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Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2018-0251.R3

Manuscript Type: Note

Date Submitted by the Author: 11-Sep-2018

Complete List of Authors: Eraclio, Giovanni; University of MilanRicci, Giovanni; University of MilanMoroni, Paolo; Univerisity of Milan; Cornell UniversitySantisteban, Carlos; Cornell UniversityPlumed-Ferrer, Carme; University of Eastern FinlandBennett, James; Northern Valley Dairy Production Medicine CenterFortina, Maria Grazia; Dipartimento de Scienze e Tecnologie Alimentari e Microbiologiche

Keyword:

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Canadian Journal of Microbiology

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1 Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms 2

3

4 Giovanni Eraclio, Giovanni Ricci, Paolo Moroni, Carlos Santisteban, Carme Plumed-Ferrer,

5 James Bennett, Maria Grazia Fortina

6

7 G. Eraclio, G. Ricci, M.G. Fortina. Università degli Studi di Milano, Department of Food,

8 Environmental and Nutritional Sciences, Division of Food Microbiology and Bioprocesses, Via

9 Celoria 2, 20133 Milan, Italy

10 P. Moroni. Università degli Studi di Milano, Dipartimento di Medicina Veterinaria, Via Celoria 10

11 20133 Milan, Italy; Cornell University, Animal Health Diagnostic Center, Quality Milk Production

12 Services, 240 Farrier Road, Ithaca, NY, 14853, USA

13 C. Santisteban. Cornell University, Animal Health Diagnostic Center, Quality Milk Production

14 Services, 240 Farrier Road, Ithaca, NY, 14853, USA

15 C. Plumed-Ferrer. Food Biotechnology, Institute of Public Health and Clinical Nutrition, University

16 of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland

17 J. Bennett. Northern Valley Dairy Production Medicine Center, 900 N Wabasha Plainview, MN

18 55964

19

20 Corresponding author: Maria Grazia Fortina (email [email protected]).

21 Phone: 39 250319131; Fax: 39 250319238

22

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mailto:[email protected]

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23 Abstract: Lactococcus garvieae is now recognized as a species with clinical significance for human

24 and veterinary medicine. The aim of this study was to evaluate the presence of this pathogen in sand

25 bedding and milk samples.. Two Minnesota farms in with problems of clinical and subclinical

26 mastitis due to streptococci-like organisms were selected. Twenty-four Lactococcus garvieae

27 isolates from sand bedding and 18 isolates from quarter milk were comparatively studied using a

28 genotypic approach. RAPD and REP-PCR experiments highlighted a similar electrophoretic profile.

29 When genes belonging to the core genome of L. garvieae were tested through a MLRT, we again

30 observed that all L. garvieae isolates coming from sand bedding and milk shared a common profile,

31 distinguishable from previously studied representative L. garvieae strains. These data indicate that

32 the L. garvieae isolated from sand bedding and milk originated from a few strains adapted to persist

33 in the same habitat. This supports the hypothesis that sand bedding can represent a reservoir of L.

34 garvieae strains and be a potential vehicle for their dissemination in dairy farms.

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36 Key words: Lactococcus garvieae, environmental dissemination, sand bedding, milk, molecular

37 typing, MLRT

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38 Environmental lactococci are now recognized as significant contributors to the incidence of

39 intra-mammary infection (IMI) (Werner et al. 2014). Among them, Lactococcus lactis and

40 Lactococcus garvieae have been isolated from bovine IMI from different dairy farms in New York

41 and Minnesota (Werner et al. 2014; Plumed-Ferrer et al. 2015). L. lactis and L. garvieae can be found

42 to cohabitate in many different ecological niches. This cohabitation could explain the close

43 phylogenetic relatedness we found between the 2 species, also mediated by efficient horizontal gene

44 transfer mechanisms (Ferrario et al. 2012; Ferrario et al. 2013; Eraclio et al. 2015). Despite the

45 growing importance of lactococci in both human and veterinary medicine, few research data are

46 available on the ecological niches from which these emerging pathogens can spread into environments

47 characterized by different selective pressures.

48 L. garvieae is mainly known as a fish pathogen, responsible for outbreaks of lactococcosis in

49 different countries (Vendrell et al. 2006). L. garvieae is not exclusive to fish and can also be found in

50 meat, vegetables, (Ferrario et al. 2012) and water (Aguado-Urda et al. 2010). L. garvieae is able to

51 spread on a dairy farm and is found in milk and cheeses, sometimes as the dominant microbial

52 population (Fortina et al. 2003; Fortina et al. 2007; Fernandez et al. 2010). L. garvieae is also

53 considered a relevant human pathogen (Russo et al. 2012; Reguera-Brito et al. 2016; Eraclio et al.

54 2018), and the role of foods as a potential source of infection for humans has been hypothesized.

55 Understanding the niche of origin of this emerging pathogen and its ability to spread into the

56 environment can be of great interest. For these reasons, in this paper, we: 1) isolated and confirmed

57 with MALDI-TOF L. garvieae from sand bedding and milk samples 2) characterized at genetic level

58 the L. garvieae isolates.

59 Farm A and B located in Minnesota, with problems of clinical (presence in the quarter of flakes, clots,

60 or other gross alterations, NMC 2001) and subclinical mastitis (when quarter somatic cell count were

61 equal to or exceed 200,000 cells mL-1 and bacteria was isolated in the absence of clinical change,

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62 NMC 2001) due to streptococci-like organisms, were chosen. The veterinary clinic (Northern Valley

63 Dairy Production Medicine Center, Plainview, MN) submitted for identification 54 quarter milk

64 samples (previous identify like streptococci species with Minnesota plate) and 25 bedding samples

65 (24 sand and 1 deep bedded pack) to Quality Milk Production Services (QMPS, Cornell University,

66 Ithaca, NY). These samples were analysed for speciation using MALDI-TOF (Bruker, The

67 Woodlands, TX, USA) technology (Randall et al. 2015). Farm A submitted 12 bedding samples and

68 31 quarter milk samples and Farm B submitted 13 bedding samples and 23 quarter milk samples.

69 Bedding samples were submitted because due to the environmental characteristics of streptococci-like

70 organisms, one of the objectives was to understand the management of bedding and the total bacteria

71 count. Bedding samples were taken on a day when bedding was to be replenished, and these were

72 collected just prior to the addition of fresh bedding, i.e. at the point of maximum contamination.

73 Farm A: a 1,693 lactating cow Holstein Friesian herd in a free stall facility with sand bedding.

74 This farm had an average daily milk production of 45 kg per cow and a bulk tank somatic cell count

75 average of 268,000 cells mL-1. From May 1, 2013 through March 31, 2014 the herd exhibited a high

76 rate of chronic subclinical mastits (13%) as determined by 2 or more consecutive monthly DHIA test-

77 days greater ≥200,000 cells mL-1. The prevalence of new infections was 8.7%, as determined by the

78 number of cows each test day with a current test-day greater than ≥200,000 cells mL-1 and a previous

79 test day linear score less than 200,000 cells mL1. Cows were milked in a 50-cow rotary parlor.

80 Farm B: a 914 lactating cow Holstein Friesian herd in a free stall facility with sand bedding.

81 The prefresh group (15 days before calving) had a deep bedded pack). This farm had an average daily

82 milk production of 39 kg per cow, and a bulk tank somatic cell count average of 365,000 cells mL-1.

83 From May 1, 2013 through March 31, 2014, the herd exhibited a high number of subclinical mastitis

84 (21.7%) and new infections were 8.8%. Cows were milked in a double 20 parallel parlour.

85 Before sampling, teat ends were carefully cleaned and disinfected with iodine followed by

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86 alcohol. First streams of fore-milk were discarded, and then approximately 10 mL of milk was

87 collected aseptically from each quarter into sterile vials.

88 Bacteriological cultures were performed according to standards of the National Mastitis Council

89 (NMC, 2017). 10 µL of each quarter milk sample were spread on blood agar plates (5% defibrinated

90 sheep blood). Plates were incubated aerobically at 37°C and examined after 24 h. Isolates were

91 identified for speciation using Matrix-Assisted Laser Desportion Ionization Time of Flight Mass

92 Spectrometry (MALDI-TOF). When a log (score) ≥ 1.7 was found this indicative a relationship at the

93 genus level and a log (score) of ≥ 2.0 was the set threshold for a match at the species level. Only 42

94 L. garvieae isolated, on the total of 51, were identified to the species level and they were retained for

95 the study.

96 For L. garvieae isolation from sand bedding, samples were enriched in 1:9 (w/w) M17 broth

97 (Difco, Detroit, MI, USA) supplemented with 10 g L-1 glucose (M17-G) and incubated at 30°C for

98 24 h. Total DNA was extracted as reported elsewhere (Borgo et al. 2013), and the presence of L.

99 garvieae verified through a species-specific PCR assay, as reported by Zlotkin et al. (1998). Positive

100 samples were then plated on M17-G agar and incubated at 30°C for 24 h. After incubation, randomly

101 selected colonies were purified, sub-culturing each colony by serially streaking, and submitted to

102 taxonomic identification, as reported previously.

103 From each sample of bedding and milk showing the presence of the pathogen, one isolate was

104 selected (Table 1) and genotypically characterized.

105 The isolates were typed by combined analysis of repetitive element PCR (REP) using primers

106 BOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′) (Versalovic et al. 1994; De Urraza et al.

107 2000) and random amplification of polymorphic DNA-PCR (RAPD) with primer M13 (5′-

108 GAGGGTGGCGGTTCT-3′) (Rossetti and Giraffa 2005). These techniques are known for their

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109 potential to discriminate between lactic acid bacteria at the strain level. The analysis was carried out

110 in comparison with 8 L. garvieae strains previously studied (Ferrario et al. 2012) and coming from

111 different environments (Table 1). Chromosomal DNA was extracted according to Fortina et al. (2003).

112 PCRs were performed using a PCR-Mastercycler 96 (Eppendorf, Hamburg, Germany) as previously

113 reported (Ferrario et al. 2012). Amplification products were separated on agarose gel stained with

114 ethidium bromide in 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.2) buffer and photographed.

115 Banding patter similarity was evaluated by construction of dendogram using the NTSYSpc software,

116 version 2.11 (Applied Biostatics Inc., NY, USA), employing the Jaccard similarity coefficient. A

117 dendogram was deduced from a similarity matrix by using the unweighted pair group method with

118 arithmetic average (UPGMA) clustering algorithm.

119 The isolates were also typed by Multilocus Restriction Typing (MLRT), a reproducible typing

120 technique for estimating overall levels of genotypic variation in population of microorganisms (Borgo

121 el al. 2007). A restriction analysis of PCR products generated from six selected housekeeping genes,

122 as previously reported (Ferrario et al. 2012) was carried out. Briefly, products from amplified atpA

123 (a-subunit of ATP synthase), tuf (bacterial elongation factor EF-Tu), dltA (D-alanine-D-alanyl carrier

124 protein ligase), als (a-acetolactate synthase), gapC (glyceraldehyde-3-phosphate dehydrogenase),

125 galP (galactose permease) and lacG (phospho-β-galactosidase) were digested with specific restriction

126 enzymes. Restriction digests were subsequently analyzed by agarose electrophoresis (2% agarose gel).

127 The relatedness of the strains was verified analyzing their profiles generated by MLRT analysis.

128 The results obtained indicate 24 sand bedding samples showed the presence of L. garvieae and

129 only the deep bedded pack was negative. This is the first evidence of the presence of L. garvieae in

130 sand bedding, even if its incidence is not quantifiable, as demonstrated by the need for enrichment

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131 procedures.Regarding milk samples, 18 were positive to L. garvieae (10 from Farm A and 8 from

132 Farm B). The 42 L. garvieae (24 from sand and 18 from milk) , were genetically compared.

133 At first, the isolates were typed by combined analysis of repetitive element PCR (REP) and random

134 amplification of polymorphic DNA-PCR (RAPD).

135 The results obtained, shown in Fig. 1, revealed interesting features. Isolates coming from sand bedding

136 and milk samples were separated at a level of similarity of 0.38% from the other L. garvieae strains

137 used for comparison, isolated from different ecological niches (Table 1). It is known that a similarity

138 coefficient of 0.70% is chosen as suitable to discriminate clusters of strains or single strains belonging

139 to different species (Rossetti and Giraffa 2005). The low correlation value for L. garvieae studied

140 suggested the existence of a marked genetic divergence. In contrast, the isolates show highly similar

141 electrophoretic profiles, easily distinguishable from the profiles obtained for the strains isolated from

142 different niches. With the exception of isolate Be15, that did not cluster together with any other

143 isolates and can probably be considered a different strain, the other isolates grouped in related

144 subgroups, the main including 52% of the sand bedding and milk isolates tested, with an inter-strain

145 level of similarity ranging from 80 to 100%.

146 When genes belonging to the core genome of L. garvieae were tested through a Multi Locus

147 Restriction Typing (MLRT), we observed again that all L. garvieae isolates coming from bedding and

148 milk shared a common profile, distinguishable from those obtained from the representative strains

149 previously studied. In Fig. 2 is reported the dendogram showing the relatedness of the strains based

150 on analysis of their restriction profiles generated by MLRT analysis, in comparison with the restriction

151 profiles obtained in a previous work for 49 L. garvieae strains (Ferrario et al. 2012). Cluster analysis

152 revealed three distinct L. garvieae groups, two of which representing two different phylogenetic

153 lineages within which L. garvieae can be genetically separated (Ferrario et al. 2013). The other group

154 contains all isolates tested in this study, together with the strain I113 that has been considered, in a

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155 previous work, a “separated strain”, constituting a different phylogenetic lineage. These results clearly

156 demonstrated that the bedding and milk isolates are highly related and easily distinguishable from the

157 other strains of the species up to now studied.

158 Moreover, the results obtained seem to indicate that the L. garvieae isolated from bedding and milk

159 originated from a few strains and adapted to persist in a same habitat. This is also confirmed by the

160 remarkable similarity showed by the isolates coming from the two different farms, highlighting that

161 the sand bedding can represent a reservoir of L. garvieae and a potential vehicle for his dissemination

162 even if bacteria can be transferred between cows via sand bedding then transferred to milk via infected

163 udders.

164

165 Funding

166 This research did not receive any specific grant from funding agencies in the public, commercial,

167 or not-for-profit sectors

168

169 Conflicts of interest

170 The authors declare no conflict of interest.

171

172

173 Acknowledgements

174 We thank Becky Callan and Belinda Gross from Cornell University, Animal Health Diagnostic

175 Center, for language editing

176

177 References

178

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179 Aguado-Urda, M., Cutuli, M.T., Mar Blanco, M., Aspiroz, C., Tejedor, J.L., Fernández-

180 Garayzábal, J.F., and Gibello, A. 2010. Utilization of lactose and presence of the phospho-β-

181 galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources. Intern.

182 Microbiol. 13: 189–193.

183 Borgo, F., Ferrario, C., Ricci, G., and Fortina, M.G. 2013. Genotypic intraspecies heterogeneity of

184 Enterococcus italicus: data from dairy environments. J. Basic Microbiol. 53: 20–28.

185 Borgo, F., Ricci, G., Manachini, P.L., and Fortina, M.G. 2007. Multilocus restriction typing: a tool

186 for studying molecular diversity within Lactobacillus helveticus of dairy origin. Int. Dairy J.

187 17: 336-342

188 De Urraza, P.J., Gómez-Zavaglia, A.M., Lozano, E., Romanowski, V., and De Antoni, G.L. 2000.

189 DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based

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191 Eraclio, G., Ricci, G., and Fortina, M.G. 2015. Insertion sequence elements in Lactococcus

192 garvieae. Gene 555: 291–296.

193 Eraclio, G., Ricci, G., Quattrini, M., Moroni, P., and Fortina, M.G. 2017. Detection of virulence-

194 related genes in Lactococcus garvieae and their expression in response to different conditions.

195 Folia Microbiol. 63(3): 291-298.

196 Fernández, E., Alegría, Á., Delgado, S., and Mayo, B. 2010. Phenotypic, genetic and technological

197 characterization of Lactococcus garvieae strains isolated from a raw milk cheese. Intern. Dairy

198 J. 20: 142–148.

199 Ferrario, C., Ricci, G., Borgo, F., Rollando, A., and Fortina, M.G. 2012. Genetic investigation

200 within Lactococcus garvieae revealed two genomic lineages. FEMS Microbiol. Lett. 332:

201 153–161.

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202 Ferrario, C., Ricci, G., Milani, C., Lugli, G.A., Ventura, M., Eraclio, G., Borgo, F., and Fortina,

203 M.G. 2013. Lactococcus garvieae: where is it from? A first approach to explore the

204 evolutionary history of this emerging pathogen. PLoS ONE. 8:e84796.

205 Fortina, M.G., Ricci, G., Acquati, A., Zeppa, G., Gandini, A., and Manachini, P.L. 2003. Genetic

206 characterization of some lactic acid bacteria occurring in an artisanal protected denomination

207 origin (PDO) Italian cheese, the Toma piemontese. Food Microbiol. 20: 397–404.

208 Fortina, M.G., Ricci, G., Foschino, R., Picozzi, C., Dolci, P., Zeppa, G., Cocolin, L., and

209 Manachini, P.L. 2007. Phenotypic typing, technological properties and safety aspects of

210 Lactococcus garvieae strains from dairy environments. J. Appl. Microbiol. 103: 445–453.

211 National Mastitis Council 2001. NMC Guidelines on Normal and Abnoraml Raw Milk Based on

212 SCC and Signs of Clinical Mastitis. NMC, 2820 Walton Commons West, Siute 131, Madison,

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214 National Mastitis Council. 2017. Laboratory Handbook on Bovine Mastitis. NMC, New Prague,

215 MN.

216 Plumed-Ferrer, C., Barberio, A., Franklin-Guild, R., Werner, B., McDonough, P., Bennett, J.,

217 Gloria, G., Rota, N., Welcome, F., Nydam, D.V., and Moroni, P. 2015. Antimicrobial

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219 Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary

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221 Randall, L.P., Lemma, F., Koylass, M., Rogers, J., Ayling, R.D., Worth D., et al. 2015. Evaluation

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224 Reguera-Brito, M., Galán-Sánchez, F., Mar Blanco, M., Rodríguez-Iglesias, M., Domínguez, L.,

225 Fernández-Garayzábal, J.F., and Gibello, A. 2016. Genetic analysis of human clinical isolates

226 of Lactococcus garvieae: relatedness with isolates from foods. Infect. Gen. Evol. 37: 185–191.

227 Rossetti, L., and Giraffa, G. 2005. Rapid identification of dairy lactic acid bacteria by M13-

228 generated, RAPD-PCR fingerprint databases. J. Microbiol. Meth. 63: 135–144.

229 Russo, G., Iannetta, M., D’Abramo, A., Mascellino, M. T., Pantosti, A., Erario, L., Tebano, G.,

230 Oliva, A., D’Agostino, C., Trinchieri, V., and Vullo, V. 2012. Lactococcus garvieae

231 endocarditis in a patient with colonic diverticulosis: first case report in Italy and review of the

232 literature. New Microbiol. 35: 495–501.

233 Vendrell, D., Balcázar, J.L., Ruiz-Zarzuela, I., de Blas, I., Gironés, O., and Múzquiz, J.L. 2006.

234 Lactococcus garvieae in fish: a review. Comp. Imm. Microbiol. Infect. Dis. 29: 177–198.

235 Versalovic, J., Schneider, M., De Bruijn, F., and Lupski, J.R. 1994. Genomic fingerprinting of

236 bacteria using repetitive sequence-based polymerase chain reaction. Meth. Mol. Cell. Biol. 5:

237 25–40.

238 Werner, B., Moroni, P., Gloria, G., Lavín-Alconero, L., Yousaf, A., Charter, M.E., Carter, B. M.,

239 Bennett, J., Nydam, D. V, Welcome, F., and Schukken, Y.H. 2014. Genotypic and phenotypic

240 identification of environmental streptococci and association of Lactococcus lactis ssp. lactis

241 with intramammary infections among different dairy farms. J. Dairy Sci. 97: 6964–6969l.

242 Zlotkin, A., Eldar, A., Ghittino, C., and Bercovier, H. 1998. Identification of Lactococcus garvieae

243 by PCR. J. Clin. Microbiol. 36: 983–985.

244

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245 Figure captions

246

247 Fig. 1. Cluster analysis of the Lactococcus garvieae isolates using combined M13 and BOXA1R

248 fingerprints (UPGMA; NTSYSpc software, version 2.11-Applied Biostatics Inc., NY, USA).

249

250 Fig. 2. Dendrogram showing the relatedness of the Lactococcus garvieae isolates based on analysis

251 of their restriction profiles generated by MLRT analysis, constructed using the UPMGA algorithm

252 in the NTSYSpc software, version 2.11 (Applied Biostatics Inc., NY, USA). The comparison with

253 MLRT data previously obtained from L. garvieae strains coming from different niches (Ferrario et

254 al. 2013) allows the differentiation of the species in three phylogenetic lineages.

255

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Fig.1. Eraclio et al.

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1 Fig.2. Eraclio et al.

Phylogenetic lineage B

Phylogenetic lineage A

Phylogenetic lineage C

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Table 1. Lactococcus garvieae isolates used in this study, and their origin.

Lactococcus garvieae strains Source of isolation

Be 1-11 Sand bedding, Farm A

Be 12-24 Sand bedding, Farm B

Mi 38-40; 42-46; 48, 49 Milk, Farm A

Mi 31-37; 47 Milk, Farm B

G07 Cow cheese

TB25 Cow cheese

Uma10 Human sample

Br3 Vegetables

Sed2 Vegetables

Ins1 Vegetables

Smp3 Meat products

Lg9 Diseased fish

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