Abstract: The present research was done for preliminary identification and serological detection of Brucella melitensis and Brucella abortus in Bengal Goats. For this purpose, samples were collected from stomach content of aborted fetus, uterine fluid, vaginal swab, blood serum and milk of 20 Bengal Goats from Bangladesh Livestock Research Institute (BLRI) goat farm, Dhaka and Lalmonirat goat farm that caused abortion. Samples were cultured and subcultured in Nutrient agar media and Blood agar media and incubated in aerobic and anaerobic incubator to obtain pure cultures of individual bacterium. As Brucella melitensis and Brucella abortus are strictly aerobic, colonies grown in aerobic incubator were subcultured to get the specific bacterium. Twelve pure isolates were obtained from aerobic growth and catalase tests were performed with those isolates and eight of them showed positive results. Then serum was collected from respective goats and serological test (serum agglutination test) was performed by using Brucella antigen (kit). Test results were negative showing the absence of Brucella melitensis and Brucella abortus into the serum samples. For further confirmation, Milk Ring test, Rose Bengal test, Complement Fixation test, ELISA test were performed with the samples of suspected goats and Brucella antigen (kit). All results from the tests imply the negativity of the presence of Brucella melitensis and Brucella abortus.
INTRODUCTION
Bengal Goat is an important economical and profitable ruminant in Bangladesh. It has specific characteristics like good quality skin, high fecundity and larger litter size. However, high kid mortality largely reduces the profitability of goat rearing. Not only kid mortality but also abortion is one of the vital reasons for non productivity. With the other physical and environmental stresses, infection with Brucella spp. is responsible for abortion of goats in many areas of the world. In Bangladesh, the incidence of this disease is not found yet but several studies and research works are going on extensively in various animal research laboratory.
The disease Brucellosis, abortion of goat, is caused by a bacterium named Brucella, a strictly aerobic, gram-negative coccobacillus. They belong to the alpha-proteobacteria group that live in close association with a eukaryotic host. This organism is sometimes carried by animals and only causes incidental infections in humans. The cattle and dairy industries seem to be the primary sources of infection. Milking breeds appear more susceptible than those kept for meat production (Corbel and Brinley-Morgan, 1984).
An epidemiological investigation of 550 strains of Brucella from all over the world has shown that the Brucella predominately affects one species: B. abortus for cattle, B. melitensis for sheep and goat, B. suis for pigs, B. canis for dogs. However, the specificity is not absolute (Meyer, 1964). So, the present study was carried out for the surveillance of both B. melitensis and B. abortus in Bengal goat.
In goats, about two thirds of acute infections of Brucella acquired naturally during pregnancy lead to infection of the udder and excretion of the bacteria in the milk during the subsequent lactation. Goat that aborts often excretes the bacteria in the milk, but generally for not more than 2 months (Alton, 1990). Exceptionally, excretion may continue for 140 days and even 180 days (Itabashi et al., 1938).
In brucellosis of goats due to Brucella, the incidence of abortion may reach 40-60%. Abortion occurs the last third of gestation and weak moribund lambs may be born at term or prematurely. The disease is contracted by pregnant sheep or goats by ingestion of food and water contaminated with organisms from aborting fetuses and their membranes of genital discharges. After intervenous inoculation of Brucella, abortion may occur from 11-21 days. (Hornitzky and Searson, 1986).
Preliminary identification of Brucella requires demonstrating colonies of short rods that are non haemolytic, catalase positive and oxides positive. Agglutination in unabsorbed antibrucella serum helps the identification of Brucella strain. Antibody (Ab) detection is commonly used for diagnosing brucellosis. Identification of Brucella can be carried out by a combination of the agar plate test, organism morphology and gram staining, colonial morphology, growth characteristics, urease, oxidase, catalase tests and the serum agglutination tests. The serological methods represent standardized and validated methods with suitable performance characteristics (MacMillan and Cockrem, 1985).
Multiplication of Brucella is slow at the optimum temperature of 37°C and enriched medium is needed to support adequate growth. Brucella colonies become visible on suitable solid media (Blood agar, Nutrient agar) in 2-3 days (Allardet-Servent et al., 1998; Ficht et al., 1990).
The colonies of smooth strains are small, round and convex. They are usually arranged singly and less frequently in pairs, or small groups. When plates are viewed in the daylight through a transparent medium they are translucent and a pale honey color. When viewed from above, colonies appear convex and pearly white. Later, colonies become larger and slightly darker (Schurig et al., 1991).
Following infection with Brucella, pregnant adult females develop a placentitis usually resulting in abortion between the 3rd and 4th month of pregnancy. Even in the absence of abortion, profuse excretion of the organism occurs in the placenta, fetal fluids, vaginal discharges and genital lymph nodes (Fensterbank, 1987).
So, present study will be helpful toward goat rearing and enhance dynamism of goat farming which not only alleviate the poverty among the marginal and land-less farmers but also boost up the national economy by increasing the goat production. Considering all above-mentioned points, the present work was designed with the following objectives:
| • | For confirming the presence of infection in herds and identifying the both species of Brucella. |
| • | To study the sero-prevalence of brucellosis in goat using serological tests. |
MATERIALS AND METHODS
The research was carried out in Bacteriology Laboratory under Animal Health Research Division (AHRD), Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka-1341, Bangladesh.
Culture Media
For isolation and identification procedures of bacteria, different kinds
of culture media were used.
Brucella Antigen (Stained Brucella Suspensions)
Stained Brucella Suspensions were used to detect, identify and quantitate
specific antibodies to Brucella in sera. They were standardized, smooth
suspensions of killed bacteria which have been stained to facilitate reading
of agglutination tests. Stained Brucella suspensions were suitable for
use in standard widal tests. The pH of the antigen was between 3.3 and 3.7 and
dark blue color.
Normal Saline (0.85%)
0.85% normal saline was used to dilute the serum sample to perform the serological
test.
Clinical History
Few goats were taken under experiments which had abortion one or several
times in their life times. Clinical signs of abortion like fever, depression,
mastitis, arthritis, sinusitis, orchitis, or nervous signs may accompany acute
infection in goats. Goats from which samples were collected showed these signs
of abortion at different time, age, kidding period. All these data related to
signs of abortion including abortion date were collected from the record books
of the farm. Most recent samples (from 2004-2005) from aborted goats were collected
to conduct the experiment. The collected data was organized according to the
order of abortion at different kidding period (Table 1).
Sample Collection Procedure
For the diagnosis of animal brucellosis by cultural examination, the choice
of samples usually depends on the clinical signs observed. The most valuable
samples include aborted fetuses (stomach contents, spleen and lung), fetal membranes,
vaginal secretions (swabs), milk, semen and uterine fluids.
| Table 1: | Collection of data according to place, sex, age, size and date of collection |
From animal carcasses, the preferred tissues, the late pregnant or early post-parturient uterus, stomach content, fetal fluid was collected. For isolation and identification, these samples were collected from my chosen animal (goat) indicated by tag no directly by using sterile plastic gloves.
Direct Microscopy Examination of the Collected Samples
Each of the samples was examined on conventional direct smear method and
to detect the fungus which was identified by their morphological features.
Culture of Samples
Samples from 15 randomly selected goats that caused abortion were examined
for isolation and identification of Brucella. Samples were streaked on
different agar medium, incubated at 37°C either in aerobic incubator (in
presence of O2) or in anaerobic incubator (containing 5% CO2).
Isolation and Final Selection of Colonies
From each Petri dish, plate culture was subcultured in Nutrient agar medium
and Blood agar medium and kept in aerobic and anaerobic condition respectively.
Subcultures were repeated on both agar plate by streak plate method until the
colonies were considered the pure separately by visually and also by microscopically.
The colonies were selected according to their size, shape, color, height, smoothness, margin and the like properties. Regarding to the above properties, colonies that possessing the highest positive signs were selected for further tests.
Preparation for Microscopic Examination
By observing the slides, comparison of the organism with the morphology
of Brucella was done by fixed smear method.
Staining
For the observation of the presence of the organisms (bacteria) and also
to study their cellular morphology, selected isolates were stained. They are
not truly acid fast but are resistant to decolorization by weak acid. Hence,
Gram’s staining method was adopted.
Catalase Test
Brucella can degrade hydrogen peroxide (H2O2)
by producing the enzyme catalase. This test was based on this characteristic.
Each experimental isolates were inoculated into appropriately labeled agar slant
tubes by means of a streak inoculation and incubated at 37°C for 24 h. After
incubation 3-4 drops of the 3% H2O2 was flown over the
entire surface of each slant. Then each slant was examined for the presence
or absence of bubbling of oxygen or foaming. Formation of bubbling due to the
accumulation of the wall indicates positive result.
Serological Test
Serological test was performed for conform identification for Brucella.
Serum Agglutination Test (SAT) and Milk Ring Test (MRT) were performed on
serum and milk samples respectively for screening and monitoring the selected
goats for brucellosis. These tests were based on the bindings of Brucella
antigen (Kit) to the Antibody (Ab) produced in serum of the infected goats.
In serological test, commercially prepared Brucella Ag (kit) was added
to serum of the test goats. Agglutination occurs if Ab is present against the
Ag (Alton, 1967).
Serum Agglutination Test (SAT)
SAT has been used with success for many years in surveillance and control
programs for detection of brucellosis. The antigen represents a bacterial suspension
in phenol saline [NaCl 0.85% (w/v)]. By preparing 0.85% normal saline, collected
serum was diluted with this (Table 2).
| Table 2: | Serial dilution of serum samples for serological test |
Serial dilutions of the serum samples were done using 1 mL micropipette. One drop of appropriate well shaken suspension of Ag was added into each tube containing the diluted samples by using the dropper provided with the Ag suspension bottle. After moderate shaking the tubes were incubated at 37°C in water bath for 24 h.
Milk Ring Test (MRT)
MRT was performed with Brucella antigen (Ag) kit. Thirty to Fifty
microliter of Ag was added to 1-2 mL volume of milk. Incubate at 37°C for
1 h with positive and negative working standard. It was kept attention that
the height of the milk column in the test tube must be at least 25 mm. To increase
the sensitivity of the test, the tubes were incubated overnight at 4°C.
Rose Bengal Test
The Rose Bengal Test (RBT) was performed on slides. The antigen was a Rose
Bengal stained suspension of a smooth attenuated strain of B. melitensis
and another strain of B. abortus.
Complement Fixation Test (CFT)
CFT was performed by the cold-fixation method. Sera were heat treated at
56°C for 30 min before testing and guinea pigs were the source of complement.
Evidence of CF at a sample dilution of ≥ 1:10 was considered positive.
Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for Antibodies in Milk
IELISA procedure was performed as previously described by Tabatabai and
Deyoe (1984). 96-well plates (Nalge Nunc International, Rochester, N.Y.) were
used. A plate reader (Bio-Tek Instruments, Winooski, Vt.) was used to read the
absorbance at 492 nm. All samples were tested in duplicate, with average absorbance
values being reported. Antigens, milk samples and the anti-species conjugate
were titrated for optimal assay performance.
RESULTS AND DISCUSSION
Preliminary Isolation and Identification of Bacteria on Agar Plate
Samples from 20 suspected goats were cultured in blood agar and nutrient
agar media in both aerobic and anaerobic incubator; 12 of them showed growths
in aerobic incubator (Table 3). As Brucella is strictly
aerobic, the growths from aerobic incubator were subjected to further tests.
Staining Characteristics
Different microscopic slides were prepared by smearing method from the isolates.
Table 4 represents staining characteristics of the bacterial
isolates that grew in aerobic incubator.
Catalase Test
Catalase test was carried out with the selected 12 samples. Table
5 represents the catalase test’s results. Eight of them showed positive
results and were further subjected to serological test.
| Table 3: | Colony characteristics of the bacteria on solid media |
| Circular (Unbroken peripheral edge), Raised (Slightly elevated) | |
| Table 4: | Microscopic studies and staining propeties of the bacterial isolates |
| (-) ve indicates negative sign, (+) ve indicates positive sign | |
| Table 5: | Result of catalase test |
Serum Agglutination Test
Serum agglutination test was performed to detect the presence of Brucella
(Table 6). None of them showed positive results.
Milk Ring Test
For further confirmation, milk ring test was performed (Table
7). There were found no Brucella in any of the samples as no positive
result was found.
Rose Bengal Tests
The RBT test results were interpreted as negative for both strains.
Complement Fixation Tests
Complement Fixation tests test was carried out with the selected 8 samples.
None of them showed positive results (Table 8).
iELISA for Antibodies in Milk
iELISA test was carried out with the selected 8 samples. None of them showed
positive results (Table 9).
| Table 6: | Results of Serum Agglutination Test (SAT) |
| Table 7: | Results of milk ring test |
| Table 8: | Complement fixation tests |
| Table 9: | iELISA for antibodies in milk |
From the above results it can be concluded that the occurrence of abortion was not associated with Brucella melitensis and Brucella abortus but other enteropathogens (single and concurrent infection) and predisposing factors may be involved. Therefore, it may be thought that more than one predisposing factors such as environmental and management factors (housing, feeding, applying different medicine), imbalance nutrition, age and immune status of the animal might involve in the occurrence of abortion in goats.
CONCLUSIONS
Because brucellosis is a disease of major economic and zoonotic importance, strategies for its control in small ruminants is essential in endemic areas. The initial aim of the strategy selected will be the reduction of infection in the animal population to such a level that the impact of the disease on human health as well as on animal health and production will be minimized. There is an occupational risk to health veterinarians and livestock related professionals who handle infected animals and aborted materials. As Brucellosis is one of the most easily acquired laboratory infections and strict safety precautions are recommended to maintain while handling Brucella suspected materials (WHO Laboratory Biosafety Manual, 1993).
Further research is needed on the identification, isolation, characterization and cloning of both inner and outer membrane proteins which could be used as diagnostic antigens for detection and elimination of brucellosis.