Cattle Practice 6.2

CATTLE PRACTICE VOL 6 PART 2 Table 2. Echographic inflammation index Days post treatment Group Inoculation + 11 days 7 14 18 21 28 Control - Trial 1 1.2 ± 0.9 0.9 ± 0.8 - 1.2 ± 0.6 - - Control - Trial 2 2.1 ± 0.6 2.5 ± 0.6 2.3 ± 0.5 - 2.4 ± 0.6 2.0 ± 0.9 Reference product 3 infusions - Trial 1 1.2 ± 0.8 1.2 ± 0.8 - 1.2 ± 0.8 - - Reference product 3 infusions - Trial 2 2.0 ± 0.6 1.2 ± 0.8 1.4 ± 0.7 - 1.3 ± 0.9 1.2 ± 0.9 Cefquinome 45mg x 2 1.2 ± 0.9 1.6 ± 0.8 - 1.0 ± 0.8 - - Cefquinome 60mg x 2 1.2 ± 0.8 1.5 ± 0.8 - 1.2 ± 0.8 - - Cefquinome 75mg x 2 1.3 ± 0.9 0.9 ± 0.9 - 1.4 ± 0.8 - - Cefquinome 75mg x 3 2.1 ± 0.7 1.4 ± 0.8 1.2 ± 0.8 - 1.4 ± 1.0 1.1± 1.0 Table 3. Echographic fibrosis index Days post treatment Group Innoculation + 11 days 7 14 18 21 28 Control - Trial 1 0.5 ± 0.8 1.2 ± 0.9 - 1.9 ± 0.4 - - Control - Trial 2 1.2 ± 0.7 1.2 ± 0.7 1.3 ± 0.5 - 1.3 ± 0.5 1.2 ± 0.6 Reference product 3 infusions - Trial 1 0.8 ± 0.9 1.5 ± 0.9 - 1.9 ± 0.3 - - Reference product 3 infusions - Trial 2 1.1 ± 0.7 1.2 ± 0.7 1.4 ± 0.5 - 1.5 ± 0.5 1.5 ± 0.5 Cefquinome 45mg x 2 1.3 ± 0.9 1.9 ± 0.3 - 1.8 ± 0.5 - - Cefquinome 60mg x 2 0.7 ± 0.9 1.3 ± 0.8 - 1.8 ± 0.4 - - Cefquinome 75mg x 2 1.2 ± 1.0 1.6 ± 0.7 - 1.6 ± 0.7 - - Cefquinome 75mg x 3 1.1± 0.7 1.1 ± 0.7 1.3 ± 0.8 - 1.1 ± 0.6 0.8 ± 0.8 Somatic cell counts remained high in the non treated infected controls (1.5 million cells per ml) although they did tend to decrease slightly towards the end of the trial. With the reference product and cefquinome at the higher dose, the somatic cell counts drop one week post treatment to approximately pre-infusion levels but are very variable and tend to rise to 0.7/1 million cells per ml. Echographic scores are summarised in Tables 2 and 3. These are derived by calculating the mean scores observed in the different qyarters of the cows at the various time points, according to the scoring system described previously. Three infusions of 75 mg of cefquinome, and the reference product do induce a decrease of the inflammation index as from one week post treatment, but, whereas the somatic cell counts tend to rise again towards the end of the trial the inflammation index stays much lower than in the control group. The fibrosis seems to be controlled to some degree in the 3x75 mg cefquinome group but increases or stays stable in the other groups, including the reference product group. The mean fibrosis index in the cefquinome group is < 1 at the end of the trial and has decreased indicating significant functional recovery. Globally in this group fibrosis is ranked slightly under the “ discrete ” score, whereas in the two other groups it is between the discrete and extensive stage. DISCUSSION AND CONCLUSION Echographical examination of the mammary gland is simple, non invasive and can be repeated as often as necessary. The appropriate pictures can be recorded on a video printer and kept if useful. Practical experience is necessary to interpret what is seen, for instance to distinguish between acini and small inflammatory nodules. Basically the nodules do not change shape or size when the probe is orientated differently whereas acini will. Fibrosis is not always easy to identify without repeated examinations (as is the case with induced disease models). Ultrasonography does not replace bacteriological criteria or somatic cell counts but gives additional information on the status of the udder. On the other hand it is much more objective than direct palpation, especially when inflammation decreases, as fibrosis is not detectable manually. It is our opinion that echographical examination is of particular value in subclinical conditions to visualize the status of the udder and to be in a position to observe the evolution of the lesions. In acute cases it is totally unhelpful as the inflammatory reaction involves practically 100% of BCVA 1998 123

CATTLE PRACTICE VOL 6 PART 2 the tissue; we have used this technique in lactating cows with induced E.coli endotoxin acute mastitis and observed a hypoechogenic mass that does not offer any additional information (unpublished data). As demonstrated by Sordillo et al. (6), 2-10 days after S.aureus challenge or chronic infection of the mammary gland, significant morphological changes can be seen that are consistent with a significant loss of secretory potential. Tissue from infected quarters exhibit less synthetic and secretory ability as indicated by lower percentages of luminal area and higher percentages of stromal area. Our echographical observations are in agreement with these morphological changes. The results of the first trial show that in lactating cows with S.aureus mastitis, antibiotics alone do not markedly modify the inflammatory and fibrotic status of the udder despite good bacteriological efficacy. In the second study the inflammation index is somewhat higher before therapy, and in this case a definite reduction can be observed both with the test product and the reference product. It is interesting to note that, as in the first trial, three infusions of the reference product do not reduce the fibrosis in contrast to the trend seen with the test compound at the optimal dose level. This would indicate a better degree of functional recovery than seen with lower doses of the compound or with the reference product used in these studies. Antibiotic treatment at drying off entails a definite decrease of the echographical scores (1) which confirms that dry cow therapy is very efficient both bacteriologically and clinically vs. S.aureus. It is possible that the persistence of the antibiotic in the udder, the physiological status during the dry period, (including the production of natural inhibitory substances - lactoferrin and lactoperoxydase) and the absence of milk, that has been shown to reduce antistaphylococcal efficacy of antibacterials ( Sandholm et al. - 1991)(4) explain this phenomenon. In practice echographical examinations are simple to perform, and appropriate portable equipment is readily available.We believe that this technique can be used to check on the status of the udder and to evaluate the extent of the morphological and functional changes despite the fact that direct correlation has not been demonstrated experimentally. In clinical field trials echographical examination of the udder would prove more objective than palpation of the gland to support claims of clinical efficacy. It would also be of interest to evaluate the effects of local antiinflammatory treatment in lactating cows by this technique and to try to justify more conclusively combination products. ACKNOWLEDGEMENT We wish to thank Hoechst Roussel Vet for supplying the test products and financial support. REFERENCES 1. Banting A. , Tranquart F., Banting S. Influence of antibiotic treatmant on the inflammatory and fibrotic staus of the mammary parenchyma of cows with induced S.aureus mastitis. 3rd International Mastitis Seminar- Tel Aviv 1995 2. Postle D.S., Roguinsky M., Poutrel B. Induced staphyococcal infection in the bovine mammary gland. Am. J. Vet. Res. 1978, 39:29-35 3. Poutrel B., Lerondelle C. Induced staphylococcal infections in the bovine mammary gland. Influence of the month of lactation and other factors relaating to the cow. Ann. Rech. Vet. 1978, 9:119-128 4. Sandholm M., Louhi M., Myllys V. Milk reduces antistaphylococcal of antibacterials..A possible explanation for failures of therapy in mastitis. Acta Vet. Scan. 1991 suppl. 87: 115-119 5. Sears P.M., Smith B.S., English P.B., Herer P.S., Gonzales R.N. Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J. Dairy Sci. 1990, 73:2785-2789 6. Sordillo L.M., Nickerson S.C., Akers R.M. Pathology of Staphylococcus aureus mastitis during lactogenesis : relationships with bovine mammary structure and function. J. Dairy Sci. 1989, 72:228-240 7.Tranquart F., Banting A., Bodard S., Grégoire S., Decorsaire F., Pourcelot L. Suivi échographique de l’inflammation mammaire chez les bovins. Rev. Française d’Echographie Animale 1991,5:12-14 BCVA 1998 124

CATTLE PRACTICE VOL 6 PART 2 Dairy Cattle Welfare – The FAWC Perspective Macpherson R., Applegrove, Conon Bridge, Dingwall, Ross-shire, IV7 8AZ INTRODUCTION As part of their on-going review of the welfare of farm animals on agricultural land the Farm Animal Welfare Council (FAWC) was asked by the GB Agriculture Departments to consider the welfare of dairy cattle and make recommendations to Ministers. Before going on to discuss the Dairy Report I thought it might be useful to refresh your memories as to what FAWC is, who serves on it and how it operates. I will then give a short résumé of our Report on the Welfare of Dairy Cattle, which was published in December 1997. THE FARM ANIMAL WELFARE COUNCIL FAWC was set up in 1979 by the then Minister of Agriculture, Peter Walker, with the remit to keep under review the welfare of farm animals on agricultural land, at markets, in transit and at the place of slaughter and to advise the Minister of Agriculture, Fisheries and Food and the Secretaries of State for Scotland and Wales of any legislative or other changes that may be necessary. The Council consists of 23 members appointed by Ministers as individuals but in such a way as to ensure that all sections of the industry and those concerned are represented. These sectors include veterinarians, farmers, local authority enforcers, consumers, animal welfarists and academics – a real mixed bag! The Council is funded by MAFF but is completely independent: • It can investigate any topic falling within its remit • Communicate and consult freely with outside bodies, the European Commission and the public, and • It can publish its findings, if it so wishes, and respond to specific Ministerial requests for advice. The Council has, over the years, advised Ministers on farm animal welfare as a basis for codes of practice and legislation and assisted in the updating of the welfare codes in the light of advances in scientific knowledge and husbandry practices. A considerable amount of Council’s time is spent in the preparation of reports similar to the Dairy Report. THE REPORT ON THE WELFARE OF DAIRY CATTLE INTRODUCTION Until fairly recently dairy farming, unlike intensively kept pigs and poultry, has conjured an image of animals at pasture with few, if any, adverse effects on their welfare. However, the need for a review became especially pertinent with the increasing intensification of dairy farming and increasing production demands. A small working party was set up to carry out the review and report their findings to Council. This group obtained oral and written evidence from experts in the dairy industry and examined scientific data wherever possible. Visits were made to both large and small commercial units and research establishments throughout the UK and seminars were held with invited experts from the industry, research establishments and the animal protection societies. The report summarises the findings of the study and makes a number of recommendations. This report could not have been produced without the help and guidance of the industry, farmers and veterinary surgeons across the country who gave us their time and spared no effort in arranging visits throughout the UK. At no time did we receive any adverse comments and all interested parties welcomed our investigations. THE REPORT The report runs to 414 paragraphs, and includes 191 recommendations, so it is not possible to give other than a brief résumé of a few sections of the report where we conclude that welfare could and should be improved as soon as possible. It should be emphasised that many of our recommendations represent good practice, are already followed by the majority of the industry and are simply included in the report to ensure observance by all. TRAINING Throughout the report considerable emphasis has been laid on training. A high degree of stockmanship is essential and the most welfare friendly system may well fail if not accompanied by good stockmanship and sound management practice. Those who look after cattle must be aware BCVA 1998 125

CATTLE PRACTICE VOL 6 PART 2 of their responsibilities to the animals and appropriate training should include on-farm instruction and training to an appropriate standard by a recognised body. If specific tasks are to be performed, e.g. foot trimming, then specific training should be given. To this end FAWC advised Government that ultrasonic scanning for pregnancy should be an act of veterinary surgery and those carrying out the procedure must be trained and certified as competent. PRODUCTION DISEASES The so called production diseases (lameness, mastitis, fertility and reproduction) give rise to a number of welfare problems and were studied in considerable detail. It was recognised that all these are extremely important but in this résumé only lameness will be discussed. Lameness is an extremely painful condition and steps must be taken as a matter of urgency to reduce the incidence. It was found that, although some farmers have taken action to reduce the level of lameness within their herds, others seem to accept it as an inevitable part of dairy farming in spite of its financial implications. This is totally unacceptable as sufficient knowledge already exists to enable the industry to reduce significantly the number of lame cows. Time and time again when discussing “on-farm” the problem of lameness the group was met with the repost “You always get a number of lame cows in a dairy (in some cases upward of 25%!) and they are milking well!!” We should take the situation very seriously before the general public becomes aware of it and the image of milk becomes tarnished. (A quote from Bob Ward at the BCVA in 1993). Major factors, contributing to lameness namely environment and management, nutrition, breeding and infectious conditions, are fully discussed in the relevant sections of the report. Whilst it is not possible to attribute accurately at the present time the part played by the various factors, the available evidence suggests that improved housing and environment would help to increase “lying time” and cow comfort and therefore help to reduce the unacceptable levels of lameness found on some farms. A number of recommendations (paragraphs 78-84) were made which if accepted by Government, will greatly improve cow welfare and the report will have served a useful purpose. The other sections of the report although not commanding such a high profile are extremely important and should be studied carefully as many recommendations have been made on such routine practices as the use of calving aids, handling facilities, lifting gear, mutilations and many others. In all of the above we have advocated a significant veterinary input to safeguard the animals’ welfare and I hope you will agree with the sentiments expressed by the group. Much of what has been discussed in the report has been said over the years but to little avail. However, with the advent of farm assurance schemes and acceptance, we hope, of our recommendations by Government, farmers will take the necessary action to improve the welfare of the dairy cow. REFERENCES FAWC Report on the Welfare of Dairy Cattle 1997 BCVA 1998 126

CATTLE PRACTICE VOL 6 PART 2 Organic Dairy and Beef Cattle Farming Browning H., Eastbrook Farm, Bishopstone, Swindon, Wilts SN6 8PW INTRODUCTION What is Organic Farming? The Principles: • To produce food of high nutritional quality in sufficient quantity. • To interact in a constructive and life-enhancing way with natural systems and cycles. • To encourage and enhance biological cycles within the farming system, involving microorganisms, soil flora and fauna, plants and animals. • To maintain and increase long term fertility of soils. • To use, as far as possible, renewable resources in locally organised agricultural systems. • To work, as far as possible, with materials and substances which can be reused or recycled, either on the farm or elsewhere. • To give all livestock living conditions which allow them to perform the basic aspects of their innate behaviour. • To minimise all forms of pollution that may result from agricultural practice. • To maintain the genetic diversity of the agricultural system and its surroundings, including the protection of plant and wildlife habitats. • To allow everyone involved in organic production and processing a quality of life conforming to the UN human rights, to cover their basic needs and obtain an adequate return and satisfaction from their work, including a safe working environment. • To consider the wider social and ecological impact of the farming system. Source: IFOAM Basic Standards HOW IS IT REGULATED? EU regulation controls crop production and processing; livestock regulation in the pipeline, due autumn ’98? Each Member State has to implement the regulation, and ensure all certification bodies (5 in UK, Soil Association certifies 70-80% of UK production) meet as a minimum the EU standards. Countries outside the EU who wish to import organic products must be approved by an EU registered certifier, and present a dossier on production and processing methodology. FOCUS ON DAIRY, BEEF AND VEAL STANDARDS 1. Dairy – Key Standards • 90% of total diet must be organically grown. • At least 60% of daily DM intake must come from forage. • No routine drugs. e.g. dry cow therapy. 2. Beef and Veal – Key Standards As dairy, plus • Calves feed on whole organic milk for at least nine weeks. • All calves when housed, including for veal, must have access to good quality forage. • No export of non-breeding stock. WHAT DOES THIS MEAN IN PRACTICE? Case Studies a) Dairying at Eastbrook Farm • Two herds of pedigree Fresian/Holsteins, 130 cows in each. • All forage produced from clover (red and white) based leys. • 3500 – 4250 litres/cow produced from forage. • Oats, peas and triticale grown on farm for concentrate feed. * 6000 litres – 7000 litres cow/annum. * 1.1 – 1.2 tonnes concentrate/cow. * 1.8 LSU/ha stocking rate. * Milk price 29.5 p/litre (net). * M.O.C. - £1450 - £1650/cow. * Vet and medical costs. * Use of homeopathy and non-drug disease control methods. b) Veal Fresian\Holstein bull calves are suckled on nurse cows, outside in summer, fed organic hay, concentrates (rolled oats, wheat, peas, soya) with normal mineral mix – i.e. no iron restrictions. Slaughtered at 5-6 months, at carcase weight approx 80 – 100 kg/dwt. Marketed as ‘rose’ veal by Eastbrook Farm Organic Meats (EFOM). The challenge has been marketing lower value cuts from forequarter. Trial with Sainsbury’s begin April ’98. BCVA 1998 127

CATTLE PRACTICE VOL 6 PART 2 The potential for growth in the UK organic sector is enormous; currently only 0.5% of land area, 70% + of product sold in the UK is imported. Demand far outstripping supply. In other countries the sector is far more advanced, e.g. in Denmark where organic milk sales now over 8% of total supply, school children receive organic milk, and premiums holding up. Prospects in the UK depend upon both domestic agricultural policy and next CAP reform, research and development, trade issues and, of course, consumer demand. THE ROLE OF ORGANIC FARMING IN A WIDER CONTEXT Irrespective of whether the UK organic industry remains tiny, or goes mainstream, the sector has had, and will continue to have, considerable influence on the rest of the industry. It’s role is to break new ground in the move towards ‘sustainability’ , and to reinforce in producers’ minds the benefits of techniques and husbandry methods which are not ‘sold’ to farmers as no-one except the farmer benefits. Examples include the use of clover and herbs, manure management, alternative veterinary techniques (most importantly of course, the benefits of excellent husbandry), balanced rotations and the basic principles of pest control. By demonstrating the potential of very low input systems, organic farming can play a major part in the regeneration of confidence in British agriculture. BCVA 1998 128

CATTLE PRACTICE VOL 6 PART 2 A Slow-Release Iodine, Selenium And Cobalt Cattle Bolus Rogers P.A.M1 , Lynch P.J2 , Porter W.L3 & Bell G.D4 1 Grange Research Centre, Teagasc, Dunsany, Co. Meath, Ireland. 2 Bayer Ltd, Industrial Est, Pottery Rd, Dunlaoghaire, Co. Dublin, Ireland. 3 Animax Ltd, Shepherds Grove West, Stanton, Bury St Edmunds, Suffolk IP31 2AR 4 Bayer Plc, Eastern Way, Bury St Edmunds, Suffolk IP32 7AH INTRODUCTION Internationally, I-deficiency is the most common traceelement deficiency in ruminants (Underwood 1962). Plasma inorganic iodine (PII) level is a very sensitive index of dietary I-status in cattle and dietary inputs of I from unsupplemented Irish forages usually are inadequate to maintain optimal blood status. Whole blood glutathione peroxidase (GPx) is a good test of dietary Se-status over the previous few months (Backall & Scholz 1979). Critical blood levels of PII and GPx, and the daily supplement of I and Se needed to maintain them, are not agreed universally. Irish research and clinical experience indicates that mean herd PII-levels of 105-285 µg/L and GPx-levels of 42- 161 iu/g Hb are optimal (Rogers & Mee 1996). Irish forage-fed cows need supplements of circa 30-60 mg I and 3-5 mg Se/d to maintain such blood levels (Mee et al 1996). Other cattle need supplementation pro-rata with bodyweight. Some authorities accept lower blood levels and correspondingly lower amounts of supplement. In either case, it is difficult to ensure that all at-risk cattle at pasture receive adequate supplementary I and Se. This is because of practical constraints of farm layout, uncontrolled water supplies and the cost of feeding concentrates at pasture1 . Slowrelease boluses are used as Co, Cu and Se supplements for ruminants. Though Ellis et al (1983) developed an intraruminal device for slow-release of I in sheep, the concept does not appear to have been exploited effectively for cattle. We report the effects of a new intraruminal bolus2 on levels of PII and GPx in steers on a farm in which unsupplemented cattle had a very low I- and Se- status in 1994-95, as asessed by blood levels of PII and GPx at that time. MATERIALS AND METHODS Irish controlled trial (Nov 95-Aug 96) Hereford X Charolais steers (n=21, mean 421kg liveweight), reared on a Kilkenny farm, were allocated at random to three groups of 7 (0= untreated controls; 1 = 1 bolus; 2 = 2 boluses). The boluses were given orally on Day 0 (16.11.95). No other I-supplement was given during the trial. For the first 4 months, the steers were fed grass-silage + concentrates low in I and Se indoors; for the last 2 months their diet was grazed grass only. Heparinised blood was drawn from each steer on Day 0 (pretreatment) and on days 26, 61, 93, 119, 159, 181, 208 and 236 post-treatment. Samples were assayed at Grange Research Centre for PII (μg/L) and GPx (iu/g Hb) by a modification of the method of Aumont & Tressol 1987 and by Blanchflower and McMurray’s modification of the method of Paglia and Valentine (1967), respectively. 1997 UK field Trial Blood from 5 treated and 5 control adult females (mainly cows, but also some heifers) in each of 47 commercial UK herds was assayed for PII pretrial and 1 month after dosing with one Ionex bolus. PII values in treated and untreated animals, and the changes in PII from pre-treatment values were compared. 1997 Irish ad-hoc observations Blood from commercial herds assayed at Grange indicated that herds given Ionex 1-3 months before blood test for routine investigation had better PII levels than unsupplemented herds. RESULTS Irish controlled trial (Nov 95- Aug 96) Table 1 shows group mean levels and changes from baseline for PII and GPx, respectively. Pre-trial mean PII - levels in Groups 0, 1 and 2 respectively were 15, 18 and 16 μg/L. This indicated a very low Istatus, not significantly different between Groups (P = .759). Throughout the trial, Group 0 maintained very low to low PII – levels. From Day 26 to Day 159, mean PII – Levels in Groups 1 and 2 were significantly (P<.001) higher than in Group 0 at all occasions, and levels in Group 2 were significantly (P<.001) higher than those in Group 1 at those times. Pre-trial mean GPx – levels in Groups 0, 1 and 2 respectively were 48, 57 and 57 iu/g Hb. This indicated a Se-status in the low-normal range, with the Groups not significantly different (P = .258) from each other. Comparison of the changes in mean GPx from pre-trial levels showed that the changes in BCVA 1998 129

CATTLE PRACTICE VOL 6 PART 2 groups 1 and 2 significantly exceeded those in Group 0 cattle by Day 26 (P<.067) and at all subsequent samplings to Day 159 (P<.01 to P<.001). The effect of 2 boluses on the change in blood levels of GPx was still significant at Day 236 (P<.012). 1997 UK field trial Table 2 summarises the results. Overall, relative to PII changes in the controls, one Ionox bolus increased PII levels by (mean±se) 117±10.7 μg/L (p<.001). CONCLUSIONS During the initial Irish controlled trial, trial levels of PII and GPx in control steers were very low and lownormal to marginal, respectively. One bolus significantly increased PII and GPx for 23 weeks; two boluses maintained normal levels of PII and GPx for 23 and 33+ weeks, respectively. The 1997 UK field-trial confirmed that one bolus very significantly increased PII levels also. Relative to changes in control cows, the net PII increase at 1 month was 117 μg/L The bolus is designed to remain in the reticulorumen during its period of trace-element release; it was specified to contain 3500 mg I, 500 mg Se and 350 mg Co/bolus. Assuming linear release over a mean time of 28 weeks, mean release would be circa 18, 2.6 and 1.8 mg I, Se and Co/bolus/d respectively. This should be adequate for yearling cattle on pastures which pose a risk of severe deficiency of I, Se or Co. If no other I, Se or Co supplements were given, adult cows would need 2-3 boluses every 5-6 months on similar high-risk pastures. REFERENCES Aumont G., Tressol J.C. (1987) Analyst 112 875-878. Backall K.A., Scholz R.W. (1979) American Journal of Veterinary Research 40 5, 733-738. Ellis K.J., George J.M., Laby R.H. (1983) Australian J of Agricultural Animal Husbandry 23 369-373. Mee J.F., Rogers P.A.M., Drennan M., O’Farrell K.J., Murphy J. (1996) Trace-element supplementation in dairy and suckler cows. Report of the Teagasc Animal Health Committee 16pp. Paglia D.E., Valentine V.N. (1967) Journal of Laboratory Clinical Medicine 70 158-169. Rogers P.A.M., Mee J.F. (1996) Proc World Buiatrics Congress, Edinburgh, July 9-12th pp394-401. Underwood E.J.. (1962) Iodine. Chapter 8 of Trace Elements in Human and Animal Nutrition (Academic Press Inc., New York & London 2nd edition) pp 218-258. 1 Optimal methods of fixed-rate mineral supplementation of grazing cattle include specially formulated Summer Feeds or a mineralised water supply, but many farmers do not use these options. 2 IonoxR (Patents pending), developed and manufactured by Animax; marketed in Ireland by Bayer. BCVA 1998 130

CATTLE PRACTICE VOL 6 PART 2 Table 1. Means, sed, mean change from pre-treatment value and sed of change for blood PII (µg/L) and GPx (iu/g Hb). In the same column, means with differing superscripts are significantly different. Inorganic iodine Day of experiment Pre-trial 26 61 93 119 159 181 208 236 Mean B0 1500a 14.29a 22.57a 25.71a 14.57a 19.00a 20.71 19.86 10.43 B1 15.71a 162.29b 124.00 b 94.43 b 63.43 b 70.43 b 40.29 32.14 8.71 B2 16.29a 382.86c 279.57c 217.86c 160.14c 104.29c 25.14 24.71 9.43 sedm 1.722 36.2 28.3 24.9 21.6 13.9 9.7 11.2 2.4 F value .759 <.001 <.001 <.001 <.001 <.001 .136 .555 .782 Sig. NS *** *** *** *** *** NS NS NS Mean change B0 -0.71 7.57a 10.71a -0.43a 4.00a 5.71 4.86 -4.57 B1 146.57b 108.29b 78.71b 47.71b 54.71b 24.58 16.43 -7.00 B2 366.57c 263.29c 201.57c 143.86c 88.00c 8.86 8.43 -6.86 Sed of change 35.8 28.3 24.8 21.9 13.6 9.03 10.3 2.35 F value <.001 <.001 <.001 <.001 <.001 .110 .526 .523 Significance *** *** *** *** ** NS NS NS Glutathione peroxidase Day of experiment Pre-trial 26 61 93 119 159 181 208 236 Mean B0 48.3a 42.3a 40.9a 51.5a 42.4a 50.2 a 48.5 a 53.6 a 45.2 a B1 57.0a 59.2b 69.4b 74.5b 68.4b 71.5 b 64.5 a 65.6 a 62.9 b B2 57.0a 55.7b 77.8b 86.7c 83.7b 88.6b 88.7 b 88.7 b 68.9 b sedm 5.86 6.25 7.96 5.73 10.09 9.65 8.80 8.04 6.58 F value .258 .034 <.001 <.001 <.002 <.003 <.001 <.001 <.006 Sig. NS * *** *** ** ** *** *** ** Mean change B0 -6.1ac -7.5a 3.2a -5.9a 1.9a 0.2 a 5.2 a -3.1 a B1 2.2b 12.5b 17.6b 11.4b 14.5a 7.5 a 8.6 a 6.0 ac B2 -1.3bc 20.8b 29.7c 26.7c 31.5b 31.7 b 31.7 b 11.8 c Sed of change 3.31 4.39 3.77 7.81 8.57 8.11 5.35 4.48 F value .067 <.001 <.001 <.002 <.01 <.003 <.001 <.012 Significance *? *** *** ** ** ** *** ** Table 2. Means ans se pre-treatment (SO) and after 1 month (S1), mean change from pre-treatment value and se of change for blood PII, and net effect of treatment (μg/L) in control (untreated) and experimental (one bolus) animals in the UK field-trials. (*** = significant at P<.OO1). Control Cows Treated Cows SO S1 Change SO S1 Change Net Effect n 234 234 234 234 234 234 234 x 80.7 109.1 28.4 80.1 225.8 145.7 117.3 se 5.7 9.4 6.9 5.9 12.1 10.9 10.7 Sig *** *** *** BCVA 1998 131

CATTLE PRACTICE VOL 6 PART 2 BCVA 1998 132

CATTLE PRACTICE VOL 6 PART 2 Making the Best Use of Bulk Milk Antibody Tests Pritchard G. C. Veterinary Investigation Centre, Rougham Hill, Bury St Edmunds, Suffolk IP33 2RX ABSTRACT Measuring antibodies for BVDV, IBRV and L.hardjo infections by ELISA using milk samples taken from the bulk tank provides a simple low cost method of assessing the disease status of dairy herds. This allows appropriate control measures to be adopted. It can also be used for disease monitoring, as a supportive diagnostic tool when investigating herd problems such as milk drop and reproductive failures, and for national surveillance. This paper discusses the use of bulk milk ELISA tests as recently introduced by the Veterinary Laboratories Agency and outlines their potential role in dairy practice. KEYWORDS: Cattle, Antibodies, Bulk milk, BVD, IBR, L.hardjo. INTRODUCTION Bovine virus diarrhoea (BVD), infectious bovine rhinotracheitis (IBR) and leptospirosis represent three of the most important endemic infectious diseases affecting cattle in Britain. In addition to the economic and welfare aspects of clinical and subclinical disease, unless controlled, these infections all tend to persist thereby presenting important implications for the movement of stock between herds of different status. Since the mid 1980s, antibody testing of bulk milk samples has been an integral part of voluntary or compulsory eradication schemes for IBR virus, BVD virus and Leptospira hardjo infections in several countries including Germany, France, the Netherlands and in Scandinavia. It has primarily been used to identify infected dairy herds with a view to implementing control measures, including vaccination. Combined with individual cow blood (or milk) tests, it is also used to detect seroreactors or individual persistently infected animals in the case of BVDV infection. In these countries, bulk milk antibody tests have provided a simple and convenient means of surveying the disease status of their national herd (Niskanen 1993, Wuijckhuise and others 1998). It was also apparent from the World Buiatrics Congress in Edinburgh 1996 and from the pre-congress workshop session on bovine abortion, that bulk milk antibody testing is used in some countries, in conjunction with individual animal blood or milk antibody testing, as a diagnostic tool for the investigation of disease outbreaks, including abortions. The recent introduction by the Veterinary Laboratories Agency (VLA) of bulk milk antibody testing by enzyme-linked immunosorbent assay (ELISA) for BVD, IBR and L.hardjo now offers similar opportunities to veterinary surgeons in Britain. Bulk milk ELISA is also used in the statutory control programmes for brucellosis and enzootic bovine leucosis. GENERAL POINTS By definition, bulk tank samples only reflect the status of cows whose milk is currently entering the tank and therefore do not include male animals, young stock, dry cows and those cows whose milk is being withheld for whatever reason. Samples will inevitably be affected by dilution and the unequal contribution of individual cows to the tank. The test measures past exposure and, like tests on sera, will not detect very recent infection or distinguish between field and vaccinal challenge. Bulk milk ELISA can be used to determine whether a herd is “positive” or “negative” for exposure to a particular infection. However, unlike its use with notifiable diseases, this qualitative approach using a fixed cut-off point, is of limited value for common endemic infections. It may also fail to identify herds with a very small number of reactors - a limitation which is of particular concern when the test is used to support national eradication schemes such as those used for IBR in mainland Europe (Hartman and others 1997). The tests offer greater potential by exploiting the basic principle that the optical density (OD) from a bulk milk ELISA gives an approximate indication of the proportion of seropositive cows, particularly with IBR and BVD since detectable titres generally persist for life. This information provides a good guide to whether infection is, or has recently been, active in the herd or whether the herd is naive. It must be appreciated that the seroreactors may all be amongst older or purchased cows and a high OD does not necessarily indicate active or recent infection. In general, to comprehensively establish herd status, at least two tests several weeks apart are required, together with an evaluation of statistically representative or specifically targeted, individual blood or bulked milk samples . Nevertheless, as a preliminary screening test, even a single bulk tank sample BCVA 1998 133

CATTLE PRACTICE VOL 6 PART 2 provides a reasonable guide to the likely situation in the herd. ADVANTAGES The main advantages of bulk milk antibody testing are cheapness, ease of collection and the ability to provide a general idea of seroprevalence without the need to blood sample large numbers of animals (particularly useful when screening for L.hardjo). The tests provide a good starting point for differential diagnosis, in addition to their value for serological monitoring. It is envisaged that veterinary practices could record the information from bulk milk tests and incorporate it into preventive medicine programmes, particularly in relation to purchased animals (Pritchard 1996a). Regular testing of bulk milk samples every few months provides a simple low cost method of confirming continuing freedom from infection in known disease-free herds; including those at potential risk of introducing new infection, and thereby allows opportunity for prompt action if indicated. For example, a positive result in a previously negative herd could herald an upsurge in infertility, abortions or milk drop. LIMITATIONS Inevitably, the bulk milk ELISA will give a biased result if the status of cows contributing to the bulk tank is significantly different to the rest of the herd. Furthermore, any estimate of within-herd seroprevalence is inevitably somewhat crude and can only provide an approximate guide. It is important not to expect too much of the test - ODs could show variations of up to 10 or 20 percent with samples collected only a few weeks apart depending on calving pattern, the number of cows contributing to the sample and their relative milk yields and antibody titres. The proportions of fat and colostrum can also have an effect. Paired (acute and convalescent) bulk milk samples taken several weeks apart are more difficult to interpret than paired individual cow blood samples because of the large number of variables involved, but this approach may still be of value in some circumstances. SAMPLE COLLECTION The sample collected should be representative of the milking herd. Wait until milking has finished and ensure that the contents of the tank are thoroughly agitated before collection. Collect sample from more than one tank if appropriate and either combine proportionately or test separately if this is likely to provide any useful information (eg. heifers and younger cows’ milk collected in one tank). Samples can also be collected from different age cohorts, for example, when it is desirable to assess the status of first or second lactation homebred cows, or unvaccinated naive animals, as a guide to current/recent infection. For this, take an equal volume of milk from 6-10 cows in this category, mix well and submit an aliquot of the composite sample for testing. Avoid collecting milk from the top of the tank to minimise the amount of cream present since this can significantly interfere with the result (although it will be removed at the testing laboratory anyway). Use a clean ladle or similar receptacle for sample collection and do not allow preservative to get into the bulk tank. Preliminary findings suggest that it is not essential that the bronopol preservative (“MMB preservative solution”) used in the tubes is added immediately. Depending on environmental temperature, it could be added at the laboratory provided the sample is received within about 48 hours of collection. Once preservative has been added, the sample is stable for several months provided it is kept refrigerated. For longer term storage the samples can be frozen. The amount of preservative used (currently 150 µl/25 ml of milk) has no effect on the OD value. THE BULK MILK ELISA TESTS The bulk milk antibody tests used by the VLA for BVD and IBR are based on the indirect ELISA tests developed by the Central Veterinary Laboratory (CVL) for use with serum samples. The BVD test was initially calibrated against the Swedish Svanova BVDV antibody ELISA which has been used extensively in Sweden (Niskanen 1993). The CVL IBR ELISA was initially trialled alongside a commercially available kit that had been validated for use on bulk milk samples. The test currently used by the VLA for L.hardjo is based on the Dutch “Ceditest” (id-dlo) ELISA. In the Netherlands, this has been used since 1989 as part of a voluntary national control scheme under which a herd is considered to be L.hardjo-free if it passes five consecutive tests over an 18 month period at a specified cut-off point. This is a relatively crude ELISA and does not distinguish between infection with L.hardjobovis (the main pathogenic species) and L.hardjoprajitno or L.saxkoebing. Since it is essentially used as a screening test this shortcoming is not particularly significant since a high bulk milk OD is unlikely to be associated with the latter infections. Within the VLA, we have modified the test to make it more robust and to identify various disease categories. With all three tests, to improve robustness and eliminate any problems with antigen standardisation, results are expressed as a signal to positive ratio (OD ratio) using reference field control positive samples unique to the VLA and are therefore not comparable with results from other laboratories. With BVD, calibration against the Swedish test allowed OD ratios to be subdivided into four main categories based on the Swedish experience. With IBR and L.hardjo, the ELISA tests were trialled within the VLA by examining milks from nearly 300 herds of known status, including completely BCVA 1998 134

CATTLE PRACTICE VOL 6 PART 2 seronegative herds and some where all the cows were individually tested. The categories developed (negative, low positive, mid positive and high positive) are designed to reflect epidemiological status. Results falling in the mid positive ranges represent the inevitable “grey area” which is a feature of most serological tests and further monitoring or more comprehensive sampling is needed to clarify the precise situation in these herds. THE TESTS IN PRACTICE 1. BVD In Britain there has been renewed interest in control measures for BVD following the recent launch of a killed vaccine and developments in Scandinavia where several schemes exist for the control and potential eradication of the infection. The recent survey by the VLA using bulk milk samples from over a thousand dairy herds in England and Wales (Paton and others 1998) highlighted the considerable regional differences in infection levels and the potential risks associated with the movement of cattle. The bulk milk ELISA provides a good starting point for BVD investigations. The categories adopted, based on OD ratios, are shown on Table 1 below which summarises interpretation, with a suggested course of action. In positive herds, particularly those in the mid to high positive ranges, individual animal or cohort blood or milk sampling of young homebred cows and uncalved heifers, will help to determine whether infection is currently active (Table 2). Table 1: Bulk milk antibody categories for BVD with brief guide to interpretation and action Category OD ratio Approx % seropos. Interpretation/Action Negative <0.10 <5 Naive. Maintain herd security/quarantine measures. Test purchases for BVD virus (including calves born to seropositive dams). Monitor bulk milks every 3 months. Low positive 0.10 - 0.35 <25 Basically naive. Proceed as above or if recent infection is suspected re-test in 3 - 5 weeks or use paired serology. Mid positive >0.35-0.70 25-65 Difficult to interpret. Re-test/monitor bulk milks or take individual blood or cohort bulk milk samples (see Table 2). High positive >0.70 >65 Likelihood of PI animals increases with OD ratio but may represent historical infection. To check for active infection blood sample young homebred cattle (Table 2). Results provide a basis for control strategy, including vaccination, if appropriate. Table 2: Main outcomes from cohort serology in BVD-positive herds, with possible control options 9-18m Heifers 1st lactation cows 2nd lactation cows Comments and main control options All negative All negative Some or all positive Active infection unlikely unless 1st lactation cows tested have only been calved for a few weeks. Persistently infected (PI) animals probably died or been culled recently. Monitor 1st lactation cohort (bloods or bulked milk) and maintain good herd security as above. All negative Some or all positive Most or all positive Active infection (acute infection/PI animals) in milking herd but no PIs amongst heifers. Consider vaccinating incoming heifers and/or test milking herd for PI animals. Some or all positive Most or all positive Most (or usually all) positive Active infection in milking herd as above plus PI animals probably present amongst heifers. Screen heifers (and younger female and male calves if retained) for virus and consider testing milking herd for PI animals. (PCR or individual bloods). Note: sample homebred animals only, 6-10 per cohort (uncalved heifers ≥9 months, first and second lactation cows). BCVA 1998 135

CATTLE PRACTICE VOL 6 PART 2 Seronegative cows/cohorts identified from these tests can be re-sampled several months later to see if seroconversion subsequently occurs, to provide a further guide to whether infection is active in the herd and whether persistently infected animals are likely to be present. The bulk milk polymerase chain reaction (PCR) test for BVD virus recently introduced by the VLA will provide additional information for use when deciding on a BVD control programme and whether, for example, it is likely to be worthwhile blood sampling the whole milking herd to identify individual persistently infected animals. 2. IBR As outlined by Pritchard (1996b), excluding completely seronegative herds, IBR status tends to fall into two fairly distinct categories. In infected herds where less than 20 percent of adult cows are seropositive (low positive herds), the seroreactors are frequently older or purchased animals and there is usually very little, if any, virus circulation or active infection. Conversely, in those dairy herds with at least 60 percent seroreactors (high positive herds) reactivation of the latent herpesvirus infection and circulation of virus (either field or vaccinal) is common. Most incoming seronegative animals (usually heifers) seroconvert to IBR within two years, sometimes accompanied by mild clinical signs such as upper respiratory infection, pyrexia or milk drop. In these herds vaccination of heifers if seronegative, is worth considering. Table 3 gives details of the bulk milk categories for IBR with a brief guide to interpretation and a suggested course of action. 3. L.hardjo The lack of L.hardjobovis specificity and the failure of leptospiral antibodies to persist in the same way as those for BVDV and IBRV, complicates interpretation of bulk milk results. Herds in the negative and low positive categories (Table 4) are unlikely to have active infection with L.hardjobovis unless very recent - note that it may Table 3: Bulk milk antibody categories for IBR with brief guide to interpretation and action Category OD ratio Approx % seropos. Interpretation/Action Negative <0.10 <5 Naive/unvaccinated. Maintain herd security/quarantine measures. Consider blood testing purchases if IBR-free status is desirable. Monitor bulk milks every 3 months. Low positive 0.10-0.40 <20 Some seropositive cows but extensive active infection unlikely. Retest in 2 weeks/monitor bulk milks as above for major changes. Maintain herd security. Mid positive >0.40-0.70 20-60 Difficult to interpret. Re-test/monitor bulk milks or take individual blood or cohort bulk milk samples, particularly from young homebred unvaccinated cows as with BVD (Table 2). High positive >0.70 >60 Heavily infected/vaccinated. Significant virus circulation likely due to reactivation and re-excretion of latent infection. Clinical recrudescence may occur sporadically in unvaccinated herds - consider vaccination, especially heifers if seronegative. Table 4: Bulk milk antibody categories for L.hardjo with brief guide to interpretation and action Category OD ratio Approx % seropos. Interpretation/Action Negative <0.07 <2 Naive/unvaccinated. Maintain herd security/quarantine measures. Review risk factors. Monitor bulk milks every 3 months. Test/ antibiotic treat purchased replacements. Low positive 0.07 - 0.40 <10 A few seropositive cows but active infection unlikely. May be waning historical infection/vaccination in old cows or cross reaction with L.saxkoebing/hardjoprajitno. Retest in 2 weeks to check for recent infection, otherwise proceed as above. Mid positive >0.40-0.70 10-30 Difficult to interpret. Re-test/monitor bulk milks or take individual blood or cohort bulk milk samples, or blood sample a statistical cross section of the herd (BCVA 1992). High positive >0.70 >30 Heavily infected/vaccinated. If unvaccinated and no obvious risk factors, re-test immediately to confirm. If vaccination has lapsed, sample (blood or pooled milk) young unvaccinated cows for evidence of active infection. Vaccinate if appropriate. BCVA 1998 136

CATTLE PRACTICE VOL 6 PART 2 take three or four weeks to develop a positive ELISA reading (mainly IgG) compared with 10 days for the microscopic agglutination test (MAT) which mainly measures IgM. The presence of a small number of seropositive cows in an otherwise uninfected herd, with no obvious risk factors (shared bulls, frequent purchases, water courses, co-grazing with sheep) is not likely to be due to infection with L.hardjobovis unless the titre is historical or herd infection is very recent. A similar situation was not infrequently encountered when testing was carried out for membership of the Cattle Health Scheme formerly operated by MAFF and proved to be a problem when allocating so called “Elite” status. In these herds, as with negative herds, regular monitoring of bulk milks provides the reassurance that disease status has not changed and it also helps to fulfil the risk assessment required by the COSHH regulations. EFFECT OF VACCINATION The present tests do not distinguish between vaccinal and field exposure. There is little point in testing herds which are already carrying out a full vaccination programme because they will inevitably give a positive result. Where vaccination has lapsed (particularly relevant with L.hardjo), there may still be a high OD ratio reflecting historical exposure or waning vaccinal titres. In such cases, in order to check whether infection is still active, collect a pooled bulk milk sample from a cohort of 6-10 unvaccinated (usually younger) cows in the herd or collect individual blood samples from the same group. If these tests are all negative, infection is not likely to be currently active although regular ongoing monitoring of unvaccinated cows is still advisable. Often individual blood samples collected from unvaccinated young cows entering a herd previously infected with L.hardjo in which vaccination has ceased, will show very high MAT titres (≥1/400) confirming active recent infection in these naive animals. THE FUTURE Losses from BVD, IBR and L.hardjo are often subclinical and many veterinary surgeons and farmers are unaware of the disease status of their herds. Bulk milk tests offer an ideal opportunity to improve this situation and stimulate an interest in monitoring infections, adopting appropriate control measures including vaccination, and screening new stock (Pritchard 1996a). Opportunities for the future include bulk milk antibody tests for Salmonella typhimurium and Neospora caninum. Knowledge of a herd’s disease status is particularly relevant at present when many previously closed herds are now purchasing replacements as a consequence of the cull for bovine spongiform encephalopathy. ACKNOWLEDGEMENTS I am grateful to colleagues in the VLA and in general practice for providing milk samples from herds of known status and for useful discussions. Special thanks are due to staff of the VICs at Sutton Bonington, Bury St Edmunds, Langford and Winchester for carrying out laboratory work during the development of these tests and to CVL for the initial work on the BVD bulk milk ELISA. REFERENCES BCVA (1992) Guidelines for the diagnosis and control of Leptospira hardjo infection in cattle. BCVA, The Green, Frampton-on Severn, Gloucestershire. Hartman, A., Van Wuijckhuise, L., Frankena, K., Franken, P., Wever, P., De Witt, J. & Cramps, J. (1997) Within-herd BHV1- prevalence prediction from an ELISA on Bulk Milk. Veterinary Record 140 484-485. Niskanen, R. (1993) Relationship between the levels of antibodies to bovine viral diarrhoea virus in bulk tank milk and the prevalence of cows exposed to the virus. Veterinary Record 133 341-344. Paton, D. J., Christiansen, K., Alenius, S., Cranwell, M. P., Pritchard, G. C. & Drew, T. W. (1998). Prevalence of antibodies to bovine viral diarrhoea virus and other viruses in bulk tank samples from dairy herds in England and Wales (In press). Pritchard, G. C. (1996a) Added animals: The challenge to preventive medicine. Cattle Practice 4 253-258. Pritchard, G. C. (1996b) Elimination of infectious bovine rhinotracheitis virus infection from a heavily infected dairy herd. Proceedings of XIX World Buiatrics Congress, Edinburgh 8 - 12 July 1996, 49-53. Van Wuijckhuise, L., Bosch, J., Franken, P., Frankena, K. and Elbers, A. R. W. (1998) Epidemiological characteristics of bovine herpesvirus 1 infections determined by bulk milk testing of all Dutch dairy herds. Veterinary Record 142, 181-184. BCVA 1998 137

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CATTLE PRACTICE VOL 6 PART 2 Testing of Individual Cow’s Milk Samples to Ensure Health and Optimum Productivity Allen M.W., Hall E., Harris C., ON MeRiT Ltd. Compton Paddock Laboratories, PO Box 91, Newbury RG20 7HB + 18 Priest Hill, Caversham, Reading RG4 7RZ ABSTRACT The analysis of milk samples from individual cows,as well as being used regularly for mastitis control, the measurement of quality and progesterone assays, also offers a cheap and practical alternative to blood sampling. The concentrations of antibodies against Bovine Virus Diarrhoea virus, Infectious Bovine Rhinotracheitis virus, Respiratory Syncytial virus and Leptospirosis can be measured in individual cows and can form the basis of disease control programmes. The measurement of the concentrations of ß-hydroxybutyrate and urea in milk can provide a rapid practical method for the assessment of nutritional adequacy during the critical first 100 days of lactation. Together with assays of milk progesterone the measurement of metabolite concentrations can form an integral part of investigations into infertility and the maintenance of health, welfare and optimum productivity. The interpretation of variations in the concentrations of minerals and trace elements in milk is complex because for some (eg. Se and Pb) milk is an excretory route whereas for others (Mg and Ca) their concentrations in milk are controlled by homeostatic mechanisms. KEYWORDS: Individual milk samples. Antibody titre BVDV, IBRV, RSV, Leptospirosis. Progesterone. ßeta hydroxybutyrate. Urea. Minerals. Trace Elements. INTRODUCTION At any time in a dairy herd it is likely that the cows will be at various stages of lactation. Thus if a bulk milk sample from the whole herd is examined it will detect a true mean value only of the parameters which do not vary with the stage of lactation. For example, antibody concentrations are unrelated to the stage of lactation and bulk milk samples may therefore be of value for detecting and interpreting disease on a herd basis, whereas fat and protein concentrations which vary with stage of lactation can be applied to genetic selection only if they are measured in samples from individual cows. It has been common practice for more than 50 years to analyse milk from individual cows, for example to detect the causes of mastitis, but individual milk samples can now provide useful data for controlling disease; for assessing the metabolic state of individual cows, and for monitoring their fertility by measuring the concentrations of hormones. This paper considers the potential value of analysing individual milk samples for antibodies to Bovine Virus Diarrhoea virus (BVDV), Infectious Bovine Rhinotracheitis virus (IBRV), Respiratory Syncytial virus (RSV) and Leptospirosis (Lepto) to assess the disease status of herds, and for metabolites, including ßeta hydroxybutyrate, urea, and minerals and trace elements, to assess nutrition, and potentially to improve the fertility of cattle herds. Measurements of antibodies have been used to assess disease status for many years. The original milk ring test was used, with a stained antigen, in the brucellosis eradication scheme and a modified ELISA test is now used on bulk milk samples to act as an early warning of the disease. Similarly the control of enzootic bovine leukosis (EBL) depends upon an ELISA measurement on a sample of bulk milk. These two tests can also be applied to individual cows but, as such, they are used less frequently than tests for antibodies to BVDV, IBRV, RSV and Lepto. MATERIALS AND METHODS: The antibodies to BVD and IBR. were measured with an ELISA kit for milk or serum (Svanova Biotech, Uppsala, Sweden) and Leptospiral antibodies by an ImmunoComb technique for cow serum/milk antibody titres against Lepto. hardjo (ImmunoComb, Biogal Galed Labs, Israel). The ßeta hydroxybutyrate (BHB) concentration was measured in TCA deproteinised milk by UV method based on the change of absorbance when NAD is converted to NADH (Randox. RANBUT RB 1007) based on the method described by McMurray, C.H. et al (1984). Oestrone sulphate in milk was measured by ELISA kit (Ridgeway Scientific Ltd) and progesterone by an ELISA kit for milk and serum (Vétoquinol, UK Ltd). OBSERVATIONS AND RESULTS 1 BVDV The measurement of antibodies to BVDV in bulk milk and individual samples has been used in Scandinavia to assess the probable exposure of cows to the virus and to estimate the prevalence of the disease since 1992 (Niskanen, R., 1993). In this BCVA 1998 139

CATTLE PRACTICE VOL 6 PART 2 laboratory individual cows have been examined only since 1996. Figure 1 shows the numbers of cows in one herd with different ranges of antibody to BVDV in their milk. The herd had no detectable antibodies in the bulk milk (Group 1), but one cow and a number of heifers were antibody-positive. The cow with high concentrations of antibody (an optical density (OD) of >0.9) had been imported from the Netherlands several years earlier and the other seropositive animals were heifers which had recently been bought into the previously antibody-negative herd. No disease had been suspected, and it was concluded that the majority of the herd were susceptible to the importation of BVDV. It was advised that the young stock should be vaccinated against BVDV and the herd should be kept strictly closed, with no further importations of stock. BVD antibody detection in milk 0 20 40 60 80 100 120 0-0.1 0.1-0.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5-0.6 0.6-0.7 0.7-0.8 0.8-0.9 0.9 + O.D. range 450 nm Number of cows Bulk milk less than 0.1 n=124 Figure 1 In contrast, the herd illustrated in Figure 2, had a bulk milk BVDV antibody titre OD of. 0.555 (group 3). Although only a medium concentration of antibody was present in the bulk sample, every milking cow was already antibody-positive, many with high concentrations (20% of the herd OD > . 0.7), suggesting that the infection had recently spread through the herd. BVD antibody detection in milk 0 5 10 15 20 25 30 35 0-0.1 0.1-0.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5-0.6 0.6-0.7 0.7-0.8 0.8-0.9 0.9 + O.D. range 450 nm Number of cows n=154 Bulk milk OD 0.555 Figure 2 An intermediate example of an antibody distribution pattern for BVDV is shown in Figure 3. Among the seven milk antibody-negative cows, which were examined several weeks later for the presence of BVDV antigens, only one potential excretor of BVDV (a persistently infected animal - or PI) was identified and removed from the herd, but the other six animals had already sero-converted, presumably after being infected by the persistently infected cow. Before BVDV Control Schemes could be recommended on the basis of milk antibody measurements, it would be necessary to determine the duration of antibody production and consequent protection induced by vaccination. The identification of PI animals would also be a prerequisite. BVD antibody detection in milk 0 5 10 15 20 25 30 0-0.1 0.1-0.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5-0.6 0.6-0.7 0.7-0.8 0.8-0.9 0.9 + O.D. range 450 nm Number of cows n = 97 Figure 3 2 & 3 IBRV and RSV A much smaller number of herds has been examined by both bulk analysis and on an individual cow basis. IBRV antibodies were present in only 50% of the milk samples examined from individual cows. For RSV however, in the few samples examined at this laboratory, the prevalence has been 100%, suggesting a widespread exposure of animals in the UK to this virus. Similarly in Sweden (Elvander, M. 1996) a very high prevalence of antibodies to RSV has been observed in milk samples. The value of measurements of IBRV and RSV antibodies in individual milk samples for the diagnosis of disease outbreaks has been recognised in Sweden and the UK. 4 Leptospirosis It is more difficult to interpret the antibody status of a cow to leptospirosis because the antibody induced by a natural infection does not persist as consistently as the antibody to the case with the viral diseases discussed above. Similarly, an animal’s antibody response to vaccination appears to persist for variable periods. Nevertheless, it may be useful to identify antibody-negative herds which cannot be isolated and maintained as completely as closed herds, so as to advise vaccination. BCVA 1998 140

CATTLE PRACTICE VOL 6 PART 2 5 Metabolite Measurements It is easier and cheaper to collect individual milk samples than blood samples, although blood samples have been used for the assessment of energy deficiency and for fertility control for many years. The measurement of progesterone in milk rather than blood is accepted as the best method for resolving many problems of infertility eg. the detection of oestrus and the diagnosis of pregnancy (Darwash A O 1997). The measurement of oestrone sulphate in milk is used to confirm that a cow is pregnant. In the 1960’s feeding cows too little energy in the period up to 100 days post partum was identified as a major cause of infertility (McClure T.J. 1970, see also Webb R et al, 1997)) A high concentration of ketone bodies in body fluids is the consequence of a dietary energy deficiency and although this may be a ‘natural’ phenomenon during the first three to four weeks of lactation, a balance between the energy requirements for lactation and the dietary intake of energy should then be reached and the concentration of ketone bodies in blood should return to normal. If the cow remains energy-deficient any longer it is likely to be infertile and the concentrations of ketone bodies increase in its blood and milk. Acetone in milk has been measured widely in Sweden and the relationship between acetone concentrations in blood and milk is shown in Table I, as is the reported relationship between blood and milk BHB. Table 1 also includes our own observations on the relationship between blood and milk BHB. Table 2 summarises data illustrating the relationship between higher than normal acetone concentrations in milk and fertility. Table 1. Relationship between metabolites in blood & milk - Individual cows RATIO MILK:BLOOD Milk acetone : blood acetone (n = 67) 0.95 s.d. 0.20 Milk BHB : blood BHB (N = 104) 0.13 s.d. 0.12 ref: ANDERSON 1984,Zbl.vet med A 31 683 Milk BHB : blood BHB (N = 23) 0.18 on MeRiT Ltd BCVA 1998 Table 2. Hyperketonaemia & Fertility. K.P. Forshell 1994 Acetone concentration in milk Low <0.7 Mm High >1.4 mM Calving – first AI (days) 81.2 89.6 Calving – last AI 103.5 116.5 No. of inseminations 1.77 1.96 Ovarian cysts (%) 0.98 4.11 There are practical difficulties in measuring the concentration of acetone in milk (and blood), including its volatility and the need for specialist analytical equipment. We have therefore evaluated the method for measuring BHB in milk, and have shown that there is a consistent relationship between blood BHB and milk BHB, indicating that milk BHB concentrations are a valid indicator of blood BHB. (Figure 4). A concentration of 0.10-0.12 mmol/l milk is thought to represent the upper value of normality, and higher concentrations are considered to be associated with dietary energy deficiency. Our preliminary investigations of a herd with a problem of inapparent oestrus involved the measurement of the concentrations of progesterone and BHB in milk. (Figure 4). correlation between milk and serum beta hydroxybutyrate concentrations y = 4.7697x + 0.8392 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 milk BHB conc (mmol/l) ser um BH B co nc (m mo l/l) serum BHB Linear (serum BHB) R= 0.787 Blood = milk X 5 23 values Figure 5 shows the milk BHB concentrations in seven of the cows over a period of two to four and a half months after calving. Despite some improvement of energy intake the milk BHB concentrations remained high. There appeared to be consistent differences between individual animals, possibly because some animals were better able to cope with the energy insufficiency than others. Within the group the single animal with the consistently high milk BHB proved to have been affected with hepatic disorder. Mean concentration in Betahydroxybutyrate in 6 cows over 3 month period and one sick animal (mmol) 0 0.1 0.2 0.3 0.4 0.5 0.6 1 3 4 5 6 7 8 9 10 12 13 14 Weeks BHB Mean 6cows Cow 383 Started Sugar Beet increased Quota exceeded Concentrates reduced Maximum normal value Figure 5 141

CATTLE PRACTICE VOL 6 PART 2 Fundamental data are still required to establish the relationship between feeding and the requirements of the higher genetic potential of the “millennium” cow. Is the genetic potential for milk production exceeding our nutritional knowledge? It has been claimed that milk urea concentrations may also be related to fertility. A review entitled “Lets keep Milk Urea Tests in perspective’ has been published in the practical journal - Hoard’s Dairyman, USA, (Garcia, A. & Linn J., 1997). The authors concluded that unless a normal base-line concentration has been established on at least 75% of a herd intermediate changes in milk urea would be difficult to evaluate although extremely high or low values might be useful. Figure 6 shows the high concentrations of urea and BHB in the milk of ten cows 30-45 days post partum. In this herd the milk protein content had fallen, and whole cereal grains were seen in the dung. The consequence was probably a dietary deficiency of potential fermentable energy for the rumen microbes to utilise the ERDP (rumen degradable protein) for the synthesis of microbial protein. BCVA 1998 Figure 6 Milk test : betahydroxybutyrate and urea 0 0.1 0.2 0.3 0.4 0.5 0.6 12 28 36 82 135 154 186 220 289 302 cow number BHB concentration (mmol/l) 0 2 4 6 8 10 12 14 16 18 Urea concentra (mmol/l) BOH Urea Table 3 summarises the likely consequences of an imbalance between the intakes of energy and protein. Table 3. Metabolite concentrations in milk 5th – 7th week of lactation Milk BHB Urea concentration Mm Concentration Low <3.5 Medium 3.5 – 6.0 High >6.0 Normal <0.1 mM Restricted intake of RDP Excess RDP or NPN High >0.1 Mm Restricted intake of energy Restricted intake of energy Excess RDP and/or restriction of energy 6 Minerals And Trace Elements In Milk Milk contains all the major minerals and trace elements. The major minerals are present in concentrations which are generally maintained within narrow ranges by homeostasis (ie. the minerals do not simply diffuse from blood to milk but are subject to active transport mechanisms which regulate their concentrations in the milk). During mastitis the inflammatory process within the gland affects secretory mechanisms causing changes in mineral concentrations. In particular the concentrations of sodium and chloride in the milk increase considerably, and as a result the conductivity of the milk increases. Recently a service measuring the concentrations of trace elements in milk has become available. Table 4 lists the average concentrations observed in milk. However, in contrast to the major minerals the concentrations of trace elements in milk do not appear to be under strict homeostatic control and high concentrations in blood often result in high concentrations in milk. For example, Figure 7 illustrates the concentration of selenium in the milk of a cow given a parenteral dose of a soluble selenium salt. Table 4.Trace Elements micromoles per litre In Milk In Blood Iron 9.5 30 Copper 1.9 12 Selenium 0.25 0.6 Lead 0.1 1.2 Manganese 0.45 0.1 Zinc 50 >10.0 Figure 7 Selenium in Milk following Injection 0.15mg/kg Se per kg bodyweight 0 50 100 150 200 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 day 8 Injected control Se ng/ ml Similarly high intakes of lead may be excreted in the milk of lactating cows. By contrast there is little evidence that low concentrations of a trace element in individual milk samples might indicate that the intake of the element is deficient. There may be homeostatic mechanisms that control the concentration of at least some of the trace elements in milk, and it would be unwise to interpret the concentrations of trace elements in milk too literally. CONCLUSION In addition to the long established quality control measurements such as somatic cell counts and bacteriological examinations, measurements of antibody concentrations and many metabolites and minerals can provide useful information at minimum 142

CATTLE PRACTICE VOL 6 PART 2 cost for improving the productivity, health and welfare of dairy cows. ACKNOWLEDGEMENTS For the compilation of data relating to individual samples to: R.W. Bostelmann, MA, VetMB, MRCVS Staffordshire; N.B. Cook, BVSc, BSc, Cert.CHP, MRCVS Royal Veterinary College; A.R.J. Davies, BVetMed,CCertCHP,MRCVS Dorset; M.E.Glover, BVSc, MRCVS Yorkshire C.M. Gould, MA, BSc, MRCVS Hampshire; N.F. Partridge, BVetMed, MRCVS Berkshire and R Mack for library info. REFERENCES Andersson L., (1984). Concentrations of Blood and Milk Ketone Bodies, Blood Isopropanol and Plasma Glucose in Dairy Cows in Relation to the Degree of Hyperketonaemia and Clinical Signs. Zbl. Vet. Med. A, 31 683-693. Darwash A O., (1997). Strategic Milk Progesterone Monitoring. Cattle Practice, BCVA< 5 353 - 355. Elvander M., (1996). Severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus. Vet.Rec. 138 101 Forshell K.P., (1994). Metabolic profiles in milk. Swedish Association for Livestock Breeding and Production. Sept 15th p526 - 536. Garcia A. & Linn J., (1997). Lets keep milk urea test results in perspective. Hoard’s Dairyman, USA, Aug.25 1997, pp.597 McClure T.J., (1970). An Experimental Study of the Causes of a Nutritional and Lactational Stress Infertility of Pasture-fed Cows, Associated with Loss of Bodyweight at about the Time of Mating. Res.vet.Sci. 11 247 McMurray C. H., Blanchflower W. J. & Rice D.A., (1984). Automated Kinetic Method for D-3-Hydroxybutyrate in Plasma or Serum. Clin Chem, 30 No 3, 421-425. Niskanen R., (1993). Relationship between the levels of antibodies to bovine viral diarrhoea virus in bulk tank milk and the prevalence of cows exposed to the virus. Vet.Rec. 133 341-344. Webb R. et al (1997). Nutritional Influence on subfertility in cattle. Cattle Practice, BCVA, 5 361-367. BCVA 1998 143

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CATTLE PRACTICE VOL 6 PART 2 Use of Trans-Thoracic Fine Needle Aspiration in the Investigation of Chronic Bovine Respiratory Disease Sturgeon B.P.R*., Doherty M.L*., Healy A.M*., Larkin H.A**., *Dept. of L.A. Studies, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. ∗∗ Dept. of Veterinary Pathology, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. ABSTRACT The diagnostic and operative techniques for trans-thoracic fine needle aspiration (TFNA) are discussed. Twelve cattle with chronic respiratory disease were investigated using this technique. Cytological examination of aspirates revealed a range in the pattern of cellular infiltration which included neutrophils, eosinophils and macrophages. In conjunction with a thorough clinical examination the results provided valuable information concerning stage of disease, choice of treatment and prognosis. KEYWORDS: Cattle, respiratory disease, transthoracic fine needle aspiration. INTRODUCTION Investigation of chronic respiratory disease in the adult bovine animal can be an unrewarding and difficult task for the practitioner. Few individual diagnostic techniques offer a high degree of sensitivity and the obvious signs of respiratory disease such as dyspnoea, tachypnoea, mouth breathing, inspiratory and/or expiratory noise and coughing may not always be evident. Of the techniques available auscultation may be unreliable as there may be little correlation between audible sounds and the severity of the lesion. For percussion to be diagnostic, lesions must be extensive before differences from the norm can be detected. Practical difficulties such as patient restraint and stress, tube entry into the trachea, and client compliance are some of the difficulties encountered in the routine use of broncho alveolar lavage and transtracheal lavage. In addition, the anatomy of the lower airways often results in the tube becoming located in the right diaphragmatic lobe, while in most cases of infectious pneumonia it is the anterior lung lobes which are involved initially. Further techniques include viral isolation from nasopharyngeal swabs and routine haematology which may add additional diagnostic and prognostic information. Arterial blood gas analysis is of limited use in the investigation of respiratory disease. Endoscopy, ultrasonography and radiology, although useful on occasion, entail the use of expensive equipment which may not be available for field cases. Trans-thoracic fine needle aspiration permits collection of material from the thoracic cavity to aid in the diagnosis of inflammatory or neoplastic conditions. It is a common diagnostic procedure in man (Kealey and others 1989) and there have been several reports of its use in small animals (Roudebush 1981, Rebar and de Nicola 1988, Teske and others 1991). It is a simple technique and is well tolerated by dyspnoeic patients. Fresh material can be collected free from contaminant cells, mucus and plant material. Examination of stained preparations gives information on the type and stage of the disease process and gives an indication of prognosis. In addition culture may be carried out on any collected material. This study reports on the contribution made by TFNA in the investigation of 12 cases of bovine respiratory disease. MATERIALS & METHODS The 12 cattle in the present study had been referred to the Veterinary Teaching Hospital, UCD during 1997. Clinical histories varied greatly; in addition to the obvious signs of respiratory disease such as coughing, tachypnoea, hyperpnoea and dyspnoea, several animals were initially presented with vague histories such as decreased milk yield, depression or potentially misleading histories such as acute colic. After preliminary examination all the cases were investigated for respiratory disease. A total of 12 animals were included in the study, all were over 10 months of age, and all had been affected for between 2-3 weeks and had recieved various antibiotic and anti-inflammatory treatments prior to admission. A clinical examination of each animal was performed daily. All animals which died or were euthanased were necropsied. BCVA 1998 145

CATTLE PRACTICE VOL 6 PART 2 SAMPLES AND ANALYSES Clinical Pathology Conventional haematological examinations were made from blood samples taken from the jugular vein immediately after admission to the hospital. Blood was collected in an EDTA tube and the haematological analyses carried out with a Coulter Counter (M530, Coulter Electronics Ltd.) particle counter. Plasma fibrinogen was determined using the routine heat precipitation technique (56°C) followed by refractometry (Benjamin, 1979). Packed cell volume (PCV), haemoglobin (Hb) concentration, red blood cell (RBC) count, and total and differential leucocyte counts were recorded. Plasma total protein concentration was determined using the biuret method (Boehringer Mannheim GmbH), and the albumin concentration with the bromcresol green reaction (BioMerieux) using an automated analyser (Cobas Mira Roche.). Plasma globulin concentration was calculated by subtracting the albumin concentration from the total protein concentration. Diagnostic Imaging Radiological examination of the thorax was performed in five of the cases. The animals were standing and a rare earth screen film combination (Kodak Lanex screen and Kodak film) and a focus grid were used. Exposure factors were 70-90 KV and 20-40 MAS. Ultrasonographical studies of the lungs and pleura were performed in three cases. To facilitate this procedure the area between the fifth and ninth intercostal spaces on right and left sides were closeclipped and an acoustic coupling gel (Aquasonics) applied. A 2.5 MHz or 5 MHz transducer was used. An ausonics Microimager 100 ultrasound machine in combination with video recordings (Panasonic) were used to obtain and store the images. Blood Gas Analysis Examinations were made from blood samples taken from the auricular artery in to a heparinised syringe, following clipping and application of surgical spirit, and analysed on an ABL 300 Blood Gas and AcidBase Analyser (Radiometer). Partial pressure of carbon dioxide (PCO2) and oxygen (PO2), acid-base analysis [ pH, bicarbonate (HCO3−), total carbon dioxide (TCO2), actual base excess (ABE), standard base excess (SBE) and standard bicarbonate (SBC)] were recorded. Broncho Alveolar Lavage (BAL) BAL was performed in four cases. A Pentax FC38LH 150cm flexible endoscope was introduced into the trachea via the nostril for visual examination and lavage of the trachea and bronchi. Trans-thoracic Fine Needle Aspiration All animals were standing and restained in a cattle crush. TFNA was carried out on all 12 cattle according to the protocol described below. Before collection, a platelet count, prothrombin and thromboplastin times were determined. A five centimetre diameter circle at the sixth to eighth intercostal space on the right hand side at the level of the costochondral junction was clipped and surgically prepared. Local anaesthetic was injected beneath the skin and into the intercostal muscles. The skin was nicked using a scalpel and a seven inch 21G needle was introduced into the muscle. A 10ml syringe was attached and the plunger pulled back. The needle was then introduced smoothly into the lung at a right angle to the chest wall, was almost removed and then the thrust repeated dorsally and ventrally. If the syringe started to fill with blood, the sample was discarded and the procedure repeated. The needle was then removed, the syringe detached, filled with air and reattached to the needle, the contents of the needle were then expelled forcibly onto a slide, smears were prepared and air dried. If fluid was aspirated this was placed in EDTA and Sterilin tubes. The samples were submitted for cytology (Gram and Leishman stains) and culture (Blood and MacConkey agar). The syringe and needle were also submitted, to permit culture if indicated by cytology. The animal was monitored (respiration, heart rate, mucosae) half hourly for three hours and then hourly for a further six hours following the procedure. Controls Smears were prepared from 20 fresh cadaver specimens to act as a control population. Aspirates were taken from the right cranial lung lobe within 30 minutes of slaughter from healthy adult cattle. RESULTS The results of the clinical investigations are summarised in Table 1. Clinical Examination Six of the cases were referred with a history of respiratory disease. On examination all 12 had variable degrees of respiratory system involvement. Haematology and Biochemistry Nine cases demonstrated inflammatory changes (fibrinoginaemia and/or globulinaemia) and four had an additional leucocytosis. Blood Gas Analysis Seven cases had hypoxia (PO2 < 80 mmHg) and four of these had a concurrent hypercapnia (PCO2 > 45). BCVA 1998 146

CATTLE PRACTICE VOL 6 PART 2 TABLE 1: Summary of the Clinical Investigation of Twelve Cases of Respiratory Disease Case / Case N° Presenting Complaint Clinical Examination Clinical Pathology Blood Gas Analysis X-ray/ U/sound BAL 10m Char bull 1 Respiratory distress TC, TP, harsh bilateral lung sounds, nasal discharge Fib 10 Glob 54 PO2 68.1 PCO2 58.2 NA NA 10m Sim bull 2 Depression, grunting Lung noise bilaterally, TP Fib 14 Glob 62 PO2 72.8 NA NA 19m Fries bull 3 Submandibular swelling Depressed, TC, TP, muffled lung sounds, jugular distension, pale mucous membranes NA PO2 77.2 Echogenic opacity on the right thoracic wall compressing the heart NA 2m Fries heifer 4 ↑ respiratory rate, bruxism, lethargy TP, ↑ lung sounds bilaterally, lethargy and stiffness Fib 10 Glob 59 PO2 98.1 PCO2 21.7 NA NA 10m Char bull 5 Coughing and ↑ respiratory rate Nasal discharge, TC, TP, crackles and wheezes bilaterally PCV 49 Fib 10 WBC 28.1 Glob 60 PO2 44.8 PCO2 64.1 Severe alveolar and interstitial changes with multiple abscessation NA 4 y Fries cow 6 RDA, grunting, ↑ respiratory rate TC, TP, thoracic fluid sounds, muffled heart sounds Fib 9 Glob 46.8 WBC 28.4 PO2 54.2 PCO2 62.1 Fluid in right hemithorax NA 4y Fries. cow 7 Depression, pain, sweating, restlessness TC, TP, ↑ normal lung sounds Fib 8 PO2 70.6 Prominent bronchial pattern NA 18m Char bull 8 Cough and nasal discharge TC, TP, ↑ normal lung sounds bilaterally Fib 9 Glob 69 NA NA Mostly epithelial with many columnar, occasional WBC 10y Angus cow 9 Cough and ↑ respiratory rate TP, crackles and wheezes bilaterally Fib 14 NA Severe mixed interstitial and alveolar pattern Mostly cell debris, few degenerate epithelial cells. Short chains of bacilli. 2y Fries. Cow 10 Occasional cough, drop in milk yield ↑ bilateral normal lung sounds WBC 18.1 Glob 63 PO2 54.3 Interstitial pattern Mostly columnar epithelial cells, few Eo 4y Fries cow 11 weight loss TP, muffled heart sounds WBC 13.5 NA Fluid in pleural space NA 2 y Fries cow 12 Occasional cough, ↑ respiratory rate TP, normal lung sounds bilaterrally Normal PO2 82.4 PCO2 16.6 Mild mixed interstitial and alveolar pattern Parasitic larvae and eggs Diagnostic Imaging Radiology identified pathological changes of the lung in five cases and ultrasonography was used in three further cases to visualise fluid. BAL Epithelial cells with occasional WBC’s predominated in three BAL samples. One further case detected parasitic larvae and eggs. TFNA and Interpretation Table 2 summarises the results of investigation using TFNA and necropsy findings. Quantitative assessment of smears collected from cadaver specimens was not possible because of the presence of significant numbers of damaged cells and bare nuclei. As a result a qualitative description of control smears and smears made from clinical cases was performed. 1. Controls The following cell types were found on the control smears : • Varying numbers of red blood cells, • Small clusters of epithelial cells; some of these were columnar, goblet cells, and others were round cells possibly from bronchiolar epithelium, • There was a mixed population consisting predominantly of macrophages and lymphocytes with occasional neutrophils, eosinophils and basophils, • There were occasional clumps of mesothelial cells. 2. Clinical Cases Cytological examination of aspirates collected by TFNA revealed six cases with bacteria and karyolysed cells. Of the remaining six; three contained a mixed population of cells including macrophages, some WBC and epithelial cells; one contained epithelial cells and eosinophils, one BCVA 1998 147

CATTLE PRACTICE VOL 6 PART 2 TABLE 2: Summary of the Investigation Using TFNA and Necropsy Findings Case N° TFNA Culture Interpretation Outcome / P.M. 1 Mostly bacteria, many karyolysed cells A.pyogenes Septic inflammation Died - necrotising pneumonia 2 Karyolysed Ne and bacterial clusters NA Septic inflammation Euth - necrotising pneumonia 3 Bacteria, few lysed cells Proteus spp A.pyogenes Septic inflammation Euth - large cavitated abscess 4 Karyolysed Ne and bacteria NSG Septic inflammation Discharged without recovery 5 Bacteria and karyolysed cells A.pyogenes Septic inflammation Euth - abscess and emphysema 6 Bacteria, few lysed cells A.pyogenes Septic inflammation Euth - pyothorax secondary to traumatic reticulitis 7 Mainly reactive macrophages, some Ne and epithelial cells NA Chronic inflammation Clinical recovery 8 Ne and some Eo NSG Inflammation Salvaged - no follow up 9 Mixed population (degenerate cells, Ne, M∅) NSG Chronic inflammation Clinical recovery 10 RBC's, epithelial cell clusters with Eo / mast cells NSG Hypersensitive / parasitic change Clinical recovery 11 M∅ and mesothelial cells Few Ne No growth Pleural fluid Euth - cardiomyopathy 12 Connective tissue and RBC’s, variable bacterial population NA Poor sample, not diagnostic Clinical recovery Key RBC Red blood cells WBC White blood cells (norm 4-14x1000/uL) Glob Globulin (norm 31-45g/dL) PCV Packed cell volume (norm 24-46%) Fib Fibrinogen (norm 3-7 g/L) PO2 Partial pressure arterial oxygen (norm 80-110 mmHg) PCO2 Partial pressure arterial carbon dioxide (norm 35-45 mmHg) TC Tachycardia TP Tachypnoea NA Not applied MØ Macrophage Ne Neutrophils Eo Eosinophils NSG No significant growth PM Post mortem Euth Euthanase RDA Right displaced abomasum m Months y Years contained pleural fluid and one specimen was not diagnostic. The working interpretation of these findings was septic inflammation for the former; chronic inflammation, hypersensitivity/lungworm and pleural transudate respectively. Culture Actinomyces pyogenes was identified in three cases, one with an additional Proteus species. Outcome Four made full recoveries and were discharged. Two cases were salvaged by the owner and no follow up was achievable. Five cases were euthanased and one died overnight. Necropsy Full post-mortems were carried out on six cases; necrotising pneumonia was diagnosed in two cases with a large cavitated abscess, abscessation and emphysema, pyothorax secondary to traumatic reticulitis, and excessive pleural fluid secondary to a cardiomyopathy were identified in the remaining cases. DISCUSSION The procedures of clinical examination, clinical pathology, blood gas analysis and diagnostic imaging techniques were limited in their ability to identify the aetiological agent, offer a prognosis or indicate treatment selection. In the cases described in this BCVA 1998 148

CATTLE PRACTICE VOL 6 PART 2 report information provided by TFNA gave a means of differentiation between such cases. In six cases (1, 2, 3, 4, 5, and 6) karyolysed neutrophils with clusters of bacteria were identified. These were interpreted as septic inflammatory pneumonia. Treatment was withheld from these cases given the chronic and progressive nature of the disease. Post-mortem examination of five of these cases confirmed the extensive nature of the lesions which would have been refractory to conventional treatment. In contrast the presence of macrophages and neutrophils in the absence of karyolysis and bacteria in cases 7, 8 and 9 was consistent with a non-septic pneumonia and a more favourable prognosis. Two of these cases achieved a clinical recovery and one was salvaged. Of the remaining cases, in case 10 eosinophils were the predominant cell type and this was interpreted as a hypersensitivity reaction. Although lungworm would be considered the commonest aetiological agent allergic pneumonitis could not be discounted. Clinical response to Levamisole may be given as reasonable evidence that verminous pneumonia was the most likely diagnosis in that case. In case 11 aspirated cells consisted predominantly of mesothelial cells. This was interpreted as a pleural transudate (examination of the fluid also detected low protein <2.5 g/dL consistent with a transudate). Postmortem identified a cardiomyopathy. The final case (case 12) identified a variable bacterial population with RBC’s and connective tissue. This was considered a non-diagnostic sample. However follow up BAL identified parasitic larvae and eggs indicating that TFNA lacks some sensitivity. BAL may be required in such cases. LIMITATIONS AND PROCEDURE COMPLICATIONS TFNA may be employed where diffuse pulmonary pathology of unknown aetiology is suspected. Although not all pulmonary diseases are diffuse in nature the chances of sampling diseased lung are enhanced by the sampling of the right cranial lobes as inhaled pathogens locate initially in this area before infiltration of the remainder of the lung. No evidence of iatrogenic damage was found at necropsy. The main theoretical complication would be shearing, with possible haemothorax, of the lung by the needle due to movement of the lung within the thorax. In practice the narrow gauge of the needle resulted in its bending with normal lung motion reducing the likelihood of iatrogenic damage. Pneumothorax would also be a possible complication although the narrow gauge of the needle reduced this risk. CONCLUSION TFNA is a relatively simple, safe and cheap technique which may be employed by the general practitioner. Patient and client compliance is good and results may be immediately visible. The information gained provides a valuable ancillary aid to the diagnosis of chronic thoracic disease in cattle and gives information concerning the stage of the disease. In the context of a thorough clinical examination TFNA provides useful information relating to the prognosis and possible treatment strategy. REFERENCES Benjamin, M.M. (1979). Fibrinogen. Outline of Veterinary Clinical Pathology, 3rd ed, pp. 116-120. Ames, Iowa : The Iowa State University Press. Kealey, W.F., Hogan, J.M. and Hurley, M.F. (1989). Percutaneous fine needle aspiration of pulmonary mass lesions - a critical examination of the accuracy of cell typing of malignant tumors. Irish Journal of Medicine and Surgery, 158, 85 - 87. Roudebush, P. (1981). Percutaneous fine needle aspiration biopsy of the lung in disseminated disease. Journal of the American Animal Association, 17, 109 - 112. Rebar, A.H. and de Nicola, D.B. (1988). The cytological examination of the respiratory tract. Seminars in Veterinary Medicine and Surgery, 3, 109 - 121. Teske, E., Stokhof, A.A., van den Ingh, T., Slappendel, R.J., de Vries, W. (1991). Transthoracic needle aspiration biopsy of the lung in dogs with pulmonic diseases. Journal of the American Animal Hospital Association, 27, 289 - 294. ACKNOWLEDGMENTS My thanks to Mary Duane for her nursing skills. To Mary O’Donaghue and Maureen Inglis for their typing, and Susan Sturgeon for her bent ear. BCVA 1998 149

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CATTLE PRACTICE VOL 6 PART 2 Ultrasonographic Examination of the Bovine Thorax Scott P.R., Royal (Dick) School of Vet. Studies, Easter Bush, Roslin, Midlothian EH25 9RG ABSTRACT Ultrasonographic examination of the thorax of 15 healthy cattle using a 5.0 MHz sector transducer revealed the lung surface as a hyperechoic (white) linear echo at a distance of 2.0 to 3.5 cm from the transducer head with equidistant reverberation artefacts below this line. Ultrasonographic examination of 12 cattle with chronic suppurative pneumonia revealed an irregular visceral pleura interspersed with hypoechoic areas extending up to 5 cm into the lung parenchyma. Pleura effusion, secondary to diffuse fibrosing alveolitis, cardiomyopathy and thymic lymphosarcoma, was readily identified as an extensive anechoic area. The consolidated ventral lung, with the sonographic appearance of liver, could be readily visualized displaced dorsally in these cattle with pleural effusion. Modern portable ultrasound machines provide the veterinary practitioner with an inexpensive, non-invasive tool with which to examine the pleural surfaces and superficial lung parenchyma of cattle on the farm. Ultrasonographic examination of the chest is helpful in chronic bovine respiratory disease investigations but it can prove difficult to differentiate lung abscessation from consolidation. Pleural effusion is readily detected by ultrasonography which aids thoracocentesis and establishment of a definitive diagnosis. KEYWORDS: Ultrasonography, Thorax, Respiratory Disease, Cattle INTRODUCTION Chronic respiratory disease is common in growing and adult cattle but the severity of the condition cannot always be diagnosed by clinical examination alone. Changes in fibrinogen and serum protein concentrations may indicate chronic inflammation in response to bacterial infection (Henshaw 1993) but these changes are not specific for respiratory disease. The investigation of respiratory disease using radiographic examination of the thorax is expensive and restricted to individual valuable pedigree cattle referred to veterinary schools. Furthermore, the position of the thoracic limbs and associated musculature in the standing animal largely restricts radiographic examinations to the caudo-dorsal thorax when pathological changes associated with aerogenous infection more commonly involve the cranioventral lung field. Ultrasonography in many farm animal veterinary practices has been restricted to early pregnancy diagnosis despite reports detailing the considerable information that can be obtained by ultrasonographic studies of the abdomen (Braun et al., 1995), liver (Braun et al., 1996a), and thorax (Braun et al., 1996b; Braun et al., 1997; Scott et al., 1997). Diagnostic ultrasonography of the equine thorax has become a particularly valuable aid to clinical investigation of the pleura and superficial lung tissue (Reimer, 1990). This report describes the ultrasonographic findings of the normal bovine thorax and some common respiratory conditions encountered in a farm animal practice in south east Scotland, and cattle referred to the Veterinary Teaching Hospital in Edinburgh. Some potential applications of ultrasonographic examination of the bovine thorax are discussed. The limitations of ultrasonography in cattle practice are emphasised. MATERIALS AND METHODS Animals Fifteen healthy yearling and adult cattle with no history or clinical signs of respiratory disease were examined using a 5.0 MHz sector transducer connected to a real-time B-mode ultrasound machine (Ausonics Opus 1). Twelve cattle with clinical symptoms of chronic suppurative pneumonia including intermittent mild pyrexia (39.0 to 39.5C) slight mucopurulent nasal discharge, productive cough, ill-thrift, tachypnoea with hyperpnoea at rest, wheezes distributed cranio-ventrally, and reduced resonance detected by percussion were examined ultrasonographically on two or more occasions at least one week apart. The definitive diagnosis was established at necropsy in six cattle and supported by histopathological and microbiological examinations as appropriate. Extensive pleural effusion was associated with individual cases of diffuse fibrosing alveolitis, dilated cardiomyopathy and thymic lymphosarcoma. Ultrasonographic examination A 5.0 MHz sector transducer connected to a realtime, B-mode ultrasound machine (Ausonics Opus 1) and thermal printer (Sanyo) were used for the ultrasonographic examinations in the present study. A 7 cm wide strip of hair was carefully clipped from both sides of the thorax extending in a vertical plane from the point of the elbow to the caudal edge of the scapula. The skin was soaked with warm tap water BCVA 1998 151

CATTLE PRACTICE VOL 6 PART 2 then ultrasound gel was liberally applied to the wet skin to ensure good contact. The transducer head was firmly held at right angles against the skin overlying the intercostal muscles of the 6th or 7th intercostal spaces. The most cranial aspect of the thorax was scanned by moving the transducer head cranially from the 6th or 7th intercostal space into the next more cranial intercostal space once or twice as the tranducer was moved down the chest wall. The ipsilateral forelimb was held forward to facilitate access to the ventral aspect of the thoracic wall. Alternatively, the ipsilateral foot was placed on a 15 cm high wooden block to allow greater access to the more cranio-ventral areas of the chest. The dorsal lung field was selected at the start of all ultrasound examinations in an attempt to visualize normal lung tissue as this area was less commonly affected in the bovine respiratory disease processes previously studied. At this stage the depth of chest wall was noted for future reference. The caudo-dorsal aspect of the thorax was examined by moving the transducer head two or three intercostal spaces more caudally from the 6th or 7th intercostal space to the 9th intercostal space. The ultrasonographic examinations in this study were made with a depth setting of 7 to 20 cm which included 2 to 3.5 cm of chest wall. Good contact between the transducer head and skin overlying an intercostal space was evident by the breadth and intensity of the ultrasound image. Interpretation of ultrasonographic findings The sonograms are presented with the chest wall at the top of the image, dorsal is to the right and ventral to the left of the image. An air interface, created by aerated lung parenchyma reflects sound waves and appears as a bright white (hyperechoic) linear echo. The sonogram below the white linear echo may contain equidistant reverberation artefacts. The area visualized below the linear echo, including the reverberation artefacts, does not represent lung parenchyma thus the initial ultrasound machine setting should be 6 to 7 cms (ie approximately 3 cms of chest wall) then pleurae, and superficial lung parenchyma. Air contained within a major airway in consolidated lung appears as a small hyperechoic area or band within the hypoechoic lung parencyma. Superficial areas of consolidated lung parenchyma or abscess transmit sound waves and appear more hypoechoic than surrounding lung tissue. Pleural fluid transmits sound waves readily and appears as an anechoic area. Gas-filled pockets within pleural fluid or abscess capsule appear as bright hyperechoic spots within the black (anechoic) fluid. Ultrasound-guided thoracocentesis Ultrasound-guided thoracocentesis was undertaken in 3 cattle with pleural effusion using 2 inch 19 gauge hypodermic needles. The overlying skin was surgically prepared and the intercostal muscles infiltrated with 2 per cent lignocaine solution (Lignavet; CVet). RESULTS The preparation of both sides of the thorax for ultrasonographic examination, including clipping the hair took approximately five minutes per animal. A further improvement in image quality was obtained if the overlying skin was shaven with a scalpel blade but this was not always needed. It was not necessary to use alcohol to remove natural oils/grease from the skin in order to achieve good quality sonograms. The ultrasonographic examination took about 10 minutes. Normal cattle The surface of normal aerated lung (visceral or pulmonary pleura) of 15 normal cattle was characterized by the uppermost white linear echo with one or two equally-spaced reverberation artefacts below this line. In normal cattle (200 to 600kg) the visceral pleura could be observed moving 2 to 4 mm in a vertical plane during respiration. No pleural fluid was visualized in the 15 normal cattle. The chest wall was approximately 2 cm thick in 200 kg calves extending to 3.5 cm in adult cattle in average body condition (body condition score 3 [scale 1 to 5]). Chronic suppurative pneumonia There was a marked irregularity of the white linear echo formed by the normal visceral (pulmonary) pleura in the antero-ventral chest of cattle with chronic suppurative pneumonia. Numerous short and irregular bright white (hyperechoic) lines could be readily visualized 2 to 6 cm from visceral pleura. These hyperechoic lines resulted from the increased sound wave transmission through more consolidated tissue (acoustic enhancement) relative to the surrounding normal lung parenchyma. It was not possible to determine by ultrasonographic examination whether the hypoechoic areas in the superficial lung tissue were either abscesses or cellular infiltration and consolidation in a lobular pattern. Indeed, few superficial lung abscess were observed at post mortem examination and the hypoechoic areas identified by ultrasonographic examination more commonly represented consolidated lung parenchyma usually with a discrete lobular appearance. Discrete lobular hypoechoic areas in the superficial lung parenchyma were visualized ultrasonographically in a cow with uterine lymphosarcoma and metastatic spread to the lungs. In some areas the visceral pleura did not move during respiration and this abnormality was attributed to adhesion between the parietal and visceral pleurae. In two cattle the visceral pleura (bright linear echo) BCVA 1998 152

CATTLE PRACTICE VOL 6 PART 2 appeared thicker than normal and was displaced 2 to 3 cm from the parietal pleura by an area of varying hyperechogenicity. At postmortem examination this area corresponded to marked fibrin exudation (pleurisy) extending up to 3 cm thick between the parietal and visceral pleurae and probably related to an earlier disease episode caused Pasteurella spp. Pleural effusion Extensive pleural effusion was associated with individual cases of diffuse fibrosing alveolitis, dilated cardiomyopathy and thymic lymphosarcoma. In two cases (DFA and thymic lymphosarcoma) the pleural effusion was primarily unilateral. In each case reduced resonance was detected on percussion of the chest but the true extent of the effusion could not be determined. The effusion extended up to 17 cm (3 cm of chest wall) and possibly beyond only the depth of field with the ultrasonographic equipment was limited to 20 cm. In all three cattle the ventral lung in the side of the chest with the pleural effusion was consolidated and displaced dorsally with the ultrasonographic appearance of liver. Ultrasound-guided thoracocentesis Ultrasound-guided thoracocentesis was relatively easy to perform with a 2 inch 19 gauge hypodermic needle when the animal was restrained in cattle stocks. After establising the aspirate as a transudate, 27 litres of fluid was removed from the right hand side of the thorax of a cow with DFA using a 12 gauge chest drain. The following day ultrasonographic examination revealed that much of this fluid had been replaced. DISCUSSION Ultrasonographic examination of the bovine chest is inexpensive, non-invasive, and unlike radiography there are no special health and safety procedures or restrictions. Furthermore, as the equipment is readily transportable ultrasonographic examinations can be performed on the farm. Ultrasonographic examination of the chest of normal cattle was wholly unrewarding as the sound waves are reflected by aerated lung forming a bright linear echo. No ultrasongraphic examinations were made in calves or growing cattle with acute respiratory disease. There was the tendency for this operator to exaggerate the significance of the ultrasonographic changes observed in cases of chronic suppurative pulmonary disease. The hypoechoic areas on the ultrasonograms more commonly represented areas of consolidation than abscess formation. Abscesses involving the pleura were clearly visualized during ultrasonographic examination in 5 lambs (Scott and Gessert 1998) and in horses with pleuropneumonia (Reimer, 1990) but appear to be much less common in cattle unless caused by migrating reticular foreign bodies (Braun et al., 1997). Many veterinary practices have purchased 5.0 MHz linear transducers primarily for early pregnancy diagnosis. While the present studies were undertaken with a 5.0 MHz sector scanner, a 5.0 MHz linear transducer was also used in some cases. Linear scanners are restricted to a lung parenchyma depth of 7 cm (3 cm of chest wall) however this should not present a problem because most pathological changes include the superficial lung parencyma. Pleural effusion can be readily detected by linear scanners but the true depth may be underestimated. Once again this limitation of image depth should not significantly alter the clinician’s interpretation as 10cm of fluid in the chest is an important finding and thoracocentesis would be indicated in such cases to differentiate between exudate or transudate. In most veterinary practice situations diagnosis of bovine respiratory disease will continue to be based upon history, clinical examination, response to antibiotic therapy, and in unsuccessful cases by post mortem examination. Ancilliary diagnostic aids such as radiography and ultrasonography will be limited to valuable pedigree animals where either a definitive diagnosis or extent of the lesion(s) is required. However, as the cost of an ultrasonographic examination only involves the veterinarian’s time, enthusiastic clinicians are encouraged to try this technology as part of their clinical investigation of chronic respiratory disease. REFERENCES BRAUN U., MARMIER O., & PUSTERLA N. (1995). Ultrasonographic examination of the small intestine of cows with ileus of the duodenum, jejunum or ileum. Veterinary Record 137 209-15. BRAUN U., PUSTERLA N., & WILD K. (1996a). Ultrasonographic examination of liver, gallbladder in cows: abnormal findings. Compendium on Continuing Education 18 1255-69 BRAUN U., SICHER D., & PUSTERLA N. (1996b) Ultrasonography of the lungs, pleura and mediastinum in healthy cows. American Journal of Veterinary Research 57 432-8 BRAUN U., PUSTERLA N. & FLUCKIGER M. (1997). Ultrasonographic findings in cattle with pleuropneumonia. Veterinary Record 141 12-7 HENSHAW C.J. (1993) Serum protein concentrations in chronic ill-thrift conditions of adult sheep. Proceedings of the Sheep Veterinary Society 18 75-8 REIMER J.M. (1990) Diagnostic ultrasonography of the equine thorax. Compendium of Continuing Education 12 1321-7 SCOTT P.R., McGORUM B. M. & ELSE R.W. (1997) Extensive pleural effusion associated with diffuse fibrosing alveolitis in an aged beef cow. Veterinary Record 141 128-9 SCOTT P.R. & GESSERT M.E.(1998) Ultra-sonographic examination of the ovine thorax. The Veterinary Journal (in press) BCVA 1998 153

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