Basic Research Journal of Animal Science Vol. 1(1) pp. 01-06 October 2013 Available online http//www.basicresearchjournals.org Copyright ©2013 Basic Research Journal

Full Length Research Paper

Detection of bovine tuberculosis in milk and serum of tuberculin reactors dairy farm animals in Assiut City,

Egypt

1Saad El-din Nasr, *1Nagah M. Saad, 2 Nasr E. A, 3Nahed M. Wahba and 3Walaa M. Elsherif

1Food Hygiene Department, Assuit University Egypt.

2Bacterial Diagnostic Products Department (Tuberculosis), Veterinary Serum and Vaccine Research Institute, Abbasia,

Cairo. 3Food Hygiene Department, Animal Health Research Institute, Assuit, Egypt.

*Corresponding author email: nahedmw@yahoo.com

Accepted 25 October, 2013

Milk and serum samples were collected from some tuberculin reactors dairy farm animals in different localities in Assiut City, Egypt. More than one method for detection of tuberculosis infection was evaluated (Tuberclin test, microscobical (by Ziehl-Neelsen stains), bacteriological examination and ELIZA test). Acid Fast Bacilli were detected microscopically in 7 (14%) of milk samples from tuberculin positive reactors and 3(6%) milk samples for tuberclin negative reactors. Bacteriological examination of processed milk samples revealed that the incidence of Mycobacteria was 4% and 2% milk samples using Lowenstein Jensen medium pyruvated and LJ medium glycerinated from positive reactors, respectively and 2% were positive from tuberculin negative reactors in both media. Concerning the site of infection, number of infected animals were generalized infection (2 cases), localized (10) and non visible lesion (38). ELISA bovine PPD results were 2 (4%), 7 (14%) and12 (24%) in serum samples of generalized infection, Localized and non-visible lesion, respectively. So, ELISA technique can be used as a complementary to the skin tuberculin test to determine the disease status of animal or as rapid screening test. Keywords: bovine tuberculosis, milk, serum and ELISA bovine PPD.

INTRODUCTION M. bovis is a member of the tuberculosis complex of organisms and represents a serious zoonotic risk to public health. Because of these ongoing economic and public health concerns, there is an urgent need for improved methods to combat bovine tuberculosis. Early diagnosis of Mycobacterial infections plays a vital role in control of tuberculosis. Diagnosis of bovine tuberculosis is under-taken by the widely spread tuberculin tests. Tuberculin tests have been used for diagnosis of tuberculosis in cattle for more than 100 years. The tuberculin test using bovine PPD tuberculin is easy to perform on a large scale on

livestock, but it has the inconvenience of having a broad range of specificity and sensitivity due to presence of common antigens in all mycobacteria and most of these antigens are skin reactive (Daniel and Janicki. 1978).

Acid-fast bacilli (AFB) microscopy and conventional Lowenstein Jensen (L-J) culture remains the cornerstone of the diagnosis of tuberculosis (Negi et al., 2005).

Despite the fact that microbiological culture is highly specific, a positive result takes a long time to be obtained (6-8 weeks). Species identification procedures extends the reporting time even further.

As the accurate diagnosis plays an important role in the control of tuberculosis, the ELISA test has the highest sensitivity and specificity than the other serological tests for diagnosis of tuberculosis and the use of ELISA as complementary to tuberculin test will greatly increase the sensitivity and specificity of the test (Orlando et al., 2010). Material and methods Tuberculin skin test (Ovdiennkop et al., 1987) A narrow zone (at the middle of neck of the tested animals) was marked by clipping the hair. The skin thickness was premeasured using caliper. The 0.1 ml mammalian PPD tuberculin was injected interdermal (I/D). 72 hours post injection, skin thickness was measured and the difference between the 1

st and 2

nd reading of skin thickness was

recorded according to General Organization of Veterinary Services (GOVS, 1992). Collection and preparation of samples (A.P.H.A., 1992) a) Milk samples (Neill et al., 1988) Milk samples collected from 50 positive tuberculin reactors and 50 negative tuberculin reactors dairy farm animals in Assiut City, Egypt. 100 ml of well mixed milk were mixed with 50 ml of sterile distilled water are centrifuged for 30 min at 3000 rpm. The resulting sediment is mixed with an equal volume of 6% HCL and incubated at 37°C for 30 min. The mixture is centrifuged for 30 min. at 3000 rpm, the sediment is neutralized with 4% sterile NaOH sol., with Phenol red as an indicator (the change in color from purple to pink indicates correct neutralization) then, re-centrifuged for further examinations. Ziehl-Neelsen stains (Cruickshank et al., 1975). The sediment of previously prepared samples was spread on clean slides. The slides were then flooded using carbolfuchsin and left on a slide holder with heating under it for 5 to 7 min. The slides washed thoroughly and decolorized using acid alcohol mixture for 1 min followed by washing with water, then flooded with Loffler's methylene blue for 3 min. Each slide was examined to detect the shape, arrangement and acid fastness. b) Serum samples (Thoen et al., 1983) From the positive reactors dairy animals, 10 ml of blood

Wahba et al. 02 were obtained aseptically from the jugular vein. The blood samples were kept at 4 °C overnight and centrifuged at 3000 rpm for 15 min. Serum was aspirated and kept at - 20 °C till used in serological tests. Isolation of tubercle bacilli (Cultural method) (Kamel et al., 1975) The obtained sediment was thoroughly mixed and distributed into slants of glycerinated and pyruvated Modified L-J media in McCartney bottles (Ernst, 1990). The bottles were incubated at 37°C and examined daily for 7 days and periodically once a week for 6-8 weeks. Suspected colonies were identified morphologically and microscopically (Cruickshank et al., 1975) Biochemical tests and confirmation of species were applied according to Ernst. (1990) ELISA test using bovine (PPD) as coating antigen (O’Reilly. 1989) Coating Round-bottomed, flexible polyvinyl microtiter plates

(Immulon II) were charged with 50 µl / well of bovine PPD

in carbonate buffer of 5µg/ml sod carbonate buffer except 4 wells which serve as blank wells, and then each well

received 50 µl of carbodiimide in carbonate buffer. Plates were then incubated overnight at 4 °C. Serum dilutions The plates were decanted, washed 3 times with washing buffer and air dried for 10 minutes. Each well received 100

µl of ammonium chloride solution except the blank wells, and incubation was done at room temperature for 30 minutes. The plates were then washed 3 times with

washing buffer. 50 µl of sera diluted to 1/10 in diluting buffer and two-fold dilutions were made. Plates were then incubated at room temperature for 30 minutes. Each plate received negative sera run in the same dilutions to calculate the cut - off value. Conjugate Plates were decanted, washed 7 times with washing buffer.

To each well, 50 µl of antibovine horseradish conjugated peroxidase diluted 1:5000 in diluting buffer was added and plates then incubated for 30 minutes at room temperature, the plates were washed 7 times with ELISA wash.

03. Basic Res. J. Anim. Sci.

Table 1. Incidence of Acid Fast Bacilli in milk samples examined microscopically and culturing

Positive samples Negative samples

No. % No. %

Mil

k f

rom

tuberculin positive reactors

50 7 14 43 86

tuberculin negative reactors

Total 100 10 10 90 90

Table 2. Incidence of Mycobacteria isolated from milk samples using conventional culture method

Lowenstein- Jensen media

pyruvated glycerinated

+ve samples % +ve samples %

Milk

fro

m

tuberculin positive reactors

50 2 4 1 2 3

tuberculin negative reactors

Total 100 3 3 1 1 4

Substrate

50 µl of freshly prepared OPD Substrate buffer was added and the plates were incubated in dark for 10 minutes. Optical density measurement The optical density OD was measured at 450 nm using ELISA reader. The serum dilution was considered positive if it yielded a mean OD of each group equal to/or greater than the cut off value (Dimitri and Mikhail, 1996). RESULTS The results of AFB in milk samples examined by direct smear using ZN stain were 7 (14%) and 3 (6%) from tuberculin positive reactors and tuberculin negative reactors, respectively (Table 1). Mycobacteria could be detected by culturing in 3 (6%) and 1 (2%) milk samples from tuberculin positive and negative reactors. The incidence of Mycobacteria was 4% and 2% using Lowenstein Jensen medium pyruvated and LJ medium glycerinated from positive reactors, respectively and 2% were positive from tuberculin negative reactors in both media (Table 2). M. bovis was isolated in percentages of 4

and 2 % of tuberculin positive and negative reactors however Mott was isolated from 2% of tuberculin positive reactors only (Table 3). ELISA results indicated that Mycobacteria could be detected among tuberculin positive reactors in percentages of 2 (4%), 7 (14%) and 12 (24%) in generalized TB, localized lesion and non-visible lesion, respectively (Table 4). DISCUSSION Bovine tuberculosis infection in cattle is usually diagnosed in the live animal on the basis of delayed hypersensitivity reactions. Delayed hypersensitivity test is the standard method for detection of bovine tuberculosis (Ereny Markos, 2011).

The I/D tuberculin test has been the widest used diagnostic technique although it has some sensitivity and specificity deficiencies and requires a second inspection of the animal for its interpretation beside the three traditional diagnostic techniques (Postmortem examination, Ziehl Neelson stain and bacteriological culture) used for tuberculosis diagnosis in slaughtered animals (Retmal and Abolas, 2004). It allows detection of cattle that have been exposed to M. bovis. However, in herds where control of TB based on the identification and removal of reactors to this test, some animals in advanced stages of the disease

Wahba et al. 04

Table 3. Identification of Mycobacteria isolated from milk samples

Type of milk samples Total No. of samples

Positive culture for AFB

Types of Mycobacteria

M. bovis MOTT

No. % No. % No. %

Milk

fro

m

tuberculin positive reactors

50 3 6 2 4 1 2

tuberculin negative reactors

50 1 2 1 2 0 0

Table 4. Correlation between the types of infection, cultural and serum ELISA test results (among tuberculin positive reactors) PM finding

No. of infected animals

Culture exam. ELISA results

% (/50)

Milk % (/50)

Generalized TB 2 1 2 2 4 Localized TB 2 4 7 14 Non visible lesion 38 0 0 12 24 Total 50 3 6 21 42

and with open lesions do not show reactivity to tuberculin (anergic) and might remain in the herd, thus constituting a potential source of infection in susceptible cattle (Diaz - Otero et al., 2003).

The results of AFB in milk samples examined by direct smear using ZN stain were higher than that recorded by culturing among tuberculin positive reactors and tuberculin negative reactors. These results may be due to the fact that ZN method was used for staining of tubercle, other acid – fast and saprophytic bacilli and can't differentiate between them (Mackie and McCartney. 1960 and Cruickshank et al., 1975).

Mycobacteria could be detected by culturing in 3 (6%) and 1 (2%) milk samples from tuberculin positive and negative reactors. These results are nearly similar to Gad et al. (2000) (5.6%) Higher results (9.3%) were detected in Canadian cattle by Ameni et al. (2003) (13.3%). Hamid et al. (2003) recorded 28.07% from milk of tuberculin positive buffaloes and from milk of 25% tuberculin positive cows.

In this study the percentage of Mycobacteria on L-J pyruvate were 4 and 2% while on L-J glycerol were 2 and 0% for tuberculin positive and negative reactors, respectively. These results indicated that L-J medium could be used for primary isolation, sensitivity testing, identification and sub-culturing of the majority of Mycobacteria as reported by Maureen (1981).

Although the number of M. bovis positive samples was low, the habit of pooling milk may still pose a public health danger to milk consumers (Schelling et al., 2000). Furthermore, M. bovis is the major cause of extra-pulmonary tuberculosis like tuberculosis of gastrointestinal tract and tuberculosis of cervical and mesenteric lymph nodes, the peritoneum and the genitourinary tract (Bonsu

et al., 2001). The distribution of the tuberculosis lesion varied greatly, being pulmonary, extra-pulmonary, mixed or generalized. The variation of distribution of the lesion may governed by the portal of entry and the possibility of dissemination (Adway, 1986). The present study revealed that the number of infected animals with generalized TB, localized lesions and non visible lesions were 2, 10 and 38 among tuberculin positive reactors (Table 4). Freitas et al. (2001) recorded 27.9% generalized lesions and 72.1% localized lesions in Brazil. The rate of recovery of M. bovis from reactor animals with visible lesions was (84%) where 105 isolates had been isolated from 125 reactor buffaloes with V.L. that nearly agree with the recorded by Jacob (1994). On the other hand Stefan et al. (2009) found that out of approximately 32,800 inspected cattle 4.7% showed suspected tuberculous lesions, Culture of suspected lesions yielded acid-fast bacilli in 11% of cases. Furthermore, the routine abattoir inspection was able to detect only 117 of the total 3322 carcasses inspected (Demelash et al., 2010). Moreover, Huitema. (1994) attributed the cause of non specific reaction to the assumption that those animals might be slaughtered at early stage of the disease where the tubercles lesions were invisible or lesions may be found in parts of the body not routinely examined in the carcass such as bone or brain.

The ELISA is one of the main important serological tests for diagnosis of bovine tuberculosis. Correlation between the types of infection, cultural of milk samples and ELISA on the sera among tuberculin positive reactors (using bovine (PPD) as coating antigen) was investigated in Table (4). It was found that 1 from 2 of generalized TB cases among animals was positive for cultural of milk samples, 2 from 10 of localized TB forms had infected milk while,

05. Basic Res. J. Anim. Sci. negative results obtained in milk of non visible lesion cases. So, culture of milk could not be used for detection of tuberculosis but there was danger for the presence of tubercle bacilli in milk to the consumer. Dunn and Hodgson. (1982) proved that milk samples with enough viable tubercle bacilli excreted can infect the milk of 100 clean cows to be of infectious level. ELISA results indicated that all serum of generalized TB cases was found positive to ELISA, only 7 from 10 of localized TB and 12 from 38 of non visible lesion cases was positive. Ereny Markos. (2011) found that out of 43 tuberculin reactor cattle with visible lesion and Out of 19 tuberculin reactor cattle with non visible lesion, 39 (90.6%) and 3 (15.7%) were positive for ELISA , respectively.

False negative ELISA results obtained in the current study (only 7 from 10 of localized TB) explained by the fact that low titer of antibodies to Mycobacterial antigens which may be associated with heavy infection and that antigens may be released into the blood circulation and cause temporary suppression of antibody formation Krambovitis. (1986) and that agree with Thorns and . (1983) who cleared that the level of specific antibodies in many M. bovis infected cattle may be low or undetectable. This is supported with Amadori et al. (1998) who pointed that antibodies to Mycobacterial antigens were investigated with various rates of success since the humeral immune response to M. bovis is late and irregular during the course of the disease.

Sensitivity of ELISA was 65.6% and specificity was 56.4%. When ELISA and caudal fold tuberculin test results in cattle were interpreted in parallel, sensitivity was 95%, specificity was 92.7%. The results of the ELISA and single cervical tuberculin test were interpreted in parallel or series. The ELISA was able to detect 47% of 68 (69%) cattle infected with M. bovis that did not have visible tuberculous lesions at P/M examination (Gaborick at al., 1996). For this Sensitivity of ELISA and specificity in comparison with prolonged time cultural consuming and false positive results of tuberculin skin test, Nasr and Osman. (2007) suggested that ELISA technique can be used with the skin test to determine the disease status of animal and reduce the frequency of misdiagnosis of animals free of bovine tuberculosis. CONCLUSION It could be concluded that Bovine tuberculosis still constitutes a public health hazard and every effort should be made to control such disease in Egypt. The aforementioned proved that culture of milk could not be used for detection of tuberculosis but there was danger for the presence of tubercle bacilli in milk to the consumer. Moreover, the ELISA technique can be used as a complementary to the tuberculin skin test to determine the disease status of animal or as a rapid screening test for

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