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturally infected ponies with the virus gave by infectious center assay a mean value of 67 infectious center/2 x 10⁶ cells as a peak titer on day 4 postinfection and 26 infectious center/2 x 10⁶ cells for equine herpesvirus 1 and equine herpesvirus 2 respectively. The mononuclear cells, in both cases, did not contain detectable infectious virus, but the infectious virus was detected from the respective cells when they were cultured in the presence of mitogen. The equine herpesvirus 1 infected mononuclear cells in culture gave a mean count of 8.05 x 10² infectious center/2 x 10⁶ cells/mL and contained 1.08 x 10⁴ plague assay/mL of infectious virus. Similarly the equine herpesvirus 2 infected mononuclear cells in culture gave a mean count of 7.1 x 10¹ infectious center/2 x 10⁶ cells/mL and contained <10¹ tissue culture infective dose₅₀/mL of infectious virus. Mononuclear cells infected in vitro with equine herpesvirus 1 gave a mean count of 9.3 x 10⁴ infectious center/2 x 10⁶ cells/mL and contained 5.75 x 10³ plaque assay/mL of infectius virus. Culturing these cells in the presence of mitogen gave a mean count of 5.5 x 10³ infectious center/2 x 10⁶ cells/mL and contained 9 x 10³ plague assay/mL of infectious virus. A correlation between infectious center assay and infectious virus assay is discussed.     

重组溶葡萄球菌酶对奶牛乳房炎的疗效研究

以85头患乳房炎奶牛共104个乳区为研究对象,随机分为4组,每组26个,分别用重组溶葡萄球菌酶粉低（200U／乳区）、中（400U／乳区）、高（800U／乳区）三个剂量,对照组为青霉素G钠（160万IU／乳区）,每天早晚挤奶后乳池灌注给药,共4d,研究不同剂量重组溶葡萄球菌酶对奶牛乳房炎的治疗效果。试验结果显示,低、中、高剂量重组溶葡萄球菌酶均能有效清除感染乳区的链球菌、葡萄球菌、化脓隐秘杆菌等G^＋菌,大幅降低牛奶中的白细胞数,提高日产奶量。其中低剂量组对隐性乳房炎、临床型乳房炎的有效率和治愈率略优于青霉素G钠（P〉0．05）;中、高剂量对隐性乳房炎、临床型乳房炎的有效率和治愈率显著优于青霉素G钠（P〈0．05）;中、高剂量的疗效相当（P〉0．05）。重组溶葡萄球菌酶是一种很好的治疗奶牛隐性乳房炎和临床乳房炎备选药物。     

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46⁺/CD3⁻) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.     

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.     

The use of polyethylene intramammary device in protection of the lactating bovine udder against experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

The susceptibility of lactating bovine udder quarters fitted with a polyethylene intramammary device to infection was investigated. Following experimental challenge with Streptococcus agalactiae or Staphylococcus aureus, the incidence of infection was significantly (p less than 0.05) lower in intramammary device-fitted quarters compared to control quarters. In general, total foremilk and strippings milk somatic cell counts for intramammary device-fitted and control quarters were not significantly (p less than 0.05) different. Differential foremilk and strippings milk somatic cell counts were significantly (p less than 0.05) higher in samples from intramammary device-fitted quarters compared to control quarters.     

Pathogenicity of two strains of Streptococcus uberis infused into lactating and non-lactating bovine mammary glands

Two strains of Streptococcus uberis, one (0140J) resistant to killing by purified bovine polymorphonuclear leucocytes suspended in milk and the other (EF20) readily killed by polymorphonuclear leucocytes were each infused into a mammary quarter of 18 lactating and 10 pregnant non-lactating cows. In the lactating cows 0140J produced clinical disease in 16 of 18 quarters whereas EF20 produced clinical disease in only two of 18 quarters. With the exception of three cows exposed to EF20, the quarters which resisted infection did so without apparent inflammatory reaction. In non-lactating cows both organisms produced clinical disease in six of 10 quarters. Two cows apart, a non-lactating udder was either resistant or sensitive to both organisms.     

牛疱疹病毒gD-PCR鉴别检测方法的建立

建立一种PCR技术,既能快速检测疱疹病毒1型成员牛传染性鼻气管炎病毒(bovine infectious rhinotracheitisvirus,IBRV),又能区分牛疱疹病毒5型和伪狂犬病毒。根据基因库中牛传染性鼻气管炎病毒的gD基因序列,应用primer 5.0软件设计了gD PCR引物,建立PCR方法,反应条件是:94℃预变性5min,94℃1min,58℃1min,72℃1min,30个循环,72℃7min,4℃5min。该方法能从IBRV阳性样本和参考毒株中扩增出372bp的目的片段,而从同属的牛疱疹病毒5型中扩增出440bp和206bp两条目的片段,从同属的伪狂犬病毒中扩增出303bp的目的片段,但不能从非疱疹病毒属成员猪呼吸与繁殖综合征病毒中扩增出目的条带。该PCR检测IBRV的灵敏度可达1PFU/mL以上。鉴于其灵敏度高、特异性好,可望在牛疱疹病毒感染快速鉴别检测方面发挥重要作用。     

Somatic cell counts in bovine milk

Factors which influence somatic cell counts in bovine milk are reviewed and guidelines for their interpretation are presented. It is suggested that the thresholds of 300 000 and 250 000 cells/mL be used to identify infected quarters and cows respectively. However, it is stressed that somatic cell counts are general indicators of udder health which are subject to the influence of many factors. Therefore the evaluation of several successive counts is preferable to the interpretation of an individual count.

Relationships between somatic cell counts and both milk production and milk composition are discussed. Subclinical mastitis reduces milk quality and decreases yield although the relationship between production loss and somatic cell count requires clarification. Finally the availability of somatic cell counting programs in Canada is presented.

     

一株MDBK细胞的低血清悬浮驯化研究

研究选取一株背景清晰、纯净的MDBK贴壁细胞,通过直接驯化和逐级降低血清相结合的办法,获得了一株可以在低血清培养基中全悬浮培养的MDBK细胞。该细胞在含0.5%胎牛血清的培养基中悬浮培养48 h后的细胞密度稳定在5.0×10⁶/mL以上,培养72 h后细胞密度最高可达1.16×10⁷/mL,且细胞形态良好,活力在93%以上,具有良好的传代稳定性。应用筛选获得的悬浮培养MDBK细胞株分别接种伪狂犬病毒和传染性牛鼻气管炎病毒后,检测病毒敏感性。细胞在24 h均发生了皱缩,72 h大部分死亡。悬浮培养的MDBK细胞对传染性牛鼻气管炎病毒的滴度在24 h达到最大值107.6TCID50/mL,对伪狂犬病毒的滴度在48 h达到最大值107.6 TCID50/mL,说明虽然细胞的培养方式发生了改变,但并没有影响上述两种病毒在该细胞上的增殖特性。对MDBK细胞的全悬浮驯化研究可为相关病毒类疫苗规模化生产及工艺的升级改进提供细胞学基础资料。     

Bovine Mastitis due to Prototheca zopfi II

The occurrence of protothecosis in a dairy herd quarantined under the National Brucellosis Eradication Program is reported. Infection was detected by milk culture and the presence of serum precipitins to a culture filtrate antigen preparation of Prototheca zopfi. The alga was always cultured from the milk when serum precipitins were present. Whey antibodies were demonstrated in infected quarters. Consumption of colostrum from an infected cow may have accounted for the brief appearance of serum precipitins in young calves. A naturally infected cow was monitored for 20 months. Serum antibodies disappeared six months after lactation ended but reappeared following parturition, with both algal cells and antibodies in the colostrum. Prototheca zopfi survived a 13 month dry period. There was no spread of infection to the calf. An experimental infection of a healthy cow was short lived but the presence of both serum and whey antibodies was demonstrated. Cross-reactions between Prototheca and Brucella abortus antigens were not observed, and the association between the diseases was found to be coincidental.     

The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

Somatic cells count in cow's bulk tank milk

The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 × 10(3) ml.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.