Animal industry and veterinary science in eastern New Guinea: World War II to Independence, 1975.

We have described the efforts of the Australian administration of the Territory of Papua and New Guinea (TPNG) to establish a veterinary service and viable animal industries. These efforts began with planning before the end of World War II in 1945 and continued until independence in 1975. Whereas pre-war cattle had mostly been used to control grass on plantations, post-war, the objective was to use the country's extensive, unoccupied grasslands for cattle production. During this period, the cattle population increased from 4000 to more than 150,000. The greatest success was achieved in herds with crosses of Bos indicus and Bos taurus owned by expatriates. The only serious disease constraint on production was myiasis due to Chrysomya bezziana, a parasite throughout New Guinea and South-east Asia. Attempts were made to increase the productivity of the indigenous pig population. Success was limited by a failure to manage inadequate nutrition and internal parasites and to fully understand the cultural aspects of pig ownership. Similar problems inhibited chicken production in villages. The serious viral infections of pigs and birds were absent from TPNG, but a border with Indonesian Papua represents a potential route for the incursion of animal diseases exotic to both Papua New Guinea and to Australia.

Animal industry and veterinary science in eastern New Guinea 1: before World War II.

Until the latter part of the nineteenth century, there were no domestic animals other than pigs, dogs and poultry in the island of New Guinea. From 1889 onwards, occupying authorities, missionaries and settlers from Germany, the UK, Japan and Australia imported ruminants, pigs and horses. Some of these importations were from Asia. This paper describes some outcomes of those importations and the potential hazards for Australia entailed in them.

Comparison of footbathing and vaccination to control ovine footrot in an experimentally infected flock.

OBJECTIVE: Compare footbathing and vaccination for control of footrot during a transmission period in a sheep flock deliberately infected with multiple strains of Dichelobacter nodosus. METHODS: The strains included a known virulent strain, a benign strain and several intermediate strains. The resulting footrot was clinically intermediate. A total of 1450 Polwarth sheep aged 1-3 years were allocated to one of five treatment groups: untreated, weekly walkthrough zinc sulfate footbathing, 1-hour stand-in Footrite® footbathing every 3 weeks, vaccination with a commercial multivalent whole-cell vaccine and vaccination with a novel recombinant DNA fimbrial vaccine. There were four replicates, in four paddocks. RESULTS: Of the untreated animals, 76% had footrot. Footbathing, either weekly or every 3 weeks, restricted the prevalence to 6/283 (2%; 97% effective) and 18/275 (6.5%; 91% effective), respectively. This was significantly lower than the prevalence in either the untreated or vaccinated group (P < 0.001). Weekly footbathing resulted in significantly fewer affected sheep than footbathing for 1 h every 3 weeks (P < 0.05). Vaccination with either whole-cell or recombinant vaccines significantly (P < 0.001) reduced the prevalence ((142/280 (51%; 33% effective), 114/278 (41%; 46% effective) respectively), with the recombinant vaccine superior (P < 0.05) to whole-cell vaccination. Significantly (P < 0.05) fewer 1-year-old sheep had footrot than older sheep. A single Footrite treatment reduced the prevalence to 12% (53/445) compared with a prevalence of 57% (27/47) for untreated sheep (79% effective). CONCLUSION: In this study footbathing was more effective than vaccination at controlling and treating multistrain footrot.

Relationship between the likelihood of footrot elimination from a flock and the virulence of the strain of Dichelobacter nodosus present.

OBJECTIVE: To assess ability to eliminate different strains of footrot in sheep using inspection and culling of affected sheep. METHODS: A flock of 1417 Polwarth sheep that had deliberately been infected with seven different strains of Dichelobacter nodosus and undergone different control measures prior to eradication, including zinc sulfate footbathing and vaccination, were subjected to visual foot inspection on a number of occasions. Sheep identified as infected or having any foot abnormalities were removed from the flock at each inspection. The experiment had three replicates and a small number of untreated control animals. Sheep were examined following the inspections to assess the effectiveness of elimination and additional measures were implemented in two of the three replicates in an attempt to eliminate all strains of D. nodosus. RESULTS: Three strains of D. nodosus were apparently successfully eliminated from all replicates (strains A, E and H). Strains B and C were detected in one replicate each, despite additional inspections. The three stains that were eliminated were the more virulent strains and the two strains that remained were the least virulent. No assessment could be made on a further two strains. CONCLUSIONS: The application of a rigorous inspection and culling program resulted in the elimination of the more virulent D. nodosus strains, but did not result in the elimination of all D. nodosus strains on all occasions.

Hydroacoustics as a tool to examine the effects of Marine Protected Areas and habitat type on marine fish communities.

Hydroacoustic technologies are widely used in fisheries research but few studies have used them to examine the effects of Marine Protected Areas (MPAs). We evaluate the efficacy of hydroacoustics to examine the effects of closure to fishing and habitat type on fish populations in the Cabo Pulmo National Park (CPNP), Mexico, and compare these methods to Underwater Visual Censuses (UVC). Fish density, biomass and size were all significantly higher inside the CPNP (299%, 144% and 52% respectively) than outside in non-MPA control areas. These values were much higher when only accounting for the reefs within the CPNP (4715%, 6970% and 97% respectively) highlighting the importance of both habitat complexity and protection from fishing for fish populations. Acoustic estimates of fish biomass over reef-specific sites did not differ significantly from those estimated using UVC data, although acoustic densities were less due to higher numbers of small fish recorded by UVC. There is thus considerable merit in nesting UVC surveys, also providing species information, within hydroacoustic surveys. This study is a valuable starting point in demonstrating the utility of hydroacoustics to assess the effects of coastal MPAs on fish populations, something that has been underutilised in MPA design, formation and management.

A multiband approach for accurate numerical simulation of frequency dependent ultrasonic wave propagation in the time domain.

Finite element (FE) simulations are popular for studying propagation and scattering of ultrasonic waves in nondestructive evaluation. For a large number of degrees of freedom, time domain FE simulations are much more efficient than the equivalent frequency domain solution. However, unlike frequency domain simulations, time domain simulations are often poor at representing the speed and the attenuation of waves if the material is strongly damping or highly dispersive. Here, the authors demonstrate efficient and accurate representation of propagated and scattered waves, achieved by combining a set of time domain solutions that are obtained for a set of frequency ranges known as bands, such that, in combination, the authors' multiband solution accurately represents the whole wave spectrum. Consequently, high accuracy is achieved, at minor computational cost, using a modest number of bands. The multiband technique is implemented for ultrasonic wave propagation in highly attenuating polyethylene material, using three frequency bands, and can yield a reduction in empirical acoustic properties fractional error compared with respective time domain simulations, in propagation duration, of a factor of 1.4, and in full-width-half-maximum, of a factor of 10. Last, the accuracy of this approach is further exemplified in a wave scattering simulation.

Artificial infection of sheep with multiple strains of Dichelobacter nodosus to induce footrot.

OBJECTIVES: To establish multiple strains of Dichelobacter nodosus in two flocks of sheep and to assess the virulence of five of these strains. METHODS: In experiment 1, sheep were challenged with five D. nodosus strains, which varied in both virulence and serotype. In experiment 2, consisting of four replicates (paddock groups), sheep were challenged with seven different strains of D. nodosus. In experiment 3, sheep were challenged with one of five D. nodosus strains. RESULTS: In experiment 1, at 28 days post challenge, four of the five challenge strains were present. Multiple-strain infections were present in 27 feet, with isolates from three serogroups being recovered from 5 feet, and four serogroups from 1 foot. Challenged hind feet were more frequently affected (P < 0.001). In experiment 2, four of the seven strains were recovered from one replicate and three strains from the remaining three replicates. Significantly more hind feet were affected (262/471, 55.6%) than front feet (198/481, 42%) (P < 0.001). Clinically, in both experiments 1 and 2 the footrot resembled an intermediate form, despite the inclusion of a virulent strain of D. nodosus. In experiment 3, this virulent strain caused a higher prevalence of more severe footrot, a greater mean total foot score and, in Merino sheep, resulted in significantly lower weight gains (P < 0.05). Interaction between D. nodosus strain and breed occurred, with Polwarth sheep being significantly more severely affected by one strain than Merino sheep. DISCUSSION: The clinical expression of multiple-strain infections has implications for both research and control of footrot. A novel method of control is proposed.

Ultrasonic attenuation and phase velocity of high-density polyethylene pipe material.

Knowledge of acoustic properties is crucial for ultrasonic or sonic imaging and signal detection in nondestructive evaluation (NDE), medical imaging, and seismology. Accurately and reliably obtaining these is particularly challenging for the NDE of high-density polyethylene (HDPE), such as is used in many water or gas pipes, because the properties vary greatly with frequency, temperature, direction and spatial location. Therefore the work reported here was undertaken in order to establish a basis for such a multiparameter description. The approach is general but the study specifically addresses HDPE and includes measured data values. Applicable to any such multiparameter acoustic properties dataset is a devised regression method that uses a neural network algorithm. This algorithm includes constraints to respect the Kramers-Kronig causality relationship between speed and attenuation of waves in a viscoelastic medium. These constrained acoustic properties are fully described in a multidimensional parameter space to vary with frequency, depth, temperature, and direction. The resulting uncertainties in acoustic properties dependence on the above variables are better than 4% and 2%, respectively, for attenuation and phase velocity and therefore can prevent major defect imaging errors.

Light-dependent maintenance of hydraulic function in mangrove branches: do xylary chloroplasts play a role in embolism repair?

• To clarify the role of branch photosynthesis in tree functioning, the presence and function of chloroplasts in branch xylem tissue were studied in a diverse range of mangrove species growing in Australia. • The presence of xylary chloroplasts was observed via chlorophyll fluorescence of transverse sections. Paired, attached branches were selected to study the effects of covering branches with aluminium foil on the gas exchange characteristics of leaves and the hydraulic conductivity of branches. • Xylary chloroplasts occurred in all species, but were differently distributed among living cell types in the xylem. Covering stems altered the gas exchange characteristics of leaves, such that water-use efficiency was greater in exposed leaves of covered than of uncovered branches. • Leaf-specific hydraulic conductivity of stems was lower in covered than in uncovered branches, implicating stem photosynthesis in the maintenance of hydraulic function. Given their proximity to xylem vessels, we suggest that xylary chloroplasts may play a role in light-dependent repair of embolized xylem vessels.

Effect on time in quarantine of the choice of program for eradication of footrot from 196 sheep flocks in southern New South Wales.

OBJECTIVE: To identify and compare programs for eradicating virulent footrot (VFR) chosen by owners of quarantined sheep flocks in southern New South Wales. METHOD: Data from 196 sheep flocks in the Wagga Wagga and Young Rural Lands Protection Boards were used to determine the program chosen, the influence of flock size on the program chosen and the effects of the program chosen and the use of contractors on the time in quarantine. RESULTS: The most popular programs in flocks using a single program were: total destocking (61/173; 35.3%) and inspection and culling of affected animals (71/173; 41.0%). Treatment of known infected animals was chosen in 41 flocks and of those, 10 (5.8%) used antibiotics for treatment and 31 (17.9%) used foot-bathing. Combined programs were used in 23 flocks and in 10 flocks a change of program occurred before eradication was achieved. The choice of program was, to some extent, affected by flock size, with owners of small flocks (<500 sheep) more likely to destock. The chosen program strongly influenced the time in quarantine, the shortest time being for destocking (mean 284 days), followed by culling of infected sheep (395 days), treatment with antibiotics (433 days) and finally foot-bathing (502 days). Time in quarantine was significantly shorter when contractors were used. CONCLUSION: All the options chosen led to the eradication of VFR. However, in this sample both the choice of program and the use of contractors influenced the time taken to achieve eradication and therefore the time in quarantine. Based on time in quarantine, foot-bathing was the least desirable option for the eradication of VFR because of the significantly greater time involved, perpetuation of risk to neighbours and increased cost of inspections. These findings were derived from flocks that were quarantined, but they are relevant to all flock owners considering eradication of VFR.

Meningitis, sepsis and endocarditis among workers occupationally exposed to pigs.

BACKGROUND: Workers exposed to pigs can develop meningitis, sepsis or endocarditis due to infection with Streptococcus suis transmitted from pigs to man. AIMS: To estimate the risk of these diseases. METHODS: We used the Occupational Hospitalization Register (OHR) which holds information about occupation and hospital treatments for all adults in Denmark. A dynamic population of male workers exposed to pigs was identified every year from 1995 to 2006 by occupational and industrial groups. First hospital treatment or death in the following year due to meningitis, sepsis or endocarditis was identified by ICD-10 codes from the OHR. By comparison with all other economically active men in Denmark, the standardized incidence ratio (SIR) was calculated for these diseases. RESULTS: Among those exposed, we observed 32 cases of meningitis, sepsis and endocarditis during 140,118 person-years. In the reference group, we observed 2680 cases during 15,209,394 person-years. The SIR of the exposed group was 1.35 (95% CI: 0.95-1.92). Among the 32 cases, 7 cases of meningitis and sepsis were specified as caused by infection with streptococci. The SIR for these seven cases was 2.4 (95% CI: 1.1-5.0). CONCLUSIONS: Our study did not find that workers exposed to pigs had an overall increased risk of developing meningitis, sepsis or endocarditis.

The apparent temperature response of leaf respiration depends on the timescale of measurements: a study of two cold climate species.

Productivity and climate models often use a constant Q10 for plant respiration, assuming tight control of respiration by temperature. We studied the temperature response of leaf respiration of two cold climate species (the Australian tree Eucalyptus pauciflora and the subantarctic megaherb Pringlea antiscorbutica, both measured in a field setting) on a short timescale (minutes) during different times within a diel course, and on a longer timescale, using diel variations in ambient temperature. There were great variations in Q10 depending on measuring day, measuring time and measuring method. When Q10 was calculated from short-term (15 min) manipulations of leaf temperature, the resulting values were usually markedly smaller than when Q10 was calculated from measurements at ambient leaf temperatures spread over a day. While for E. pauciflora, Q10 estimates decreased with rising temperature (corroborating the concept of a temperature-dependent Q10), the opposite was the case for P. antiscorbutica. Clearly, factors other than temperature co-regulate both leaf respiration rates and temperature sensitivity and contribute to diel and seasonal variation of respiration.

Freeze/thaw-induced embolism depends on nadir temperature: the heterogeneous hydration hypothesis.

Freeze/thaw-induced embolism was studied in leaves of field-grown snow gum (Eucalyptus pauciflora) subject to frequent morning frosts. Juvenile trees were grown in buried pots, brought to the laboratory at different stages of acclimation and subjected to simulated frost-freezes (at 2 degrees C h(-1)) to nadir temperatures of -3 or -6 degrees C, which snow gums commonly experience. Frost-frozen and subsequently thawed leaves were cryo-fixed to preserve the distribution of water and were then examined by cryo-scanning electron microscopy. No embolisms were found in leaves frozen to -3 degrees C and thawed. In contrast, 34% of vessels were embolized in thawed leaves that had been frozen to -6 degrees C. This difference was seen also in the extent of extracellular ice blocks in the mid-vein expansion zones in leaves frozen to -3 and -6 degrees C, which occupied 3 and 14% of the mid-vein area, respectively. While the proportion of embolism depended on nadir temperature, it was independent of season (and hence of acclimation state). From the observation that increased embolism at lower nadir temperature was related to the freeze-induced redistribution of water, we hypothesize that the dehydration of cell walls and cells caused by the redistribution exerts sufficient tension on xylem water to induce cavitation on thawing.

Distribution and prevalence of footrot in Bhutan.

The first cases of footrot in Bhutan were reported in sheep in 1990 at the National Sheep Breeding Centre (NSBC), which supplies breeding animals to village sheep flocks throughout Bhutan. Despite the presence of footrot at the Centre the distribution of apparently disease-free sheep continued. Cases of footrot were reported in village flocks soon after the disease was diagnosed at NSBC. A national survey was designed to establish the distribution and prevalence of footrot in Bhutan. This detected footrot in 19/94 village sheep flocks surveyed. The 19 affected flocks were distributed among nine different administrative districts whereas the villages selected were in 13 of a total of 16 sheep growing districts. The highest within-flock prevalences were among the seven flocks sampled in Bumthang district (mean 20.4%). The prevalence of the disease within flocks was generally much lower in other affected districts and in three districts a single affected animal was identified in the sample of 14 sheep examined in each village. Nationally, footrot prevalence was estimated to be 3.1% (95% CI 2.16-4.04%). There was a positive association between the receipt of animals from NSBC and the presence of footrot. The prevalence of the disease was higher in flocks with a migratory system of management than in those using a sedentary system. The relative risk of there being footrot in a migratory flock was nine-times higher than in a non-migratory flock. Only one strain of Dichelobacter nodosus (serogroup B) was identified among the 234 isolates obtained from the 19 affected flocks. Sheep with footrot healed quickly when treated with a vaccine made from this strain.

The use of an autogenous Dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in Bhutan.

An outbreak of virulent footrot was investigated in a flock of 605 Merino cross-bred sheep in Bhutan. Conventional control methods in the preceding eight years had reduced its prevalence from 36-79% in different components of the flock to about 15% overall. Only one serogroup (B) of Dichelobacter nodosus was identified among 40 isolates cultured from affected sheep. A vaccine prepared from this strain was used in a pilot trial to compare the response of 14 treated and 14 untreated sheep. All affected, vaccinated animals in this trial healed quickly and were protected against re-infection while additional cases developed among untreated sheep during a period favourable for the spread of footrot. The serogroup B vaccine was administered to the whole flock for two successive years. No other footrot treatment was given during these or subsequent years. The whole flock was examined three times, foot by foot, for two years and twice yearly for another two years. When vaccination began there were 88 affected sheep in the flock, an affected sheep being defined as an animal with a foot-score of 2 or greater in one or more feet. There were neither affected sheep in the flock 30 days after the first dose of vaccine nor were any identified in later inspections. Virulent footrot, originating from the farm under investigation, persisted in neighbouring village flocks during this period. It was concluded that whole flock specific D. nodosus vaccination made a major contribution to the elimination of all clinical signs of footrot from the flock of 605 sheep where the condition had previously persisted for 10 years.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist.

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Effect of climatic region on the clinical expression of footrot of lesser clinical severity (intermediate footrot) in sheep.

OBJECTIVE: To determine if the clinical classification of intermediate footrot (IFR) is changed to virulent footrot (VFR) by a transfer of the infected flock to a region where climatic conditions are more favourable for the transmission of the disease. DESIGN: Clinical examination of two groups of Merino wethers infected with IFR; one group of 309 in a region considered less favourable for footrot and another group of 343 at a second site considered more favourable. PROCEDURES: After characterising the form of footrot at the first site, infection was established at the second site by mixing 142 wethers from the first site with 201 unrelated wethers considered to be free of IFR and VFR. Observations of clinical characteristics were made over a 16 month period during which an outbreak of footrot occurred. Clinical assessments were made by inspecting every foot of every sheep at regular intervals and allocating a footscore. Evidence that the same clonal lines of D. nodosus were responsible for the footrot at both sites was provided by serotyping of isolates and using omp gene RFLP as a molecular epidemiological tool. RESULTS: The disease at the first site was classified as IFR because 7% of the sheep developed a maximum footscore (MFS) of 4, the most severe category, despite relatively low rates of transmission. When the outbreak occurred at the second site, which was more suitable for footrot transmission, the maximum proportion of the flock that developed a MFS of 4 was 3.6%, confirming the initial classification of IFR. CONCLUSIONS: When a flock infected with IFR was moved to a region where climatic conditions were more favourable for footrot transmission, the clinical classification of the disease remained the same in both the original flock and in sheep exposed to the infection for the first time.

Eradication of footrot of lesser clinical severity (intermediate footrot).

OBJECTIVE: To determine if intermediate footrot (IFR) can be eradicated from a flock of sheep by inspection and culling of cases during a non-transmission period and if prior antibiotic treatment or vaccination increases the likelihood of eradication. PROCEDURE: A replicated field experiment that compared the three eradication strategies was followed by an observational study of the best of these applied in a commercial flock of 3000 sheep. RESULTS: In the replicated experiment, IFR was eradicated either by inspection and culling alone, or when combined with vaccination. Eradication failed when the sheep were treated with parenteral antibiotics before inspection and culling during the non-transmission period. In the whole-flock program, eradication by repeated inspection and culling of footrot cases during the non-transmission period was successful and the flock remained free of infection 3 years later. CONCLUSIONS: IFR can be eradicated by inspection and culling but latent infections, which may persist undetected for at least 34 weeks, require surveillance inspections to be repeated during the non-transmission phase of the program. The use of parenteral antibiotics as an aid to the eradication of IFR is contraindicated.

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination.

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test for D. nodosus. A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.