Microbial agents in macroscopically healthy mammary gland tissues of small ruminants

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Microbial agents in macroscopically healthy mammary gland tissues of small ruminants

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Microbial agents in macroscopically healthy mammary gland

tissues of small ruminants

Liliana Spuria 1 _{, Elena Biasibetti} Corresp., 1 _{, Donal Bisanzio} 2 _{, Ilaria Biasato} 1 _{, Daniele De Meneghi} 1 _{, Patriza} Nebbia 1 _{, Patriza Robino} 1 _{, Paolo Bianco} 3 _{, Michele Lamberti} 4 _{, Claudio Caruso} 5 _{, Alessia Di Blasio} 5 _, Simone Peletto 5 _{, Loretta Masoero} 5 _{, Alessandro Dondo} 5 _{, MariaTeresa Capucchio} 1

1

Department of Veterinary Sciences, University of Turin, Grugliasco, Italy 2

Big Data Institute, Nuffield Department of Medicine, University of Oxford, c/o Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom 3

ASLTo4, ASL, Torino, Italy 4

ASLCn1, ASL, Barge, Italy 5

Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d'Aosta, Torino, Italy Corresponding Author: Elena Biasibetti

Email address: elena.biasibetti@unito.it

Background. Health of mammary glands is fundamental for milk and dairy products

hygiene and quality, with huge impacts on consumers welfare. Methods. This study aims to investigate the microbial agents (bacteria, fungi and lentiviruses) isolated from 89 macroscopically healthy udders of regularly slaughtered small ruminants (41 sheep, 48 goats), also correlating their presence with the histological findings. Multinomial logistic regression was applied to evaluate the association between lesions and positivity for different microbial isolates, animal age and bacteria. Results. Twenty five samples were microbiologically negative; 138 different bacteria were isolated in 64 positive udders. Coagulase-negative staphylococci were the most prevalent bacteria isolated (46.42%), followed by environmental opportunists (34.76%), others (10.14%) and pathogens

(8.68%). Most mammary glands showed coinfections (75%). Lentiviruses were detected in 39.3% of samples. Histologically, chronic non-suppurative mastitis was observed in 45/89 glands, followed by chronic mixed mastitis (12/89) and acute suppurative mastitis (4/89). Only 28 udders were normal. Histological lesions were significantly associated with the animal species and lentiviruses and coagulase-negative staphylococci infections. Goats had significantly higher risk to show chronic mixed mastitis compared to sheep. Goats showed a significantly lower risk (OR=0.26; 95% CI: 0.06-0.71) of being infected by

environmental opportunists compared to sheep, but higher risk (OR=10.87; 95% CI: 3.69-37.77) of being infected with lentiviruses. Discussion. The results of the present study suggest that macroscopically healthy glands of small ruminants could act as a reservoir of microbial agents for susceptible animals, representing a potential risk factor for the

1 _{Microbial agents in macroscopically healthy mammary gland}

2 _{tissues of small ruminants}

3

4 _{L. Spuria}1_{, E. Biasibetti}1_{, D. Bisanzio}2_{, I. Biasato}1_{, D. De}

5 _Meneghi1_{, P. Nebbia}1_{, P. Robino}1_{, P. Bianco}3_{, M. Lamberti}4_{, S.}

6 _Peletto5_{, C. Caruso}5_{, A. Di Blasio}5_{, L. Masoero}5_{, A. Dondo}5_{, M. T.}

7 _Capucchio1

8

9 1_{Department of Veterinary Sciences, University of Torino, Largo}

10 _{Braccini 2, 10095 Grugliasco (Torino), Italy}

11 2_{Big Data Institute, Nuffield Department of Medicine, University}

12 _{of Oxford, c/o Wellcome Trust Centre for Human Genetics,}

13 _{Roosevelt Drive, Oxford, OX3 7BN, United Kingdom}

14 3_{ASLTo4, via Po 11, 10034 Chivasso (Torino), Italy}

15 4_{ASLCn1, Piazza Statuto 2, 12032 Barge (Cuneo), Italy, and}

16 5_{Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle}

17 _{d'Aosta, Via Bologna 148, 10154 Torino, Italy}

18

19 _{Corresponding author:}

20 _{Elena Biasibetti}1

21 _{E-mail address: elena.biasibetti@hotmail.it}

22

23

24

25 _Abstract

26 _{Background. Health of mammary glands is fundamental for milk}

27 _{and dairy products hygiene and quality, with huge impacts on}

28 _{consumers welfare.}

29 _{Methods. This study aims to investigate the microbial agents}

30 _{(bacteria, fungi and lentiviruses) isolated from 89 macroscopically}

31 _{healthy udders of regularly slaughtered small ruminants (41 sheep,}

32 _{48 goats), also correlating their presence with the histological}

33 _{findings. Multinomial logistic regression was applied to evaluate}

34 _{the association between lesions and positivity for different}

35 _{microbial isolates, animal age and bacteria.}

36 _{Results. Twenty five samples were microbiologically negative; 138}

37 _{different bacteria were isolated in 64 positive udders.}

Coagulase-38 _{negative staphylococci were the most prevalent bacteria isolated}

39 _{(46.42%), followed by environmental opportunists (34.76%),}

40 _{others (10.14%) and pathogens (8.68%). Most mammary glands}

41 _{showed coinfections (75%). Lentiviruses were detected in 39.3% of}

42 _{samples. Histologically, chronic non-suppurative mastitis was}

43 _{observed in 45/89 glands, followed by chronic mixed mastitis}

44 _{(12/89) and acute suppurative mastitis (4/89). Only 28 udders were}

45 _{normal. Histological lesions were significantly associated with the}

46 _{animal species and lentiviruses and coagulase-negative}

47 _{staphylococci infections. Goats had significantly higher risk to}

48 _{show chronic mixed mastitis compared to sheep. Goats showed a}

49 _{significantly lower risk (OR=0.26; 95% CI: 0.06-0.71) of being}

50 _{infected by environmental opportunists compared to sheep, but}

51 _{higher risk (OR=10.87; 95% CI: 3.69-37.77) of being infected with}

52 _{lentiviruses.}

53 _{Discussion. The results of the present study suggest that}

54 _{macroscopically healthy glands of small ruminants could act as a}

55 _{reservoir of microbial agents for susceptible animals, representing a}

56 _{potential risk factor for the widespread of acute or chronic infection}

57 _{in the flock.} 58 59 60 61 62 63 64 65 66 67

Manuscript to be reviewed

68 69 70 71 72 73 74 _Introduction

75 _{The importance of small dairy ruminants has significantly}

76 _{increased during last decades, especially in developing countries}

77 _{where they represent fundamental economic and social livelihoods}

78 _{of a large human population (Oluwatayo and Oluwatayo, 2012). In}

79 _{high income economies, they are also becoming an important}

80 _{alternative to supply dairy products for human consumption (Lérias}

81 _{et al., 2014).}

82 _{Functionality and health of the mammary gland are fundamental for}

83 _{dairy farms and directly correlated with milk production, milk and}

84 _{dairy products hygiene and quality, and consequently farmers}

85 _{profits. Mastitis is the inflammation of the mammary gland}

86 _{characterized by an increase in the number of somatic cells in the}

87 _{milk and/or by pathological changes in the mammary tissue}

88 _{(Schlafer & Foster, 2016). It is generally caused by microbial}

89 _{agents invasion, mainly represented by bacteria, and in a small}

90 _{extent by mycoplasmas, viruses (Small Ruminant Lentiviruses -}

91 _{SRLV), fungi and algae.}

92 _{According to Bergonier and Berthelot (2003a), mastitis is important}

93 _{from three perspectives. Firstly, there are economic implications,}

94 _{because of reduced growth of lambs and kids and their mortality,}

95 _{treatment costs, reduced milk production and milk prices dependent}

96 _{on cellular quality in particular areas. Furthermore, hygienic issues}

97 _{related to the risk of infection or intoxication of consumers by milk}

98 _{bacteria such as Escherichia coli, Staphylococcus aureus, Listeria}

99 _{monocytogenes, Salmonella spp., etc can occur. Finally, the E.U.}

100 _{Directive 46/92 (modified by Directive 71/94), which defines the}

101 _{milk bacteriological quality, must be considered (Bergonier et al.,}

102 _{2003b; Olechnowicz &} JK@B@  2014). In sheep, mammary

103 _{infections are also of great welfare concern (European Food Safety}

104 _{Authority, 2009), since clinical mastitis is a disease that leads to}

105 _{anxiety, restlessness, changes in feeding behavior and pain in}

106 _{affected ewes (Fthenakis & Jones, 1990). Even in subclinical}

107 _{mastitis, normal behavioral patterns of sheep are modified}

108 _{(Gougoulis et al., 2008; Gougoulis, Kyriazakis & Fthenakis, 2010),}

109 _{hence rising potential welfare concerns (Gelasakis et al., 2015).}

110 _{Mastitis of small ruminants are commonly divided into clinical and}

111 _{subclinical forms. Clinical mastitis is the term used for}

112 _{inflammatory process of the mammary gland, mostly from}

113 _{bacterial infection, which is accompanied by evident clinical}

114 _{symptoms. These visible signs of clinical mastitis include swelling,}

115 _{redness or necrosis of one or more half udders and/or abnormal}

116 _{discharge of milk. Affected animals can also present with anorexia,}

117 _{fever or agalactia. Evolution of clinical mastitis might be toxaemia}

118 _{and gangrenous necrosis of the udder (Omaleki et al., 2011), which}

119 _{could result in a fatal event. Conversely, subclinical mastitis is}

120 _{characterized by inflammation of the udder that can be diagnosed}

121 _{only by enumeration of inflammatory cells in milk or inflammatory}

122 _{markers evaluation (White & Hinckley, 1999; Olechnowicz &}

123 JK@B@ , 2014; Addis et al., 2016; Bramis et al., 2016). Among

124 _{the organisms associated with clinical mastitis, Staphylococcus}

125 _{aureus has been reported to be the most common in both meat and}

126 _{dairy ewes milk samples. Mannheimia haemolytica, Escherichia}

127 _{coli and various streptococci are other important causative}

128 _{organisms isolated from udder secretions (Mørk et al., 2007; Smith}

129 _{et al., 2015; Arsenault et al., 2008; Dore et al., 2016).}

Coagulase-130 _{negative staphylococci (CNS) are the most prevalent bacteria}

131 _{isolated in subclinical mastitis of sheep and goats (Contreras et al.,}

132 _{2007; Ergün et al., 2009; Onni et al., 2010; Addis et al., 2016;}

133 _{Bramis et al., 2016; Dore et al., 2016), with a prevalence in dairy}

134 _{ewes ranging from 25 to 93%. On the contrary, they are less}

135 _{frequently observed in meat ewes (12-34%) (Olechnowicz &}

136 JK@B@ , 2014). Small Ruminant Lentivirus infection has also

137 _{been reported to be an important cause of chronic subclinical}

138 _{mastitis predisposing the gland to secondary infections (Cutlip et}

139 _{al., 1985; Brodie et al., 1995; Arsenault et al., 2003; Arsenault et}

140 _{al., 2008).}

141 _{Despite mammary gland representing the specialized milk}

142 _{production organ, no studies about the microbiological flora}

143 _{isolated from healthy mammary gland tissue of small ruminants}

144 _{and, particularly, the correlation between microbiological data and}

145 _{histological findings are currently available.}

146 _{For this reason the aim of the present study was to investigate the}

147 _{microbial agents (bacteria, fungi and SRLV) isolated from}

148 _{macroscopically healthy mammary parenchyma of regularly}

149 _{slaughtered small ruminants and to correlate their presence with the}

150 _{histological findings.}

151

152 _{Materials & Methods}

153 _{Sample collection}

154 _{A total of 89 macroscopically healthy small ruminants udders (n =}

155 _{41 sheep - 23 lactating and 18 non lactating- and n = 48 goats - 36}

156 _{lactating and 12 non lactating) belonging to a total of 25 ovine and}

157 _{27 caprine flocks, respectively, were collected between October}

158 _{2013 and February 2016 in two slaughterhouses of northern Italy,}

159 _{Piedmont region. Animals enrolled in the study were slaughtered}

160 _{for different causes unrelated to the mammary glands involvement.}

161 _{The sheep aged from 1 to 15 years (median 5) and were mainly}

162 _{Biellese (n = 20; 48.8%) and cross-breed (n = 17; 41.5%), with}

163 _{Frabosana (n = 2; 4.9%), Lacaune (n = 1; 2.4%) and Sambucana}

164 _{breed (n = 1; 2.4%) being less represented. The goats aged from 2}

165 _{to 13 years (median 5.5) and were mainly Saanen (n = 13; 27.1%)}

166 _{and cross-breed (n = 25; 52.1%), with a lower prevalence of}

167 _{Camosciata delle Alpi breed (n = 10; 20.8%). Animals were}

168 _{classified in three groups according to their age: the first group}

169 _{included 27 animals which were up to 3 years old (n = 14 sheep}

170 _{and n = 13 goats), the second group was composed of 44 animals}

171 _{aged from 4 to 8 years (n = 20 sheep and n = 24 goats) and the}

172 _{third group was composed of 18 animals over 8 years (n = 7 sheep}

173 _{and n = 11 goats).}

174 _{Only udders considered free of clinical mastitis and any other}

175 _{alterations at gross examination (inspection and palpation for}

176 _{macroscopic signs of abnormalities) were included in this study.}

177 _{After gross examination, all the udders were stored at 4°C and}

178 _{transported to the laboratory of the Department of Veterinary}

179 _{Sciences of University of Turin. The skin of the udder was washed,}

180 _{dried and disinfected with cotton soaked with 70% alcohol. Two}

181 _{fragments of parenchyma of approximately 2.5 cm}3_{, were collected}

182 _{in two different points and fixed in vials containing 10% neutral}

183 _{buffered formalin for histopathological examination, one sample}

184 _{was frozen at} O-N% as tissue bank, and the remaining tissue was

185 _{sent to the Istituto Zooprofilattico Sperimentale of Piemonte,}

186 _{Liguria and Valle d’Aosta (Turin) for bacteriological, mycological}

187 _{and virological investigations.}

188

189 _Bacteriology

190 _{The udder surface was sterilized through cauterization and then cut}

191 _{using scalpel until the glandular tissue was exposed. A swab was}

192 _{inserted into the incision and then it was streaked on several agar}

193 _{plates. Columbia agar containing 5% sheep blood (Liofilchem) and}

194 _{Chocolate agar (Liofilchem) plates were used and incubated at}

195 _{37°C aerobically and 5% CO}2_{-enriched atmosphere, respectively,}

196 _{for 24-48 hours. Bacteria identification was carried out by}

197 _{phenotypic identification methods; for this purpose a growth-based}

198 _{automatic methods (Vitek 2 System, bioMérieux) that measures}

199 _{either turbidity or colored products by various metabolic activities}

200 _{such as acidification, alkalinization, enzyme hydrolysis, and growth}

201 _{in the presence of inhibitory substances was used. According to}

202 _{Carter and Cole (1990) it has been selected only the colonies}

203 _{numerically representative, in pure culture or showing}

204 _{phenotypically features as haemolysis. In order to obtain a clear}

205 _{interpretation of the results, a parallel control for the identification}

206 _{of inhibiting substances in the udders was performed. Therefore,}

207 _{two fragments of parenchyma were placed on an agar plate}

208 _{containing an inoculum of Bacillus subtilis and incubated}

209 _{aerobically for 24 hours at 37°C. The test was considered positive}

210 _{if a ring around the fragment, which indicates the inhibition of}

211 _{bacterial growth, was observed.}

212 _{For the detection of Mycoplasma spp., a PPLO Selective Agar}

213 _{(Microbiol) was incubated at 37°C in 5% CO}2_-enriched

214 _{atmosphere. A pre-enrichment was performed in order to promote}

215 _{the isolation of Mycoplasma spp. In particular, a fragment of}

216 _{parenchyma was inoculated in PPLO Broth (Microbiol) and}

217 _{incubated at 37°C in 5% CO}2_{-enriched atmosphere. After 72 hours,}

218 _{0.1 ml of broth was inoculated on PPLO agar. Mycoplasma’s}

219 _{growth was monitored for 10 days by microscopic examination}

220 _{with the aim to identify any typical tiny “fried egg” or finely}

221 _{granular (“ground glass”) colonies that penetrate the agar surface.}

222 _{Bacteria were classified in four groups:}

223  pathogens (PATO): according to the literature, pathogenic bacteria

224 _{reported in clinical mastitis;}

225  coagulase-negative staphylococci (CNS): opportunistic bacteria

226 _{reported in the normal skin microbial flora usually responsible for}

227 _{subclinical mastitis. Their pathogenicity is highly variable from}

228 _{asymptomatic to mild forms. Sporadic cases of clinical mastitis,}

229 _{especially in immunocompromised animals, are also described;}

230  environmental opportunists (EO): ubiquitous microorganisms

231 _{detected in the gastro-enteric tract, soil, water and bedding. Once}

232 _{penetrated within the udder, they are able, under particular}

233 _{conditions, to become pathogenic and cause mastitis of different}

234 _severity;

235  other microorganisms (OM): bacteria not isolated in clinical or

236 _{subclinical mastitis.}

237

238 _Mycology

239 _{Sabouraud agar plate (Biolife) was used for the isolation of several}

240 _{types of fungi and filamentous bacteria, directly streaking the swab,}

241 _{as previously described, on the surface on the plate. The plate was}

242 _{incubated for 10 days at room temperature. In case of growth of}

243 _{suspect colonies, a microscopic examination was carried on in}

244 _{order to obtain an identification at genus-level.}

245

246 _Virology

247 _{For SRLV detection, DNA was extracted from 50-100 mg of udder}

248 _{tissue by using ReliaPrep™ gDNA Tissue Miniprep System}

249 _{(Promega), following manufacturer’s instructions. DNA was}

250 _{amplified following a slightly modified version of the protocol}

251 _{described in Grego et al., 2007. Briefly, two forward degenerate}

252 _{primers, GAG F1}

(5’-TGGTGARKCTAGMTAGAGACATGG-253 _{39) and GAG F2 (5’-CAAACWGTRGCAATGCAGCATGG-3’),}

254 _{and two reverse degenerate primers POL R1}

(5’-255 _{CATAGGRGGHGCGGACGGCASCA-3’) and POL R2}

(5’-256 _{GCGGACGGCASCACACG- 3’) were designed, based on}

257 _{conserved regions (Grego et al., 2007).}

258 _{The first PCR reaction was carried out in a total volume of 50 µl}

259 _{containing 1X PCR Buffer (Invitrogen, Platinum Taq DNA}

260 _{Polymerase), 0.2 mM of dNTPs, 3 mM of MgCl2, 0.3 µM of each}

261 _{primers, 1U Taq Polymerase (Invitrogen). The PCR was performed}

262 _{under the following conditions: initial template denaturation step at}

263 _{95°C for 15 min; 35 cycles of denaturation at 94°C for 30 s,}

264 _{annealing at 55°C for 1 min, and elongation at 72°C for 2 min,}

265 _{followed by a final elongation step at 72°C for 10 min. Second}

266 _{PCR was carried out in the same conditions (using primers GAG}

267 _{F2 and POL R2), with a lower annealing temperature (55°C) for a}

268 _{total of 45 cycles. Amplification products were loaded and checked}

269 _{on a 2 % agarose gel.}

270

271 _Histology

272 _{Formalin fixed fragments of parenchyma were paraffin embedded}

273 _{and cut at 5 µm thickness sections. The sections were stained with}

274 _{haematoxylin and eosin (H&E) and examined by light microscope}

275 _{at different magnifications. Microscopically, lesions were classified}

276 _{as negative when they showed a normal pattern; affected by}

277 _{chronic non-suppurative mastitis when they showed interstitial}

278 _{and/or alveolar mononuclear inflammatory cells (lymphocytes,}

279 _{plasmacells, macrophages) and mild to severe fibrosis; affected by}

280 _{chronic mixed mastitis when they showed interstitial and/or}

281 _{alveolar mononuclear inflammatory cells and a variable number of}

282 _{neutrophils; affected by acute suppurative mastitis when they}

283 _{showed interstitial and/or alveolar neutrophils (Figure 1).}

284

285 _{Statistical analysis}

286 _{Multinomial logistic regression performed using generalized linear}

287 _{mixed model (GLMM) was applied to evaluate the association}

288 _{among the histological findings, infection and animal attributes.}

289 _{The GLMM included as covariates the positivity for CNS, EO,}

290 _{PATO and SRLV to investigate relationship between infection}

291 _{status and histological lesions. The animal attributes included in}

292 _{GLMM were age and species which could be linked with}

293 _{histological lesions. Each analyzed udder was considered positive}

294 _{or negative for each investigated agent if one of its isolate showed}

295 _{positivity during laboratory tests.}

296 _{A logistic regression, adjusted for animal age and presence of each}

297 _{bacteria class, was performed to investigate relationship between}

298 _{presence of follicles at histological examination and infections of}

299 _{SRLV. This model was adjusted for infection of bacterial groups}

300 _{(EO, CNS), age and species. The possible effect of age, species and}

301 _{SRLV positivity on colonization of udder by CNS, EO was tested}

302 _{with a series of logistic regressions performed using GLMM. Given}

303 _{the low number of udders showing positivity to PATO, it was not}

304 _{possible to perform such investigation for this class of bacteria. All}

305 _{regression models contained farm as random effect to adjust for}

306 _{sampling more individuals from same farm.}

307

308 _Results

309 _{Among the 89 macroscopically healthy udders, 25/89 (28.09%;}

310 _{sheep = 6; goats = 19) were microbiologically negative, while at}

311 _{least one bacterial species was isolated in the remaining 64/89}

312 _{(71.91%; sheep = 35; goats = 29). In the positive udders, 138}

313 _{different bacterial species were isolated. The bacterial species}

314 _{isolated from udders of sheep and goats are reported in Table 1.}

315 _{Coagulase-negative staphylococci (CNS) were the most prevalent}

316 _{bacteria isolated from both goats and sheep. Among the 138}

317 _{isolated bacteria, 64 (46.42%) belonged to this group. The detail of}

318 _{the species isolated are summarized in Table 1.}

319 _{Environmental opportunists were the second most frequently}

320 _{identified bacterial group (48/138; 34.76%). Aerococcus viridans}

321 _{was the predominant strain in both species being isolated in 29}

322 _{samples and thus representing 21.01% of the total isolates. A total}

323 _{of 14/138 microorganisms (10.14%) were classified as “other}

324 _{microorganisms”. The most frequent microorganisms were}

325 _{Acinetobacter lwoffii and Kocuria rosea, with 3 cases each}

326 _{(2.17%). Pathogen bacteria were isolated in a lower percentage}

327 _{(12/138 samples; 8.68%). Staphylococcus aureus was found in 5}

328 _{cases (3.62%), while Streptococcus uberis was identified in 4}

329 _{(2.9%). Streptococcus dysgalactiae, Pseudomonas aeruginosa and}

330 _{Escherichia coli represented the 0.72% of isolated strains.}

331 _{Co-infections has been detected in 48/64 (75%) of the mammary}

332 _{glands; specifically, co-infections caused by two (42.2%) and three}

333 _{(26.5%) different bacterial species were demonstrated in 27/48 and}

334 _{17/48 samples respectively. Four bacterial species (4.7%) were}

335 _{identified in 3/48 samples and five (1.6%) were found in 1/48}

336 _{udder only. Only the 25% (16/64) of the positive udders were}

337 _{characterized by a single infection.}

338 _{In 4 udders (4.5%) inhibiting substances were identified. Only 1}

339 _{Aspergillus spp. (1.1%) grew on Sabouraud agar plate.}

340 _{Small Ruminant Lentivirus infection was detected in 35/89 samples}

341 _{(39.3%). Most of them were isolated along with bacteria (21/35;}

342 _{60%). Goats were more infected than sheep (29/35; 82.9% versus}

343 _{6/35; 17.1%).}

344 _{Histologically, 45/89 udders showed chronic non-suppurative}

345 _{mastitis (50.56%; sheep n = 17 and goat n = 28), 12 chronic mixed}

346 _{mastitis (13.48%; sheep n = 2 and goat n = 10), 4 acute suppurative}

347 _{mastitis (4.5%; sheep n = 1 and goat n = 3) and 28 did not show}

348 _{any lesions (31.46%; sheep n = 21 and goat n = 7).}

349 _{In chronic mastitis the inflammatory cells were mainly located in}

350 _{the interstitium; alveolar neutrophils were detected in mixed (66%)}

351 _{or suppurative (100%) mastitis only. However necrosis and loss of}

352 _{the alveolar epithelium were never observed.}

353 _{Microbiological and histological findings observed in the present}

354 _{study are reported in Table 2. In chronic non-suppurative mastitis,}

355 _{mixed mastitis and normal mammary glands, coagulase-negative}

356 _{staphylococci and environmental opportunists were the most}

357 _{frequently detected bacteria. On the contrary, acute suppurative}

358 _{mastitis showed a greater number of pathogenic microorganisms.}

359 _{Microbial agents were absent in 19/28 normal mammary glands}

360 _only.

361 _{Within SRLV positive udders, 25/35 (71.5%) showed a chronic}

362 _{non-suppurative mastitis (Figures 1A, B); 8/35 (22.9%) had a}

363 _{chronic mixed mastitis (Figure 1C); 1/35 (2.8%) showed an acute}

364 _{suppurative mastitis (Figure 1D) and only 1/35 (2.8%) did not show}

365 _{any lesions (Figure 1E). Lentiviruses positive udders (68.6%;}

366 _{24/35) revealed scattered hyperplastic lymphoid follicles}

367 _{throughout the parenchymal tissue and particularly around the}

368 _{lactiferous ducts (Figure 1F).}

369 _{Histological lesions were significantly associated to species and}

370 _{SRLV and CNS infections. Small Ruminant Lentivirus positive}

371 _{udders had significantly high risk to show both chronic mixed}

372 _{mastitis and chronic non-suppurative mastitis at histological}

373 _{examination (Table 3). Goats had significant higher risk to show}

374 _{chronic mixed mastitis compared to sheep (Table 3). CNS infection}

375 _{significantly increased the risk of chronic non-suppurative mastitis.}

376 _{Presence of PATO did not increase the risk of presenting one}

377 _{particular histological lesion (Table 3). Lentiviruses positive udders}

378 _{also showed a 12.3 (95% CI: 3.8-45.2; p<0.01) times higher risk to}

379 _{have lymphoid follicles at histological examination. Goats had}

380 _{significant lower risk to be infected by EO compared to sheep}

381 _{(Table 4), but higher risk of being positive for SRLV (Table 5).}

382 _{Age and co-infections did not show any effects on bacterial or viral}

383 _{infections (Table 4).}

384

385 _Discussion

386 _{This is the first study investigating the presence of microbial agents}

387 _{and histological features in macroscopically healthy mammary}

388 _{glands of small ruminants.}

389 _{Coagulase-negative staphylococci prevail, (64/138; 46.42%), in}

390 _{agreement with the literature. Already Bergonier et al. (2003b)}

391 _{reported a CNS prevalence in subclinical mastitis ranging from 25}

392 _{to 93%. Similar results (42.9%) were recently observed also in}

393 _{Portugal in milk samples from clinically affected sheep udders}

394 _{(Queiroga (2017), in Iran in subclinical ovine mastitis (Narenji Sani}

395 _{et al., 2015), in Greece, in dairy goats (Gelasakis et al., 2016) and}

396 _{in Italy in both species (Dore et al., 2016). The identification of}

397 _{Staphylococcus xylosus in sheep and Staphylococcus caprae in}

398 _{goats is also in agreement with the literature (Bergonier et al.,}

399 _{2003b; Contreras et al., 2007; Ergün et al., 2009), while}

400 _{Staphylococcus equorum was isolated only by Hariharan et al.}

401 _{(2004) in sheep and by Contreras et al. (2003) in goats. Differently}

402 _{from previous studies reporting a common isolation (Bergonier et}

403 _{al., 2003b; Contreras et al., 2007), Staphylococcus chromogenes}

404 _{was observed in two samples only. According to the literature}

405 _{(Contreras et al., 2007; Gelasakis et al., 2015), the other species of}

406 _{CNS identified in the present research were observed with a lower}

407 _{frequency (Table 1).}

408 _{The role of OM in the epidemiology of small ruminants mastitis is}

409 _{still unclear. Only Aerococcus viridans and Enterococcus spp. have}

410 _{previously been described as a sporadic cause of mammary}

411 _{infection in sheep and goats (Clements, Taylor & Fitzpatrick, 2003;}

412 _{Marogna et al., 2012; Queiroga, 2017).}

413 _{Goats had a significant lower risk to be infected by EO compared}

414 _{to sheep. Since EO prevalence increases when deficiencies in}

415 _{mechanical milking system or milking hygiene in small ruminants}

416 _{flocks (particularly in sheep flocks) occur, milking routines as well}

417 _{as milking equipment must be improved and periodically checked.}

418 _{The low rate of fungi isolation Aspergillus spp. (1.1%) can be}

419 _{related to their common detection in clinical mastitis (Bergonier et}

420 _{al., 2003b), while the present study reports the isolation in}

421 _{macroscopically unaffected tissues.}

422 _{The SRLV identification (35/89; 39.3%) agrees with the literature}

423 _{(Fras et al., 2013). Our work also showed that SRLV were more}

424 _{likely detected in goat udders than sheep (29/35; 82.9% versus}

425 _{6/35; 17.1%). The high prevalence of SRLV infection in goats}

426 _{could be caused by the horizontal transmission with colostrum and}

427 _{milk. Indeed, in sheep industry, lambs usually suckle only by their}

428 _{mothers. On the contrary, kids are separated from mothers at birth,}

429 _{housed together, and hand-fed colostrum and milk, often from a}

430 _{common pool. So, a SRLV presence in the mammary tissue of a}

431 _{single animal may infect all the young animals. This occurrence has}

432 _{been reported to be the most reliable reason for the high prevalence}

433 _{of CAEV infection in goats in Western Europe, Australia, and}

434 _{North America (Narayan & Cork, 1985; Fras et al., 2013).}

435 _{Small Ruminant Lentivirus infections have been associated with a}

436 _{spectrum of immune-mediated diseases and opportunistic}

437 _{infections (Brodie et al., 1995). In goats, several studies observed}

438 _{that SRLV infected animals appear to be more predisposed to}

439 _{subclinical bacterial infections by non-haemolytic staphylococci}

440 _{(Smith & Cutlip, 1988; Ryan, Greenwood & Nicholls, 1993). Also}

441 _{in the present study, goats showed a significant higher risk to show}

442 _{chronic mixed mastitis compared to sheep.}

443 _{In sheep, in vivo relationship between SRLV and bacteria is not}

444 _{known, but previous in vitro studies provided evidence for}

445 _{increased bacterial adherence and decreased phagocytosis in}

446 _{macrophages infected by SRLV (Monleón et al., 1997). In the}

447 _{present study, chronic mastitis observed in SRLV positive udders is}

448 _{frequently associated with CNS and EO infections. Although the}

449 _{SRLV positive samples showed a significantly higher risk to be}

450 _{affected by both mixed and non-suppurative mastitis, co-infections}

451 _{did not show any effects on the virus detection.}

452 _{Despite the udders considered in the present study being}

453 _{macroscopically healthy, the absence of lesions at the histological}

454 _{screening was observed in 28 samples only. The remaining 61}

455 _{udders showed different types of mastitis; most of them were}

456 _{chronic (64%) and mild in severity. Mild to severe fibrosis was}

457 _{observed in all the samples (28%: slight; 25.8%: moderate; 46%:}

458 _{severe). However, this finding cannot be related to the chronicity of}

459 _{the disease only, because the ratio between parenchyma and stroma}

460 _{shows individual proportions and changes during the different}

461 _{phases of the reproductive cycle of the animals and of the lactation}

462 _{cycle (Lérias et al., 2014). The mastitis chronicity was mainly}

463 _{evaluated on the basis of the inflammatory cells type, diagnosing a}

464 _{chronic mastitis when the exudate was composed by macrophages,}

465 _{histiocytes and plasmacells. An interesting finding is represented}

466 _{by the identification of these non-suppurative cells mainly in the}

467 _{interstitium; alveolar and ductal cells (neutrophils) were detected in}

468 _{mixed or suppurative mastitis only. In these udders, an increased}

469 _{number of desquamated cells was also observed in the alveoli.}

470 _{The correlation between microbiological and histological findings}

471 _{suggests that the animals in which CNS were isolated had a}

472 _{significantly increased risk to develop a chronic non-suppurative}

473 _{mastitis. There were no other significant correlations. However, in}

474 _{acute suppurative mastitis pathogen bacteria were always isolated,}

475 _{except in one case in which the identification of inhibiting}

476 _{substances justifies a negative microbiological examination}

477 _{(Zavanella, 2011).}

478 _{At histological examination, SRLV positive udders revealed}

479 _{scattered hyperplastic lymphoid follicles. Indeed, SRLV generally}

480 _{causes induritive and non-suppurative mastitis characterized by}

481 _{periacinar interstitial diffuse mononuclear cell infiltration,}

482 _{interstitial intense fibrosis and hyperplastic lymphoid follicle}

483 _{formation scattered throughout the parenchymal tissue particularly}

484 _{around the lactiferous ducts (van der Molen, Vecht & Houwers,}

485 _{1985; Brodie et al., 1995; Monleón et al., 1997). Eleven animals}

486 _{affected by chronic non-suppurative mastitis and bacteriologically}

487 _{negative were positive for SRLV, thus confirming the role of}

488 _{SRLV in sub-clinical chronic mastitis (Plummer and Plummer,}

489 _{2012). The statistical model results further support this result.}

490

491 _Conclusions

492 _{The results of the present study demonstrate that macroscopically}

493 _{unaffected glands of small ruminants do not represent a sterile}

494 _{substrate, but may be a reservoir of microbial agents whose role}

495 _{remains to be better evaluated. The detection of a large number of}

496 _{bacterial species improves the current knowledge of the species}

497 _{capable of colonizing and potentially infecting mammary glands of}

498 _{small ruminant. Many bacteria are able, at least temporarily, to be}

499 _{sequestered inside the mammary tissue. This ability might be one}

500 _{of the mechanisms that these bacterial species adopt to by-pass host}

501 _{defenses and treatments, allowing them to persist within individuals}

502 _{and the flock.}

503 _{Therefore, from a flock management perspective, small ruminants}

504 _{with subclinical chronic mastitis have a potential risk to develop}

505 _{acute or chronic clinical disease. Given the paucity of the sample}

506 _{size of our data, the magnitude of risk cannot be determined from}

507 _{the current study. The high number of EO and OM suggests the}

508 _{need to develop and apply procedures for controlling microbial and}

509 _{environmental contamination and consequently to improve hygiene}

510 _{and quality of milk and dairy products and finally the consumers’}

511 _welfare.

512 _{Despite some histological lesions being associated with SRLV and}

513 _{CNS infections (hyperplastic lymphoid follicles and}

non-514 _{suppurative mastitis), it seems difficult to correlate each}

515 _{histological pattern with specific microbial isolation.}

516 _{Possible future investigations could aim to screen macroscopically}

517 _{affected mammary glands in order to evaluate the relationships}

518 _{between the isolated biological agents in clinical acute and chronic}

519 _{mastitis with histological patterns.}

520

521

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Figure 1

Figure 1. Histological findings in healthy mammary glands of small ruminants.

mononuclear inflammatory cells (lymphocytes, plasmacells and macrophages).

Haematoxylin& Eosin (H&E) stain. B) Particular of the mononuclear inflammation in chronic non suppurative mastitis. H&E stain. C) Chronic mixed mastitis with interstitial mononuclear inflammatory cells (arrow) and alveolar neutrophils (arrowhead). H&E stain. D) Acute

suppurative mastitis characterized by interstitial and alveolar neutrophils (arrows). H&E stain. E) A normal mammary tissue. H&E stain. F) Small Ruminant Lentiviruses positive mammary gland with wide and focal hyperplastic lymphoid follicle (arrow) around a lactiferous duct. H&E stain.

Table 1

Table 1. Number and frequency of bacterial species isolated.

1 Number and frequency of bacterial species isolated. Microorganisms Species Isolates N° % Sheep Goats Pathogens Staphylococcus aureus 5 3.62% 3 2 Streptococcus uberis 4 2.90% 2 2 Streptococcus dysgalactiae 1 0.72% 1 0 Pseudomonas aeruginosa 1 0.72% 0 1 Escherichia coli 1 0.72% 0 1 total of group 12 8.68% 6 6 Coagulase-negative staphylococci Staphylococcus arlettae 2 1.46% 2 0 Staphylococcus auricularis 5 3.62% 2 3 Staphylococcus capitis 1 0.72% 0 1 Staphylococcus caprae 7 5.07% 0 7 Staphylococcus chromogenes 2 1.46% 2 0 Staphylococcus cohnii 2 1.46% 1 1 Staphylococcus equorum 7 5.07% 4 3 Staphylococcus haemolyticus 2 1.46% 1 1 Staphylococcus kloosii 1 0.72% 1 0 Staphylococcus lentus 1 0.72% 1 0 Staphylococcus muscae 2 1.46% 1 1 Staphylococcus sciuri 2 1.46% 1 1 Staphylococcus simulans 2 1.46% 1 1 Staphylococcus vitulinus 4 2.90% 3 1 Staphylococcus warneri 5 3.62% 3 2 Staphylococcus xylosus 18 13.04% 9 9 Staphylococcus spp. 1 0.72% 0 1 total of group 64 46.42% 32 32 Environmental opportunists Aerococcus viridans 29 21.01% 19 10 Aeromonas hydrophila 3 2.17% 3 0 Aeromonas sobria 1 0.72% 1 0 Bacillus pumilus 6 4.34% 1 5

Manuscript to be reviewed

Escherichia coli 2 1.46% 1 1 Enterococcus cecorum 1 0.72% 1 0 Enterococcus durans 1 0.72% 0 1 Enterococcus faecalis 1 0.72% 0 1 Klebsiella oxytoca 2 1.46% 2 0 Pseudomonas fluorescens 1 0.72% 0 1 Streptococcus constellatus 1 0.72% 1 0 total of group 48 34.76% 29 19 Other microorganisms Acinetobacter lwoffii 3 2.17% 1 2 Acinetobacter genomospecies 1 0.72% 0 1 Acinetobacter spp. 1 0.72% 0 1 Alcaligenes faecalis 2 1.46% 2 0 Bordetella trematum 1 0.72% 1 0 Kocuria rosea 3 2.17% 3 0 Paenibacillus spp. 1 0.72% 0 1 Vagococcus fluvialis 2 1.46% 1 1 total of group 14 10.14% 8 6 Total 138 100% 75 63 2

Manuscript to be reviewed

Table 2

Table 2. Summary of bacterial species identified within each histological type of

mastitis.

1 _{Summary of bacterial species identified within each histological type of mastitis.}

Histological type of mastitis Bacteriological data Virological data PATOa _CNSb _EOc _OMd _{Negative SRLV}e

Chronic non suppurative mastitis 6 34 22 8 13 25

sheep 3 14 11 2 2 5

goat 3 20 11 6 11 20

Chronic mixed mastitis 2 10 8 0 3 8

sheep 1 2 3 0 0 0

goat 1 8 5 0 3 8

Acute suppurative mastitis 3 1 2 0 1 1

sheep 1 0 1 0 0 0 goat 2 1 1 0 1 1 Negative 1 19 16 6 8 1 sheep 1 16 14 6 4 1 goat 0 3 2 0 4 0 2 3 4 a_{PATO = Pathogens} 5 b_{CNS = Coagulase-negative staphylococci} 6 c_{EO = Environmental opportunists} 7 d_{OM = Other microorganisms}

8 e_{SRLV = Small Ruminant Lentiviruses}

9 10 11 12 13

Manuscript to be reviewed

Table 3

Table 3

Results from multinomial logistic regression applied to evaluate association among

histological lesions in mammary gland of goat/sheep and bacterial infection, SRLV infection, age and species. Udders without sign of histological lesions were set as reference class for the multinomial regression.

1 _{Results from multinomial logistic regression applied to evaluate association among histological}

2 _{lesions in mammary gland of goat/sheep and bacterial infection, SRLV infection, age and}

3 _{species. Udders without sign of histological lesions were set as reference class for the}

4 _{multinomial regression.}

5

Covariate Chronic mixed mastitis

[OR (95% CI)]

Chronic non suppurative mastitis [OR (95% CI)]

Caprine (Ref: Ovine) 13.7 (1.75, 34.1)* 5.21 (0.83, 17.1)

Age (Ref: 1-3 years of age):

4-8 years of age 3.14 (0.46, 22.65) 2.21 (0.74, 12.19) >8 years of age 1.32 (0.14, 10.46) 0.82 (0.15, 4.42) EOa _{2.61 (0.46, 17.16) 2.18 (0.41, 4.79)} CNSb _{7.25 (0.59, 22.10) 6.72 (1.83, 28.51)*} PATOc _{9.21 (0.68, 25.67) 7.8 (0.56, 16.71)} SRLVd _{47.5 (3.15, 80.16)** 43.52 (4.30, 104.52)**} 6 _{*p<0.05;**p<0.01} 7 a_{EO = Environmental opportunists} 8 b_{CNS = Coagulase-negative staphylococci} 9 c_{PATO = Pathogens}

10 d_{SRLV = Small Ruminant Lentivirus}

11

12

Table 4

Table 4. Results from logistic regressions applied to evaluate factors in mammary gland

of sheep/goat which can favour infection of CNS and EO.

1 _{Results from logistic regressions applied to evaluate factors in mammary gland of sheep/goat}

2 _{which can favour infection of CNS and EO.}

Covariate CNSb _{[OR (95% CI)] EO}a _{[OR (95% CI)]}

Caprine (Ref: Ovine) 0.12 (0.32, 4.2) 0.26 (0.06, 0.71)*

Age (Ref: 1-3 years of age):

4-8 years of age 1.25 (0.61, 3.32) 1.27 (0.27, 3.805) >8 years of age 1.34 (0.48, 5.76) 2.09 (0.38, 7.45) EOa _{2.16 (0.79, 4.98)} -CNSb _{- 3.51 (0.59, 5.31)} PATOc _{1.23 (0.21, 4.67) 2.31 (0.42, 6.34)} SRLVd _{1.52 (0.42, 3.78) 2.97 (0.60, 4.24)} 3 _{*p<0.05;**p<0.01} 4 a_{EO = Environmental opportunists} 5 b_{CNS = Coagulase-negative staphylococci} 6 c_{PATO= Pathogens}

7 d_{SRLV = Small Ruminant Lentivirus}

Table 5

Table 5. Results from logistic regressions applied in mammary gland of goat/sheep to

evaluate factors that can favour SRLV infection.

1 _{Results from logistic regressions applied in mammary gland of goat/sheep to evaluate factors}

2 _{that can favour SRLV infection.}

Covariate CNSb _{[OR (95% CI)]}

Caprine (Ref: Ovine) 10.87 (3.69, 37.77)**

Age (Ref; 1-3 years of age):

4-8 years of age 0.66 (0.18, 2.33) >8 years of age 0.46 (0.11, 1.82) EOa _{1.67 (0.41, 3.29)} CNSb _{1.15 (0.58, 5.18)} PATOc _{0.67 (0.34, 4.89)} 3 _{*p<0.05;**p<0.01} 4 a_{EO = Environmental opportunists} 5 b_{CNS = Coagulase-negative staphylococci} 6 c_{PATO= Pathogens} 7 8 9 10 11 12

Manuscript to be reviewed

13

14