Transmission of Mannheimia haemolytica from the tonsils of lambs to the teat of ewes during sucking

Veterinary Microbiology 148 (2011) 66–74

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Research article

Transmission of Mannheimia haemolytica from the tonsils of lambs to the teat of ewes during sucking I.A. Fragkou a, D.A. Gougoulis a, C. Billinis a, V.S. Mavrogianni a, M.J. Bushnell b, P.J. Cripps c, A. Tzora d, G.C. Fthenakis a,* a

Veterinary Faculty, University of Thessaly, P.O. Box 199, 43100 Karditsa, Greece The Royal Veterinary College, University of London, North Mymms AL9 7TA, United Kingdom Faculty of Veterinary Science, University of Liverpool, Neston, South Wirral CH64 7TE, United Kingdom d TEI Epirus, 48100 Arta, Greece b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 January 2010 Received in revised form 6 August 2010 Accepted 16 August 2010

Objective of the work was to study whether Mannheimia haemolytica may be transmitted from the mouth of the lambs into the teat of the dam during sucking. We compared bacterial populations within the teat duct and milk of ewes immediately before and immediately after sucking by the lambs. Tonsils of lambs of the ewes were swabbed. M. haemolytica strain DAG21T recovered from a teat duct of a ewe was compared to strain DAG21R recovered from the tonsils of her lamb by using 16s rRNA sequencing. We used those two isolates and another one of known pathogenicity, for challenging ewes: (i) 2mm deep into healthy teats, (ii) 2-mm deep into teats with chapping lesions or (iii) into the cistern of healthy mammary glands. Of samples collected before suckling, 20/792 were bacteriologically positive, and of those after, 50/792 were bacteriologically positive (P < 0.001); in 37 cases, a negative sample became positive. One M. haemolytica (DAG21T) was recovered after suckling from a teat duct of a ewe. The organism was isolated from 57/ 90 tonsillar swabs from lambs. Risk of infection of ewe’ teats was 0.004 throughout lactation, being greatest (0.021) during the 3rd week of lactation. The 16s rRNA sequences of strains DAG21T and DAG21R were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica. Organism deposition into healthy teats caused subclinical mastitis; deposition into teats with lesions or directly into mammary gland caused clinical mastitis. When results of inoculation of the three strains were compared between them, statistical significance was always P > 0.9. Results provide clear evidence that suckling by lambs can lead to transmission of M. haemolytica into the teats of the ewes; the bacteria have the potential to cause mastitis if circumstances are favourable. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Mastitis Sheep Teat Bacterial transmission Mannheimia haemolytica

1. Introduction In studies of ovine mastitis, it has been postulated that sucking is associated with transfer of microorganisms into the teat duct (Scott and Jones, 1998; Jones and Watkins, 2000; Mavrogianni et al., 2005). There could be three

* Corresponding author. E-mail address: [email protected] (G.C. Fthenakis). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.08.008

different sources of bacteria for the teat duct: the lamb (mouth, nasopharynx), the ewe (udder skin) or the environment. The organisms may subsequently ascent to the mammary gland and cause mastitis. Knowledge of the source of the cause of the disease is important for implementation of appropriate control strategies. Mannheimia haemolytica is an important mastitis causal agent (Bergonier and Berthelot, 2003). Previous researchers have suggested the possibility of transmission of the organism during sucking by lambs. M. haemolytica is a

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common cause of ovine mastitis in suckling, but not in dairy, ewes (Jones and Lanyon, 1987; Jones and Watkins, 1998). It has been isolated from the teat skin of suckling, but not of pregnant, ewes (Scott and Jones, 1998). As most lambs in a group carry M. haemolytica in their tonsils (AlSultan and Aitken, 1985), they have been suggested to act as the potential source of the organism for the mammary gland (Scott and Jones, 1998; Jones and Watkins, 2000). Hitherto however, this hypothesis has not been confirmed. Moreover, previous experimental work in ovine mastitis has been limited to using bacterial isolates recovered from the mammary gland of ewes (El-Masannat et al., 1991; Watkins et al., 1991; Mavrogianni et al., 2005, 2006b). The potential pathogenicity of strains of M. haemolytica of respiratory origin has not been evaluated. During the study, we carried out two experiments. Objectives of the first experiment were (i) to determine if M. haemolytica would be isolated from the teat duct of ewes immediately after sucking by lambs and (ii) to compare ‘‘mammary’’ (from the teat duct) and ‘‘respiratory’’ (from tonsils of lambs) isolates of the organism. The objectives of the second experiment were (i) to establish whether ‘‘respiratory’’ isolates are pathogenic for the mammary gland of ewes and (ii) to compare their pathogenicity to that of known pathogenic ‘‘mammary’’ isolates. 2. Materials and methods 2.1. Overview of the study In Experiment I, we used 11 multiparous Karagounikobreed lactating ewes introduced into the experiment within 5 days of lambing. In order to monitor bacterial populations within the teat duct and the milk of the ewes, paired-samples were collected immediately before and after sucking by lambs. The tonsils of the 15 lambs of these ewes, were swabbed as described by Al-Sultan and Aitken (1985). Finally, a M. haemolytica isolate recovered from the teat duct of a ewe (‘‘mammary’’ isolate: DAG21T), was compared to an isolate from the tonsils of her lamb (‘‘respiratory’’ isolate: DAG21R) by using molecular techniques (16s rRNA sequencing). In Experiment II, we used these two M. haemolytica (DAG21T and DAG21R), as well as a M. haemolytica isolate of known pathogenicity for the mammary gland of ewes (ES26L). Animals were allocated at random into group ‘‘A’’ (challenged 2-mm deep into their healthy teat), ‘‘B’’ (challenged 2-mm deep into chapped teat) or ‘‘C’’ (challenged directly into the gland cistern); then, they were allocated into subgroup ‘‘m’’ (challenged with strain DAG21T), ‘‘r’’ (challenged with strain DAG21R) or ‘‘c’’ (challenged with strain ES26L). Therefore, nine (3  3) subgroups (Am/Ar/Ac, Bm/Br/Bc, Cm/Cr/Cc) were formed. Challenge isolates and procedures are summarised in Table 1. All animals were sequentially euthanised after challenge (Table 1). All experimental procedures were carried out under an appropriate license issued by the Greek Ministry of Agriculture, based on EU guidelines. The whole work was carried out under successive blinding procedures.

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Although the principal investigator (GCF) supervised the whole work, parts of it were carried out by other researchers (ewe monitoring and sampling in Experiment I: DAG, bacterial identifications: AT, lamb sampling: VSM, confirmation of identity of M. haemolytica: MJB, molecular techniques: CB, Experiment II: IAF), who were not aware of the other persons’ findings. Statistical analysis was carried out by an independent external epidemiology advisor (PJC). 2.2. Experiment I 2.2.1. Samplings from ewes Paired-samples were obtained from both mammary glands of each ewe. Samples were collected on three occasions weekly, for a period of six weeks during lactation (2nd to 7th after lambing). Lambs were separated from their dams for 60 min. On each occasion, the first sample of each pair (‘‘A’’) was collected before sucking by the lambs. Then, up to 30 s later, lambs joined their dams and immediately (<7 s) started sucking. The second sample of each pair (‘‘B’’) was collected within 30 s after termination of the sucking activity. All lambs were observed, in order to confirm that both teats of the dam had been sucked. Before sampling, detailed clinical examination of mammary glands and teats of ewes, was performed (Fthenakis, 1994; Mavrogianni et al., 2005). The teat apex and the lower (1 cm) part of the teat skin were disinfected with povidone iodine scrub solution. A sterile, plastic, 20 G catheter (Abbocath1, Abbott Laboratories Inc., Abbott Park, USA) was used for sampling the teat duct. The catheter stylet was taken out and the plastic catheter was cut with a sterile blade to a length of 2 mm. The catheter was held by the investigator from the cannula hub; it was inserted into the teat, rolled around the internal teat wall, in order to sample the mucosa, and then withdrawn. Description and validation details of the method have been presented by Mavrogianni et al. (2006a). Subsequently, secretion samples were obtained. Standard procedures previously described in detail (Fthenakis, 1994; Mavrogianni et al., 2005), were used during the study. Duct material and mammary secretion samples were plated onto Columbia 5% blood agar; the media were incubated aerobically at 37 8C for up to 72 h. Organisms isolated were identified by using standard microbiological techniques (Barrow and Feltham, 1993; Euzeby, 1997); for staphylococcal identification, the ‘‘API-Staph SYSTEM’’ quick identification strips (BioMerieux SA, Marcy-l’-Etoile, France) were also used. 2.2.2. Samplings from lambs Samples were obtained on weekly occasions. Disposable cotton swabs (Sterilin Ltd., Stone, United Kingdom) were used to swab the tonsils of the 15 lambs of these ewes; the procedures described by Al-Sultan and Aitken (1985) were followed. Each tonsillar swab was smeared over a blood agar plate and then placed into Soy-broth (BioMerieux SA, Marcy-l’-Etoile, France). Agar plates and broth bottles were examined after 24 h incubation at 37 8C; if plate cultures were negative, a loopful from the broth was subcultured onto a blood agar plate, which was

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inoculated for up to 72 h. From positive blood agar plates, colonies morphologically and microscopically resembling M. haemolytica were identified by using standard bacteriological techniques (Barrow and Feltham, 1993; Euzeby, 1997). 2.2.3. Molecular studies of M. haemolytica Two strains, one (DAG21T) isolated from the teat duct of a ewe and one (DAG21R) isolated from the tonsils of her lamb, which had been provisionally identified as M. haemolytica, were subjected to molecular identification. Isolate ES26L (isolated in England from the mammary secretion of a ewe with mastitis [El-Masannat et al., 1991]) was also included in the study. DNA was isolated from a blood agar colony of the Mannheimia isolates using a commercial kit (Gentra Systems, Minneapolis, USA). PCR amplification was carried out according to the guidelines described by Kwok and Higuchi (1989). The sequence of the primers and the PCR conditions to amplify a part of 16s rRNA gene, were the same as previously described (Angen et al., 1999). Following amplification, 10 ml of each PCR product was analysed by electrophoresis on 2% agarose gel and stained with ethidium bromide (0.5 mg ml 1). A 100 bp DNA ladder was analysed on the same gel to serve as a size marker. As a negative control, DEPC treated H2O was used instead of DNA in PCR assay to exclude any contamination. The specificity of the PCR products was verified after direct PCR product sequencing (MDG Biotech). PCR products were gel-purified (QIAquick Gel Extraction Kit, Qiagen) and sequenced in both directions commercially by MWG Biotech (Germany). All samples were analysed twice and only high-quality 16s rRNA sequences were used. Phylogenetic and molecular evolutionary analyses were conducted using program MEGA 3.1 (Kumar et al., 2004). Neighbor-joining tree was constructed from a difference matrix employing the Kimura 2parameter correction. One thousand bootstrap pseudoreplicates were used to test the branching (shown as percentages). 2.3. Experiment II 2.3.1. Challenge procedures of ewes In total, 30 lactating Karagouniko-breed ewes were used. After lambing and until inoculation (22nd day after lambing), a thorough clinical examination was carried out in the ewes. The teat duct was sampled under aseptic

conditions; then mammary secretion samples were obtained. Lambs of these ewes were weaned 18 days after lambing and subsequently, the animals were hand-milked. They were allocated at random into one of the groups and challenge procedures (Table 1). Before challenge, chapping lesions were inflicted in both teats of ewes of subgroups Bm/Br/Bc, by immersing the lower 3.0–3.5 cm of both teats into a 1 N solution of NaOH for 1 min; the procedure was repeated on the following day (Fox et al., 1991). The resulting chapping was scored ‘‘2’’: ‘‘Less than half of the teat chapped, with few lesions present and much of the ulcerative tissue covered with scabs or absent; areas of normal skin apparent; no sensitivity of the animal to skin palpation’’. For inoculation, each M. haemolytica strain was grown on Columbia blood agar and checked for purity; then it was inoculated into Soy-broth and incubated aerobically at 37 8C for 5 h. Serial dilutions of the broth culture into PBS were carried out. Inocula contained 1200–1300 c.f.u., as estimated by the method of Miles and Misra (1938). For challenge of ewes in groups A or B, a sterile plastic fine catheter (Abbocath1) 2-mm long, was inserted into the teat; the syringe was attached to the catheter and the bacterial suspension was deposited inside the teat (Mavrogianni et al., 2005, 2006b). Ewes in groups C were inoculated directly into the mammary gland cistern. The same technique was used to inject 0.2 ml of PBS into the other udder side (teat or mammary gland) of each ewe, in order to be used as control. 2.3.2. Samplings of experimental ewes Detailed examinations of the mammary glands and teats were carried out daily. The teat ducts of the ewes were sampled initially; mammary secretion samples were then collected. All samples were cultured onto Columbia blood agar; the media were incubated aerobically at 37 8C for up to 72 h. The California Mastitis Test (CMT) was carried out in all secretion samples; the test was performed under quality assurance procedure, which confirmed that it was a reliable proxy measurement for somatic cell counts (Fragkou et al., 2007a). Secretion films made by directly smearing 20 ml from each sample on a microscope objective plate, were stained by the Giemsa method; the percentage of leucocyte subpopulations was determined by counting at least 200 cells therein and distinguishing their type. Two ewes of each of groups Am/Ar/Bm/Br/Cm/Cr, as well as one ewe of each of groups AC/Bc/Cc were

Table 1 Challenge isolates and procedures employed in Experiment II. Group

M. haemolytica isolate

Procedure (inoculum: 1200–1300 c.f.u.)

Time-points after challenge, when ewes were euthanised

Am (n = 4) Ar (n = 4) Ac (n = 2) Bm (n = 4) Br (n = 4) Bc (n = 2) Cm (n = 4) Cr (n = 4) Cc (n = 2)

DAG21T DAG21R ES26L DAG21T DAG21R ES26L DAG21T DAG21R ES26L

2-mm deep into healthy teat

2 2 2 2 2 2 2 2 2

2-mm deep into chapped teat

Into the gland cistern

days (2), 4 days (2), 4 days, 4 days days (2), 4 days (2), 4 days, 4 days days (2), 4 days (2), 4 days, 4 days

days (2) days (2) days (2) days (2) days (2) days (2)

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euthanised 2 days after challenge; the other ones within each group were euthanised 4 days after challenge (Table 1). A detailed dissection of the mammary glands and the teats started immediately and was carried out by using the aseptic technique (Mavrogianni et al., 2005, 2006b). Samples were obtained for bacteriological and histological examination. All samples obtained were plated onto Columbia blood agar; the media were incubated aerobically at 37 8C for up to 72 h. Bacteria isolated were identified by using standard microbiological techniques (Barrow and Feltham, 1993; Euzeby, 1997). Tissue samples were fixed in 10% neutralbuffered formalin and embedded in paraffin wax, using conventional techniques. Haematoxylin and eosin standard staining procedures were performed for histopathological studies. 2.4. Data management and statistical analysis In Experiment I, analysis of results was carried out by comparing changes in infection status following sucking by lambs for duct material and mammary secretion samples. Duct material and mammary secretion samples were assessed separately. Statistical significance was assessed by the Sign Test, which allowed for the readings to be paired and ignored any effect of collecting samples on multiple occasions. The critical probability was set at P = 0.05, on a two-sided null hypothesis of no difference. In order to calculate risk of infection of ewes’ teats, the following assumptions were made. If a teat was not infected before a sucking bout, then it was at risk during that bout. For infection by M. haemolytica a ewe was considered to be at risk, only when her lamb(s) had been found to harbour the organism in the tonsils (as demonstrated by isolation in tonsillar swabs). Then, the risk of a teat being infected was calculated as the number of infections divided by the number of occasions that this teat was at risk during a sucking bout. Risk of infection by M. haemolytica was calculated for each week of lactation, as well as for the period 2nd to 4th week, for the period 5th to 7th week and for the whole experimental period (2nd to 7th week). Where probabilities were estimated, they were accompanied with exact 95% two-sided binomial confidence intervals (CI), or exact 97.5% CI as appropriate (where the observed proportion was exactly 0% or 100%). In Experiment II, a scoring system (Fragkou et al., 2007a) was used and numerical values were assigned for the pathological findings in the udder of experimental animals. A separate score (0–4 scale) was given for macroscopic and for histological findings in teats and mammary parenchymas; these were then added to a 0–16 scale to produce a pathology score for the findings in each side of the udder of a ewe. For analysis of results of inoculation between the various M. haemolytica strains within each group, the Chi-square test was used. Statistical significance was defined at P < 0.05. All statistical analyses were performed in Minitab 15 (Minitab Inc., State College, PA, USA) and Stata 9 (Stata Corp., College Station, TX, USA).

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3. Results 3.1. Experiment I 3.1.1. Bacterial isolations from ewes None of the ewes included in the study had a history of mastitis. No bacteria had been isolated from any teat duct material or mammary secretion samples obtained from the ewes before inclusion into the study. Neither changes in mammary secretion, nor mammary abnormalities were detected in ewes during the study. In total, 396 pairs of samples were collected. Of samples collected before sucking, 14/396 (3.5%) teat duct material and 6/396 (1.5%) mammary secretion were bacteriologically positive. Of samples collected after sucking, 42/396 (10.6%) teat duct material and 8/396 (2.0%) mammary secretion were bacteriologically positive. After sucking, there was a significant increase, by 200% (from 14 to 42), of infected teat ducts (P < 0.001). No effect was found on mammary secretion: from 6 infected samples to 8 (P > 0.6). The risk of a teat becoming bacteriologically positive (with any one microorganism) consequent to sucking activity was 0.089 (95% confidence intervals = 0.062–0.122). In 40 (10.1%) cases, there was a change of bacteriological status of teat duct material samples in-between the sucking activity: in 6 cases a positive sample became negative, whilst in 34 cases a negative sample became positive. In 4 (1.0%) cases there was a change of bacteriological status of mammary secretion samples inbetween suckling: in 1 case a positive sample became negative, whilst in 3 cases a negative sample became positive. Bacteria were always isolated in pure culture. The majority of isolates were coagulase-negative staphylococci (Staphylococcus epidermidis, S. simulans, S. xylosus, S. chromogenes, S. sciuri, S. caprae, S. schleiferi). These organisms accounted for 44/56 (79%) and 9/14 (64%) of total isolates from teat duct material and mammary secretion samples, respectively. Other organisms isolated were: streptococci, Escherichia coli, Bacillus spp., M. haemolytica, Arcanobacterium pyogenes, Klebsiella sp. and S. aureus (Table 2). There were no significant differences in the proportion of staphylococci recovered before (14/ 20) or after suckling (40/50) (P = 0.410). One M. haemolytica isolate (DAG21T) was recovered from the teat duct of one ewe, after sucking by her lamb, during the 3rd week of lactation. Finally, of the 14 bacteriologically positive mammary secretion samples obtained during the study, in 10 (69%) the same organisms as those from the respective duct material sample, were isolated. 3.1.2. M. haemolytica isolations from lambs and risk of infection of ewes’ teats In total, M. haemolytica was isolated from 57/90 (63%) tonsillar swabs from lambs (Table 3). Overall risk of infection of ewes teats with M. haemolytica was 0.004 throughout the six weeks of the experiment. Risk of infection was greatest (0.021) during the 3rd week of lactation (Table 4).

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Table 2 Frequency of isolation of bacterial species from duct material or mammary secretion samples from ewes. Samples before sucking by lambs

Coagulase ve Staphylococci Streptococci E. coli Bacillus spp. M. haemolytica A. pyogenes Klebsiella sp. S. aureus Total a

Samples after sucking by lambs

All samples

DMa

MSa

DM

MS

DM

MS

11 1 1 0 0 0 1 0 14

3 2 0 0 0 1 0 0 6

33 0 2 4 1 1 0 1 42

6 2 0 0 0 0 0 0 8

44 1 3 4 1 1 1 1 56

9 4 0 0 0 1 0 0 14

DM: duct material, MS: mammary secretion.

Table 3 Proportion of tonsillar swabs from lambs, which yielded M. haemolytica. Age of lamb (weeks)

Total

2

3

4

5

6

7

4/15

10/15

9/15

10/15

11/15

13/15

57/90

Table 4 Risk of infection of the ewes’ teats with M. haemolytica, according to week of lactation. Week of lactation 2nd

3rd

Overall (2nd to 7th week) 4th

5th

6th

7th

2nd to 4th week

5th to 7th week

0.000 0.021 0.000 0.000 0.000 0.000 0.009 0.000 0.004 [0.000–0.142]a [0.001–0.110]b [0.001–0.085]b [0.000–0.074]a [0.000–0.066]a [0.000–0.061]a [0.002–0.043]b [0.000–0.023]a [0.001–0.020]b a b

One-sided 97.5% confidence intervals. Two-sided 95% confidence intervals.

3.1.3. Comparison of M. haemolytica isolates M. haemolytica (our strains and from GenBank) formed a distinct cluster (Fig. 1), whereas other Mannheimia species (GenBank) formed separate clusters on separate branches. By using 16s rRNA sequence analysis, the identity of the strains as M. haemolytica was confirmed. The 16s rRNA sequences of the two Greek strains were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica; for this analysis, a fragment of 491 bp was used, which was common to strains previously described in the literature. This analysis revealed that the homology of the nucleotide sequences between the two Greek strains and the English strain ES26L was 95.5%. 3.2. Experiment II 3.2.1. Clinical, bacteriological and cytological findings Before chapping or challenge, the mammary glands and the teats of all ewes were clinically healthy. The teats were soft with no external abnormalities. No bacteria were isolated from any duct material or mammary secretion samples. The CMT was always negative; in Giemsa-stained secretion films, no leucocytes were observed. After

chapping of teat skin (Bm/Br/Bc ewes), the CMT became positive (score ‘‘1’’) and leucocytes (80–90% neutrophils, 10–20% macrophages) were observed in Giemsa-stained secretion films. After challenge, ewes in groups A developed subclinical mastitis. M. haemolytica was isolated in pure culture from duct material and mammary secretion samples 1 day after challenge and thereafter; isolation rates were: 12/24 from Am, 13/24 from Ar, 7/12 from Ac ewes (Table 5). Leucocytes were seen in Giemsa-stained secretion films: up to the 2nd day after challenge, their majority (>80%) was neutrophils, with fewer macrophages and lymphocytes also present; subsequently, the percentage of neutrophils decreased (60–70%) and that of macrophages and lymphocytes increased. After challenge, all ewes in groups B and C became systemically ill and developed clinical mastitis; rectal temperature up to 40.6 8C and indifference were evident. Mammary secretion was abnormal (serous or purulent, with flakes or clots); mammary signs (enlarged, hot, hard and painful mammary gland) were seen. M. haemolytica was isolated in pure culture from duct material and secretion samples 1 day after challenge and thereafter; isolation rates were: 24/24 from Bm and Br, 12/12 from Bc ewes, 12/24 from Cm and Cr ewes, 7/12 from Cc ewes

[(Fig._1)TD$IG]

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Fig. 1. Phylogenetic analysis was performed on a 491-bp fragment of the 16sRNA of M. haeemolytica strains. Two strains isolated from the teat duct of a ewe (DAG21T) and the tonsils of her lamb (DAG21R) were compared with 28 Mannheimia spp. isolates retrieved from the EMBL database (species name and isolate designation presented). Boostrap confidence values are indicated as percentages.

(Table 5). Leucocytes were seen in Giemsa-stained secretion films: up to the 3rd day after challenge, their majority (>85%) was neutrophils, with fewer macrophages and lymphocytes also present; subsequently, the percentage of neutrophils decreased (65–75%) and that of macrophages and lymphocytes increased. No clinical changes were recorded in the contralateral teats and mammary glands of A and C ewes. Chapping lesions were evident in the contralateral teats of B ewes. No bacteria were isolated from any duct material or secretion samples obtained from the contralateral side (isolation

rate: 0/24 for all groups). CMT was negative for all samples obtained from A and C ewes (0/12 for all A and C groups) and was mildly positive (score ‘‘1’’) in samples from all contralateral gland of B ewes. 3.2.2. Pathological findings 3.2.2.1. Groups Am/Ar/Ac. M. haemolytica was isolated in pure culture from tissue samples; isolation rates were: 9/ 12 from Am, 9/12 from Ar and 4/6 from Ac ewes. There were some folds, rough internal lining and mild thickening

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Table 5 Isolation of M. haemolytica, CMT results and scores for findings of post-mortem examination, regarding the challenged side of the udder of ewes, by various M. haemolytica strains. Group

Am Ar Ac Bm Br Bc Cm Cr Cc

Isolation of M. haemolyticaa Duct material

Mammary secretion

Tissue samplesb

7/12 8/12 4/6 12/12 12/12 6/6 0/12 0/12 1/6

5/12 5/12 3/6 12/12 12/12 6/6 12/12 12/12 6/6

9/12 9/12 4/6 12/12 12/12 6/6 6/12 7/12 3/6

CMT results (score 1)b

Scores for findings of post-mortem examinationc

7/12 8/12 5/6 12/12 12/12 6/6 12/12 12/12 6/6

3.5 + 0.5 3.75 + 0.5 4.0 + 1.0 6.5 + 6.5 6.25 + 6.25 6.0 + 7.0 0.25 + 6.0 0.5 + 6.5 0.25 + 6.5

a

n/m: number of samples which yielded the challenge organism out of total number of samples collected. n/m: number of samples with positive CMT (1) out of total number of samples collected. k + l: scores for findings of post-mortem examination of teat + scores for findings of post-mortem examination of mammary parenchyma (mean values for animals in each group), maximum possible values: 8 + 8. b c

in the teat duct, which was clearly distinguished from the teat cistern as a separate anatomical structure. Histologically, there was leucocytic infiltration prominent at the border between teat duct and teat cistern in the ewes euthanised 2 days after challenge (neutrophils, lymphocytes, plasma cells) and at the teat cistern in the ewes euthanised 4 days after challenge (lymphocytes, plasma cells); the leucocytes were observed under the epithelium of the teat. Hyperplastic lymphoid nodules consisting of lymphocytes and plasma cells were observed at the border between the teat duct and teat cistern. No gross pathological findings were evident in the mammary parenchyma or the supra-mammary lymph nodes; mild leucocytic infiltration was seen only in some ewes. Total scores for findings of post-mortem examination, summed over all ewes and days, were: 16 for Am, 17 for Ar (maximum possible: 64) and 10 for Ac ewes (maximum possible: 32) (Table 5). 3.2.2.2. Groups Bm/Br/Bc. M. haemolytica was isolated in pure culture from all tissue samples from Bm, Br and Bc ewes. There were folds, hyperaemia and thickness of the mucosa in the teat duct. Histologically, we recorded principally extensive subepithelial neutrophilic infiltration, presence of lymphocytes and plasma cells, with exocytosis through the epithelium and into the lumen, lysis of neutrophils and destruction of epithelial cells; a conspicuous lymphoid area was observed at the border between teat duct–teat cistern. In the skin of all teats studied, erosion and ulceration with presence of serocellular crusting and leucocytic accumulation, were evident; however, these lesions did not extend into the teat duct or cistern; their maximal estimated depth was 2.5 mm and the underlying tissues were unremarkable. Macroscopic lesions in the respective mammary parenchyma included subcutaneous oedema and sanguineous fluid exuding from sections of the reddened parenchyma; the supra-mammary lymph nodes were enlarged. Histologically, neutrophilic infiltration, extravasation, intra-alveolar live and exhausted neutrophils, destruction of epithelial cells, alveolar destruction, lymphocytic infiltration and

haemorrhages were evident. Total scores for findings of post-mortem examination, summed over all ewes and days, were: 52 for Bm and 50 for Br (maximum possible: 64) and 26 for Bc ewes (maximum possible: 32) (Table 5). 3.2.2.3. Groups Cm/Cr/Cc. M. haemolytica was isolated in pure culture from tissue samples; isolation rates were: 6/ 12 from Cm, 7/12 from Cr and 3/6 from Cc ewes. No macroscopic or histological lesions were observed in the teats. Macroscopic lesions in the inoculated mammary parenchyma included subcutaneous oedema and sanguineous fluid exuding from sections of the reddened parenchyma, as well as enlarged supra-mammary lymph nodes. Histologically, neutrophilic infiltration, extravasation, intra-alveolar live and exhausted neutrophils, destruction of epithelial cells, alveolar destruction, lymphocytic infiltration and haemorrhages were evident. Total scores for findings of post-mortem examination, summed over all ewes and days, were: 25 for Cm, 28 for Cr (maximum possible: 64) and 13.5 for Cc ewes (maximum possible: 32) (Table 5). 3.2.3. Comparison of results between strains of M. haemolytica When results of inoculation of the various strains (recovery rates of M. haemolytica, CMT results, scores for post-mortem findings) were compared between them, the statistical significance was always P > 0.9. 4. Discussion 4.1. Experimental model In previous studies of ovine mastitis, it has been confirmed that the teat is the portal of entry of the causal agents, the most important of which are Staphylococcus spp. and M. haemolytica, together accounting for over 80% of isolates (Bergonier et al., 2003). Staphylococci are considered to originate from milkers’ hands or from the skin of the udder (Bergonier and Berthelot, 2003). Scott and Jones (1998) and Jones and Watkins (2000) proposed

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that M. haemolytica possibly originated from the tonsils of the sucking lambs; however, this hypothesis has never been confirmed. The experimental model that we used, i.e. pairedsamples immediately before and immediately after suckling, minimized the time in-between sampling to suckling to sampling. Thus, it ensured that bacterial isolations reflected the actual dynamics of infection throughout. Swabbing of lambs confirmed that challenge of the teat with M. haemolytica was perennial during lactation. By using molecular techniques, the identity of the two M. haemolytica strains (from the teat duct of a ewe and from the tonsils of the lamb sucking her) was compared. Finally, in Experiment II we used the ‘‘teat-challenge’’ model (Mavrogianni et al., 2005, 2006b), in order to test the pathogenicity of the above two strains for the mammary gland of ewes, as well as to compare them with an isolate of known pathogenicity for the mammary gland. 4.2. Dynamics of infection We found significantly increased teat duct infection rates after suckling, but no strong evidence of increased intramammary infections. Infection of the teat would have occurred soon after initiation of suckling, given that the mean duration of sucking bouts by lambs is around 45–50 s (Gougoulis et al., 2007). The possibility that some bacteria enter into the teat, subsequently being withdrawn during the same sucking bout cannot be ruled out. In a previous experimental study (Mavrogianni et al., 2005), deposition of pathogenic organisms into the duct of clinically healthy ewes did not always result to clinical mastitis; thus the protective role of the intrinsic defences of the teat was confirmed. In the current study, the bacteria entering into the teat duct during sucking provided a ‘‘natural’’ means of inoculation of the teat duct, which resisted invasion upwards into the parenchyma. The present findings provide field corroboration of the protective role of the healthy teats of ewes, especially after application of a challenge factor (i.e. sucking). 4.3. Transmission of M. haemolytica One M. haemolytica strain was recovered from teat duct material after sucking by lambs; the organism had not been isolated from the same site immediately before suckling. This is clear evidence that the organism was transmitted to the teat duct during sucking by the lamb. The results of the molecular techniques (16s rRNA sequencing) showing that M. haemolytica strains were identical, confirm that the organism originates from the nasopharynx of lambs. Incidence of M. haemolytica mastitis is greatest during the first month of lactation (Jones and Lanyon, 1987); this coincides with the increased risk of teat infection during the same period. We postulate that as the lower part of the teat comes into contact with the pharynx of the lamb (Titchen, 1977), the organism is attached thereon, subsequently entering into the duct; perhaps the tongue of the lamb may ‘‘push’’ the bacteria upwards into the duct. Isolation of the organism after a sucking bout (duration: 45–50 s) indicates

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the speed by which the whole process can take place. Vilela et al. (2004) have documented M. haemolytica’s requirement for attachment on the mammary cells, in order to exhibit its pathogenicity. Perhaps the same sucking activity by the lamb would subsequently remove from the teat bacterial cells not yet attached onto mammary epithelial cells. 4.4. Experimental mastitis with a ‘‘respiratory’’ M. haemolytica In previous experimental studies, researchers used M. haemolytica isolates from the mammary gland of ewes (ElMasannat et al., 1991; Watkins and Jones, 1992; Vilela et al., 2004; Mavrogianni et al., 2005). Hitherto, no attempts have been made to study the pathogenicity of ‘‘respiratory’’ strains for the mammary gland of ewes. The establishment of similarity between ‘‘respiratory’’ and ‘‘mammary’’ strains called for an in vivo study of these strains, in order to confirm their ability to cause mastitis. No differences in pathogenicity have been found between the ‘‘mammary’’ and the ‘‘respiratory’’ strain. The ‘‘respiratory’’ isolate (from the tonsils of a lamb) was able to cause mastitis (corroborated by the results of Experiment II). However, it seems that presence of hostrelated predisposing factors is required for development of clinical disease. In studies of experimental respiratory infection with M. haemolytica spray-challenge, it has always been necessary to apply a predisposing factor for development of pneumonia. Preliminary challenge with Mycoplasma ovipneumoniae (Buddle et al., 1984) or Para-influenza 3 Virus (Biberstein et al., 1971) was usually applied; alternatively, application of cold stress (Bakima et al., 1991), promotion of the toxinogenic effect of the challenge organism with iron (Al-Sultan and Aitken, 1985) or depletion of immunological competence of the host (Confer, 1993), was carried out. In contrast to the above, direct intra-thoracic inoculation of the M. haemolytica successfully induced pneumonia (Purdy et al., 1998). The results of Experiment II are in line with results of similar-type experiments in the respiratory tract. Deposition of the organism into the teat duct did not result to clinical mastitis; however, when factors reducing teat defences, for example teat lesions (Mavrogianni et al., 2006b), were present or when these defences have been by-passed by direct inoculation into the gland cistern, acute clinical mastitis developed. Moreover, teat lesions have been found to predispose to increased attachment of M. haemolytica on the affected teat skin (Fragkou et al., 2007b). Mucosa-associated lymphoid tissues are secondary lymphoid organs and represent the first encounter of antigens breaching mucosal surfaces with the immune system. Lymphoid nodules described in the teat duct–teat cistern border are considered of increased significance for the protection the mammary gland (Mavrogianni et al., 2005, 2006b, 2007; Fragkou et al., 2010). The bronchal epithelium is the interface between the lungs and the outside world; the teat duct plays a similar function for the mammary gland.

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4.5. Concluding remarks The results provide clear evidence that sucking by lambs can lead to transmission of M. haemolytica into the teat duct of ewes. Strains of the organism from the respiratory tract of lambs have the potential to cause mastitis. The healthy teat duct can withstand the infection; the lymphoid nodules present at the border between teat duct–teat cistern play an important defensive role. Presence of predisposing factors (e.g. teat lesions), which increase bacterial colonisation on teat skin and deplete the defences of the teat, results to clinical mastitis. M. haemolytica from the nasopharynx of ewes is transmitted to the newborn lambs during post-natal grooming (Al-Sultan and Aitken, 1985) and colonises their nasopharynx. Occasionally, these organisms can descent to the respiratory tract and cause pneumonia (Sargison, 2008). The same organisms, during sucking, are transmitted to the teat of the dam and, under certain circumstances, cause mastitis. It is therefore interesting that strains form the respiratory tract of a ewe may infect (via the offspring) her mammary gland. Blood et al. (1994) have mentioned that cases of M. haemolytica-associated mastitis in ewes can occur simultaneously to cases of pneumonia in lambs. No detailed studies exist, but, in theory, this can be possible, as it would be caused by the same strain(s) of M. haemolytica, which would be pathogenic. In view of the above, but notwithstanding the limitations of classical-type vaccines against mastitis (Fragkou et al., 2010), M. haemolytica vaccines protective against pneumonia can perhaps be somehow protective against mastitis caused by the same organism. References Al-Sultan, I.I., Aitken, I.D., 1985. The tonsillar carriage of Pasteurella haemolytica in lambs. J. Comp. Pathol. 95, 193–201. Angen, O., Mutters, R., Caugant, D.A., Olsen, J.E., Bisgaard, M., 1999. Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA–DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb, nov., Mannheimia granulomatis comb, nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov., and Mannheimia varigena sp. nov. Int. J. Syst. Bacteriol. 49, 67–86. Bakima, M., Kaeckenbeeck, A., Meniai, K., Arendt, J., Lomba, F., Lekeux, P., 1991. Experimental induction of pneumonic pasteurellosis in goats— study of physiopathological changes. Ann. Med. Vet. 135, 431–436. Barrow, G.I., Feltham, R.K.A., 1993. Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge University Press, Cambridge. Bergonier, D., Berthelot, X., 2003. New advances in epizootiology and control of ewe mastitis. Liv. Prod. Sci. 79, 1–16. Bergonier, D., De Cremoux, R., Rupp, R., Lagriffoul, G., Berthelot, X., 2003. Mastitis of dairy small ruminants. Vet. Res. 34, 689–716. Biberstein, E.L., Shreeve, B.J., Angus, K.W., Thompson, D.A., 1971. Experimental pneumonia of sheep. Clinical, microbiological and pathological responses to infection with Myxovirus parainfluenzae 3 and Pasteurella haemolytica. J. Comp. Pathol. 81, 339–351. Blood, D.C., Radostits, O.M., Gay, C.C., 1994. Veterinary Medicine. A Textbook for the Diseases of Cattle, Sheep, Pigs, Goats and Horses, 8th edn. Bailliere Tindall, London. Buddle, B.M., Herceg, M., Davies, D.H., 1984. Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica. Vet. Microbiol. 9, 543–548. Confer, A.W., 1993. Immunogens of Pasteurella. Vet. Microbiol. 37, 353– 368.

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