Albanian j. agric. sci. ISSN: 2218-2020, (2012), (Special Edition) Copyright © Agricultural University of Tirana

THE USE OF LABORATORY TECHNIQUESFOR THE DIAGNOSIS OF CLINICAL MASTITIS AND SUB-CLINICAL OF DAIRY CATTLE KETI KUMBE MARGARITI1*, PRANVERA ÇABELI KUSI2, DOMENICO BUONAVOGLIA3, TANA SHTYLLA, SONILA ÇOÇOLI4 1

Albanian National Food Authority

2

Faculty of Veterinary Medicine, Agricultural University of Tirana

3

Universita degli Studi ”Aldo Moro”, Facolta Medicina Veterinaria, Bari, Italy

4

Institute of Food Safety and Veterinary

*Autor of correspondence: E-Mail: ketikumbe@yahoo. it

Abstract: The diseases of bovine udder in general and those of the mastitis in particular remains a major problem for many farmers in the dairy herd farm. Mastitis continues being one of the most expensive diseases, the most complex and the most wide-spread in the dairy herd. Studies made confirm that from mastitis the farmers lose approximately 180-200$ during a year in a cow, as a result of a sharp fall of the production of milk, the sharp fall of prices and the increasing of the veterinaries medicines and services. A great part of this loss, aproximately 70-80% comes from subclinical mastitis and 20-30% as a result of clinical mastitis. In our studies, from 264 samples controlled with CMT test, 25% of them resulted with mastitis, where 16. 3% were subclinical forms and 8. 7 of them were clinical forms. Statistical studies of the dates, presented very high qualities of the CMT test (specificity 99. 4% and diagnostically values 98. 4%), which proves that this test is easy, simple, fast and not expensive can be applied directly in herd of cattles. Our studies, also presented that % of bacterial positive animals is very high, 40. 3%, which proves that the udder infection of cattle’s are very wide-spread and the hygienic qualities of milk is not very well. Our results prove that field test and laboratory test combined with each other secures a useful control, diagnose and treatment of the all mastitis forms. The systematic and programmed control and treatment of mastitis in cattle is dictated from their actual situation of spreading Keywords: Whiteside, subclinical mastitis, clinical mastitis, SCN

1. Introduction Mastitis term means an inflammatory of the mammary gland and udder tissue, associated with changes in physical, chemical, cytological and bacteriological milk. Mastitis is responsible for maintaining breast milk, creating a suitable environment for the development of different microorganisms. Mastitis caused by pathogenic microorganisms, and as such of this is considered with nature infection disease. These analyzes have indicated that in 70-80% of cases, accumulated milk from the affected quarter breast with mastitis cows results in microfloresmastitogenes, which demonstrates the prevalence of infectious nature of the disease [6]. Mastitis directly affects breast tissue, provoking significant reduction of milk production or complete interruption of milk production. Besides reducing milk production, mastitis causing a change in the physical and chemical composition of milk, International Conference 31 October 2012, Tirana

resulting to the reduction of its nutritional value and making it inadequate for human consumption and industrial processing. A basic requirement for combating and preventing mastitis is the establishment of an early diagnosis. For this reason it is imperative that experts familiar with all the techniques of diagnosis of mastitis should decide quickly which practice will be used on such case. The correct diagnosis of clinical mastitis is based on the assessment and control of breast milk and the general condition of the organism. 2. Material and Methods The study was conducted during the period of year 2010-2011. To achieve the objectives of this study were taken 264 milk samples, at random, from 67 cows of different ages, in early lactation, mid lactation and dry period. This study was carried out in the municipalities of Kashar, Farke, Petrele, Zall-Herr and Paskuqan, which have almost the same levels of

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thhe feeding, breeding, hoousing and thhe same clim matic c conditions. Innitially a clinical audit was w conducted to identify breeast clinicaal mastitis cases andd to d differentiate them from m sub-clinical forms. For d determination n of clinical mastitis casees we were based b inn examinatioon of visiblee breast abnoormalities succh as thhe presence of edema, high h temperaature, pain while w touching the breasts, andd abnormal seecretion of breast b ( (change in color c and coomposition of o the milk). For e each animal included inn the study were taken milk f from each quarter q of udder, u whicch went thrrough d different techhniques, as defined d in thhe method off this p paper. Sampples were appplied stricttly hygienic and s sanitary meassures. Also, before b takingg first portioons of m sample,, the first droops were takken away andd not milk u used, as they normally are contam minated by micro m f flora locateed in the suction channel. For b bacteriologic cal analysis we w used sterille bottles, annd we took among 5-10 ml milk m from evvery quarter. The c collected sam mples were chilled, c and transported t t the to laboratory foor immediatee analysis. Each E milk saample t four quaarters of thee animals inn the taken from the s study underw went in thee same tim me a) Califfornia M Mastitis Teest, b) miicroscopic examinationn to d determine thhe number of somaticc cells andd the p percentage o neutrophhils and c)) bacterioloogical of a analysis. ults and Disscussion 2. Resu 264 millk samples from f 67 diffferent cowss, for e each quarterr breast in particular analyzed too the C California M Mastitis Test.. The data obtained o shoowed thhat 66 of 264 2 controllled samplesbbelonging to 27 c cows or 25% % of total conntrol sampless resulted possitive w with CMT. Six of these cows, or 23 sam mples, r representing 8.7% of the total samples were contrrolled a animals withh signs of breeast gland innflammation,, thus p practically w were cows with w clinicall mastitis. FortyF thhree milk saamples of 21 cows appaarently "norm mal", o 16. 3% of the sam or mples controolled with CMT C b belonged too animals without visible signss of innflammationn breast glannd, thus theyy were cows with m mastitis sub--clinical. Thhe followingg chart provvides C CMT results in controlledd animals. s that thee modified CMT C The dataa obtained show e evidence reveals not onlyy effectivelyy clinical maastitis c cases, but alll those casess dealing witth the increaase in thhe rate of cell contennts, which at the tim me of p performing thhe test is stiill hidden, which w meanss that thhis test is able a to diaggnose sub-cllinical or hiidden International Conference C 3 October 2012, Tirana 31

forrms of masttitis cows, as is it meentioned in the liteerature are inn an higher ppercentage in n herds of coows forr milk produuction than inn the usual cases c of cliniical maastitis final [6, [ 9, 13, 144]. As a rule,, CMT posittive reaaction trackss start at the level of about a 500, 000 0 cellls/ml. Accoording to staatistical processing, whhich allows to excllude all possible changees for differrent breeeds of catttle, the coontent of 150000-2000000 som matic cells per 1 ml.. milk can genuinely be con nsidered norrmal, and w when it exceeeds 500, 0000 a celll / ml, the giivenquarter oof statistical data is infeccted [5,, 7, 8, 9, 12].

25% 75%

Figure1:CMT test results of controlled c samples

Also, via CMT we hhave distingu uished also the positivity expressed in vvarying deg grees of it, to cattegorize the severity s of clinical or sub b-clinical forrms of mastitis, which w repressent an eassy or alarm ming ndition of this t infectionn. Thus, in the follow wing con graaphs are prresented inffection ratess according to varrious levels of positivityy and frequeency of masttitis acccording to thhe affected quuarters.

14% 9% 36%

41%

Figure 2: CMT Test rresults accord ding to the p level of positivity

As noted in the chaart above, CMT C evidennce sho ows varying degrees of positiviity in conttrol sam mples. Different percentaages expresssed in our chhart sho ows once aggain that thiis simple tesst helps us not only in establisshing a rapidd diagnosis, but also guides us in the prophhylaxis of thiis infection. This T means that t if mastitis m sub--clinical alw ways begins with w a plus, we neeed to interveene as soonn as possiblee with hygieenic pro ophylaxis to prevent worrsening of itss further spreead

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of infection in other healthy nipples, and perhaps even in normal animals. If CMT evidence showsincreased degree of positivity (+, + +, +++, ++++) then a specific therapy intervention would be decisive [4, 15]. The same milk samples (264 samples taken from 67 cows), controlled by CMT, and is also used for the determination of cellular content. It is known that the increase in the number of somatic cells in milk runs parallel with the increase in the number of bacteria in it. Persistent high number of somatic cells in a herd is usually a sign of high-level mastitis sub-clinical [1, 3, 11, 12]. According to known methods, for each milk sample lying on the microscopic glass, it is estimated the number of somatic cells and the percentage of neutrophils. Our research data show that cows with mastitis or in a state where functions breast gland are vulnerable it grows significantly NSC, as well as PN (percentage of neutrophils). Taking into consideration the group of SCC over 500, 000 a cell / ml and PN, over 20% of results that 71 samples or 26% of the samples taken in the study and 80 samples or 30. 3% belongs to animals with clinical mastitis or subclinical,and such data almost match those achieved by simple field evidenceof CMT. According to this statement it is clear that the percentage of positive samples in CMT test is less than 2% lower than that of the cell contents, and about 5% lower than that of samples containing neutrophils with greater than 20%. This small and acceptable difference of (some errors even subjective) CMT,

compared with microscopic analysis to determine the NSC and PN, allows to consider it (test CMT) a very practical test, a simple, rapid and inexpensive, in the detection of affected breast quarters by mastitis in terrain. All milk samples from 264 quarters among 67 cows in the study underwent bacteriological analysis, making their planting based on cultural grounds (agar simple, simple blood agar). Bacteriological analysis showed that the percentage of animals with positive bacterial charge is quite high, 40. 3%. This indicator shows that cow’s breast infections are extensive and that the quality sanitary-hygienic milk leaves a lot to be desired. For this reason the study of breast infections and determination of micro qualitative composition causes is a necessary condition not only for the clarification of the nature and etiology of mastitis, but also to determine the measures to combat them. Our data for the samples taken in the study show that there is a very good concordance between the presence of different micro isolated and the presence of mastitis, diagnosed from CMT evidence. Discrepancy exists only in two cases, when is not isolated micro-flora and when is not diagnosed mastitis. Microorganisms isolated, as shown in the table below belong to both cases of mastitis clinical as well as in their sub-clinical forms which proves once again that Etiology of mastitis is generally the same, since there is no specific agents for one or another form of mastitis.

Table 1: Bacterial causes of mastitis in dairy cows Causality S. aureus S. epidermitis(1 case not mastitis) S. agalactiae Diplococcus E. coli Coliforme

Number of Cases

Clinical Cases

Sub-Clinical Cases

11 2 2 4 3 4

3 0 0 0 2 1

8 1 2 4 1 3

Causes of mastitis in cows in the study were staphylococci, which are found in 40. 74% of positive samples.Their apparent dominance in micro-flora dairy cows with mastitis is a peculiar fact that should be taken in considerationin organizing relevant measures to combat them. Streptococci are relatively rarely encountered in 7. 4% of cases with mastitis.Bacteriological analysis data of this study show that the cows’ mastitis under examination is infectious by nature (microbial). This proves that International Conference 31 October 2012, Tirana

positive cows resulted bacteria positive in CMT bacteriological analyzes also show that Etiology of infectious mastitis agents, either clinical or subclinical form is just what we know. As such, the bacteriological research which are difficult to conduct in any case, must be planned and programmed well and combine with other methods of diagnosis to determine the exact type of treatment and the efficiency of it.

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Thus, complex field and laboratory evidence combined between them provides an investigation, diagnosis and effective treatment for all forms of mastitis. Tracking and systematically programmed combating the cows mastitis is dictated by the current

state of their proliferation. The data obtained from this work, as a result of performing a complex tests described above are comprehended in the correlation between them, to verify the reliability.

Table 2: The compatibility between CMT test and bacteriological analysis T+ T-

D+ (bacteriologicallypositive) 64a (Positive cases in CMT) 14 c (Negative cases in CMT ) 78a + c

D- (bact. negative) 1 b (posit. in CMT) 197 d(neg. in CMT ) 198 b + d

In the above table, are presented the data obtained from CMT evidence and bacteriological analysis, considering the latter as evidence of a higher level "Gold Standard". According to the OIE manual there are calculated epidemiological elements, which show compatibility between the two tests used [7, 13,]. Our data set in prevalent reports show a high satisfactory evidence of compared results. CMT test specificity reached 99. 4% and its diagnostic value are 98. 4%, which shows once again that this test is easy and simple and can be applied directly to cattle herds to assess the state of their breast and in particular order, for tracing and detection of mastitis sub-clinical form. 4. Conclusions •

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•

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In our study, from 264 samples checked, 25% of them resulted in mastitis, where 16. 3% of presented mastitis sub-clinical and 8. 7% of samples, clinical mastitis, which means that evidence in CMT is able to diagnose format various of cow mastitis; Different rates of positivity, that CMT evidence states, not only helps in the diagnosis of mastitis, but also directs the prophylaxis of infection; Statistical processing of the data from CMT showed extensive and enough high field test value (99. 4% specificity and 98. 4% diagnostic value), which proves that this test is easy, simple, quick and inexpensive and can be applied directly in flocks cows to assess the condition of the breast in particular and for the detection of mastitis subclinical form; As CMT evidence is also the indication of counting cell content in cow milk, helps to determine the status of the breast gland, as well as to diagnose so-called mastitis sub-clinical; Bacteriological analysis data showed that the cows’ mastitis taken in examination is infectious by nature (microbial). This proves that positive

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Total 65a + b 211 c + d 276 a +b+ c+ d

•

cows at CMT, resulted bacterial positive in bacteriological analysis; Etiology of infectious mastitis agents, either clinical or sub-clinical form is just what we know. As such, the bacteriological research difficult to conduct in any case, must be well planned and programmed and combined with other methods of diagnosis to determine the exact type of treatment and the efficiency of it. 5. References

1. Allison J R D: Antibiotic residues in milk. BritishVeterinary Journal 1985, 141(1): 9-11 2. Bell R C, Wilesmith J W, Johnston K G: Bovine mastitis caused by Bacillus cereus. Veterinary Record 1981, 198(108): 404-408 3. Booth J M, Harding F: Testing for antibiotic residues in milk. Veterinary Record 1996, 119 (34): 565-569. 4. Chaming R J: Incidence of clinical mastitis in dairy herds in a mastitis control scheme. Veterinary Record, 1983, 112(18): 438 5. Dinsmore R P, English R: Use of augmented cultural techniques in the diagnosis of the bacterial cause ofclinical bovine mastitis. J. Dairy Sci, 1992, 75(10): 2706- 2712. 6. Golodetz C L, White M E: Prognosis for cows with severe clinical coliform mastitis. Veterinary Record, 1975, 112(17): 402-403. 7. Harmon R J: Physiology of mastitis and factors affecting somatic cell count. J. Dairy Sci. 1994, 77 (7): 2103- 2112. 8. Ogden N H: Teat factorsin E. coli mastitis. Vet. Rec, 1986, 118: 507-509 9. Shawkat Q Lafi, Nabil Q Hailat: Bovine and ovine mastitis in Dhuleil Valley ofJordan. Vet. Arhiv, 1998, 68: 51-57. 10. Shuster E, Harmon R J, Jackson J A: Endotoxin mastitis in cows milked four times daily. J. Dairy Sci, 1991, 74(5): 1527-1538.

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11. Tola V, Çabeli P: Permbajtjaqelizore e qumeshtittelopeve, Bul. i Shk. Zoo. Vet. 1987, 6: 161-170

13. Tola V, Çabeli P: Prova me hidroksidnatriumi ne gjurmimin e mastitevetelopeve. Bul. Shk. Zoot. Vet., 1989, 2: 110-117

12. Tola V, Çabeli P: Pasojat negative temastiteve ne prodhimtarine e qumeshtittelopeve. Buletini i Shkencave Zoo. Vet., 1989, 1: 35-41

14. Watts J L: Etiological agents of bovine mastitis. Vet. Microbiol.,1988, 16: 41-66.

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15. ZecconiAlfonso: Una mastite per tutte le stagioni. Scienza Veterinaria, 1987, 31: 129-138

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