an epidemiological study of listeriosis in dairy cattle

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were also compared with those obtained at 4 0 C in the absence of medium. For this study the 20 faecal samples collected in October 1994 (20) and an additional 79 samples collected in March 1995 were used. Cold enrichment was carried out by keeping samples in either saline, LSEB, nutrient broth (NB) or no medium at 4°C and culturing periodically for up to 7 weeks. Isolation and identification was carried out according to the methods given below (Figure 5. 1). b) Identification procedure: (Appendix 3) At least five colonies of Listeria were picked off the LSA plates and sub- cultured on 5% Sheep Blood Agar (SBA) or Horse Blood Agar (HBA) plates at 37 0 C overnight. The following tests were carried out to identify the isolates as Listeria species:- Gram staining, Catalase test, Motility test, Haemolytic activity, CAMP test and Carbohydrate utilisation. Information provided in Table 1. 1. was used to aid identification. Fresh sample Re-culture after Cold enrichment 3 weeks at 4 0 C for 7 weeks 135
culturing negative LSEB (1/10w/v) samples at 4 week at 30 0 C for 48 h and 7 weeks intervals Plating on LSA at 30 0 C for 48 h Identification tests: Gram staining Catalase Oxidase Motility Sub-culturing Haemolytic on SBA CAMP at 37 0 C for 24h Sugar utilisation Figure 5. 1. Isolation and identification procedure for Listeria spp. 1) Gram staining: Bacterial colonies smeared onto a clean microscope slide were air dried, fixed by passing through the Bunsen flame two or three times and placed on a staining rack. The slides were flooded with Crystal violet for 1 minute, washed under a running tap, stained with Gram’s iodine for 1 minute and then washed a second time. The slides were then decolourised with acetone for 15 seconds, washed and counter stained with Safronin for 30 seconds. The slides were washed, dried and then examined under a light microscope. Listeria were identified as Gram positive rods with a purple- blue colour. 2) Haemolytic test: The haemolytic activity of the bacteria was assessed visually after culture on 5% Sheep (Horse) Blood Agar. Some members of the genus Listeria induce varying degrees of erythrocyte lysis (L. monocytogenes, L. ivanovii more haemolytic, 136
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AN EPIDEMIOLOGICAL STUDY OF LISTERI
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In the second part of the study a l
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I would like to thank to Metin Oztu
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DECLARATION I declare that apart fr
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2. 2. Materials and Methods 47 2. 2
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4. 4. Discussion 125 CHAPTER 5 A pi
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CHAPTER 1 LIST OF TABLES AND FIGURE
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Table 3. 11. The relationship betwe
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CHAPTER 5 Table 5. 1. Date of visit
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Figure 6. 4. The monthly faecal exc
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Listeralla hepatolytica. In the sam
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Listeriolysin O (Fernandaz-Garayzab
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L. monocytogenes was the only recog
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Table 1. 2. Serovar distribution of
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Serotyping is based on the identifi
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electrophoresis. Different strains
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and others (1995) came to the concl
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animals suggests that L. monocytoge
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Immunity reducing Septicaemia facto
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monocytogenes and a prerequisite to
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a variable number of cases of encep
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The relationship between age and Li
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a reservoir from which it spreads t
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1. 8. Clinical signs and pathology
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is seldom clinical illness in the d
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Bacterial culture is widely used fo
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Differential diagnosis: Listerial m
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Since elimination of L. monocytogen
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first description, it was only 1980
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and meat products have resulted in
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infectious dose for the infection i
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a longitudinal study involving five
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in Britain. In this part of the stu
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groups were excluded from analysis.
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The proportions of farms with cases
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Table 2. 3. The proportion of anima
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percentage 50 45 40 35 30 25 20 15
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Milking cows (%) Rep. heifers (%) D
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Abortion 274 (57.8%) 28 (21.9%) Ner
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with others (Kanuk and Berenson 197
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occurred in replacement heifers and
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CHAPTER 3 The relationship between
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(ii) Cases in milking cows: Farms r
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To assess the relationship between
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) Duration of housing: If animals w
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d) Presence of moles: Farmers were
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listeriosis. Maize silage feeding a
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found between the duration of feedi
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a) Type of housing : Housing animal
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Herd sizes n Y N OR P value x Y N O
Page 101 and 102: of disease. Similarly storing feed
Page 103 and 104: a) Type of forages: Feeding maize s
Page 105 and 106: ) Use of bedding: Using straw beddi
Page 107 and 108: ) Source of forages: Home made gras
Page 109 and 110: 1.29-18.22) whereas straw bedding i
Page 111 and 112: 100 2 34 2.33 0.01 X 2 22 3.35 0.01
Page 113 and 114: a) Dung disposal: Storage of manure
Page 115 and 116: vaccination against Leptospirosis +
Page 117 and 118: suppression of the growth of Lister
Page 119 and 120: 1989, Sargison 1993). It is also po
Page 121 and 122: L. monocytogenes has been isolated
Page 123 and 124: association (Kirkwood 1988, Thrusfi
Page 125 and 126: The predictor variables that met ou
Page 127 and 128: Forage feeding Maize silage Hay Str
Page 129 and 130: indoor feeding fed ad libitum on th
Page 131 and 132: cases of Listeriosis in sheep 2.9 1
Page 133 and 134: and controlling moles in fields wer
Page 135 and 136: Cases of Listeriosis in beef cattle
Page 137 and 138: This model was similar to the Model
Page 139 and 140: Table 4. 9. The multivariate relati
Page 141 and 142: 5) Nervous signs: Cases of Listerio
Page 143 and 144: feeders, maize silage feeding in ri
Page 145 and 146: A contradictory finding was made ab
Page 147 and 148: selective enrichment broth, at refr
Page 149 and 150: growth of some strains of Listeria
Page 151: 5. 2. 2. Bacteriology : a) Culture
Page 155 and 156: 6) Sugar tests: Listeria are capabl
Page 157 and 158: uffered saline (PBS), pH 7.2, centr
Page 159 and 160: Investigation of non-specific bindi
Page 161 and 162: and 0.94 for L. monocytogenes (17/1
Page 163 and 164: Detection limit of the method: A si
Page 165 and 166: Relationship between housing, silag
Page 167 and 168: 5 13 3 3 1 3 1 1 1 6 14 1 4 5 3 1 0
Page 169 and 170: Table 5. 11. The relationship betwe
Page 171 and 172: frequency of isolation of L. monocy
Page 173 and 174: CHAPTER 6 A study of the dynamic of
Page 175 and 176: There were no sample size calculati
Page 177 and 178: 4 of the farms had their silage ana
Page 179 and 180: a) Faecal samples: Faecal samples w
Page 181 and 182: • • • 1 5 9 6. 2. 5. Sample p
Page 183 and 184: A total of 944 isolates of L. monoc
Page 185 and 186: UK), 0.5μM DNA primer, 1 unit Supe
Page 187 and 188: entered and left the herd over the
Page 189 and 190: Table 6. 7. Monthly frequency of ex
Page 191 and 192: Table 6. 8. The relationship betwee
Page 193 and 194: (MA) mean age, (n) number, (R) rang
Page 195 and 196: frequency of excretion (P
Page 197 and 198: There was no evidence from this far
Page 199 and 200: ) Environmental samples: Both L. in
Page 201 and 202: monocytogenes. By April the proport
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A total of 211 milking cows were ex
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100 % 90 80 70 60 50 40 30 20 10 0
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The number of new cases each month
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monocytogenes was also isolated fro
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Table 6. 18 The isolates, their ori
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Table 6. 19. The distribution of th
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On farm C pattern 1 was detected in
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Figure 6. 8. The repeatability of R
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Figure 6. 10. The distribution of s
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Figure 6. 12a. The RAPD pattern obt
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1988, Husu 1990) and from zero to a
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experimentally been shown that ther
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periods. However, on farms B and E
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A large number of L. monocytogenes
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Figure 6. 13. Animal-environment cy
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Some important farming practices we
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chance finding of L. monocytogenes
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Asahi, O., Hosoda, T. and Akuyama,
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Bourry, A. and Poutrel, B. (1996) B
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Chakraborty, T., Ebel, F., Wehland,
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delGarso, L. and Wallop, W. (1975)
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Eveland, W.C. (1963) Demonstration
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Fraser, J.A. and Sperber, W.H. (198
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Golden, D.A, Beuchat, L.R. and Brac
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humans. In Miller, A.J.,. Smith, J.
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Jones, F.S. and Little, R.B. (1934)
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Listeria innocua by oral inoculatio
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Low, J.C. and Donachie, W.A. (1997)
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McBride, M.E. and Girard, K.F. (196
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Nieman, R.E. and Lorber, B. (1980)
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Peters, M., Pohlenz, J., Jaton, K.,
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Bakteriologie, Mikrobiologie und Hy
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Seeliger, H.P.R. (1981) Apathogenic
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Tilney, L.G. and Portnoy, D.A. (198
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Walker, J.K., Morgan, J.H., McLauch
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APPENDIX 3 PREPARATION OF BACTERIOL
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Cool to 50 0 C Mix Solution A with
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Phosphate buffered saline (PBS; 10x
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APPENDIX 2 THE OVERALL RESULTS OF U
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Grass silage Y N OR (95% CL) p Valu
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storage Y N OR (95% CL) p Value cla
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source home made 38 182 0.6 (0.1-16
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source home made 34 345 0.8 (0.3-2.
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source barley 27 251 0.93(0.6-1.6)
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source potatoes 2 11 1.4 (0.-7.7) 0
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in a covered barn 73 595 0.8 (0.4-1
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sawdust 0 16 0.0 (0.0-2.73) 0.3 str
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APPENDIX 5 THE QUESTIONNAIRE AND TH
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Abbreviations: 1 first blood sampli
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B 113 1 114 1 115 1 116 1 117 1 118
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B 104 1 104 2 104 3 105 1 105 2 105
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B 20 1 20 2 20 3 21 1 21 2 21 3 19
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0.169 1.157 1.134 1.467 1.356 0.145
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Plate 4 Layout and ODs B 73 1 73 2
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UNIVERSITY OF BRISTOL LISTERIOSIS I
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PART C : HERD SIZE :--BETWEEN JULY
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(please circle one for each) 3. Wha
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etween which months were your milki
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not stored beneath the slats compos
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STUDY OF LISTERIOSIS IN DAIRY CATTL
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STUDY OF LISTERIOSIS IN DAIRY CATTL
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Now I would like to get some detail
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Root crops : ______________________
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In this section I will be asking qu
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33. Which days of the week bulk tan
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-what was used to treat? -what was
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an epidemiological study of listeriosis in dairy cattle

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