an epidemiological study of listeriosis in dairy cattle

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The primary aim of this pilot study was to develop and standardise techniques which would allow cattle infected with L. monocytogenes to be detected in a longitudinal study. A three stage procedure, cold enrichment prior to LSEB (Lovett and others 1987) and LSA (Curtis and others 1989a) was used to identify animals shedding Listeria spp. in their faeces. No comparison of LSEB and LSA was made with any other Listeria isolation media. However the superiority of LSEB and especially LSA has been acknowledged by other researchers. LSA was found to be more effective in isolating L. monocytogenes from artificially seeded clinical specimen such as faeces and vaginal swabs (Curtis and others 1989a) and from food (Tiwari and Aldenrath 1990, Curtis and Lee 1995) but less effective in isolating the bacteria from silage (Fernandez-Garayzabal and others 1992b) Cold enrichment was also reported to be inefficient in isolating L. monocytogenes from food (Pini and Gilbert 1988), faecal samples (Hayes and others 1991) and autopsy material (Eld and others 1993) when compared with liquid selective enrichment at higher temperatures. The variability between the isolation techniques and the failure of our attempts to isolate L. monocytogenes from faeces without exposure to cold storage led us to combine cold enrichment with selective enrichment and selective plating at higher temperature. This application enabled us to identify more Listeria positive samples. This is in agreement with other findings. The successful use of cold enrichment followed by selective media and plating at higher temperature for the isolation of L. monocytogenes from food by Lewis and Corry (1991) and clinical specimens by Gray (1948) and Pittman and others (1967), and for epidemiological investigation by Husu (1990) has been reported. In conventional cold enrichment procedures a liquid medium (selective or non selective) has always been used (Gray and Killinger 1966 and Hayes and others, 1991). The use of NB, Saline and LSEB as cold enrichment media failed to improve the 153
frequency of isolation of L. monocytogenes from faeces when compared with the samples held at 4 0 C without any medium. Both NB and LSEB increased the number of positive samples after the first week of cold enrichment but there was no further increase during the cold enrichment period. In contrast, the use of saline as a cold enrichment medium had an adverse effect on the growth of Listeria organisms. A similar effect of saline was stated by Gray and Killinger (1966). Our results indicate that holding faecal samples at refrigeration temperature without any liquid media was the better application. The duration of cold enrichment used by other researchers varied between 1 to several months (Gray 1948, Pittman and others 1967). We attempted to reduce this time by determining the sensitivity of the isolation technique at different times of cold enrichment. The best result was obtained after 7 weeks of cold enrichment when the sensitivity was 100% and 94% for Listeria spp. and L. monocytogenes respectively. Investigation of the lowest limit of detection revealed that it was 3.17 cfu/ml for broth spiked with Listeria organisms and 7 cfu/g for the known Listeria positive faeces sample. The difference between the two figures could be explained with the nature of samples as the faecal sample would contain many more bacteria other than Listeria. After the second week of cold enrichment of faecal samples inoculated with 3.17x10 -1 cfu/ml of L. monocytogenes revealed growth. This supports the idea that cold enrichment favours the multiplication of Listeria organisms (Gray and Killinger 1966). This pilot study revealed some preliminary information about the behaviour of Listeria spp. especially L. monocytogenes. Unlike other researchers we found a very high excretion level of Listeria spp. and more importantly L. monocytogenes (Skovgaar and Morgen 1988, Husu 1990). There was also a seasonal pattern of excretion which coincided with silage feeding, housing and the seasonal occurrence of clinical listeriosis (Chapter 2). This seasonal variation may have been the result of different feeding 154
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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
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of disease. Similarly storing feed
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a) Type of forages: Feeding maize s
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) Use of bedding: Using straw beddi
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) Source of forages: Home made gras
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1.29-18.22) whereas straw bedding i
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100 2 34 2.33 0.01 X 2 22 3.35 0.01
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a) Dung disposal: Storage of manure
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vaccination against Leptospirosis +
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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 and 152: 5. 2. 2. Bacteriology : a) Culture
Page 153 and 154: culturing negative LSEB (1/10w/v) s
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: Table 5. 11. The relationship betwe
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
Page 203 and 204: A total of 211 milking cows were ex
Page 205 and 206: 100 % 90 80 70 60 50 40 30 20 10 0
Page 207 and 208: The number of new cases each month
Page 209 and 210: monocytogenes was also isolated fro
Page 211 and 212: Table 6. 18 The isolates, their ori
Page 213 and 214: Table 6. 19. The distribution of th
Page 215 and 216: On farm C pattern 1 was detected in
Page 217 and 218: Figure 6. 8. The repeatability of R
Page 219 and 220: 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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