Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection.

Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1ß, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno-histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post-seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II+ cells in heart, pancreas and pyloric caeca, IgM+ cells in heart and pancreas, and CD3+ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD-affected tissues were MHC II+ and CD3+ cells suggesting that antigen-presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.

Immune gene profiles in Atlantic salmon (salmo salar L.) post-smolts infected with SAV3 by bath-challenge show a delayed response and lower levels of gene transcription compared to injected fish.

Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post-smolts were infected by either i.m.-injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.-injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT-qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN-stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.-injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time-points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m-injected fish at later time-points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups.

Atlantic salmon (Salmo salar L.) post-smolts challenged two or nine weeks after seawater-transfer show differences in their susceptibility to salmonid alphavirus subtype 3 (SAV3).

BACKGROUND: Pancreas disease (PD), caused by salmonid alphavirus (SAV), is an important disease affecting salmonid aquaculture. It has been speculated that Atlantic salmon post-smolts are more prone to infections in the first few weeks following seawater- transfer. After this period of seawater acclimatization, the post-smolts are more robust and better able to resist infection by pathogens. Here we describe how we established a bath immersion (BI) model for SAV subtype 3 (SAV3) in seawater. We also report how this challenge model was used to study the susceptibility of post-smolts to SAV3 infection in two groups of post-smolts two weeks or nine weeks after seawater - transfer. METHODS: Post-smolts, two weeks (Phase-A) or nine weeks (Phase-B) after seawater- transfer, were infected with SAV3 by BI or intramuscular injection (IM) to evaluate their susceptibility to infection. A RT-qPCR assay targeting the non-structural protein (nsP1) gene was performed to detect SAV3-RNA in blood, heart tissue and electropositive-filtered tank-water. Histopathological changes were examined by light microscope, and the presence of SAV3 antigen in pancreas tissue was confirmed using immuno-histochemistry. RESULTS: Virus shedding from the Phase-B fish injected with SAV3 (IM Phase-B) was markedly lower than that from IM Phase-A fish. A lower percentage of viraemia in Phase-B fish compared with Phase-A fish was also observed. Viral RNA in hearts from IM Phase-A fish was higher than in IM Phase-B fish at all sampling points (p < 0.05) and a similar trend was also seen in the BI groups. Necrosis of exocrine pancreatic cells was observed in all infected groups. Extensive histopathological changes were found in Phase-A fish whereas milder PD-related histopathological lesions were seen in Phase-B fish. The presence of SAV3 in pancreas tissue from all infected groups was also confirmed by immuno-histochemical staining. CONCLUSION: Our results suggest that post-smolts are more susceptible to SAV3 infection two weeks after seawater-transfer than nine weeks after transfer. In addition, the BI challenge model described here offers an alternative SAV3 infection model when better control of the time-of-infection is essential for studying basic immunological mechanisms and disease progression.

The effects of low and high glycemic index 24-h recovery diets on cycling time trial performance.

AIM: The present study investigated the effects of high and low glycemic index (GI) 24 h recovery meals on the physiological responses and subsequent athletic performance, following a glycogen depleting protocol. METHODS: Ten well trained cyclists (age, 33.6±7.4y, height, 175.3±7.6 cm, weight 74.5±8.2 kg, and VO(2max), 60.5±6.0 ml∙kg(-1)∙min(-1)) participated in two trials in a randomized cross- over design. On day 1, subjects performed a glycogen depleting protocol after which they then consumed either high or low GI recovery diets over the next 24 h, which provided 8 g.kgBW(-1) of carbohydrate. On day 2, the subjects returned to the laboratory, 2- 3 h postprandial, to perform a 40 km time trial (TT) on the Velotron cyclePro© ergometer. RESULTS: No difference was observed in TT performance times between the high GI (93. 5±9.29 min) trial and the low GI (90.7±11.1 min) trial (t=1.1; P=0.35). Additionally, no differences in carbohydrate (F=1.1, P=0.37) fat (F=1.1, P=0.40) oxidation or blood glucose concentration (F=0.9, P=0.5) was observed. DISCUSSION: The results of the present study suggest that the ingestion of a high GI carbohydrate 24 h recovery diet following glycogen depleting exercise, has no greater effect on endurance performance than consuming a low GI carbohydrate 24 h recovery diet. It may be concluded from these results that, provided enough carbohydrate is consumed during a 24 h recovery period, there is no difference in subsequent endurance performance.

Laser resonance ionization mass spectrometry.

The analysis of inorganic atomic species is greatly facilitated by the coupling of lasers with mass spectrometers. A tunable dye laser, alone or in combination with a pump laser, ionizes atoms by resonant excitation processes; the ions are then analyzed in the mass spectrometer. The laser-mass spectrometer system promises to overcome traditional limits of sensitivity and selectivity and to have diverse applications in analytical chemistry.

CD4 homologues in Atlantic salmon.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.

Admissions processes at the seven United Kingdom veterinary schools: a review.

The major challenge in veterinary undergraduate admissions is to select those students with most suitability for veterinary training and careers from a large and diverse pool of applicants with very high academic ability. This paper describes a review of the admissions processes of the seven veterinary schools in the UK. There was significant commonality in the entry requirements and the criteria upon which the schools made decisions on candidates. There was some variation in the procedures used by individual schools to select candidates, but common themes existed within these processes. All of the schools evaluated both academic and non-academic factors for individual applicants, and all used interviews in some format as a selection tool after an initial short-listing process. The procedures and approaches to selection processes are compared and discussed.

Looking after the individual to reduce disease in the flock: a binomial mixed effects model investigating the impact of individual sheep management of footrot and interdigital dermatitis in a prospective longitudinal study on one farm.

A total of 160 ewes on one farm in England were studied for 18 months. The incidence of footrot and interdigital dermatitis in individually identified sheep and treatment and flock control measures were recorded. A binomial mixed effect model with the incidence of footrot or interdigital dermatitis as the outcome was used to investigate patterns of association between treatments, flock control measures and the incidence of footrot or interdigital dermatitis. In this one flock, the incidence of footrot and interdigital dermatitis was positively associated with the incidence of footrot and interdigital dermatitis and with trimming of feet and negatively associated with the use of parenteral antibiotics and topical antibiotic sprays in either the first and/or second 2-week period prior to the incidence of interest. These results provide two hypotheses: one that 'footrot and interdigital dermatitis are infectious diseases that can be controlled, in part, through the use of antibiotic therapy, which acts to reduce the infectious period of diseased sheep' and two, that 'routine trimming of diseased and healthy feet exacerbate disease, through environmental contamination and/or through increased susceptibility of sheep with recently trimmed feet'.

Characterisation of salmon and trout CD8alpha and CD8beta.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

A novel association of neuropilin-1 and MUC1 in pancreatic ductal adenocarcinoma: role in induction of VEGF signaling and angiogenesis.

We report that Mucin1 (MUC1), a transmembrane glycoprotein that is overexpressed in >80% of pancreatic ductal adenocarcinoma (PDA), induced a pro-angiogenic tumor microenvironment by increasing the levels of neuropilin-1 (NRP1, a co-receptor of vascular endothelial growth factor (VEGF)) and its ligand VEGF. Expression of tumor-associated MUC1 (tMUC1) positively correlated with NRP1 levels in human and mouse PDA. Further, tMUC1hi PDA cells secreted high levels of VEGF and expressed high levels of VEGF receptor 2 (VEGFR2) and its phosphorylated forms as compared with tMUC1low/null PDA. This enabled the tMUC1hi/NRP1hi PDA cells to (a) induce endothelial cell tube formation, (b) generate long ectopic blood vessels and (c) enhance distant metastasis in a zebrafish xenograft model. Concurrently, the proteins associated with epithelial-to-mesenchymal transition, N-cadherin and Vimentin, were highly induced in these tMUC1/NRP1hi PDA cells. Hence, blocking signaling via the NRP1-VEGF axis significantly reduced tube formation, new vessel generation and metastasis induced by tMUC1hi PDA cells. Finally, we show that blocking the interaction between VEGF165 and NRP1 with a NRP1 antagonist significantly reduced VEGFR signaling and PDA tumor growth in vivo. Taken together, our data suggest a novel molecular mechanism by which tMUC1 may modulate NRP1-dependent VEGFR signaling in PDA cells.

The occurrence of treponemes in contagious ovine digital dermatitis and the characterisation of associated Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is a recently recorded, apparently new infection of the ovine hoof, which differs clinically from footrot caused by Dichelobacter nodosus and which fails to respond well to accepted treatment practices for footrot. Despite the welfare implications of such an infection, very little research has been performed on CODD to date and the aetiology remains confused. Suggestions have been made that there is a potential role for treponemes in the pathogenesis of CODD but that D. nodosus is apparently not involved. Six farms were therefore targeted in this study to provide a more in-depth investigation into the bacterial flora of CODD lesions. Dark ground microscopy, culture and PCR techniques were used, concentrating on the presence of D. nodosus and spirochaetes, particularly those of the genus Treponema. The results demonstrated that isolates of D. nodosus were indeed present in a high percentage (74%) of CODD lesions compared with 31% of apparently healthy feet. The isolates were shown to be of similar virulence type to those reported previously in cases of footrot, and the range of serogroups was also found to be similar to footrot, with serogroup H being prevalent. Treponemes were present in 70% of CODD lesions and 38% of apparently healthy feet, supporting a possible association between CODD and treponemes. However, any further progress on the aetiology of CODD and the potential for novel, effective treatment will depend on an improved ability to culture these organisms routinely in the laboratory thereby enabling their complete characterisation.

The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales.

Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.

Footrot and interdigital dermatitis in sheep: farmers' practices, opinions and attitudes.

In 1999, a study was initiated to improve the treatment and control of footrot and interdigital dermatitis in sheep flocks in England and Wales. In November 2000, a retrospective cross-sectional study was conducted in which 392 sheep farmers were asked to estimate the prevalence of footrot and interdigital dermatitis in their flock in the previous 12 months, whether they considered these diseases to be a problem, how they treated and controlled them and their opinion on the success of the treatment and control measures that they used; 209 of them provided usable responses. The farmers tended to be more concerned as the prevalence of the diseases increased; 91 per cent of the farmers with a prevalence of footrot of less than 5 per cent considered it a small or very small problem on their farm, but 51 per cent of the farmers with a prevalence of 5 per cent or more also considered it to be a small or very small problem. Approximately 60 per cent of the farmers who used parenteral antibiotics considered that they were good or excellent at treating footrot, and this treatment was associated with a prevalence of less than 5 per cent. A similar proportion of farmers also considered topical foot sprays and footbathing to be good or excellent for controlling footrot or interdigital dermatitis, but these treatments were not associated with a lower prevalence of footrot or interdigital dermatitis. Of the 29 farmers who used a footrot vaccine, 20 (69 per cent) considered it good to excellent and this was associated with a prevalence of footrot of less than 5 per cent in their flock; however, vaccination was not associated with lower levels of footrot across the whole sample. Farmers spent approximately 34 minutes per sheep per year treating and controlling footrot; 31 per cent were prepared to spend more money and more time to manage footrot, 27 per cent were prepared to spend more time, 19 per cent were prepared to spend more money and 23 per cent were not prepared to do either. The farmers who were willing to invest more money or more money and time had a higher prevalence of footrot.

The prevalence of posttraumatic stress disorder and its clinical significance among Southeast Asian refugees.

All 322 patients at a psychiatric clinic for Indochinese refugees were surveyed to determine the presence of posttraumatic stress disorder (PTSD). If PTSD was not diagnosed at the time of initial evaluation, a structured reinterview was performed. Seventy percent of the patients (N = 226) met the criteria for a current diagnosis of PTSD, and an additional 5% (N = 15) met the criteria for a past diagnosis. The Mein had the highest rate of PTSD (93%) and the Vietnamese the lowest (54%). Of the patients with PTSD who were enrolled in the clinic before March 1988, 46% (N = 87) were given a diagnosis of PTSD only after the reinterview. PTSD is a common disorder among Indochinese refugees, but the diagnosis is often difficult to make.

Reduction of HSV-1 binding to BHK cells after treatment with phosphatidylinositol-specific phospholipase C.

Baby-hamster kidney cells were treated with unspecific and phosphatidylinositol-specific phospholipase C (PI-PLC) prior to infection with herpes simplex virus type 1 or 2. Subsequent to PI-PLC treatment, a 30% reduction of infectivity and receptor binding was observed for type 1 virus, while type 2 was unaffected. Treating the cells with unspecific phospholipase C did not affect subsequent infection with either virus. Treatment of the virus particles with the unspecific phospholipase reduced its infectivity, probably due to loss of the viral envelope. PI-PLC treatment of virus particles did not have any such effect on virus infectivity.

Tooth allografts fail to stimulate immunologic memory in mice.

Tooth transplants should not preclude future transplants in the recipient. In this study teeth failed to stimulate immunologic memory in weakly disparate mice. The teeth did carry transplantation antigens and were rejected after second-set skin grafts. The teeth probably enhanced their own survival through some immunoregulatory mechanism.

Treating psychiatric disorders among Mien refugees from highland Laos.

The Mien, a Southeast Asian hill people, have immigrated to various countries throughout the world since the mid-1970s. They have brought their ancient culture with them, including beliefs and practices related to health, illness, and healing. During the last several decades they have suffered much war-related trauma, including extensive human, material and symbolic losses. This report describes our clinical experience with Mien refugees in the Indochinese Psychiatric Program of the Oregon Health Sciences University. We discuss symptom presentation among this group of patients, diagnostic and treatment issues, and the impact of cultural health beliefs upon illness and treatment. Major depression and post-traumatic stress disorder have been the most commonly encountered psychiatric diagnoses, usually revealed through somatic symptoms. Not only must clinicians take careful medical and psychiatric histories, but they must also be alert to the high probability of an extensive history of trauma. Clinical improvement in response to psychotropic medications has been limited. As a result, psychosocial and psychotherapeutic approaches to treatment have been developed and expanded and now are commonly employed, effectively combining support and education in the creation of a holding environment that includes both individual and group formats. Two case histories are presented which describe symptom presentation, health belief systems and therapeutic issues involved in treating Mien patients. They also illustrate that traditional and Western healing approaches can co-exist in the optimal care of these patients.

A unified serotyping scheme for Moraxella bovis.

Fifty-three Australian, seven British, two American and two New Zealand isolates of Moraxella bovis were classified into seven serogroups on the basis of their variable fimbrial (pilus) antigens using whole cell slide agglutination (SA), enzyme-linked immunosorbent assays (ELISA) and tandem-crossed immunoelectrophoresis (TCIE). Although results of serogroup classification by SA and ELISA were identical in 68.7% of isolates, it was found necessary to resolve the discrepancies between the two systems using TCIE. Results suggest that world-wide variation in the potentially host-protective fimbrial antigens of M. bovis may be relatively limited. It is proposed that the previous numerical classifications of British and Australian serogroups are appropriately amalgamated as a result of this latest study and are designated as serogroups A to G inclusive. A protocol for the further serotyping of fresh, fimbriate isolates of M. bovis is suggested.