1株猪鼻支原体的分离鉴定及致病性

猪鼻支原体是猪场感染率极高的一种支原体,可引起多发性浆膜炎、关节炎等症状。目前尚未建立标准的猪鼻支原体发病模型,也缺乏标准强毒株。本研究从某猪场发病猪的关节液中分离得到一株支原体,通过PCR检测、菌落形态观察、菌体形态电镜分析、菌株MLST分型等方法对其进行了鉴定,确认其为猪鼻支原体。为评价菌株致病性,将该分离株接种1月龄自然分娩不吃初乳猪（SF-pCD猪）,试验猪在感染后出现关节肿胀、跛行等临床症状,日增重显著低于对照组,并出现部分死亡。剖检结果显示感染组出现胸膜炎、腹膜炎、心包炎及关节炎,肺部组织病理分析显示为间质增宽,无虾肉样病变。从发病猪的扁桃体、肺、心及关节积液中可再次分离到攻毒株。综上,本研究分离获得一株猪鼻支原体,人工感染猪后可出现典型的发病症状,猪鼻支原体发病模型的建立为致病机制及疫苗研究奠定了重要的基础。     

Serological evidence of Mycoplasma cynos infection in canine infectious respiratory disease

A high proportion of dogs suffer from respiratory disease when they are placed in kennels for vacation or re-homing. The role of Mycoplasma cynos as an initiating agent in canine infectious respiratory disease was investigated by examining the serological response of dogs to this organism at the time of entry into a large re-homing kennel. Forty-two paired serum samples from dogs (21-day interval) were examined for antibody to M. cynos using Western blotting. The development of antibody in the serum was related to clinical disease recorded over the same period. Sixty seven per cent of the dogs showed a two-fold or greater rise in antibody to M. cynos during the first 3 weeks in the kennel. Reactivity with a 45 kDa antigen was dominant. Of those showing a positive serological reaction, 80% had recorded clinical respiratory disease while 20% remained healthy. The findings of this study show that an antibody response to M. cynos is common in dogs entering the re-homing kennel and is positively related to the development of clinical respiratory disease.     

犊牛支原体肺炎继发牛A型多杀性巴氏杆菌感染的诊治

某奶牛场犊牛相继发生肺炎和关节炎,为确诊该牛场犊牛群发病的原因并提出防控方案,本试验剖检新生犊牛并采集病料,分别开展牛支原体及其他病原菌的分离培养、PCR鉴定及药敏分析;进行牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒PCR检测;制作犊牛肺脏组织病理切片并进行观察和评估。从犊牛肺脏组织分离到牛支原体和牛A型多杀性巴氏杆菌;牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒检测均为阴性;肺脏组织病理切片可见肺泡结构破坏、出血及大量炎性细胞浸润;药敏试验结果显示,牛支原体和牛A型多杀性巴氏杆菌分别对泰乐菌素和头孢唑啉敏感,但对青霉素、庆大霉素、林可霉素和氨苄西林均呈现耐药。该犊牛群确诊为牛支原体肺炎继发牛A型多杀性巴氏杆菌感染,采用泰乐菌素联合头孢唑啉肌肉注射,配合对症治疗和规范管理,有效控制了该场犊牛疾病。     

新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份,用牛支原体特异性引物进行PCR检测,将检测为阳性的样本进行病原培养纯化,对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析,通过测定颜色变化单位(CCU)测定分离株生长曲线,并对分离株进行药物敏感性试验。【结果】PCR结果显示,10份鼻拭子中检测出7份牛支原体阳性样本,1份病死牛肺脏组织也检测为阳性;在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落,纯化后分离株菌落形态为典型的煎蛋状;Dienes染色可见明显的深蓝色中心脐;生化试验结果显示,分离株不水解明胶、精氨酸、七叶苷,不发酵乳糖、葡萄糖和甘露醇,不分解尿素,可还原氯化三苯基四氮唑;16S rRNA测序结果显示,分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高,均为99.9%;生长曲线测定结果显示,分离株在培...     

Growth and interaction of Mycoplasma bovirhinis and Mycoplasma dispar in vitro

Two mycoplasmas were grown in pure and mixed cultures in glucose calf-serum broth with initial pH 7.8. Sensitivity to pH was also tested. The main data for Mycoplasma bovirhinis and Mycoplasma dispar, respectively, in pure cultures were as follows: lag phase, days: <1, 1–2; log phase: 1–2, 1–4; relatively stationary phase: 0–1, 2–4; decline phase (to the extent roughly logarithmic): 2–6, 5–10; maximal titers: 5 × 10⁷ to 10⁹, 5 × 10⁶ to 10⁸ color changing units per 0.2 ml; highest pH, approximately, at which decline started: 7.7 and 7.0; definitely toxic initial pH: 5.6, 6.8; relative production of acidity; less, more. Decline either shortly ended in loss of viability or, correlated with higher pH levels, led to a prolonged maintenance in lower numbers. The decline of M. bovirhinis was postulated to be essentially caused by an autotoxic product/products other than H⁺.

In mixed cultures an antagonistic effect due to the faster growing M. bovirhinis against M. dispar was recorded. The effect varied according to the initial numbers of organisms and their ratio. Two mechanisms seemed to be active: 1. decrease of pH somewhat below neutrality led to the death of the sensitive M. dispar; 2. M. dispar, when present in relatively high initial numbers, was inhibited by M. bovirhinis during the latter's logarithmic growth at pH-levels above 7.0, the inhibition ending shortly afterwards. A rapidly inactivated product/products of M. bovirhinis metabolism, inhibitory to M. dispar was posited. The results offer an improved insight into diagnostic practices for M. dispar.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

Mycoplasma hyopneumoniae (M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio (R_n). This R_n-value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates.

Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R_n-values of the highly virulent and the low virulent isolates were estimated to be 1.47 (0.68–5.38) and 0.85 (0.33–3.39), respectively. No significant difference between the groups was found (P = 0.53). The overall R_n was estimated to be 1.16 (0.94–4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae, assessed for the first time by a reproduction ratio, shows that one piglet infected before weaning will infect on average one penmate during the nursery phase.     

Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4–7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5–9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.     

Genomic, protein and antigenic variability of mycoplasma bovis

Restriction endonuclease analysis (REA) with three enzymes SmaI, PstI, BamHI- was used to identify 13 different genomic groups among 37 Mycoplasma bovis strains. One genomic group was comprised of 14 strains. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) patterns for one strain chosen from each genomic group and an international reference strain PG45 were all similar. Antigenic variability in M. bovis species was investigated by immunoblotting, using serum from a calf that had been naturally infected with M. bovis and three M. bovis-specific monoclonal antibodies — mAbs N₂, I₂ and 5D7. Twenty M. Bovis field strains were tested, comprising one from each genomic group, six from the same genomic group and the reference strain. Antigenic profiles obtained with calf serum differed markedly one from the other, the heterogeneity being equally great among the strains belonging to the same genomic group as those coming from different groups. A stable antigen common to 164 out of 168 strains was detected by mAb N₂, whilst with mAbs I₂ and 5D7, two different membrane antigenic systems were demonstrated that were strikingly variable. These variations in expression occurred not only from one strain to another, but also within the same lineage of clones from a single cell.     

Insertion sequence profiling of UK Mycoplasma bovis field isolates

Mycoplasma equigenitalium and Mycoplasma subdolum have been associated with infertility, endometritis, vulvitis and abortions in mares, and with reduced fertility and balanoposthitis in stallions. Despite their role in equine genital disorder, determinants of virulence and pathogenesis as well as factors provoking specific host immune responses have not been identified, so far. To establish the major immunogenic components of Mycoplasma (M.) equigenitalium and M. subdolum, antigen profiles of their type strains as well as 30 clinical isolates were compared by SDS-PAGE and immunoblot analysis using hyperimmune rabbit sera and equine sera from clinical cases. To define the major protein antigens of both mycoplasma species, detergent-phase fractionation with Triton X-114 was performed. Western blot analysis of 30 clinical isolates revealed a high similarity of their overall antigen profile with only slight differences. In contrast, monospecific polyclonal antibodies raised against detergent-phase proteins of the two mycoplasma species identified three prominent proteins (pST17, pST42, and pET45) undergoing variation in expression and size among clinical and clonal isolates.     

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97 kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97 kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.     

Helminths of wild boar in the isolated population close to the northern border of its habitat area

One hundred wild boars (Sus scrofa) from a geographically isolated population on the island Saaremaa of western Estonia were examined for visceral helminths. Seven helminth species, Metastrongylus pudendotectus, M. salmi, M. elongatus, Ascaris suum, Trichuris suis, Dicrocoelium dendriticum and Taenia hydatigena larva, were found. The predominant helminths discovered were lung nematodes (prevalence 82%, mean intensity 96.2 per animal). A significant negative correlation was observed between the weight of wild boars and the number of lungworms and helminth species. The number of helminth species found in the wild boar population on the island was lower compared to that of the adjacent mainland.     

A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds

Sixteen litters of seven pigs from each of nine Danish farrow-to-finish herds were followed to investigate the serological patterns caused by natural infection with Mycoplasma hyopneumoniae, Pasteurella multocida toxin and Actinobacillus pleuropneumoniae serotypes 2, 5–7, 12. In seven of the herds, pigs were followed as two separate cohorts started 4 weeks apart, and in two herds only one cohort was followed.

A total of 999 pigs were included in the study. The pigs were blood sampled at weaning and subsequently every fourth week until slaughter. All pigs were examined for antibodies against M. hyopneumoniae (enzyme-linked immunosorbent assay), P. multocida toxin (enzyme-linked immunosorbent assay) and A. pleuropneumoniae serotypes 2, 5–7, 12 (complement-fixation tests). The most-common pattern (28%) of seroconversion was that of pigs first seroconverting to A. pleuropneumoniae serotype 2, followed by seroconversion to M. hyopneumoniae. Each herd had a dominant serotype of A. pleuropneumoniae to which most pigs seroconverted. Seroconversion to the respiratory pathogens occurred mainly in the growing-to-finishing units (8–24 weeks). The risk of seroconversion to the P. multocida toxin was very low (<20%) and occurred late.

None, four and seven herds tested seropositive to PRRS and to swine influenza virus subtypes H3N2 and H1N1, respectively, when testing 10 pigs per herd (selected randomly among the study pigs) at the age of 20 weeks.     

3种生态型野生扁蓿豆种质资源ISSR与SSR遗传多样性分析

遗传多样性的量化与分类是收集和利用种质资源的重要前提。运用SSR和ISSR技术分析扁蓿豆种质资源的遗传多样性。从供试材料中筛选到具有多态性的SSR引物18对,ISSR引物18条。SSR引物共扩增到109条清晰的多态性条带,多态性比率为80.09%,相似系数变化范围为0.669～0.885。ISSR引物共扩增到125条清晰的多态性条带,多态性比率为87.08%,相似系数变化范围为0.448～0.811。对2种标记结果进行UPGMA聚类分析,结果表明,扁蓿豆材料部分聚为1类,黄花扁蓿豆材料大部分聚为1类。Mantel检测显示,2种标记的遗传相似性呈显著相关(r=0.019 6,t=0.121 2),扁蓿豆比黄花扁蓿豆的遗传多样性水平稍高,细叶扁蓿豆之间的遗传差异较大。     

遮阴对三种豆科牧草光合特性和叶绿素荧光参数的影响

为了探讨豆科牧草在遮阴条件下生态适应性及寻求适度的遮阴比例,系统研究了不同程度的遮阴处理(0%,48%,70%,95%)对3种豆科牧草的光合特性和叶绿素荧光参数的影响。结果表明,随着遮阴强度的提高,白三叶的光补偿点和光饱和点减小,紫花苜蓿与草木樨在适度遮阴条件下可维持较低的光补偿点和光饱和点;3种豆科牧草叶片叶绿素a、叶绿素b和叶绿素a+b的含量逐渐降低,且相同遮阴处理下白三叶均高于其他2种豆科牧草,紫花苜蓿在Y3处理条件下表现为叶绿素a/b值最大;3种豆科牧草PSⅡ受到损伤的程度为紫花苜蓿＞草木樨＞白三叶;遮阴会使白三叶与草木樨的ΦPSⅡ和qP略有降低,但在Y3处理条件下,白三叶的ΦPSⅡ和qP会略有增加,而紫花苜蓿随着遮阴强度的提高,其ΦPSⅡ和qP呈先增加后减小的趋势。3种豆科牧草的耐阴强度顺序为:白三叶＞草木樨＞紫花苜蓿,其中,白三叶在70％以上的遮阴强度下仍然可以长势良好,而草木樨在48％的遮阴强度下有利于生长,紫花苜蓿则受遮阴影响较大。     

铝和酸胁迫对苜蓿根瘤菌生长和抗氧化酶系的影响

在铝和酸胁迫下,比较分析了天蓝苜蓿和紫花苜蓿根瘤菌的生长及SOD(超氧化物歧化酶)、CAT(过氧化氢酶)、POD(过氧化物酶)、及GR(谷胱甘肽还原酶) 等抗氧化酶系响应特点。铝胁迫设5个梯度,即0,25,50,75,100 μmol/L;酸胁迫设4个pH水平,即4,5,6,7。结果表明,铝和酸胁迫下,2种苜蓿根瘤菌的OD_A600均显著降低,生长受到抑制,且天蓝苜蓿根瘤菌的OD_A600显著高于紫花苜蓿根瘤菌。在pH水平为4和5时,紫花苜蓿根瘤菌没有生长。在铝毒胁迫下,天蓝苜蓿根瘤菌的SOD酶活性一定程度减小后缓慢上升,而CAT、POD和GR酶活性随铝浓度增大显著下降;紫花苜蓿根瘤菌的SOD,CAT及GR酶活在低Al胁迫下无显著变化,在高铝胁迫下显著下降。随着pH水平的下降,天蓝苜蓿和紫花苜蓿根瘤菌均表现为SOD、CAT、POD、GR酶活性的显著下降。在不同铝及酸胁迫下,天蓝苜蓿根瘤菌各抗氧化酶活性显著高于紫花苜蓿根瘤菌。综合分析认为天蓝苜蓿根瘤菌较之紫花苜蓿根瘤菌有较强的耐酸、耐铝性。     

黄腐酸对紫花苜蓿根瘤菌内生定殖及幼苗生长的影响

黄腐酸（Fulvic acid,FA）是一种植物生长调节剂,可促进植物生长。为探索FA对根瘤菌生长、菌株内生定殖及幼苗生长的影响,本试验以3株青色荧光蛋白标记根瘤菌株Rhizobium LH3436f（3436f）、Rhizobium GN5f（gn5f）和Ensifer meliloti 12531f（12531f）为材料,筛选出有利于各标记根瘤菌生长的FA浓度,添加到根瘤菌液中并接种于苜蓿幼苗根系,检测根瘤菌结瘤、苜蓿幼苗生长和菌株内生定殖情况。结果表明：添加适宜浓度FA可显著促进标记根瘤菌侵染根系和在苜蓿体内定殖,12531f根瘤菌添加0.02% FA后接种苜蓿,其在根中的内生定殖数量达到单独接种的11.18倍,差异显著（P<0.05）;根瘤菌添加FA接种可明显促进苜蓿结瘤和生长,效果最佳的是3436f添加0.01% FA接种;根瘤菌添加FA接种也使苜蓿叶绿素含量显著提高,gn5f添加0.02% FA时苜蓿叶片叶绿素含量最高,显著高出WCK 111.41%（P<0.05）。因此,FA可促进根瘤菌在苜蓿幼苗内的定殖,二者可协同促进苜蓿幼苗生长。     

Experimental studies of chronic pneumonia of sheep

Strains of Mycoplasma ovipneumoniae and Pasteurella haemolytica isolated from sheep affected with chronic pneumonia were inoculated by endobronchial route to conventionally-reared and SPF (Specific Pathogen-Free) lambs. Changes resembling those of the naturally-occurring disease were produced in most lambs given the organisms in combination and in some given M. ovipneumoniae alone. Similar but less extensive changes were seen in SPF lambs and fewer animals were affected. Different strains of M. ovipneumoniae did not affect the extent of changes produced in SPF lambs. M. ovipneumoniae became established in the lungs of both types of sheep; P. haemolytica did so less readily.

It was concluded that chronic pneumonia may be reproduced in conventional animals by combined inoculation of M. ovipneumoniae and P. haemolytica. Age and status of immunity to mycoplasmas may account for the different responses of conventional and SPF lambs.     

羊蜱蝇携带巴尔通体和斑点热群立克次体PCR检测及遗传关系分析

为了解四川省石渠县羊蜱蝇巴尔通体和斑点热群立克次体感染情况,采集藏绵羊体表羊蜱蝇,经形态学鉴定后,提取羊蜱蝇总DNA,PCR扩增巴尔通体gltA和rpoB基因、斑点热群立克次体OmpA和OmpB基因,对阳性产物测序、比对及构建进化树,从而确定羊蜱蝇感染巴尔通体和斑点热群立克次体的种类及感染率。在石渠县的4个乡镇总计采集到407只羊蜱蝇成虫,4个乡镇均检出了Bartonella melophagi,总感染率为14.0%（57/407）。在阿日扎镇、呷衣乡和长沙贡马乡检出了Rickettsia raoultii,总感染率为11.1%（45/407）,且长沙贡马乡感染率显著高于阿日扎镇和呷衣乡（P<0.05）;在新荣乡检出了Rickettsia sp.,感染率为6.6%（8/121）。本次试验中,未发现混合感染。石渠县藏绵羊源羊蜱蝇携带巴尔通体和立克次体,首次在石渠县藏绵羊源羊蜱蝇中检出了B.melophagi、R.raoultii和Rickettsia sp.。     

Management-related risk factors for M. paratuberculosis infection in Michigan, USA, dairy herds

A cross-sectional study was conducted from June through December 1996 to identify management-related risk factors for herd-level M. paratuberculosis infection. Data were collected from 121 participating herds. A two-part questionnaire was administered to gather data on current and previous management practices and herd productivity. A random sample of cows aged ≥24 months was selected from each herd and tested for antibodies to M. paratuberculosis using the IDEXX Antibody ELISA (sensitivity 64%, specificity 96%). A positive herd was one in which ≥2 animals tested positive for antibodies to M. paratuberculosis. A negative herd was one in which no animal tested positive. Herds in which only one animal tested positive were dropped from statistical analysis to reduce the risk of including false-positive herds in the statistical analyses.

There were 80 herds with one or more positive animals and 41 herds with no positive animals in the sample (66% herd-level prevalence). Twenty-six herds (21%) were dropped from further analyses because they had only one positive cow. Twelve herds (10%) were dropped from analysis because of missing data. The resulting sample used for statistical modeling included 46 positive herds and 37 negative herds (55% herd-level prevalence). A multi-variable logistic-regression model was used to evaluate the results. The variable ‘use of an exercise lot for lactating cows' was associated with a three-fold increase in odds of a herd being positive for M. paratuberculosis infection (O.R.=3.01, C.I.=1.03–8.80); ‘cleaning of maternity pens after each use' was associated with a three-fold reduction in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.28, C.I.=0.08–0.89); ‘application of lime to pasture areas in 1993' resulted in a ten-fold decrease in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.10, C.I.=0.02–0.56).