Printer Friendly

Comparison of [alpha]1-antitrypsin, [alpha]1-acid glycoprotein, fibrinogen and NOx as indicator of subclinical mastitis in riverine buffalo (Bubalus bubalis).

INTRODUCTION

Riverine buffalo milk production in the Indian sub continent has long been accepted as the backbone of the rain-fed agrarian socio-economic fabric. Sustainability of buffalo milk production even during dry spells has contributed to a lower suicide rate amongst farmers in drought stricken terrain. The quality of milk lies in its hygienic status. Milk production involves rapid physical, chemical and biological changes right from galactopoiesis to let down. Mastitis, complex multi-factorial inflammatory reaction, which often results from an intra-mammary bacterial infection entails losses due to reduced milk production, treatment costs, increased labor, milk withheld for human consumption due to residues in the form of antibiotics and micro-organisms and pre-mature culling. Consequently, an early detection at the sub clinical stage is necessary to prevent production loss and to enhance prospects of recovery (Guha et al., 2010).

Subclinical mastitis, a herd problem, affects the normal functioning of the mammary gland epithelial cells' ability to convert circulating nutrients into milk components (Gera and Guha, 2012). It often goes unnoticed due to absence of visually apparent changes in udder and milk. Detection of SCM is also difficult due to pooling of milk for sale from different milk collection points so that the source of SCM cannot be determined after collection (Gera et al., 2011; Guha et al., 2012). Subclincial mastitis is also a depot of micro-organisms that lead to the spread of infection to the other animals within the herd.

Acute phase proteins (APPs) are an assortment of blood hepatic glycoproteins that change in concentration due to external or internal challenges, such as infection, inflammation, surgical trauma, or stress. Quantification of APP concentration in body fluids can provide valuable diagnostic information in the detection, prognosis, and monitoring of disease in several animal species (Gonzalez et al., 2008). The recent recognition, that APPs are produced in the bovine mammary gland in response to bacterial mastitis has made it obligatory to consider them as alternative biomarkers for mastitis. An increase in concentration of APPs precedes the onset of clinical signs even in the absence of macroscopic changes in the ruminant milk (Safi et al., 2009).

Macrophages, a somatic cell fraction of milk, are the source of nitric oxide (NOx) in bovines. In intra-mammary infection (IMI), macrophages are the initially predominant cell type to travel from the peripheral circulation to the mammary gland in response to inflammatory insults and contribute to the pathophysiology of the mammary gland. NOx is produced in large amounts by inducible nitric oxide synthase (iNOS) and its derivatives, such as peroxynitrite and nitrogen dioxide, and plays a role in inflammation (De and Mukherjee, 2009).

The diagnostics based on physical and chemical changes in SCM milk is not satisfactory. A confirmatory diagnosis of SCM according to International Dairy Federation (IDF) recommendations is based on the microbiological status and inflammatory reactions i.e., somatic cell count (SCC [greater than or equal to] 2 x [10.sup.5] cells/ml of milk) of the quarter. However, the logistic and financial considerations involved with sampling all animals in a herd have precluded these techniques from being widely adopted (Guha et al., 2010). One of the principles of detecting inflammation within the mammary gland is to study the mammary epithelial integrity (Gera and Guha, 2011). For this reason alternative parameters to indicate inflammation are used to identify trends in the development of the udder health in dairy herd (Guha et al., 2010). Several superior breeds of milch buffaloes are being developed on the Indian subcontinent where buffaloes are foremost dairy animal. Thus, the present study was undertaken to investigate the effectiveness of the aforesaid APPs and NOx in detecting SCM and recognizing them as indicators for bubaline SCM for further development of kit for diagnosing SCM in herds. In the present study their concentration in healthy and SCM milk was analyzed both statistically and epidemiologically and further correlated with [Log.sub.10]SCC.

MATERIALS AND METHODS

Collection of milk samples

Fifty ml of milk samples each were collected from 776 Murrah buffaloes over 3096 quarters under aseptic condition in sterile containers. The quarters were marked as right-fore (RF), right-hind (RH), left-fore (LF) and left-hind (LH).

Bacterial culture examination

The milk samples collected aseptically were shaken thoroughly. A 4 mm diameter platinum loop was used to streak 0.01 ml of the sample on 5% ovine blood agar plates. The plates were incubated aerobically at 37[degrees]C for 24 h. The resulting growth from the respective plates of media was purified and identified on the basis of morphology, colony characteristics and Gram's reaction (Gera and Guha, 2011).

Somatic cell count

The somatic cell count (SCC) of the milk samples was determined microscopically (Gera and Guha, 2011). Following through mixing, a 4 mm diameter platinum loop was used to evenly spread 0.01 ml of milk over four 1.0 [cm.sup.2] area template outlines. Slides were stained for 30 s in Newman-Lampert stain, with the composition as follows:
Methylene blue        1.2 gm.
95% ethyl alcohol     54 ml.
Tetrachloroethane     40 ml.
Glacial acetic acid   6 ml.


Somatic cells were stained with deep blue nuclei against a light blue background. The working factor of the microscope was calculated to be 35,400 by using a stage micrometer, calculating diameter of the microscopic field (0.012 cm) and the field per square cm (8850) for the given microscope. Total no. of cells was obtained by multiplying the total no. of cells counted in 25 fields with the working factor.

Estimation of fibrinogen

The fibrinogen was estimated by the tyrosine method as described by Varley et al. (1980). The fibrinogen was precipitated with calcium in casein free skimmed milk samples. The blue coloured complex developed due to the reduction of phosphomolybdate and phosphotungstate by tyrosine residues of polypeptide was estimated spectrophotometrically at 680 nm.

Estimation of [[alpha].sub.1]-acid glycoprotein

The [[alpha].sub.1]-acid glycoprotein protein was estimated by the tyrosine method as described by Varley et al. (1980). Casein of skimmed milk was removed by acid precipitation and the heat coagulable protein by percholoric acid. The [[alpha].sub.1]-acid glycoprotein protein was finally precipitated with phosphotungstic acid. The tyrosine content of the precipitate was estimated by the above mentioned procedure.

Estimation of [[alpha].sub.1]-antitrypsin

The [[alpha].sub.1]-anti trypsin was measured by the Benzoyl arginine p-nitroanilide (BAPNA) method as described by Fritz et al. (1974), with little modification as described by Guha and Gera (2011). Casein and fat were removed by clearing solutions containing rennet and polyethylene glycol. The trypsin residue formed a yellow coloured complex 4 nitroaniline after reacting with BAPNA which was measure spectrophotometrically at 405 nm. The intensity of colour was inversely proportional to [[alpha].sub.1]-anti trypsin concentration.

Estimation of NOx

The NOx (nitrate+nitrite) was estimated by Griess reaction as described by Bouchard et al. (1999). Nitrate was converted to nitrite by nitrate reductase. The acidified nitrite produced nitrosating agent which reacted with sulfanilic acid to produce diazonium ions. The diazonium ions coupled with N (1-naphthyl) ethylenediamine to form choromophoric azo-dye whose intensity was measured spectrophotometrically at 550 nm.

Calculation of percent sensitivity, specificity, accuracy, predictive values and likelihood ratios

Percent sensitivity, specificity, accuracy, predictive values and likelihood ratios were found taking bacterial growth in culture media and SCC [greater than or equal to] 2 x [10.sup.5] cells/ml of milk as the benchmark (Katsoulos et al., 2010; Guha et al., 2010; Gera and Guha, 2011). The cut-off values for each significantly altered parameter were obtained from Receiver Operator Characteristic (ROC) analysis curve with the aid of the MedCalc software. The percent sensitivity, specificity were calculated by the formulae of Thrusfield (2005). The percent accuracy was calculated by the formula of Reddy et al. (2001). Percent positive and negative predictive values, likelihood ratios (both positive and negative) were also measured by the methods of Petrie and Watson (2008).

Statistical analysis

Analysis of variance (ANOVA) and Duncan Multiple Range Test (DMRT) were carried out to compare the milk components. Comparison of means of estimated concentration of different parameters in healthy and SCM milk, irrespective of the etiology, was done by t-test. SCC was converted to [Log.sub.10]SCC. Pearson's correlation coefficient among milk components showing substantial alternation in concentration between healthy and SCM milk samples including [Log.sub.10]SCC was also calculated. All statistical analysis was done with SPSS statistical software (Petrie and Watson, 2008).

RESULTS

Etio-prevalence of SCM

In the present study, on the basis of bacterial culture examination and SCC it was observed that 347 riverine buffaloes (496 quarters) were SCM positive. Milk samples showing SCC [greater than or equal to] 2 x [10.sup.5] cells/ml and growth in culture media were considered positive for SCM. The SCC was observed to increase significantly (p<0.01) in SCM milk irrespective of the etiological agents (Tables 2 and 3). The mean SCC in SCM milk was 2.05 [+ or -] 0.056 (Table 3). From Table 1 it is evident that the most prevalent etiological agent was Staphylococcus spp. followed by Streptococcus spp. And Escherichia coli. A few instances of mixed infection and (Corynaebacterium spp. and Bacillus spp.) were also encountered during the investigation (Table 1). Together the frequencies of Gram positive infections were >79%.

Effect of SCM on milk components

In the present study there was a statistically significant (p<0.01) increase in the concentration of [[alpha].sub.1]-anti trypsin in infected milk samples irrespective of the causative agents. A significant (p<0.05) increase of [[alpha].sub.1]-acid glycoprotein concentration in the SCM milk was also observed. Fibrinogen was below detection levels in both healthy and infected milk samples. NOx also showed significant increase in SCM milk (Tables 2 and 3).

Percent sensitivity, specificity, accuracy, predictive values and likelihood ratios

After calculating the percent sensitivity, specificity, accuracy, predictive values and likelihood ratios for all the significantly altered parameters, [[alpha].sub.1]-anti trypsin was most in agreement with IDF criteria for SCM (i.e. bacterial growth in culture media and SCC [greater than or equal to] 2 x [10.sup.5] cells/ml), followed by [[alpha].sub.1]-acid glycoprotein. The value for NOx was at par (Table 4). However, the values for the same parameters were high when the causative agent was only Gram positive bacteria (Table 5).

Udder profile correlation coefficient

From Table 6 it can be observed that [Log.sub.10]SCC is strongly correlated (p<0.01) with [[alpha].sub.1]-antitrypsin only in SCM milk. With NOx, [Log.sub.10]SCC is correlated at (p<0.05) and (p<0.01) in healthy and SCM milk, respectively. [Log.sub.10]SCC is correlated with [[alpha].sub.1]-acid glycoprotein at p<0.05. NOx is correlated with [[alpha].sub.1]-antitrypsin and [[alpha].sub.1]-acid glycoprotein at p<0.01 and p<0.05, respectively.

DISCUSSION

The present study was carried out to compare the usefulness of [[alpha].sub.1]-antitrypsin, [[alpha].sub.1]-acid glycoprotein, fibrinogen and NOx in detecting SCM, with special reference to bubaline SCM.

SCM milk samples were those that showed bacterial growth in culture media and had a SCC of [greater than or equal to] 2 x [10.sup.5] cells/ml (IDF, 2005). Gram positive bacterial agents were the most prevalent (Table 1). Similar observations were reported by Sharma et al. (2010) who attributed the contamination to the presence of organisms in the sub-continent atmosphere. The mean SCC in the SCM milk were significantly (p<0.01) high (Tables 2 and 3) owing to inflammatory reactions (Guha et al., 2010).

The significant increase of [[alpha].sub.1]-anti trypsin in SCM milk (Tables 2 and 3) could be due to bacterial infection. The APP showed a substantial increase in SCM milk caused by all types of organisms. The increase in the concentration of [[alpha].sub.1]-anti trypsin was attributed to breach in the blood milk barrier by the action of inflammatory modulators and bacterial toxins, thus, it is a serum derivative (Gera and Guha, 2011).

A significant (p<0.05) increase of [[alpha].sub.1]-acid glycoprotein concentration was also observed for all types of infections (Tables 2 and 3). Up to 2006 there was no report of the presence of this [[alpha].sub.1]-acidglycoprotein in healthy or mastitic milk of dairy animals. Mansson et al. (2006) was first to report [[alpha].sub.1]-acid glycoprotein in healthy as well as in milk showing higher SCC in cows. A weaker and negative correlation of [[alpha].sub.1]-acid glycoprotein with SCC was reported by these authors. But, in the present investigation it was observed that the concentration of [[alpha].sub.1]-acid glycoprotein had a strong positive correlation with [Log.sub.10]SCC. The increase could be due to excess of somatic cells in SCM. Two isoforms of [[alpha].sub.1]-acid glycoprotein, a low MW group (44 kDa), produced in the mammary gland (MG-AGP), and a higher MW group (55 to 70 kDa), produced by somatic cells (SC-AGP), were isolated by Ceciliani et al. (2007). Identical SC-AGP isoforms can be found both in milk and blood polymorpho-nuclear cells. Hence, an increase in the concentration of [[alpha].sub.1]-acid glycoprotein can be attributed to increased synthesis by the somatic cells as well as by mammary gland cells as an immuno-protective measure. Gera and Guha (2011) also reported a similar observation in crossbred cow SCM milk.

In the present study, fibrinogen was not detected in either healthy or SCM milk (Table 1). Our observation agrees with Tabrazi et al. (2008) and Gera and Guha (2011); who reported fibrinogen, a mild APP, appears in the milk during acute or chronic stage as a blood clotting factor or indicator of fibrosis. Fibrinogen was not taken up for further investigation.

From Table 3 it can be observed that NOx in the infected milk samples increased significantly (p<0.01). A similar observation was made by Bulbul and Ylmaz (2004) and Gera and Guha (2011). They attributed the increase to increased macrophages, a fraction of SCC.

We perused percent sensitivity, specificity, accuracy, predictive values and likelihood ratios of [[alpha].sub.1]-anti trypsin, [[alpha].sub.1]-acid glycoprotein and NOx as predictors of mastitis, taking the IDF criteria as the bench mark. It was observed that % sensitivity, specificity, accuracy were better for [[alpha].sub.1]-anti trypsin, followed by [[alpha].sub.1]-acid glycoprotein and NOx (Table 4) for all kind of infections. Our observation concurs with the reports of Gera and Guha (2011) in crossbred cows. These values were more in milk samples infected with Gram positive bacteria. The predictive values and likelihood ratios for positive tests are observed to be greater for [[alpha].sub.1]-anti trypsin (83.00%; 15.28) than [[alpha].sub.1]-acid glycoprotein (74.02%; 8.90) and NOx (69.64%; 7.44) (Table 4). The percent positive predictive values and likelihhod ratio (positive) when calculated in SCM milk infected with Gram positive bacteria for [[alpha].sub.1]-anti trypsin, [[alpha].sub.1]-acid glycoprotein and NOx were found to be 85.13; 21.88, 81.13; 16.21, and 72.46; 9.92, respectively (Table 5). The variation in these values was due to the fact that the concentration of the same parameters were lesser in SCM milk infected with E. coli than those milk samples which were infected with Staphylococcus or Streptococcus, though the level of significance were same for all the cases when compared with healthy milk (Table 2). The elevation of the parameters in gram positive SCM samples might be due to the fact that gram positive bacteria are more pathogenic in destroying the mammary gland epithelia whereas E. coli are relatively less severe on mammary gland cells (Wenz et al., 2006).

Likelihood test of a positive test result >10 indicates that the test can be used to rule in the disease. Likelihood ratio of negative results describes how much more likely the animal has a negative test result when it has the disease (Petrie and Watson, 2008). The likelihood ratio for a positive test for [[alpha].sub.1]-anti trypsin was found to be greater than 10 irrespective of the bacterial agent causing SCM. For [[alpha].sub.1]-acid glycoprotein the ratio was greater than 10 when SCM causative agents were Gram positive bacteria. The likelihood ratio (positive) for NOx was lesser than 10 irrespective of the mastitogenic agents. The purpose of separately considering Gram positive bacterial agents is that they are the most prevalent mastitogenic agents in the tropical countries as discussed above. To the best of our knowledge no such studies for [[alpha].sub.1]-acid glycoprotein and NOx were conducted previously. This is the first of its type. Hence, it can be considered as a pioneer work with special reference to bubaline SCM.

To prevent any ambiguity, double statistical evaluation for each presumed indicator was done in this study by correlating with [Log.sub.10]SCC (Gold Standard test) separately in healthy and SCM milk. From Table 6 it can be observed that [[alpha].sub.1]-anti trypsin was also found strongly correlated (p<0.01) with [Log.sub.10]SCC in SCM milk (0.795), while it was insignificant in healthy milk (0.092). The [[alpha].sub.1]-acid glycoprotein had a positive correlation, significant at p<0.05 (0.098 vs 0.559, healthy vs infected milk) with [Log.sub.10]SCC (Table 6). It is also evident that the correlation between [Log.sub.10]SCC and NOx were significant at p<0.05 and p<0.01 respectively in healthy and infected milk (0.546 vs 0.845, healthy vs infected milk). This may be due to the fact that the source of NOx is macrophages, a somatic cell fraction as discussed above. Apart from Log10 SCC, NOx was also significantly correlated with [[alpha].sub.1]-antitrypsin and [[alpha].sub.1]-acid glycoprotein at p<0.01 and p<0.05, respectively. Similar observations were reported by Gera and Guha (2011) in cow milk.

CONCLUSION

It can be reasonably concluded that though the concentration of two APPs, viz. [[alpha].sub.1]-antitrypsin and [[alpha].sub.1]-acid glycoprotein as well as NOx was significantly higher in milk during subclinical form of the inflammatory reaction, but, only [[alpha].sub.1]-anti trypsin was in agreement with the IDF criteria for SCM diagnosis for all kind of bacterial infections. The [[alpha].sub.1]-acid glycoprotein indicates SCM when caused by Gram positive bacteria alone. NOx did not attest to be a good indicator of SCM. The threshold value for [[alpha].sub.1]-antitrypsin and [[alpha].sub.1]-acid glycoprotein is fixed at 6,396.22 U/L and 2.92 mg/ml, respectively. Standardizing easy qualitative methods for estimating these indicators is recommended to enable the development of a kit for diagnosing SCM in the field. A pathophysiological explanation of the ascertained association is also recommended for further study.

http://dx.doi.org/10.5713/ajas.2012.12261

REFERENCES

Bouchard, L., S. Blais, C. Desrosiers, X. Zhao and P. Lacasse. 1999. Nitric oxide production during endotoxin induced mastitis in cows. J. Dairy Sci. 82:2574-2581.

Bulbul, A. and B. Ylmaz. 2004. Relationship between the level of nitric oxide and somatic cell count in the milk of cows with mastitis. Veteriner Bilimleri Dergisi 20:95-102.

Ceciliani, F., V. Pocacqua, C. Lecchi, R. Fortin, R. Rebucci, G. Avallone, V. Bronzo, F. Cheli and P. Sartorelli. 2007. Differential expression and secretion of alpha1-acid glycoprotein in bovine milk. J. Dairy Res. 74:374-380.

De, U. K. and R. Mukherjee. 2009. The inhibitory response of Azadirachta indica extract on nitric oxide production by milk leukocytes during clinical mastitis. Vety. Archiv. 79:41-50.

Fritz, H., I. Trautschold and E. Werle. 1974. Methods in Enzymatic Analysis, 2nd ed. Academic Press New York, USA, p. 234.

Gera, S. A. Guha. 2011. Assessment of acute phase proteins and nitric oxide as indicators of subclinical mastitis in Holsteinx Haryana cattle. Ind. J. Anim. Sci. 81:1029-1031.

Gera, S. and A. Guha. 2012. Effect of sub clinical mastitis on milk biochemical constituents in crossbred cows. Ind. Vet. J. 89:3334.

Gera, S., A. Guha, A. Sharma and V. Manocha. 2011. Evaluation of trace element profile as an indicator of bovine sub-clinical mastitis, Intas Polivet 12:9-11.

Gera, S., A. Sharma, R. S. Dabur, V. K. Jain and S. L. Garg. 2006. Studies on changes in milk composition and chemotherapeutic sensitivity in camel (Camelus dromedarius) in subclinical mastitis. In: Proceedings of the Internation Scientific Conference in Camels (Vol-II pp. 937-946) Quassim University Saudi Arabia.

Gonzalez, F. D. H., F. Tecles, S. Marti'nez-Subiela, A. Tvarijonaviciute, L. Soler and J. J. Cerone. 2008. Acute phase protein response in goats. J. Vet. Diagn. Invest. 20:580-584.

Guha, A. and S. Gera. 2012. Evaluation of milk trace elements, lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activity of subclinical mastitis milk as and indicator of subclinical mastitis in riverine buffalo (Bubalus bubalis). Asian Australas. J. Anim. Sci. 25:353-360.

Guha, A., S. Gera and A. Sharma. 2010. Assessment of chemical and electrolyte profile as an indicator of subclinical mastitis in riverine buffalo (Bubalus bubalis), Har. Vet. 49:19-21.

International Dairy Federation (IDF). 2005. Diagnostic potential of California Mastitis Test to detect subclinical mastitis 26. Maastricht, Netherlands. pp. 15-19.

Katsoulos, P. D., G. Christodoulopoulos, A. Minas, M. A. Karatzia, K. Pourliotis, K. Spyridon and S. K. Kritas. 2010. The role of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase in the diagnosis of subclinical intramammary infections in dairy sheep and goats. J. Dairy Res. 77:107-111.

Mansson, H. L., C. Branning, G. Alden and M. Paulsson, 2006. Relationship between somatic cell count individual leukocyte populations and milk components in bovine udder quarter milk. Int. Dairy J. 16:717-727.

Petrie, A. and P. Watson. 2008. Statistics for Veterinary and Animal Science, (Blackwell Publishing, London).

Reddy, L. V., P. C. Choudhuri and P. A. Hamza. 2001. Comparative efficacy of different tests in the diagnosis of subclinical mastitis in crossbred cows. Ind. Vet. J. 78:903-905.

Safi, S., A. Khoshvaghti, S. R. Jafarzadeh, M. Bolourchi and I. Nowrouzian. 2009. Acute phase proteins in the diagnosis of bovine subclinical mastitis. Vet. Clin. Pathol. 38:471-496.

Sharma, N., V. Pandey and N. A. Sudhan. 2010. Comparison of some indirect screening tests for detection of SCM in dairy cows. Bulg. J. Vet. Med. 13:98-103.

Tabrazi, A. D., R. A. Batavani, S. A. Rezaei and M. Ahmadi. 2008. Fibrinogen and ceruloplasmin in plasma and milk from dairy cows with subclinical and clinical mastitis. Pak. J. Biol. Sci. 11: 571-576.

Thrusfield, M. 2005. Veterinary epidemiology, 3rd ed, Blackwell Science, United Kingdom., p. 158.

Varley, H., A. H. Gowenlock and M. Bell. 1980. Practical clinical biochemistry. 5th ed. William Heinemann Medical Books Ltd., London: United Kingdom. pp. 574-575.

Wenz, J. R., G. M. Barrington, F. B. Garry, R. P. Ellis and R. J. Magnuson. 2006. Escherichia coli isolates' serotypes, genotypes and virulence genes and clinical coliform mastitis severity. J. Dairy Sci. 89:3408-3412.

Anirban Guha *, Ruby Guha (1) and Sandeep Gera (2)

Dept. of Animal Resource Development and Animal Husbandry, Govt. of West Bengal, India

* Corresponding Author: Anirban Guha. Tel: +919836341948, E-mail: archies76@gmail.com

(1) Dept. of Biochemistry, College of Basic Sciences, Chaudhary Charan Singh, Haryana Agricultural University, Hisar-125004, Haryana, India.

(2) Veterinary Physiology and Biochemistry, Lala Lajpat Rai University of Veterinary and Animal Sciences (erstwhile. Chaudhary Charan Singh Haryana Agricultural University), Hisar--125004, Haryana, India.

Submitted May 8, 2012; Accepted Jul. 17, 2012; Revised Aug. 17, 2012
Table 1. Prevalence of bacterial agents in subclinical mastitis milk
of riverine buffalo (Bubalus bubalis)

Genus                                 Animal
                                Number   Percentage
Staphylococcus spp.              146       42.07
Streptococcus spp.               118       34.00
Escherichia coli                  69       19.88
Others (Corynaebacterium spp.     14        4.05
  and Bacillus spp.)
  +Mixed infection
Total                            347        100

Genus                                Quarters
                                Number   Percentage
Staphylococcus spp.              210       42.33
Streptococcus spp.               172       34.67
Escherichia coli                  95       19.15
Others (Corynaebacterium spp.     19        3.85
  and Bacillus spp.)
  +Mixed infection
Total                            496        100

Table 2. Effect of different bacterial agents on acute phase proteins
and NOx in healthy and subclinical mastitis milk of riverine buffalo
(Bubalus bubalis)

Parameters                           Mean [+ or -] SE of
                                        healthy milk
                                         (n = 496)

Somatic cell count                  0.93 * [+ or -] 0.007
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti               3,340.43 * [+ or -] 138.54
  trypsin (U/L)
[[alpha].sub.1]-Acid                0.176 (a) [+ or -] 0.026
  glycoprotein (mg/ml)
Fibrinogen (g/dl)                        Not detectable
NOx (Nitrate+nitrite) ([micro]M)     11.09 * [+ or -] 0.19

Parameters                           Mean [+ or -] SE of subclinical
                                        mastitis milk (n = 496)
                                         Staphylococcus spp.
                                               (n = 210)

Somatic cell count                      2.08 ** [+ or -] 0.020
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                   6,982.13 ** [+ or -] 122.72
  trypsin (U/L)
[[alpha].sub.1]-Acid                     3.08 (b) [+ or -] 0.02
  glycoprotein (mg/ml)
Fibrinogen (g/dl)                            Not detectable
NOx (Nitrate+nitrite) ([micro]M)         18.07 ** [+ or -] 0.14

Parameters                           Mean [+ or -] SE of subclinical
                                        mastitis milk (n = 496)
                                          Sterptococcus spp.
                                               (n= 172)

Somatic cell count                      2.05 ** [+ or -] 0.013
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                   6,074.22 ** [+ or -] 102.87
  trypsin (U/L)
[[alpha].sub.1]-Acid                     2.99 (b) [+ or -] 0.01
  glycoprotein (mg/ml)
Fibrinogen (g/dl)                           Not detectable
NOx (Nitrate+nitrite) ([micro]M)         17.09 ** [+ or -] 0.11

Parameters                           Mean [+ or -] SE of subclinical
                                        mastitis milk (n = 496)
                                           Escherichia coli
                                               (n = 95)

Somatic cell count                      2.01 ** [+ or -] 0.015
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                    5,798.76 ** [+ or -] 94.78
  trypsin (U/L)
[[alpha].sub.1]-Acid                      2.88 (b) [+ or -] 0.08
  glycoprotein (mg/ml)
Fibrinogen (g/dl)                            Not detectable
NOx (Nitrate+nitrite) ([micro]M)         16.28 ** [+ or -] 0.14

Parameters                           Mean [+ or -] SE of subclinical
                                        mastitis milk (n = 496)
                                             Others+mixed
                                              infection
                                               (n = 19)

Somatic cell count                      2.03 ** [+ or -] 0.012
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                   5,998.73 ** [+ or -] 99.34
  trypsin (U/L)
[[alpha].sub.1]-Acid                      2.89 (b) [+ or -] 0.03
  glycoprotein (mg/ml)
Fibrinogen (g/dl)                             Not detectable
NOx (Nitrate+nitrite) ([micro]M)          16.89 ** [+ or -] 0.24

Mean having different superscripts * and ** horizontally differ
significantly (p<0.01).

Mean having different superscripts (a) and (b) horizontally differ
significantly (p<0.05).

Table 3. Mean [+ or -] SE of SCC, acute phase proteins and NOx in milk
from healthy and subclinical mastitis (irrespective of the causative
agent) in riverine buffalo to decide the threshold limit (n = 496)

Parameter                               Healthy milk

Somatic cell count                  0.93 * [+ or -] 0.007
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                3,340.43 * [+ or -] 138.54
  trypsin (U/L)
[[alpha].sub.1]-Acid                0.176 (a) [+ or -] 0.026
   glycoprotein
  (mg [ml.sup.-1)
Fibrinogen (g/dl)                       Not detectable
Nox (nitrate+nitrite) ([micro]M)    11.09 * [+ or -] 0.19

Parameter                         Subclinical mastitis milk

Somatic cell count                  2.05 ** [+ or -] 0.056
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti               6,397.71 ** [+ or -] 150.99
  trypsin (U/L)
[[alpha].sub.1]-Acid                2.92 (b) [+ or -] 0.032
   glycoprotein
  (mg [ml.sup.-1)
Fibrinogen (g/dl)                       Not detectable
Nox (nitrate+nitrite) ([micro]M)     17.33 [+ or -] 0.29

Parameter                         Cut-off points

Somatic cell count                      -
  (x [10.sup.5] cells/ml)
[[alpha].sub.1]-Anti                 6,396.22
  trypsin (U/L)
[[alpha].sub.1]-Acid                  2.92
  glycoprotein
  (mg [ml.sup.-1)
Fibrinogen (g/dl)                       -
Nox (nitrate+nitrite) ([micro]M)      17.34

Mean having different superscripts * and ** horizontally differ
significantly (p<0.01).

Mean having different superscripts (a) and (b) horizontally differ
significantly (p<0.05).

Table 4. Evaluation of [[alpha].sub.1]-anti trypsin,
[[alpha].sub.1]-acid glycoprotein and NOx as an indicator for
diagnosis of subclinical mastitis in riverine buffalo

Name of                          Total        Test
the parameter                   samples     positive
                                examined    buffaloes
                                   (N)        (a+b)

[[alpha].sub.1]-Anti trypsin      2,048        494
[[alpha].sub.1]-Acid              2,048        508
  glycoprotein
NOx                               2,048        514
Positive bacterial                2,048        496
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml
                                Test reaction as compared to
                                cultural examination
Name of                           True        False
the parameter                   positive    positive
                                   (a)         (b)

[[alpha].sub.1]-Anti trypsin       410         84
[[alpha].sub.1]-Acid               376         132
  glycoprotein
NOx                                358         156
Positive bacterial                 496          -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                Test reaction as compared to
                                cultural examination
Name of                           True        False
the parameter                   negative    negative
                                   (c)         (d)

[[alpha].sub.1]-Anti trypsin      1,468        86
[[alpha].sub.1]-Acid              1,420        120
  glycoprotein
NOx                               1,396        138
Positive bacterial                1,552         -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

Name of                          Percent       Percent      Percent
the parameter                   sensitivity   specificity   accuracy
                                 a/(a+d)       c/(b+c)      (a+c)/N
                                   x100          x100         x100

[[alpha].sub.1]-Anti trypsin       82.66         94.59       91.70
[[alpha].sub.1]-Acid               75.81         91.49       87.70
  glycoprotein
NOx                                74.18         89.95       85.64
Positive bacterial                  100           100         100
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

Name of                          Positive      Negative
the parameter                   predictive    predictive
                                value (%)     value (%)
                                 a/(a+b)       c/(c+d)
                                   x100          x100

[[alpha].sub.1]-Anti trypsin       83.00         94.47
[[alpha].sub.1]-Acid               74.02         92.21
  glycoprotein
NOx                                69.64         91.00
Positive bacterial                   -             -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

Name of                           Likelihood          Likelihood
the parameter                   ratio (positive)        ration
                                 sensitivity/         (negative)
                                     (100-         (100-sensitivity)/
                                  specificity)        specificity

[[alpha].sub.1]-Anti trypsin         15.28                0.18
[[alpha].sub.1]-Acid                  8.90                0.26
  glycoprotein
NOx                                   7.44                0.29
Positive bacterial                     -                   -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

Table 5. Evaluation of [[alpha]1.sub.1]-anti trypsin,
[[alpha]1.sub.1]-acid glycoprotein and NOx as an indicator for
diagnosis of subclinical mastitis in riverine buffalo caused by gram
positive bacteria only

                                Total      Test
Name of                         samples    positive
the parameter                   examined   buffaloes
                                (N)        (a+b)

[[alpha]1.sub.1]-Anti trypsin   1,964      429
[[alpha]1.sub.1]-Acid           1,964      424
  glycoprotein
NOx                             1,964      454
Positive bacterial              1,964      412
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                Test reaction as compared to
                                cultural examination
                                True       False
Name of                         positive   positive
the parameter                   (a)        (b)

[[alpha]1.sub.1]-Anti trypsin   366        63
[[alpha]1.sub.1]-Acid           344        80
  glycoprotein
NOx                             329        125
Positive bacterial              412        -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                Test reaction as compared to
                                cultural examination
                                True       False
Name of                         negative   negative
the parameter                   (c)        (d)

[[alpha]1.sub.1]-Anti trypsin   1,489      46
[[alpha]1.sub.1]-Acid           1,472      68
  glycoprotein
NOx                             1,427      60
Positive bacterial              1,552      -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                   Percent       Percent     Percent
Name of                          sensitivity   specificity   accuracy
the parameter                     a/(a+d)       c/(b+c)      (a+c)/N
                                    x100          x100         x100

[[alpha]1.sub.1]-Anti trypsin       88.83         95.94       94.45
[[alpha]1.sub.1]-Acid               83.50         94.85       92.46
  glycoprotein
NOx                                 79.85         91.95       89.41
Positive bacterial                   100           100         100
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                  Positive      Negative
Name of                          predictive    predictive
the parameter                    value (%)     value (%)
                                   a/(a+b)       c/(c+d)

[[alpha]1.sub.1]-Anti trypsin       85.31         97.00
[[alpha]1.sub.1]-Acid               81.13         95.58
  glycoprotein
NOx                                 72.46         95.97
Positive bacterial                    -             -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

                                 Likelihood ratio        Likelihood
Name of                              (positive)       ration (negative)
the parameter                      sensitivity/       (100-sensitivity)
                                 (100- specificity)     /specificity

[[alpha]1.sub.1]-Anti trypsin          21.88                0.12
[[alpha]1.sub.1]-Acid                  16.21                0.17
  glycoprotein
NOx                                     9.92                0.22
Positive bacterial                       -                    -
  culture and
  SCC [greater than or equal
  to] 2 x [10.sup.5]
  cells/ml

Table 6. Correlation coefficient of milk biochemical components in
healthy and SCM milk of riverine buffalo (n = 496)

                                      [Log.sub.10] SCC
                                    Healthy    Infected
                                     milk       milk

[Log.sub.10] SCC                      -          -
[[alpha].sub.1]-Anti trypsin          -          -
[[alpha].sub.1]-Acid glycoprotein     -          -
NOx (nitrate+nitrite)                 -          -

                                    [[alpha].sub.1]-Anti trypsin
                                    Healthy    Infected
                                     milk       milk

[Log.sub.10] SCC                    0.019      0.795 **
[[alpha].sub.1]-Anti trypsin          -          -
[[alpha].sub.1]-Acid glycoprotein     -          -
NOx (nitrate+nitrite)                 -          -

                                    [[alpha].sub.1]-
                                    Acid glycoprotein
                                    Healthy    Infected
                                     milk       milk

[Log.sub.10] SCC                    0.098      0.559 *
[[alpha].sub.1]-Anti trypsin        0.064      0.028
[[alpha].sub.1]-Acid glycoprotein     -          -
NOx (nitrate+nitrite)                 -          -

                                    NOx (nitrate+nitrite)

                                    Healthy    Infected
                                     milk       milk

[Log.sub.10] SCC                     0.546 *   0.845 **
[[alpha].sub.1]-Anti trypsin         0.022     0.803 **
[[alpha].sub.1]-Acid glycoprotein    0.056     0.644 *
NOx (nitrate+nitrite)                  -          -

* Indicate significant at p<0.05. ** Indicate significant at p<0.01.
COPYRIGHT 2013 Asian - Australasian Association of Animal Production Societies
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2013 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Guha, Anirban; Guha, Ruby; Gera, Sandeep
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Jun 1, 2013
Words:5539
Previous Article:Brief biography of awardees for 2012 AJAS/CAPI Outstanding Research Award.
Next Article:Regulation of interferon-stimulated gene (ISG)12, ISG15, and MX1 and MX2 by conceptus interferons (IFNTs) in bovine uterine epithelial cells.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters