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Item Bovine pestivirus disease : an investigation of a severe outbreak of bovine viral diarrhoea virus infection in calves in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Infectious Diseases at Massey University, Palmerston North, New Zealand(Massey University, 2000) Anderson, Peter DAn outbreak of bovine pestivirus disease, in which there was high mortality (37%) in 102 calves, was investigated. It was postulated that the severity of the outbreak may have been due to the presence of a highly virulent strain of bovine viral diarrhoea virus. Nine calves from the field outbreak were transported to Massey University for detailed clinical, post-mortem and laboratory examination. Samples were also submitted from a further three animals on the farm. The results of immunological and virological studies indicated that seven calves had acute bovine viral diarrhoea virus infection and five calves had mucosal disease. Although the mucosal disease cases showed more severe clinical signs, lesions were widespread in both groups. A non-cytopathic bovine viral diarrhoea virus isolate from one calf was used as the challenge virus in a transmission experiment designed to investigate the pathogenicity of this strain. The 11 calves used in this experiment comprised of four unvaccinated, challenged calves, four vaccinated calves (two challenged, two in-contact), two unvaccinated, in-contact calves and a control (neither vaccinated nor in-contact). The experiment took place over a month, allowing multiple clinical examinations and sampling procedures to be carried out before necropsy. The challenge virus caused mild disease, with lesions similar to those reported in experiments in which Type 1 bovine viral diarrhoea virus isolates were used. Following experimental challenge, virus was not recovered from the calves, but a serological diagnosis of bovine viral diarrhoea virus infection was made by demonstrating a greater than fourfold rise in titre of bovine viral diarrhoea virus antibody in all challenged calves. There were only minor changes in haematological indices in challenged calves. The six challenged calves showed two distinctive lesions in intestinal sections. These were crypt necrosis (of glands of Lieburkuhn) and cryptal prolapse (herniation of crypts into the submucosal site of Peyer's patches depleted of lymphocytes). In the disease outbreak, these lesions were only observed in the mucosal disease cases. Focal haemorrhages at sites of lymphocytic nodules were found in the nasal cavity of all challenged and vaccinated calves in the transmission experiment, but not in the unvaccinated, in-contact calves or the control calf. These lesions have not been reported in natural infections. Vaccination was only partially protective, and there was evidence of spread of bovine viral diarrhoea virus infection to one vaccinated, in-contact calf. Scoring of histological lesions allowed a measurement of the effect of vaccination. There was a 60% reduction in the total histological lesion score in the four vaccinated calves (two challenged, two in-contact) when compared with the four unvaccinated, challenged animals. It was concluded that the high mortality seen in the calves in the field outbreak was due to mucosal disease, and that this was consequential to a high infection rate in the dams during pregancy at a time when the foetuses were at risk of becoming persistently infected (45-125 days of gestation). The pathological "fingerprint" for bovine viral diarrhoea virus infection was found to be the concomitant finding of three lesions at necropsy. Firstly, erosive lesions in the squamous epithelium of the upper alimentary tract. Secondly, catarrhal enteritis, with the distinctive and characteristic microscopic lesions of crypt necrosis and cryptal prolapse. Thirdly, lymphoid tissue lesions, especially lymph node enlargement, lymphoid depletion and inflammation of Peyer's patches. Despite the difficulties in pathotyping the challenge virus in the transmission experiment, there was little evidence that it was a Type 2 strain. Genetic typing of this virus, by sequencing of polymerase chain reaction products, would be useful in determining its place in the phylogeny of bovine pestiviruses.Item Epidemiology of coccidiosis in calves and control of coccidiosis using toltazuril at the time of weaning : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Parasitology at Massey University, Palmerston North, New Zealand(Massey University, 2005) Gaddam, Mary JonesTwo separate studies were conducted to investigate the impact of coccidiosis in young calves. In one study calves were reared to weaning (100kg liveweight) by feeding meal with or without monensin added. The oocyst counts were low in both groups up to weaning and there was no statistically significant (p<0.05) improvement in terms of body weight or a decline in oocyst counts in the monensin-treated group At weaning a single dose of toltrazuril (20mg/kg) was given to half the calves in both groups. A similar treatment regime was given in a second study where calves had been raised to weaning by commercial calf rearers. Half of these were treated with toltrazuril (20mg/kg) and half not. In both studies there was a statistically significant (p<0.001) reduction in oocyst counts in treated calves which remained very low for 4-5 weeks post treatment. The treatment also significantly increased (p<0.001) weight gains in treated calves by 3-5kgs at 5-6 weeks post treatment. The coccidial status of other calves on a variety of farms were also monitored including a group of organic beef farms. High oocyst counts were noted on occasions where calves were not on anti-coccidial treatment. Low oocyst counts were noted in adult cows where they were examined. The two most prevalent species overall were Eimeria zuernii (95%) and E. bovis (87%) followed by E. auburnensis (62%), E. cylindrica (42%), E. canadensis (31%), E. wyomingensis (23%), E bukidnonensis (36%), E. ellipsoidalis (24%) E. alabamensis (12%), E. brasiliensis (12%), and E. subspherica (27%). The most predominant species, measured as the most numerous oocysts overall, were E. bovis (31%) followed by E. zuernii (27%), E. auburnensis (13%), E. bukidnonensis (7%), E. cylindrica (6%), E. wyomingensis (5.3%), E. canadensis (4.4%), E. ellipsoidalis (3.3%), E. brasiliensis (1.9%), E. subspherica (1.5%), and E. alabamensis (1%). The most prevalent species were also the most pathogenic species. On many occasions calves were infected with more than one species, sometimes as many as 5-6 Eimeria species. A redescription of the 11 species of Eimeria in cattle identified from New Zealand Farms was made.Item Diagnosis of bovine venereal campylobacteriosis in New Zealand : a thesis presented in partial fulfilment of the requirements for the Master of Applied Science at Massey University, Palmerston North, New Zealand(Massey University, 2005) Sansone, Natalia BenquetBovine venereal campylobacteriosis (BVC) is a venereal disease causing infertility in cows due to early embryonic loss. Caused by Campylobacter fetus subsp venerealis (Cfv), which lives in the preputial crypts of older asymptomatic carrier bulls and the vagina of carrier cows. Diagnosis of the disease is difficult as it is a fastidious organism to culture and the serological methods which have been developed to diagnose BVC, exhibit substantial cross-reactivity with Campylobacter fetus subsp fetus (Cff), due to the close genomic and phenotypic relationship of the two subspecies. Definitive differentiation between Cfv and Cff requires molecular biology techniques. In New Zealand, Cfv was last isolated in 1993 and suspected again in 2001/2002 after the use of a recently-developed IgA ELISA test that claimed to be 98.5% specific for Cfv. A nation wide study showed that there was no relationship between the test's results and the reproductive performance of the herds. Most of the positive results obtained were false probably due to cross-reactions with Cff The lack of positive isolations of Cfv raised questions about the sensitivity of the isolation methods used. Five experiments were undertaken. Experiments 1-3 examined the microbiological methods that are used to isolated Cfv. Results indicated that the optimal samples and culture conditions were preputial washes or scrapes from bulls, or vaginal washes (20 ml PBS) from cows, enriched in Lander's medium for three days before subculture onto blood agar. All cultures need to be undertaken under microaerophilic conditions. These experiments also addressed the reliability of the IgA ELISA for animals managed according to the husbandry conditions that prevail in New Zealand. Results showed that there was a great deal of within- and between- animal variation in ELISA values and that there was substantial cross reactivity with Cff. (ICC ranged from 0.29 to 0.03 depending on the Group and all heifers challenged with Cff became positive to IgA ELISA). In consequence, the test appears to be of limited diagnostic value in situations where cattle may be cross contaminated with Cff. Experiment 4 evaluated a PCR method for identification of Cfv in preputial washings that had been inoculated with small numbers of the organism, whilst Experiment 5 used the same method to attempt to identify Cfv in preputial washings or scrapings that had been collected from experimentally-infected bulls. Positive results were obtained with as little as 880 Cfv cells/ml of preputial wash after samples had been concentrated by centrifugation and filtration (5 μm and 0.8μm pore size filters). The PCR also detected the presence of Cfv in two bulls, 48 h after infection. It was concluded that the IgA ELISA test is unlikely to be suitable for use in New Zealand, due to the risk of cows being contaminated with Cff from the sheep with which they are co-managed. Microbiological identification, whether through PCR or culture and isolate, although difficult, remains the definitive diagnostic method. If the presence of the disease in New Zealand is to be confirmed or refuted, careful and methodical collection of samples from animals, whose history suggests the possibility of the condition, will be required.Item Epidemiological aspects of infectious bovine keratoconjunctivitis in New Zealand : this thesis is presented in partial fulfilment of the requirements of the degree of Master of Veterinary Science at Massey University(Massey University, 1982) Sinclair, Jane AnnInfectious bovine keratoconjunctivitis (IBK) has been recognised in New Zealand only in the past 7 years and there is little information available concerning epidemiological features of the disease under local conditions. There was an urgent need for rational control measures to be formulated and for a preliminary investigation which might indicate particular aspects of the disease deserving more detailed study. The first of two projects was a postal survey involving 400 beef farmers in the Hunterville and Gisborne districts. A questionnaire was sent out to determine management practices which might influence the occurrence of IBK and to obtain an impression of the impact of the disease on the farming industry. The survey results (72% response rate) showed that IBK is widespread in both areas, appears to have been noticed only within recent years and the annual incidence is still rising (1% in 1975 to 28% in 1980 in the Gisborne area). Outbreaks follow a seasonal pattern with most occurring in late summer. All age groups of cattle may be affected, but morbidity rates differ (young stock 18%; adults 10%). A greater susceptibility of the Hereford.breed to the disease was observed only in the Gisborne district. Difficulties with stock management and lower sale prices were two major consequences of IBK reported and concern was expressed over control measures. Only 11% of farmers routinely treated cattle and yet they reported that treatment improved the recovery rate. The other project involved a study of haemolytic strains of Moraxella bovis isolated from cattle eyes and the relationship of infection rates to clinical IBK cases. Three local beef farming properties were chosen because of their previous history of IBK and over a 1 year period identified animals were observed for clinical signs and conjunctival sac samples were collected for culture. Moraxella bovis was identified by its β-haemolysis, colony and organism morphology, and Gram negative staining characteristics, and confirmed biochemically using five tests including alkaline peptonization of litmus milk. Only one farm experienced an outbreak of IBK. The latter property operated a feedlot system, and early results implicated "carrier animals" in the initiation of a new outbreak. The prevalence of M.bovis isolations increased approximately one month before any clinical signs were apparent and infections remained at a higher level throughout the outbreak. A large number of animals which became infecte never developed signs of the disease. (Peak figures in one pen 87% infected and 55% IBK). Seventy-eight percent of those cattle were infected with M.bovis in both eyes at some time, whereas only 23% of the cases of IBK were bilateral. Moraxella bovis infections decreased slowly after the peak, but many cattle remained infected for at least 5 months. The widespread nature of M.bovis infections indicated the desirability for treatment of both eyes of all animals in a group if control of an IBK outbreak is to be attempted, but such treatment is sometimes impracticable. More detailed investigation is needed into the local factors that are responsible for converting a latent infection of M.bovis into IBK.Item Specificity of a hand-held immunochromatographic assay for anthrax in cattle : a thesis presented in partial fulfilment (50%) of the requirements for the degree of Master of Veterinary Studies in Infectious Diseases at Massey University, Turitea Campus, Palmerston North, New Zealand(Massey University, 2002) Muller, JanineThe purpose of this study was to determine whether the hand-held immunochromatographic assay was a reliable method for field based diagnosis of anthrax. This study was designed and conducted with an emphasis on obtaining an estimate of performance accuracy, in terms of specificity for the protective antigen (PA) component of the anthrax toxin. To examine specificity the hand-held assay had to be assessed under similar circumstances to that experienced in the field from typical animals in which anthrax may be suspected. To achieve this, blood samples were collected post-mortem from 240 cattle at the Stanhope and Camperdown knackeries in Victoria. Blood smears were prepared, hand-held assays were performed on-site and a sample of blood transported back to the laboratory for bacterial culture. All 240 samples gave negative results in the hand-held assay and B. anthracis was not detected in any sample by culture or blood smear, which were considered the definitive diagnostic tests. Thus the hand-held assay was regarded as 100% specific (98.5-100%; 95% CI) for these cattle examined in Victoria. The purpose of the second study was to determine whether the live Sterne strain 34F2 vaccine for anthrax would result in false positives arising in the hand-held assay in cattle recently vaccinated. Ten cattle were vaccinated with the 34F2 vaccine and monitored for 15 days. No PA was detected in the blood of vaccinated cattle in the hand-held assay or on culture within this time. These results show that the hand-held assay does not give false positive test results in cattle post-vaccination with live 34F2 B. anthracis vaccine. The hand-held assay can be used with confidence on samples from recently vaccinated cattle that have died when it is necessary to know whether they had succumbed to anthrax or not. The hand-held assay has the potential to be adopted as a routine test for the preliminary assessment of sudden death in cattle. The simplicity of the assay enables it to be used by unskilled lay people, which means that it could be used by knackery workers for surveillance in areas with a previous history of disease or by veterinarians as a preliminary routine tool in investigating sudden death in cattle. However the study described in this thesis only assesses the specificity of the assay and a further study, involving a similar number of cattle affected with anthrax, needs to be conducted to assess the sensitivity of the assay.Item Serological diagnosis of Brucella infection :a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University, New Zealand(Massey University, 1978) Timbs, Derek VernonThe automated complement fixation test (CFT) and the brucellosis card test (BCT) have been widely used as official tests in the New Zealand Bovine Brucellosis Eradication Scheme. During the course of the eradication programme it was observed that a significant proportion of cattle reacted to the BCT yet remained negative to the CFT and this often occurred on more than one occasion for any particular animal. Twenty cows, from reactor herds, that had been BCT+/CFT- on at least three successive occasions were slaughtered. Despite extensive sampling, attempts at isolating Brucella abortus organisms from tissues of these animals were unsuccessful. Serum from one cow was found to be positive to a wide range of serological tests and it also caused a strong prozone reaction in the CFT, which could easily have been overlooked. The possibility that the automated CFT, which is essentially a one dilution test, was unable to detect such prozoning sera was investigated. It was shown that providing a suitable choice of antigen concentration was made, such sera would be detected by the automated test. Brucella - specific IgG1, IgG2, and IgM levels in Prozoning and non prozoning sera were measured using the single radial immunodiffusion test. It was shown that serum containing a high proportion of specific IgG2 was likely to exhibit prozoning and that various degrees of prozoning could be induced by varying the ratio of specific IgG1 to specific IgG2. Cattle, previously sensitized by calfhood Br. abortus strain 19 vaccination, were experimentally inoculated with killed Br. abortus. It was shown that although serum agglutination test (SAT) and CFT titres appeared for a short period, titres to the BCT in some cattle tended to remain longer thus allowing an animal to be BCT+/CFT-. An analysis of herd testing data indicated that BCT+/CFT-animals were more likely to exist in infected herds than in non-infected herds. In heavily infected herds up to 16% of CFT- animals were BCT+ whereas in non-infected or very lightly infected herds less than 4% were CFT-/BCT+. It was concluded that in sensitized cattle at least exposure to the organism without true infection is capable of stimulating antibody which is detected by the BCT, but not necessarily able to provoke positive CFT titres. The performance of the Auto-Analyzer adaptation of the CFT as used in the New Zealand eradication scheme was assessed. Various prozoning sera from known infected animals were tested and the effect of varying antigen concentrations on these and other sera was noted. Significant differences in antigen concentration required for optimal complement fixation were detected. Prozoning sera required more antigen than non-prozoning sera and even sera that did not exhibit prozoning had varying optimal antigen requirements. By using I125 labelled bovine gama-globulin the dilution gradient of serum within the Auto-Analyzer system was estimated. Knowledge of the serum dilution gradient being obtained was essential for proper understanding of unusual traces given by prozoning sera.Item A study of some endemic viruses of cattle, with particular reference to enteric viruses : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University, New Zealand(Massey University, 1990) Durham, Peter John KeithThis investigation was undertaken to isolate and characterise a number of viruses from diarrhoeic cattle faeces, as a preliminary step in the systematic investigation of viruses as possible causes of diarrhoea in cattle, and more particularly calves. A further and important aim was to gain further experience in a number of virological procedures. Using 3 passages of inocula in each of secondary foetal bovine kidney and lung cells, and monkey kidney (Vero) cells, 7 viruses were isolated from 56 faeces, 2 intestinal samples, and 1 spleen, all from scouring animals. Five of the isolates were found to produce a rapid and complete cytopathic effect in a variety of cell cultures, and their biological and physicechemical properties were subsequently studied in some depth. One of these isolates was also studied with the immunofluorescent technique, and its buoyant density was determined in a caesium chloride gradient. These isolates were concluded to be bovine enteroviruses, and were found in further studies to be separable into 2 types on the basis of cross-neutralisation tests, fluorescent antibody tests, and behaviour in the presence of low concentrations of hydroxybenzyl benzimidazole. These 2 serotypes on further cross-neutralisation tests were found to be serologically distinct from the 7 U.S. standard serotypes that were available. Another of the isolates was found to cause a slow growing and relatively nonprogressive type of cytopathic effect in only Vero cells, and was consequently harder to study. On the basis of limited studies of this isolate, it was concluded to be probably a member of the diplornavirus group, possibly having some affinity with the "reo-like" viruses. Further more precise studies will be needed to confirm or refute this relationship. The remaining virus, which was isolated from the spleen, was identified as being bovine viral diarrhoea (BVD) virus on the basis of its cytopathic effect and neutralisation by standard BVD antiserum. A limited survey for neutralising antibodies to infectious bovine rhinotracheitis and BVD viruses, and haemgglutinating-inhibiting antibodies to parainfluenza 3 virus and reoviruses 1, 2, and 3 was undertaken. It showed that antibody to all the viruses was present in a considerable proportion of the North Island cattle population. This work can only be regarded as a preliminary study, as it is probable on the basis of overseas work that a number of other viruses remain to be isolated from diarrhoeic cattle faeces. It is hoped to continue this investigation and to eventually establish a better understanding of the relationships between viruses and bovine gastrointestinal disease, and more particularly to establish their possible economic significance.Item Two studies on the control of wildlife-derived tuberculosis : farmer views and model validation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Epidemiology at Massey University, Palmerston North, New Zealand(Massey University, 2002) Corner, Rene AnneThe two studies included in this thesis are part of a larger research programme evaluating tools to aid in the eradication of bovine tuberculosis (TB) from cattle in New Zealand. The first study was a survey of farmer’s attitudes towards the National Pest Management Strategy (NPMS) and tuberculosis control. The second study validated the population component of PossPOP, a spatial stochastic simulation model of TB in a wild possum population. A postal questionnaire survey identified variation of attitudes of 404 farmers in four regions of varying TB status within New Zealand. Farmers were stratified by region, enterprise type, area TB classification and herd TB status. Of the farmers contacted by telephone prior to sending out the questionnaire 91% agreed to participate in the study and 83% of these farmers returned a completed questionnaire. The questionnaire contained questions on farmer demographics, TB status, herd TB history, farm management practices and attitudes towards the control of TB. Farmers with infected herds were generally positive about the control program and believed that TB could be eradicated from their herds. A number of farmers were concerned about proposed changes to the NPMS, such as the implementation of direct payment of TB testing costs by farmers and removal of compensation for infected cattle. An important finding was that the majority of farmers were not aware that the Animal Health Board was in charge of the NPMS. PossPOP was built using the first 22 months’ data from a longitudinal study of a possum population run at Castlepoint in the lower North Island of New Zealand. Data from the remaining 9 years of the study was used set for model validation. PossPOP was validated by comparing age distribution, sex structure and the proportion of births, deaths and immigrations in the modelled population against the field population. There was general agreement between the model and the field population and also published population patterns. PossPOP produced a stable population over time at different densities, with similar temporal patterns to the field population. Emergent biological properties were examined, such as rate of population rebuilding after a major population cull, the removal of immigration from both populations and age specific mortality. The field population grew much more rapidly following a cull compared with the PossPOP population due to home range expansion of possums that were living on the periphery of the study site, which was not programmed into the model. These results showed that while PossPOP models a small area, it reflects patterns of control over large areas making it a useful tool to evaluate large scale possum control strategies.Item Investigations into the control of neosporosis in cattle : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Clinical Science, Massey University, Palmerston North, New Zealand(Massey University, 2011) Weston, Jennifer FaithThe research presented in this thesis was undertaken to further understanding of the control of neosporosis in cattle. A prospective cohort study of primiparous heifers on a farm with a history of Neospora-associated abortion found a 0.65 risk of abortion among seropositive heifers, suggesting that identification and culling of seropositive heifer replacements may be cost-effective. A clinical trial of a registered Neospora caninum vaccine utilising 2,246 cattle from five farms with endemic N. caninum infection was assessed for efficacy in preventing abortion and vertical transmission. Overall vaccine efficacy was 0.25 (p=0.12) and vaccination increased the risk of vertical transmission. Histopathological and serological results from 148 cases of abortion from this trial were compiled to establish aetiological diagnoses. Nine of 34 cases where the fetus was examined had histopathological evidence of N. caninum infection. Histopathology revealed dual infectious aetiologies in 2 cases and serology suggested that, in another 17 cases, there had been recent exposure to a second infectious agent capable of causing abortion in conjunction with N. caninum lesions in the fetus or fetal bacteraemia. As a prelude to cattle challenge trials, a challenge study conducted on pregnant sheep revealed a strong dose-response for abortion and that indirect fluorescent antibody test results did not correlate well with infection status or pregnancy outcome. A novel challenge method of applying tachyzoites to an abraded oral mucosa was undertaken in pregnant heifers to establish whether oral lesions could facilitate the direct horizontal transmission of N. caninum between cattle. One of eight heifers seroconverted, her calf and one other were seropositive when sampled within 12 hours of birth, and three other heifercalf pairs had at least one positive polymerase chain reaction result at parturition. This method of transmission between cattle may be responsible for only a small proportion of infections but is a major new finding in the epidemiology of N. caninum infection and warrants further investigation. Finally, inoculation with mouse-passaged N. caninum tachyzoites prior to mating did not prevent abortion when heifers were challenged again on Day 70 of gestation, suggesting that live inoculation may not be a suitable control option.Item A study of pencillin concentrations in bovine conjunctival sac fluid as it pertains to the treatment of Moraxella bovis infection : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 1984) Abeynayake, PreeniInfectious bovine keratoconjunctivitis is one of the commonest eye diseases of cattle. A specialised organism Moraxella bovis, is generally held to be responsible for the often serious damage to the cornea and the conjunctiva. Temporary blindness is common but even without treatment, most cattle eventually regain their vision. Although the disease has been recognised for more than 30 years in most cattle-farming areas of the world, only in the past 8-10 years has it become evident in New Zealand. A wide range of antibacterial products has been used for treatment but there has been very little definitive work undertaken which would form a sound basis for any schedule of medication. In view of the information lacking in this respect, it was decided to study the pharmacokinetics of an antimicrobial drug in the conjunctival sac fluid after different formulations of the drug had been administered by different routes. Penicillin was chosen as the model antibiotic because it is remarkably free of side effects, effective against M. bovis and available in a range of products suitable for administration by various means. The overall aim of the work was to determine a concentration-time profile for penicillin in conjunctival sac fluid (CF) and it was reasoned that this data could then be used to establish a schedule of treatment that would produce an optimum effect against M. bovis. The preliminary requirement of the research programme was to investigate a suitable method of collecting unchanged samples of CF over a number of days (i.e. 1-7 days). As soon as the project was initiated, it became clear that any substantial distress to the animals caused lachrymation, and the possibility of the CF then containing endogenous antibacterial substances could not be discounted. Of the three methods of collection tested, the use of blunted capillary tubes was found to be best for the purpose because the method avoided any local tissue irritation and the cattle soon learned to tolerate any associated handling and minor restraint. Special safeguards were built into the experiment to confirm the absence of antibacterial substances other than penicillin. The specificity of the inhibitory substance in CF, namely penicillin, was regularly confirmed by testing for parallelism against standard dilutions of penicillin, and periodically by neutralizing all antibacterial activity in a sample, using penicillinase. Estimations of the penicillin concentration in CF samples were carried out by means of the agar-well diffusion technique. Minor modifications to the basic assay system were required to ensure that the sensitivity would cover the range of penicillin concentrations expected to appear in CF. After a series of titrations involving the size of the inoculum of the indicator organism, the depth and volume of the agar medium, the volume of the test solution for each well and the incubation schedule, each variable was standardized for all subsequent assays. A large plate (28 x 28 cm, containing 64 wells 4.5 mm in diameter, 2.5 cm apart) permitted the assay of 12 CF samples alongside four standard dilutions of penicillin under uniform conditions. Using Bacillus subtilis as the indicator organism, the assay system was sensitive in the range between 10 and 0.07 iu/ml penicillin, with a standard error of predicted values of 0.04-0.17. In order to nominate a penicillin concentration in CF that would be effective against M. bovis, the penicillin sensitivity of several strains of the organism were measured in terms of their minimum inhibitory concentration (MIC). The four New Zealand isolates tested were highly sensitive; most having a MIC of penicillin of 0.03 iu/ml, and this was identical for the bactericidal concentration. After making allowance for an in vivo safety factor of 5, the minimum therapeutic concentration (MTC) of penicillin against M. bovis was defined for this series of experiments to be 0.15 iu/ml. The length of time that the penicillin concentration in CF remained equal to or above the MTC following treatment with any particular product, was considered as the duration of therapeutic concentration (DTC; hours) for that particular treatment. The major experiments using clinically normal cows involved the estimation of penicillin concentration in CF following systemic, subconjunctival or topical administration. Every treatment was repeated in five or six animals but without exception any variation in the DTC between eyes and animals was found to be not significant. In all experiments, the decline in penicillin concentration in CF followed an exponential pattern, irrespective of the route of administration. A series of systemic injections was carried out by the appropriate route using three different products of penicillin at a standard dose-rate of 20,000 iu/kg. Penicillin concentrations observed in CF (± SEM) following the intravenous injection of sodium benzyl penicillin (peak 2.0 iu/ml and DTC 5.5 ± 0.25 hours) and the intramuscular injection of procaine penicillin (peak 1.0 iu/ml and DTC 16.5 ± 1.25), were considered inadequate for the treatment of IBK. Penethamate hydriodide, administered by either the intramuscular or the subcutaneous route, achieved an approximate peak concentration of 3.0 iu/ml and produced a minimum therapeutic duration of 61 ± 5.57 hours. Such a difference between the kinetics of penethamate hydriodide and benzyl penicillin was attributed to the greater lipid solubility of the diethylaminoethyl ester of penicillin. Although the profile of penicillin in CF following penethamate administration seems favourable, the cost of the product would probably prohibit its regular use. In a further experiment in which half the dose was used, the DTC was reduced to 23.5 ± 4 hours. A subconjunctival injection of procaine penicillin at a dosage of 6 x 105 iu in 2 ml , administered either through the skin or through the conjunctiva, achieved an approximate peak of 8 iu/ml for both routes and DTCs of 67.6 ± 5.0 hours and 40 ± 2.6 hours respectively. The faster rate of penicillin decay following an injection given through the conjunctiva, is possibly attributable to the back diffusion of the drug through the needle puncture. In general, the penicillin profile in CF following a subconjunctival injection is conducive to an extended bactericidal effect and the trial results tend to confirm the clinical impressions of its usefulness in the field. Treatment by this means is relatively cheap and easily undertaken. If a more prolonged effect is desirable, another dose might be administered two days after the first. Topical application of sodium benzyl penicillin in aqueous solution at a concentration isotonic with 0.9% saline, produced a DTC in CF for 12.6 ± 1.5 hours. This duration is considered long for a water soluble salt in an aqueous base. When this salt and other less water soluble ones were formulated in an ointment base,the time of effect was significantly prolonged. Sodium benzyl penicillin and procaine penicillin in the ointment base, produced DTCs of 39.8 ± 2 hours and 37 ± 4 hours respectively, while the ointment formulation of benethamine penicillin produced a DTC of 56 ± 4.5 hours. The prolonged decline observed for all eye ointments can be partly accounted for by the viscous nature of the base but other differences may be dependent on the relative water solubility of each penicillin salt. In addition, the various structures of the surface mucosae such as crypts and specialised cells, are likely to retard free diffusion and therefore retain penicillin, in CF. The extent of dissociation of a substance depending on its pKa may also influence the overall rate of decline. Regular examination of the treated eyes and cell counts undertaken on CF samples, did not indicate any inflammatory reaction even after repeated application of ointments. The various penicillin profiles observed in CF now provide a sound basis for establishing treatment schedules. Optimum treatment schedules can be advocated for different penicillin products, based on concentrations and durations that could be expected to control M. bovis infection in superficial tissues of the eye. However, in order to confirm the therapeutic effectiveness of such schedules, controlled clinical trials using infected animals are obligatory.