COMPARISON OF SOME RAPID TECHNIQUES USED FOR DIAGNOSIS OF BRUCELLOSIS OF ABORTED COWS COMPARED WITH CONVENTIAL METHOD

COMPARISON OF SOME RAPID TECHNIQUES USED FOR DIAGNOSIS OF BRUCELLOSIS OF ABORTED COWS COMPARED WITH CONVENTIAL METHOD

ABOU GAZIA, K.A.; EMAN, SH. RAMADAN and MONA M. SOBHY

Dept. of Reproductive diseases, ARRI, ARC, Giza, Egypt.

Email: khalidabougazia@yahoo.com

INTRODUCTION

Brucella has a significant economic impact on our livestock business. It has severe consequence on production of cattle that influences two of the greatest infertility and sterility problems, (Parker, 2003).

Brucellosis is a highly infectious bacterial disease that mainly affects cattle, sheep, pigs and goats. The organism causing brucellosis primarily infects the reproductive organs and thrives in the uterus of pregnant animals, often leading to late abortion (OIE 2001). The organism can remain undetected for prolonged periods as there are no clinical signs until abortion occurs (AHVLA, 2013).

Bovine brucellosis is the best known and most controversial infection of the bovine reproductive system. It is one of the core profiles of economic consideration in livestock production enterprises since loss of calf due to abortion and its squeal lead to infertility (Verma et al., 2000).

Brucellosis infection of cattle causes abortion or premature calving of recently infected animals, the foetus, placenta and uterine fluid contain large quantities of Brucella organisms which can infect other animal coming into contact with an infected animal around the time of calving (Nielsen et al., 2005).

The gold standard technique for diagnosis of brucellosis is isolation and identification of the causative bacterium Brucella species. Isolation of Brucella organisms requires a high secured laboratory facilities (biological containment level 3), an extended time for results, highly skilled personnel and hazardous procedure. Brucellosis is generally diagnosed by detection of antibodies in serum or other body fluids. Subsequently, various modification of agglutination test and numerous other tests have been developed to increase test accuracy (Nielsen and Yu, 2010).

Brucellosis was firstly diagnosed by using a simple tube agglutination test by Wright and Smith (1897). The other tests have been developed to increase test sensitivity. However no test is 100% accurate. So, generally serological diagnosis consists of testing sera by several tests, usually as screening test of high sensitivity followed by a confirmatory test of high specificity (Nielsen et al., 2005).

The present study was contemplated to reveal some rapid diagnostic techniques used for diagnosis of brucellosis of aborted cows as DFAT and DOT-ELISA compared with convential method

MATERIALS and METHODS

In the present study a total of 158 specimens were collected from aborted cows of different farms in Egypt (102 uterine discharges and 56 lochia). Also 158 blood samples of the same aborted cows were collected for serological tests.

The conventional bacteriological methods (Alton      et al., 1988) were applied for isolation and identification of Brucella organisms from the all specimens.

Primary inoculation was done on sheep blood agar plates in duplicate by directly streaking the swabs to be cultivated. The plates were incubated at 37°C in the presence of 5% CO₂ in Carbon dioxide incubator for 72-120 hrs. The isolates were initially recognized on the basis of their cultural and morphological features. They were also biochemically characterized as described by (Carter and Cole, 1995).

DOT-ELISA was applied on the colonies plates for detection of Brucella microorganisms in the aborted materials as described by (Nielsen et al., 2004).

Direct Florescence Antibody Technique (DFAT) was applied on the specimens of uterine discharges and lochia. Samples coated on the slides as antigens and then Brucellaspecific antibodies labeled with a fluorescein conjugate were added (Nicoletti and Tanya, 1993).

The serological tests applied on these studies were Rose Bengal tset (RBT), Standerd Tube agglutination test (SAT), Rivanol test (RT) and Enzyme linked Immunoasorbant Assay (ELISA) according to (Nielsen, 2002).

RESULTS

From 102 uterine discharge samples only 8 brucella isolates could be identified, also 4 brucella isolates could be identified from 56 lochia samples, but when we used DFAT on uterine discharge and lochia gave 25 positive brucella cases.

Serological test applied on serum obtained from 158 blood samples showed better detection of brucella antibody by ELISA  66 sample than Rose Bengal 58 positive sample.

Table 1: Brucella isolates encountered from aborted cows by culture.

  * based on cultural, morphological and biochemical features.

Table 2: The incidence of Brucella in samples of aborted cows by DFAT and Dot ELISA.

Table 3: The prevalence of Brucella in serum samples of aborted cows by serological test.

Fig.1: Positive DFAT applied on lochia of aborted cow.

DISCUSSION

Brucellosis remains a major worldwide zoontic disease (Cutler and Whatmore, 2003). It is a bacterial disaease of global importance that may affect different mammals. The disease primarily affects the reproductive system with concomitant loss in productivity of animals (Young, 1995).

Brucellosis is considered as an emerging problem in developing countries where there is an increasing incidence of B. melitensis in cattle (Corbel, 1997). The organisms survive within the environment for prolonged periods (Moreno and Gorvel, 2004). Interaction with placental trophoblasts suggests that the ability to acquire iron is vital as the Brucella enter their acute replicative stage within the placental disruption resulting in fetal loss or birth of weak and/or infected off spring (Eschenbrenner et al., 2002 and Cutler et al., 2005).

The accurate diagnosis of brucellosis in any species goes straight forward but may be very difficult in some cases (Nielsen and Yu, 2010). Brucelladiagnostic tests were developed based on agglutination methods. These assays have been played with problems of both sensitivity and specificity (Alton et al., 1988 and Nielsen, 2002).

Diagnosis of brucella infection can be made by isolation and identification of the organisms by convential methods (Bercovich, 2000). In the present study, the rate of isolation of B.melitensis from aborted cows was 12 isolates; 8 from uterine discharges and 4 from lochia by cultural bacteriological method and Dot- ELISA (Table, 1 & 2). These findings agree with (Zowghi and Ebadi 1988), that all the isolates of brucella encountered in this study were identified by biochemical tests as described by (Carter and Cole, 1995).

The obtained results revealed Brucella positive by direct fluorescent antibody test applied through specific binding of antibody to the provide antigen conjugated with fluorescein conjugate. The results were 25positive cases of DFAT; 16 from uterine discharges and 9 from lochias. These findings coincide with (Samartino et al., 1999) and (Bahn and Nockler 2005). The higher incidence rate of Brucellaorganisms was done by DFAT which is a simple, rapid diagnostic test, relatively inexpensive and accurate (Nielsen et al., 2004).

Serodiagnonostic methods for brucellosis have primarily been based on serology with lipopolysaccharides (LPS) from smooth strains producing greatest immunological response (Kittelberger et al., 1997). In this study, different serological tests were applied on the serum samples; RBT gave 64, SAT gave 58 RT gave 55 and ELISA test gave 66 positive results (Table, 3). These findings agreed with (Verma et al., 2000 and Nielsen et al., 2005). The higher positivity of ELISA test generally has very high sensitivity and excellent screening assays for diagnosis of brucella especially in individual animal test of serum (Wright et al., 1997 and Gall et al., 2001 and McGiven et al., 2003).

CONCLUSION

Finally, we conclude that in order to eradicate and control brucellosis, we must apply good surveillance reliable diagnostic test. The practical serological diagnosis must be based on screening test of high sensitivity followed by a confirmatory test as ELISA test of high specificity. A combination of serological test with DFAT is usually needed for diagnosis of brucella organisms in aborted cases.

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