Clinical findings in sheep farms affected by recurrent bacterial mastitis

Small Ruminant Research 88 (2010) 119–125

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Clinical findings in sheep farms affected by recurrent bacterial mastitis夽 Gavino Marogna, Sandro Rolesu, Stefano Lollai, Sebastiana Tola, Guido Leori ∗ Centro di Referenza Nazionale per le Mastopatie degli Ovini e dei Caprini (C.Re.N.M.O.C.), Istituto Zooprofilattico Sperimentale della Sardegna “G. Pegreffi”, Sassari, Italy

a r t i c l e

i n f o

Article history: Available online 12 January 2010 Keywords: Mastitis Sheep Infection

a b s t r a c t This study was aimed to investigate the relationships existing between clinical findings and bacterial entities isolated from milk of dairy sheep affected by mastitis. The influence of other parameters on the clinical picture, such as age, nutritional state, breeding conditions, and milking techniques, was also evaluated. All sheep belonged to flocks suffering from serious and repeated outbreaks of infectious mastitis. A total of 2198 Sarda dairy sheep were subjected to a detailed clinical examination, and at least one clinical sign of mastitis was detected in 1666 sheep (75%). Bacteriological examination of milk samples collected from all animals produced 1093 positive results (49.7%). Of bacterial species identified, three accounted for 55.3% of all isolates: Streptococcus uberis (25.6% of positives and 12.7% of total), Staphylococcus epidermidis (16.2% of positives and 8% of total), and Staphylococcus aureus (13.5% of positives and 6.7% of total). Upon investigation of correlations existing among clinical signs and bacterial species responsible for the outbreak, S. uberis showed a statistically significant correlation with serous appearance of milk, presence of clots in secretions, and reactivity of supramammary lymph nodes (p < 0.05); S. epidermidis showed a statistically significant correlation with presence of pustules and ulcers (p < 0.05); and S. aureus showed a statistically significant correlation with clinical signs of chronic mastitis: nodules, abscesses, and atrophy (p < 0.05%). Manual milking techniques were more associated to udder infections than mechanical milking. However, an interesting correlation emerged between presence of S. uberis and mechanical milking with small portable devices. In conclusion, this study revealed interesting and unprecedented correlations among clinical signs, bacterial species isolated from infected milk, and farm management techniques. The results reported here emphasize the primary role played by clinical practice in managing infectious ovine mastitis outbreaks, and strengthen its relevance for recovery of affected flocks. © 2009 Elsevier B.V. All rights reserved.

1. Introduction

夽 This paper is part of a special issue entitled “SIPAOC Congress 2008” Guest Edited by Antonello Carta, Giovanni Garippa and Giuseppi Moniello. ∗ Corresponding author. Tel.: +39 0792892293. E-mail addresses: [email protected] (G. Marogna), [email protected] (S. Rolesu), [email protected] (S. Lollai), [email protected] (S. Tola), [email protected] (G. Leori). 0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.12.019

Currently, mastitis outbreaks of bacterial etiology are the prominent health problem affecting dairy sheep farms (Contreras et al., 2007; Conington et al., 2008). The occurrence of a mastitis outbreak in a farm gives rise to a plethora of economical, sanitary, and legal problems (Bergonier et al., 2003). Infectious mastitis outbreaks of small ruminants are usually caused by Gram positive bacteria, mostly Staphylococcus and Streptococcus species (Bergonier et al.,

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2003; Contreras et al., 2007). In small ruminants, the annual incidence of clinical mastitis episodes is estimated to be under 5% in normal conditions, while problematic flocks can reach incidences higher that 30–50%, sometimes peaking up to 70% due to the mortalities or to adoption of culling procedures (Bergonier et al., 2003; Contreras et al., 2007). In sheep and goats, mastitis episodes are the main reason for culling because of sanitary problems, which occur mainly during the first 2–3 months of lactation (Malher et al., 2001; Bergonier et al., 2003; Leitner et al., 2008). The deleterious consequences of mastitis on quality and quantity of milk have been reported to be more significant in small ruminants compared to dairy cows (Leitner et al., 2004a). A consistent reduction of milk production in sheep and goats following udder infections has been reported by many authors, with varying intensities depending on involvement of one or both half-udders. Upon involvement of both half-udders, a reduction of 58% and 30% in milk production has been reported in sheep and goats, respectively (Torres-Hernandez and Hohenboken, 1979; McCarthy et al., 1988; Gonzalo et al., 1994, 2002; Cuccuru et al., 2002; Leitner et al., 2003, 2004a,b). Other authors reported overall development problems and significant weight gain decrease in lambs as a consequence of the reduced amount and lower nutritional quality of mastitic milk (Robinson et al., 1969; Warren and Rembarger, 1963; Torres-Hernandez and Hohenboken, 1979). Alterations in suckling behavior were also reported (Gougoulis et al., 2008) and, in the most severe cases, an increase in mortality has been directly related to mastitis in the mothers (Watson and Buswell, 1984). Usually, diagnosis of infectious mastitis occurs upon clinical examination and, if necessary, by means of microbiological tests (Conington et al., 2008). In the field practice, clinical findings are paramount for designing effective recovery plans within affected farms. The term mastitis indicates presence of an inflammatory process in the mammary gland; usually, a bacterial infection triggers an inflammatory reaction which, in turn, leads to the development of variably detectable clinical signs. The clinical syndrome can follow a variable course, ranging from hyperacute to chronic. The occurrence of disease chronicization may produce clinical pictures which become gradually less clinically evident. Depending on the clinical course, mastitis can be conventionally differentiated into: hyperacute, presenting a serious udder inflammation accompanied by an evident systemic response; acute, when serious udder inflammation is present but there is no systemic involvement; chronic, when there is no systemic involvement and only fibrous lesions, sometimes poorly detectable, are present. In some cases, mastitis can be further differentiated into subacute, when clinical signs are less evident compared to classical acute forms, and subclinical, when clinical signs are not present and mastitis can only be detected by means of laboratory examination procedures. Purpose of this study was to investigate correlations among clinical signs and bacteria isolated from mastitic milk. The investigation was also extended to other parameters, such as age, nutritional state, breeding conditions, and milking techniques, in order to evaluate their influence on

mastitis outbreaks. This study should be considered as representative of the clinical picture in affected flocks; in fact, all animals belonged to problematic farms, and are not to be considered as a randomized sample. 2. Materials and methods 2.1. Study area Sardinia is the Italian region with the largest number of dairy sheep, accounting to about 3.5 million with a density of 139.49/km2 . In Sardinia, dairy sheep farming represents a cornerstone of the island economy, and finding effective strategies for mastitis diagnosis and control is of outstanding importance. 2.2. Sample selection A clinical and microbiological investigation was carried out on dairy sheep of the Sarda breed in order to elucidate the role of clinical examination in flocks suffering from recurrent mastopaties, aimed to finding relationships among bacterial species involved and the spectrum of clinical signs. Therefore, only flocks showing recurrent episodes of bacterial mastitis were included in this study. A sample of 2198 sheep were selected for clinical examination using the database of the Regional Breeders Association of Sardinia (ARAS), which measures and records somatic cell counts of bulk tank milk produced in about 90% of Sardinian farms. All sheep were subjected to a detailed clinical examination within 1 h from milking. In fact, in our experience, clinical examination of the udder is better performed soon after milking in order to facilitate evaluation of volume differences between the two halves, presence of edema, sclerosis and atrophy. The sheep included in this study belonged to fifteen farms that were selected according to the following requirements: (i) detection of a bulk tank milk somatic cell count (SCC) higher than 5 × 106 /mL in at least 5 determinations performed along two years (2004–2005); (ii) flocks larger than 50 sheep; (iii) persistence of a mammary health problem in the farm. Clinical examinations were performed during full lactation between January and April 2006. Milk samplings and clinical examinations were performed on all lactating animals in the farm at the time of the inspection. 2.3. Medical history and clinical examination The following general data were recorded in the farm: composition of the farmland, farming style, milking technique, size of the flock, amount of milk produced the day before inspection (2 milkings), recent introduction of new animals, prophylactic and therapeutic interventions adopted, other animal species present in the farm. The following data were recorded for every sheep: age, state of the sensorium, nutritional state upon estimation of the Body Condition Score (BCS), alopecia, diarrhea, foot rot, polyarthritis, generic lameness, and keratoconjunctivitis, plus any antibiotic treatment performed during the previous lactation. Animals were identified trough tattoos or ear tags. BCS was evaluated by means of an arbitrary scale (2–2.25–2.50–2.75–3–3.25) corresponding to the following nutritional states, respectively: very lean, lean, medium nutritional state, good nutritional state, fat, very fat. The milking technique was defined as manual milking and mechanical milking, and the latter was further differentiated into mechanical milking with fixed plants (by means of fixed large or medium-sized plants installed within milking rooms and allowing the simultaneous milking of 24–48 sheep per cycle), and mechanical milking with portable devices (by means of a “trolley”, a movable milking device with a simpler design allowing the milking of 1–2 sheep per cycle). Clinical examination was performed on each half-udder within 1 h after milking, and included a general inspection, evaluation of half-udder consistency, macroscopic examination of milk, and palpation of supramammary lymph nodes. Presence or absence of “lùpia” (lacteal cysts) Marogna et al. (2008) was also assessed. During clinical examination, presence of pustules, scabs, corneal growths, ulcers, nodules, abscesses, rubor, calor, and dolor (by warmth on palpation) was assessed. Nodules, abscesses, and “lùpie” were differentiated as follows: a nodule was defined as a relatively hard, roughly spherical, often painless, abnormal structure; an abscess was defined as a collection of pus and infected material in or on the skin, that has accumulated in a cavity formed by the tissue as a result of an infectious process; a lùpia was

G. Marogna et al. / Small Ruminant Research 88 (2010) 119–125 defined as a retented subcutaneous cyst in the mammary gland resulting from closure of a lactiferous duct. Consistency of udder upon palpation was classified as: normal, edematous, sclerotic, or atrophic. The macroscopic appearance of milk was classified as: normal, serous, hemorrhagic, with pus, with clots, or absence of secretion.

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Table 1 Relationship between presence/absence of clinical signs in the udder and results of bacteriological cultures of milk. Bacterial culture

Clinical signs Present

2.4. Milk sampling and microbiological examination A minimum of 5 mL of mixed milk from both half-udders was collected in a sterile container, after cleaning the teat with denatured 90% ethanol and discarding the first milk drops. Samples were refrigerated and subjected to microbiological examination in the laboratory within 24 h from collection. For microbiological cultures, 10 ␮L of milk were seeded in 5% sheep blood agar and incubated at 37 ± 1 ◦ C for 24–48 h. Upon growth of several morphologically different colonies, samples were classified as “mixed bacterial flora” and bacterial identifications were not carried out. Upon growth of a strongly predominant type of bacterial colony, Gram stain, catalase and coagulase tests were performed, colonies were reisolated in suitable media, and biochemical identification was carried out (API, BioMérieux, Marcy l’Etoile, France). Biochemical profiles were identified using the apiweb System® (BioMérieux). Bacterial colonies were inspected for presence of: haemolysis in 5% blood agar, growth and fermentation in Mannitol Salt Agar (MSA), production of “free” coagulase (BBL Coagulase Plasma Rabbit with EDTA, Becton Dickinson, Heidelberg, Germany), presence of “clumping factor” (Staphylase Test, Oxoid, Cambridge, UK), growth and reaction in Baird-Parker RPF Agar (Rabbit Plasma Fibrinogen Agar, Microbiol, Uta, Italy). Coagulase-negative staphylococci (CNS) were subjected to PCR-RFLP using gap and 16S rRNA as targets, and then analyzed by agarose gel electrophoresis and by Pulsed-Field Gel Electrophoresis (PFGE) for separation of DNA macrofragments, as described previously (Onni et al., 2007). Identification of streptococci was confirmed using two different multiplex polymerase chain reaction (multiplex-PCR) tests, the first with primers specific for the most frequently isolated Streptococcus uberis, Enterococcus faecalis, and Enterococcus faecium, and the second with primers specific for Streptococcus bovis and Streptococcus suis (Wahner et al., 2007; Campesi et al., 2008). Milk was also subjected to PCR for specific detection of Mycoplasma agalactiae (Tola et al., 1996) and seeded in Hayflick agar without enrichment, considering the elevated method sensitivity. All Mycoplasma isolates were cloned and identified using a specific PCR test (Tola et al., 1997). Gram negative bacteria were not subjected to species identification. 2.5. Statistical processing of data Statistical analysis of data was carried out with SPSS© for Windows V.15.0.1 (22 November 2006) (Microsoft, Redmond, WA) through binomial logistic regression in “forward conditional” mode, in which the dependent variable was positivity or negativity to bacteriological tests, and the independent variables were the parameters recorded for each farm and for each animal. The variables under examination were first analyzed individually, in order to verify their significance, and then in combination, in order to assess the effect of the single variables against all others. The acceptable level of significance for a single variable to be inserted into the general model was p < 0.05. Significant variables were then assessed together in a single model. The same procedure was carried out for every bacterial species isolated from milk samples, in order to search for differences among the individual pathogenic agents in determining different sets of symptoms.

3. Results 3.1. Clinical examination At least one mammary clinical sign was detected in 1666 sheep (75%). The most common clinical finding was enlargement of supramammary lymph nodes. When considering clinical signs as edema, rubor, calor, dolor, and presence of blood in milk as indicative of an acute infection, and sclerosis, atrophy, and presence of nodules as indicative of a chronic infection, the clinical examination of mammary glands revealed at least one clinical sign of acute

Positive Negative Total

Absent

Total

918 736

175 369

1093 1105

1654

544

2198

mastitis in 430 sheep (19%), and at least one clinical sign of chronic mastitis in 1006 sheep (46%). Clinical signs indicating the concomitant presence of both acute and chronic mastitis were detected in 175 sheep (8%). When considering localization, clinical signs of both chronic and acute mastitis were detected in the left half-udder of 39 sheep (1.8%), and in the right half-udder of 45 sheep (2%). 3.2. Bacterial identifications Milk collected from a total of 2198 udders was subjected to bacteriological examination, producing 1093 (49.7%) positive results. Table 1 reports the relationship between clinical findings and microbiological examination. About half of the samples showed presence of bacteria in milk. Samples classified as “mixed microbial flora” accounted for 12.3% of positive samples and for 6.1% of total samples. The most frequently detected bacterial genus was Staphylococcus. A total of 13 species were identified, representing 41.7% of all isolates. Staphylococcus spp. were present in 20.7% of samples. Table 2 lists all Staphylococcus species identified and presence/absence of symptoms in the affected animal (Table 2). Streptococci were the second most common finding in milk samples; in combination, Streptococcus spp. and Enterococcus spp. represented 33.3% of all isolates, and were present in 16.6% of samples. Table 3 lists all Streptococcus and Enterococcus species identified and presence/absence of symptoms in the affected animal. Contagious Agalactia (CA) was diagnosed only in one out of 15 farms. In the farm affected by CA, 35.5% of lactating sheep showed positivity to M. agalactiae. Three bacterial species accounted Table 2 Staphylococcus species isolated from milk and their relationship to clinical signs. Species

Clinical signs Present

Total

Absent

Staphylococcus aureus Staphylococcus epidermidis Staphylococcus chromogenes Staphylococcus xylosus Staphylococcus warneri Staphylococcus simulans Staphylococcus sciuri Staphylococcus intermedius Staphylococcus hyicus Staphylococcus hominis Staphylococcus caprae Staphylococcus capitis Staphylococcus auricularis Staphylococcus spp. (unidentified)

129 138 31 12 3 5 2 2 5 2 22 1 1 15

19 39 7 4 1 0 1 0 2 0 3 2 0 4

148 177 38 16 4 5 3 2 7 2 25 3 1 19

Total

368 (81.8%)

82 (18.2%) 450

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Table 3 Streptococcus and Enterococcus species isolated from milk and their relationship to clinical signs. Species

Clinical signs

Total

Present

Absent

Streptococcus uberis Streptococcus acidominimus Streptococcus bovis Streptococcus constellatus Streptococcus dysgalactiae Streptococcus suis Streptococcus spp. (unidentified) Enterococcus faecalis Enterococcus faecium Enterococcus durans Enterococcus spp. (unidentified)

248 9 8 5 1 2 4 34 6 1 7

32 0 3 1 0 0 0 5 2 0 2

280 9 11 6 1 2 4 39 8 1 9

Total

325 (87.8%)

45 (12.2%)

370

for 55.3% of all isolates: S. uberis (25.6% of positives and 12.7% of total), Staphylococcus epidermidis (16.2% of positives and 8% of total), and Staphylococcus aureus (13.5% of positives and 6.7% of total). Gram negative bacteria in general accounted for 4.2% of all isolates. The genus Pseudomonas accounted for 0.3% of all isolates. Other bacterial species were detected; however, because of prevalence or other microbiological aspects, these were considered not significant and/or peripheral. 3.3. Correlations among clinical variables and bacterial positivity Clinical signs showing a significant correlation with presence/absence of bacteria are listed in Table 4. The degree of such correlation is expressed as odds ratio and is represented by the value exp(B). Significant correlations of the input variables were found with the dependent variable (positivity to bacterial culture), as follows: (1) Subjects with reactive supramammary lymph nodes were 38% more likely to be positive to bacterial culture, compared to subjects with non-reactive supramammary lymph nodes. (2) Subjects with visible abscesses upon clinical examination were 142% more likely to be positive to bacterial culture, compared to subjects not bearing this symptom. (3) Subjects with sclerosis and nodules were 105% and 115% more likely to be positive to bacterial culture, respectively, compared to subjects without these clinical signs. (4) Subjects producing milk with a serous appearance or with clots were 154% and 317% more likely to be positive to bacterial culture, respectively, compared to subjects producing macroscopically normal milk. Conversely, other clinical variables included in this study did not show a significant correlation with bacterial positivity. Among the most important variables taken into consideration were age, mammary atrophy, and presence of skin scabs and ulcers. The correlation of bacterial positivity with milking techniques was evaluated, with the following results: (1) Manual milking was associated with a 62% higher risk of bacterial positivity compared to mechanical milking.

(2) Mechanical milking with portable devices (“trolley” systems) was associated with a 40% higher risk of bacterial positivity compared to mechanical milking with fixed plants. Interestingly, isolation of S. uberis was 40% more likely to correlate with portable milking devices (“trolleys”) compared to fixed mechanical milking. Moreover, S. uberis was less frequently isolated in farms using manual milking techniques; indeed, the likelihood of S. uberis isolation in manually milked samples was 14% lower compared to mechanically milked samples (OR = 0.14). Correlation of BCS with clinical signs of acute mastitis (mammary edema, calor, rubor, dolor, and hemorragic secretion) was investigated in order to assess if the fullblown disease manifestations were related to alterations of the nutritional state. A significant correlation was found for values between 2.00 and 2.25 (corresponding to very lean and lean, respectively), only for the variables calor, rubor, and dolor (p < 0.05). Conversely, the variables edema and hemorrhagic milk did not show a significant correlation with BCS (p > 0.05). The relationship of BCS with chronic signs of mastitis was also investigated, in order to assess if chronicization of lesions and the consequent drop in milk production were related to high BCS values (3.00 and 3.25, corresponding to fat and very fat, respectively). However, no correlations were found between BCS and the clinical signs of chronic mastitis examined in this study (p > 0.05%). The correlation between clinical signs and the bacterial species isolated was also examined, in order to investigate possible useful correlations between a specific pathogen and a given clinical picture. Such investigation was limited to the three most frequently isolated bacterial species: S. uberis, S. epidermidis and S. aureus. Sheep with a specific isolation were classified as y = 1, as opposed to y = 0 for all other sheep. As a result, isolation of S. uberis from milk showed a statistically significant correlation with: serous appearance of milk, presence of clots in secretions, and reactivity of supramammary lymph nodes (p < 0.05). Isolation of S. epidermidis from milk showed a statistically significant correlation only with presence of pustules and ulcers (p < 0.05). Finally, isolation of S. aureus from milk showed a statistically significant correlation with several clinical signs of chronic mastitis: nodules, abscesses, sclerosis, and atrophy (p < 0.05%). Interesting results were also obtained upon

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Table 4 Variables of the equation processed: significant variables. Variables

B

S.E.

Wald

Sig.

Exp(B)

Udder Nodules Abscesses Rubor Sclerosis

0.765 0.884 −0.639 0.720

0.175 0.303 0.260 0.101

19.137 8.502 6.045 51.345

0.000 0.004 0.014 0.000

2.150 2.419 0.528 2.055

Milk Serous Clots Absence of secretion

0.936 1.430 −1.218

0.255 0.644 0.184

13.430 4.924 43.939

0.000 0.026 0.000

2.549 4.177 0.296

Other Supramammary lymph nodes Hand milking Constant

0.308 −0.469 −0.300

0.092 0.102 0.078

11.310 21.086 14.637

0.001 0.000 0.000

1.361 0.626 0.741

B: coefficient of the variable; S.E.: standard error of B; Wald: Wald statistics; Sig.: significance value; Exp(B): estimated odds ratio of the variable.

investigation of the presence of “lùpia” in the 2198 udders examined in this study. This lesion was found in 11 out of 15 farms, involving 2.1% of all animals examined. However, no significant correlations were found between presence of “lùpia” and isolation of bacteria from milk (p > 0.05%). 4. Discussion During clinical and microbiological examination of a large sample of Sarda dairy sheep within flocks suffering repeated mastopathy problems, coagulase-negative staphylococci (CNS) and streptococci were the bacterial entities most frequently isolated from milk. Such finding has been reported previously, and seems to be associated to changes occurred in the breeding style, which is gradually shifting from extensive, to semi-extensive, and finally to intensive. Increased susceptibility to environmental pathogens may be linked to their ability to thrive in the microclimate generated into the sheepfold, which is warm, humid, and protected by the direct exposure to ultraviolet radiations. Inside the sheepfold, the crowded conditions and the decubitus during rumination and sleep increase exposure of the udder to contact with the environmental microbial flora. Exposure to environmental pathogens is also directly related to poor hygienic conditions. Mechanical milking procedures seem to contribute to the decrease of udder health problems; however, if cleaning of milking devices and premises is not performed correctly, it may encourage the spread of mastopaties. Notably, during our visits to the sheepfolds we did not find significant differences between hygienic procedures adopted for cleaning fixed or portable milking devices. However, we did observe a strong correlation between mechanical milking with portable devices and the spread of a S. uberis infection within the flock. The reason for this enhanced spread might well be linked to higher survival skills of this microorganism on metal and plastic surfaces, but it might find a more likely explanation in the specific mechanism used for generating vacuum and in the pulsation frequency of the milking device. In fact, these small milking machines (at least the most popular models) have lower performance characteristics compared to large fixed milking plants, and are not able to warrant a constant pulsation and a continu-

ous vacuum inside the milking device. This discontinuous stimulation may induce an uneven and incomplete closure of the teat sphincters, facilitating backward flows and, consequently, bacterial colonization. Moreover, these small devices are used for milking one to two sheep at a time; therefore, general wearing problems and presence of small cracks in nozzles facilitate bacterial colonization, act as fomites, and quickly mediate the spread of infections among all animals within the flock. Such observations further confirm that, when an outbreak of S. uberis infection is detected, the separation of infected animals from the flock should also be accompanied by adoption of manual milking procedures. In this study, S. uberis was the single bacterial species showing the highest incidence. Infections caused by S. uberis have long been underestimated and underrated in the sanitary management of dairy sheep, although serious damages to the mammary gland may result from infection. It is interesting to notice the low incidence of Gram negative bacteria observed in dairy sheep (4.2% of isolates). Indeed, the diffusion of Gram negative bacteria in ovines is considerably lower than in bovines, where one of the most “classical” forms of mastitis is caused by Escherichia coli. Concerning the flock where M. agalactiae was detected, the most significant finding is related to the clinical picture observed in animals affected by CA. The clinical picture of CA shows signs of endemicization (the first report of CA in Sardinia dates back to 1980), leading to attenuation of the “classical” clinical picture. Keratoconjunctivitis and polyarthritis were not detected; symptoms, localized only in the udder, were generally mild and subacute. Identification of CA was complicated by the difficulties in making a differential diagnosis with streptococcal infections. The definition of the bacterial species responsible for the clinical picture required confirmation through laboratory diagnosis. Several interesting conclusions may be drawn upon analysis of the clinical pictures observed during this study. For instance, the presence of clinical signs indicative of both chronic and acute mastitis in the same half-udder (about 2% of all half-udders examined) might be linked to recrudescence of older episodes (as a result of stressful events or immunodepression), as well as to novel bacterial infections overlapping with the old, chronicized ones. However, the

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concomitant detection of both pictures should not be considered surprising. A high correlation between presence of clinical signs in the udder and positivity to microbiological examination was found in this study; conversely, the number of clinically negative sheep with positivity to bacterial culture was relatively low (Tables 1–3). In previous reports describing similar studies performed on Spanish flocks, with SCCs of 250,000 and 1 × 106 cells/mL, prevalence of subclinical mastitis was estimated to be of 16% and 35%, respectively (Romeo et al., 1998; Bergonier et al., 2003). In a further report from Spain, Gonzalo et al. (2002) described a 24.6% prevalence of subclinical mastitis in dairy sheep, while Arsenault et al. (2008) reported isolation of potentially pathogenic bacteria in 28.8% of subjects with clinically normal udders. Such findings differ significantly from our observations; further relevance to this discrepancy is given when considering that the flocks selected for the present study had SCCs higher than 5 × 106 cells/mL (with average values of 7 and 8 × 106 cells/mL). In light of these results, it becomes important to point out how research on ovine mastitis still leverages strongly on studies performed on bovines. Dairy cattle farming has long been economically crucial for most developed countries, and important efforts have been made for understanding and fighting this detrimental disease. On the other hand, small ruminant farming has been traditionally considered less relevant, and research on ovine mastitis has lagged behind. Nevertheless, this picture is now changing, and the gap among dairy species is being filled. However, scientific conclusions drawn for cattle are often erroneously considered to be directly transferable to small ruminants. For instance, the relevance of clinical examination as an instrument for mastitis diagnosis and control is considerably higher in small ruminants. In fact, physical examination of the sheep udder is considerably easier, since both anatomical features and dimensions of the lactating organ allow for a deeper examination of all tissues, and enable detection of even minimal anomalies. Conversely, in cows the large dimensions of the lactating udder do not allow a comprehensive physical examination of deep lesions, and enlargement of the supramammary lymph nodes is only detectable when alterations are significant. Considering such limitations, it remains necessary to rely also on field tests (Californian Mastitis Test, CMT) and laboratory tests (SCC) for diagnosis. The inability to detect less significant alterations at the clinical examination is the most probable reason for the high number of subclinical mastitis cases usually reported in dairy cows. In our opinion, the systematic application of CMT and SCC to dairy sheep can lead to a more superficial clinical examination of the ovine udder. As a result, the notification of subclinical mastitis in ovines after diagnosis with CMT and SCC is a common finding in the scientific literature, without any reference to methodology, strategy, and details concerning clinical examinations. On the other hand, we suggest that a correctly performed clinical examination of the sheep udder may allow to gather enough useful diagnostic information, providing better insights than either CMT or SCC. As an example, some forms of chronic mastitis presenting parenchimal atrophy and scle-

rosis can go undetected upon CMT and SCC, but are easily detectable upon clinical examination (presence of these lesions often requires sheep culling). Furthermore, clinical examination enables detection of acute and chronic lesions close to lambing, as well as during the first and last days of lactation, when CMT and SCC do not produce reliable results. This is especially relevant when considering that during these lactation stages the risk of infection is highest, in sheep as well as in cows. Studies performed on dairy cows reported that the classification error of CMT can range between 25% and 50% (Ruegg and Reinemann, 2002; Pyörälä, 2003), and that SCC should always be supported by microbiological examination, since results are often ambiguous (Pyörälä, 2003). It is important to highlight that the reference values for SCC established by the European Regulation (CE) N. 853/2004 are intended for use in cows and not in small ruminants, for which reference values have yet to be established. A complete and extensive diagnosis and an appropriate therapy require the combination of clinical examination and microbiological tests for identification of the etiological agent responsible for the mastitis outbreak. However, we believe that diagnosis, definition, and classification of mastitis outbreaks could also be performed directly by means of the clinical examination alone, providing a secure, rapid, and economically advantageous process for controlling the mastitis problem in dairy sheep. Acknowledgement A large part of this work was funded by the Italian Ministry of Health, project IZS SA 05/04. References Arsenault, J., Dubreuil, P., Higgins, R., Bélanger, D., 2008. Risk factors and impacts of clinical and subclinical mastitis in commercial meatproducing sheep flocks in Quebec, Canada. Preventive Veterinary Medicine 87 (3–4), 373–393. Bergonier, D., De Crémoux, R., Rupp, R., Lagriffoul, G., Berthelot, X., 2003. Mastitis of dairy small ruminants. Veterinary Research 34, 689–716. Campesi, F., Marogna, G., Uzzau, S., Leori, G.S., 2008. Allestimento e uso di un metodo Multiplex-PCR per la diagnosi di Streptococcus uberis e Enterococcus faecalis da latte ovino. In: Atti del X Congresso Nazionale della Società Italiana di Diagnostica di Laboratorio Veterinaria (S.I.Di.L.V.), Alghero (SS). Conington, J., Cao, G., Scott, A., Bunger, L., 2008. Breeding for resistance to mastitis in United Kingdom sheep, a review and economic appraisal. Veterinary Record 162, 369–376. Contreras, A., Sierra, D., Corrales, J.C., Marco, J.C., Paape, M.J., Gonzalo, C., 2007. Mastitis in small ruminants. Small Ruminant Research 68, 145–153. Cuccuru, C., Preti, C., Meloni, M.G., Moroni, P., 2002. Riduzione delle cellule somatiche nella specie ovina. Obiettivi & Documenti Veterinari 23 (1), 21–26. Gonzalo, C., Ariznabarreta, A., Carriedo, J.A., Primitivo, F.S., 2002. Mammary pathogens and their relationship to somatic cell count and milk yield losses in dairy ewes. Journal of Dairy Science 85, 1460–1467. Gonzalo, C., Carriedo, J.A., Baro, J.A., Primitivo, F.S., 1994. Factors influencing variation of test day milk yield somatic cell count, fat, and protein in dairy sheep. Journal of Dairy Science 77, 1537–1542. Gougoulis, D.A., Kyriazakis, I., Papaioannou, N., Papadopoulos, E., Taitzoglou, I.A., Fthenakis, G.C., 2008. Subclinical mastitis changes the patterns of maternal-offspring behaviour in dairy sheep. Veterinary Journal 176, 378–384. Leitner, G., Chaffer, M., Shamay, A., Shapiro, F., Merin, U., Ezra, E., Saran, A., Silanikove, N., 2004a. Changes in milk composition as affected by subclinical mastitis in sheep. Journal of Dairy Science 87, 46–52.

G. Marogna et al. / Small Ruminant Research 88 (2010) 119–125 Leitner, G., Chaffer, M., Caraso, Y., Ezra, E., Kababea, D., Winkler, M., Glickman, A., Saran, A., 2003. Udder infection and milk somatic cell count, NAGase activity and milk composition – fat, protein and lactose – in Israeli-Assaf and Awassi sheep. Small Ruminant Research 49, 157–164. Leitner, G., Merin, U., Silanikove, N., 2004b. Changes in milk composition as affected by subclinical mastitis in goats. Journal of Dairy Science 87, 1719–1726. Leitner, G., Silanikove, N., Merin, U., 2008. Estimate of milk and curd yield loss of sheep and goats with intrammamary infection and its relation to somatic cell count. Small Ruminant Research 74, 221– 225. Malher, X., Seegers, H., Beaudeau, F., 2001. Culling and mortality in large dairy goat herds managed under intensive conditions in western France. Livestock Production Science 71, 75–86. Marogna, G., Rocca, S., Cabras, F.L., Leori, S.G., 2008. La “lùpia”: mastopatia fibrocistica degli ovini da latte di razza Sarda. In: Atti XVIII Congresso Nazionale della Società Italiana di Patologia ed Allevamento degli Ovini e dei Caprini (SIPAOC), Trezzo sull’Adda (MI). McCarthy, F.D., Lindsey, J.B., Gore, M.T., Notter, D.R., 1988. Incidence and control of subclinical mastitis in intensively managed ewes. Journal of Animal Science 66, 2715–2721. Onni, T., Cubeddu, G.F., Marogna, G., Sanna, G., Cillara, M.G., Lollai, S., Leori, G., Tola, S., 2007. Caratterizzazione di ceppi di stafilococchi coagulasi negativi (SCN) isolati da latte ovino mediante PCR RFLP e PFGE. In: Atti del IX Congresso Nazionale S.I.Di.L.V., Roma. Pyörälä, S., 2003. Indicators of inflammation to detect mastitis. Veterinary Research 34, 565–578.

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Robinson, J.J., Foster, W.H., Forbes, T.J., 1969. The estimation of the milk yield of a ewe from body weight data on the suckling lamb. Journal of Agricultural Science, Cambridge 72, 103. Romeo, M., Ziluga, I., Marco, J., 1998. Diagnòstico in situ de la infecciòn mamaria mediante palpaciòn, californian mastitis test y su seguimiento mediante recuento de celulas somàticas. Ovis 59, 61– 77. Ruegg, P.L., Reinemann, D.J., 2002. Milk quality and mastitis tests. Bovine Practice 36, 41–54. Tola, S., Angioi, A., Rocchigiani, A.M., Idini, G., Manunta, D., Galleri, G., Leori, G., 1997. Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction. Veterinary Microbiology 54, 17– 22. Tola, S., Idini, G., Manunta, D., Galleri, G., Angioi, A., Rocchigiani, A.M., Leori, G., 1996. Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction. Veterinary Microbiology 51, 77–81. Torres-Hernandez, G., Hohenboken, W., 1979. Genetic and environmental effects on milk production, milk composition and mastitis incidence in crossbred ewes. Journal of Animal Science 49, 410–417. Wahner, N., Marogna, G., Bacciu, D., Campesi, F., Cubeddu, G.P., Lollai, S., Uzzau, S., Rubino, S., Leori, S.G., 2007. Allestimento di un sistema Multiplex PCR per l’identificazione di Streptococcus agalactiae, S. dysgalactiae, S. uberis e Enterococcus faecalis. In: Atti del IX Congresso Nazionale S.I.Di.L.V., Roma. Warren, E.P., Rembarger, 1963. Some factors affecting milk yield of ewes and growth of lambs. Journal Animal Science 22, 866. Watson, D.J., Buswell, J.F., 1984. Modern aspects of sheep mastitis. British Veterinary Journal 140, 529–534.