Evaluation of Four DNA Extraction Protocols for Brucella abortus Detection by PCR in Tissues from Experimentally Infected Cows with the 2308 Strain

Abstract

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

Introduction

D espite the implementation of control programs, brucellosis is a global public health problem with severe economic consequences for cattle production (Sreevatsan et al. 2000). The etiologic agent of brucellosis is an immobile intracellular facultative coccobacillus belonging to the class Proteobacteria and genus Brucella (Redkar et al. 2001). The Brucella genus is composed of 10 species, of which 7 infect terrestrial animals—B. abortus, B. melitensis, B. suis, B. ovis, B. canis, B. neotomae, and B. microti (Scholz et al. 2008)—and 2 infect marine mammals—B. ceti and B. pinnipedialis (Foster et al. 2007). The most recently described species is B. inopinata, which was isolated from an infected human breast implant (Scholz et al. 2010). B. melitensis, B. suis, and B. abortus are smooth, highly pathogenic species that cause severe disease in goats, sheep, swine, bovines, and humans (Corbel et al. 2006).

Brucellosis is transmitted from animals to humans via ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols (Christopher et al. 2010). Brucella localizes in the supramammary lymph nodes and mammary glands of 80% of the infected animals, which consequently secrete the pathogen in milk throughout their lives (Hamdy and Amin 2002). Most infected cows abort only once, but the placenta will be heavily infected at subsequent normal calvings (Morgan 1969, Godfroid 2010).

B. abortus is responsible for bovine brucellosis, which causes abortion; additionally, when brucellosis is detected in a herd, flock, region, or country, international veterinary regulations restrict animal movements and the trade of animal products, resulting in significant economic losses (Bricker and Halling 1994, Sreevatsan et al. 2000). For these reasons, eradication programs focus mainly on B. abortus, and epidemiological studies require techniques that identify the genus and species of field isolates (Bricker and Halling 1994, Newby et al. 2003). Brucellosis is diagnosed by indirect and direct methods. The indirect ones are used in the free herd certification routines (Brasil 2012). Unfortunately, serological tests have low sensitivity, especially in the early stages of the disease when antibody production is limited (Araj 2010). Bacterial isolation and identification use the morphological and biochemical characteristics of Brucella, and the culture of blood and tissues is the standard technique for the direct diagnosis of Brucella spp. (Ewalt and Bricker 2000, Ocampo-Sosa et al. 2005, Godfroid et al. 2010). Direct diagnosis is essential for confirmation of the infection and characterization of the circulating species. Factors such as the sample type, sampling time (e.g., disease stage), and sample preparation equipment used for manipulation and isolation of bacteria affect the success rates of culture methods (Mantur et al. 2007, Alişkan 2008).

In contrast, classical methods, such as the bacterial isolation and identification, are expensive, time-consuming, and hazardous for laboratory personnel (Redkar et al. 2001). Molecular techniques, mainly PCR, are good alternatives for the routine diagnosis of Brucella (Paulin and Ferreira Neto 2003). PCR is highly sensitive, specific, trustworthy, and reproducible, and it minimizes the risks associated with handling potentially infectious specimens (Mullis and Faloona 1987, Dauphin et al. 2009). PCR-based tests are faster and more sensitive than the traditional methods (Morata et al. 1998, Christopher et al. 2010). However, the disadvantages of PCR are its high cost and potential inhibition of DNA amplification by serum proteins, somatic cell debris, polysaccharides, and other components of body tissues and fluids (Wilson 1997). The PCR diagnostic method would be improved by a DNA extraction protocol that eliminates inhibitors of Taq DNA polymerase and thus reduces false-negative results. Many factors influence the sensitivity of PCR assays, including quality of the test DNA, which should be of high purity and yield and with little or no damage after extraction from tissues (Dauphin et al. 2009). The present study compared 4 protocols for DNA extraction from homogenates of different organs from cows infected with the 2308 strain of B. abortus.

Materials and Methods

Samples and experimental design

The positive and negative gold standard groups were composed of organ homogenates originating from a brucellosis vaccine clinical trial. All samples were from cows inoculated with 3.5×10⁷ of B. abortus strain 2308 bacteria in the conjunctival sac at approximately the fifth month of pregnancy. The positive gold standard group included tissue samples from the following: 27 placental cotyledons, 12 supramammary lymph nodes, 17 prescapular lymph nodes, 6 livers, 10 spleens, and 7 udders. In all of these samples, B. abortus 2308 was isolated by classical bacteriological methods. The negative gold standard group included 27 samples from each organ that were negative for Brucella isolation. All samples were processed using 4 different DNA extraction protocols but with the same amplification and detection methods. The volume of homogenates used for DNA extraction protocols was 400 μL. For each protocol, the relative sensitivity and confidence interval were calculated. P values were calculated by the chi-squared test.

DNA extraction protocols

Lysis with proteinase K (PK) and phenol–chloroform

DNA was extracted according to Leal-Klevezas et al. (1995) with some modifications described by Matrone et al. (2009).

Lysis with guanidine isothiocyanate followed by treatment with carrier suspension (diatomaceous earth) (Boom)

The DNA extraction method described by Boom et al. (1990) was used with some modifications by Matrone et al. (2009).

Lysis with guanidine isothiocyanate (GT)

DNA was extracted as described by Chomkzynski (1993) and Matrone et al. (2009).

Lysis by heating and freezing with liquid nitrogen (Santos) (adapted from Santos et al. 1995)

Briefly, 400 μL of homogenized samples were clarified at 14,000× g for 5 min at 4°C, and the supernatant was decanted. The pellet was suspended in 50 μL of 0.5 M NaOH with agitation for 10 min before 25 μL of 1 M NaH₂PO₄ was added. The sample was centrifuged at 14,000× g for 10 min at 4°C, and the supernatant was discarded. Tris–EDTA buffer (TE 1% Triton X-100, 50 μL) was added, and the tubes were boiled in a dry bath at 100°C for 10 min and then centrifuged for 15 s. The tubes were stored in liquid nitrogen for 5 min and centrifuged for 15 s. The boiling and freezing steps were repeated twice. Phenol (150 μL) was added; the sample was vortexed for 20 s and centrifuged at 14,000× g for 5 min at 4°C. A total of 100 μL of the aqueous phase was carefully pipetted to avoid the organic interface and transferred to a new tube. The supernatant was extracted with 200 μL phenol–chloroform–isoamyl alcohol (25:24:1). The tubes were vortexed for 10 s and clarified at 14,000× g for 5 min at 4°C. A total of 75 μL of the aqueous phase was transferred to a new tube, avoiding pipetting of the organic interface, and 75 μL of propanol was added to the supernatant and then homogenized and stored at −20°C for 4 h. Afterward, the tubes were centrifuged at 14,000× g for 10 min at 4°C. The supernatant was collected, and 400 μL of 70% ethanol was added. After centrifugation at 14,000× g for 10 min at 4°C, the supernatant was removed, and the pellet was dried at room temperature. Finally, 30 μL of TE buffer was added, vortexed for 10 s, and dried at 56°C for 15 min. The extracted DNA was stored at −20°C until examination.

DNA amplification

The primers B₄ (5′-TGGCTCGGTTGCCAATATCAA-3′) and B₅ (3′-CGCGCTTGCCTTTCAGGTCTG-5′) described by Baily et al. (1992) were used to amplify a 223-bp fragment. PCR was performed in a volume of 50 μL, consisting of 20 μL of ultrapure water, 5 μL of 10× reaction buffer (500 nM KCl, 15 nM MgCl₂, 100 nM Tris- HCL, pH 9.0), 8 μL of deoxyribonucleotide triphosphates [dNTP] mixture (200 nM of each nucleotide [dCTP, dATP, dGTP, dTTP]), 1.5 μL of MgCl₂ (50 nM), 5 μL of each primer (10 pmol/μL), 0.5 μL of Taq DNA polymerase (5 units/μL), and 5 μL of diluted DNA.

Serial dilutions were performed for each tissue and for each protocol. The dilution with the highest number of positive samples was selected for subsequent use. For the PK method, a dilution of 1:3 was selected for cotyledon, supramammary lymph node, prescapular lymph node, liver, and udder, whereas the 1:10 dilution was selected for spleen. For the Boom method, a dilution of 1:2 was selected for liver. For cotyledon, supramammary lymph node, prescapular lymph node, udder, and spleen, 10 μL of the undiluted DNA and 40 μL of PCR mix were used in the reaction. For the GT method, the best dilutions were 1:4, 1:15, 1:15, 1:2, 1:10, and 1:4 for cotyledon, supramammary lymph node, prescapular lymph node, liver, spleen, and udder, respectively. For the Santos method, the best dilutions were 1:2, 1:12, 1:10, 1:12, 1:5, and 1:5 for cotyledon, supramammary lymph node, prescapular lymph node, liver, spleen, and udder, respectively.

The amplification was performed with an initial denaturation of 94°C for 5 min, 40 cycles of 94°C for 1 min (denaturation), 60°C for 1 min (annealing), and 72°C for 1 min (extension) with a final extension of 72°C for 10 min. Finally, all amplification products were analyzed by electrophoresis in an agarose gel containing 1.5% ethidium bromide (0.5 μg/mL) and subsequent observation with an ultraviolet transilluminator.

Results

Table 1 shows the results of the PCR assay for the B. abortus 2308 strain after each of the DNA extraction protocols. Table 2 shows the relative sensitivity values for the same DNA extraction protocols.

Table 1.

Brucella abortus Detection by PCR from the Organs of Cows That Were Experimentally Infected Using Different DNA Extraction Methods, According to the Classical Isolation Method

	Results of PCR according to DNA extraction protocol
	PK			BOOM			GT			SANTOS			Cumulative results
Results of isolation by organs	Pos	Neg	Total	Pos	Neg	Total	Pos	Neg	Total	Pos	Neg	Total	Pos	Neg	Total
Cotyledon
Positive	27	0	27	27	0	27	27	0	27	24	3	27	105	3	108
Negative	6	21	27	2	25	27	7	20	27	1	26	27	16	92	108
Total	33	21	54	29	25	54	34	20	54	25	29	54	121	95	216
Supramammary^a
Positive	10	2	12	7	5	12	8	4	12	11	1	12	36	12	48
Negative	2	25	27	2	25	27	2	25	27	1	26	27	7	101	108
Total	12	27	39	9	30	39	10	29	39	12	27	39	43	113	156
Prescapular^a
Positive	10	7	17	7	10	17	8	9	17	15	2	17	40	28	68
Negative	2	25	27	5	22	27	5	22	27	1	26	27	13	95	108
Total	12	32	44	12	32	44	13	31	44	16	28	44	53	123	176
Liver
Positive	5	1	6	3	3	6	4	2	6	6	0	6	18	6	24
Negative	0	27	27	3	24	27	2	25	27	0	27	27	5	103	108
Total	5	28	33	6	27	33	6	27	33	6	27	33	23	109	132
Spleen
Positive	3	7	10	3	7	10	4	6	10	7	3	10	17	23	40
Negative	1	26	27	2	25	27	2	25	27	2	25	27	7	101	108
Total	4	33	37	5	32	37	6	31	37	9	28	37	24	124	148
Udder
Positive	7	0	7	4	3	7	6	1	7	5	2	7	22	6	28
Negative	8	19	27	1	26	27	5	22	27	0	27	27	14	94	108
Total	15	19	34	5	29	34	11	23	34	5	29	34	36	100	136
Cumulative results
Positive	62	17	79	51	28	79	57	22	79	68	11	79
Negative	19	143	162	15	147	162	23	139	162	5	157	162
Total	81	160	241	66	175	241	80	161	241	73	168	241

lymph node.

Table 2.

Relative Sensitivity of Brucella abortus PCR Detection of 4 DNA Extraction Protocols from the Organs of Cows That Were Experimentally Infected, Considering the Bacteriological Isolation As Gold Standard

	DNA extraction protocol
	PK		Boom		GT		Santos		Cumulative results
Organs	rS (%)	CI 95%(%)	rS (%)	CI 95%(%)	rS (%)	CI 95% (%)	rS (%)	CI 95% (%)	rS (%)	CI 95% (%)
Cotyledon	100	[84,5;100]	100	[84,5;100]	100	[84,5;100]	89	[69,7;97,1]	97	[91,5;99,3]
Supramammary^a	83	[50,1;97,0]	58	[28,6;83,5]	67	[35,4;88,7]	92	[59,7;99,6]	75	[60,1;85,9]
Prescapular^a	50	[33,4;80,6]	41	[19,4;66,5]	47	[23,8;71,5]	88	[62,2;97,9]	59	[46,2;70,4]
Liver	83	[36,5;99,1]	50	[13,9;86,0]	67	[24,1;94,0]	100	[51,7;100]	75	[52,9;89,4]
Spleen	30	[8,1;64,6]	30	[8,1;64,6]	40	[13,7;72,6]	70	[35,4;91,9]	57	[41,0;72,6]
Udder	100	[56,1;100]	57	[20,2;88,2]	86	[42,0;99,2]	71	[30,2;94,9]	79	[58,5;91,0]
Cumulative results	78	[67,5;86,6]	64	[52,9;74,8]	72	[60,7;81,4]	86	[76,0;92,5]

Lymph node.

PK, proteinase K lysis; Boom, guanidine isothiocyanate lysis with the carrying suspension diatomaceous earth; GT, guanidine isothiocyanate lysis; Santos, lysis by boiling and freezing with liquid nitrogen; rS, relative sensitivity; CI, confidence interval.

Tables 1 and 2 show the cumulative PCR results for each DNA extraction protocol. The proportion of positive samples detected by the Santos protocol was significantly higher than that of the Boom (p=0.003) and GT methods (p=0.0506) and equal to that of the PK method (p=0.2969). No differences were found among the other protocols. In contrast, the cumulative results for each organ show that the number of PCR-positive cotyledon samples was significantly higher than those of the supramammary lymph node (p=0.0001), prescapular lymph node (p<0.0001), liver (p=0.0006), spleen (p<0.0001), and udder (p=0.0019). The number of positive samples in spleen was significantly lower than in the liver (p=0.0233), udder (p=0.0067), and supramammary lymph node (p=0.0039). There were no other differences between organs in the number of positive PCR samples.

Discussion

This study compared 4 DNA extraction protocols for PCR diagnosis of B. abortus. Each protocol has a different capacity for recovering Brucella DNA, and these differences influence the sensitivity of PCR assays, as previously noted (Dauphin et al. 2009).

The Santos DNA extraction protocol had a higher relative sensitivity (86%) compared with the GT (72%) and Boom protocols (64%) when classic bacteriological isolation was considered the gold standard. However, no significant difference was found between the Santos and PK protocols (78%) (Table 1).

In a previous study, we compared the performance of the Boom, GT, and PK extraction methods using organs of aborted fetuses and calves born from cows infected with B. abortus (Matrone et al. 2009). In that study, the Boom protocol was the best method when experimental infection was used as the gold standard, but no significant differences were observed when classical bacteriology was used as the reference method.

The Santos protocol was used for PCR detection of Mycobacterium leprae in hair from cutaneous injuries and nasal secretions (Santos et al. 1995). However, Ribeiro (2006) could not detect Mycobacterium bovis using this protocol. Although this method has low costs, it requires handling of liquid nitrogen, which can make this method impractical.

The PK DNA extraction protocol was used in several studies. Fekete et al. (1990) verified that this protocol is efficient for PCR detection of B. abortus in liver. Richtzenhain et al. (2002) found 100% sensitivity for Brucella spp. detection in homogenized organs of aborted fetuses, and Leal-Klevezas et al. (1995) successfully detected B. abortus in blood samples from infected bovines. Vinodh et al. (2008) tested a method similar to our PK protocol that had higher analytical sensitivity than the boiling method. The results obtained in the present study corroborate those previously reported because when classical bacteriological isolation was used as gold standard, the PK protocol provided the best detection of positive samples.

Queipo-Ortuño et al. (2005) found that the diatom–guanidinium isothiocyanate protocol (i.e., the Boom method) removes the inhibitors from a variety of clinical specimens and may improve the sensitivity and reproducibility of the PCR assay. In our study, the low sensitivity of the Boom protocol can be explained by the adherence of DNA to the carrier suspension (diatomaceous earth), which prevented DNA elution.

DNA purified by the GT protocol may contain a high concentration of factors inhibiting DNA amplification, such as serum proteins, somatic cell debris, polysaccharides, and other components of body fluids (Wilson 1997), or a high concentration of host DNA. The GT protocol does not include the prewashing of samples, which is performed in the PK and Boom protocols. Morata et al. (1998) demonstrated that increasing the number of washes reduces amplification inhibitors in the final product. In our hands, the Santos method performed better than those including sample prewashing, suggesting that the prewashing step does not influence amplification of the purified DNA.

Positive results in the PCR and classical bacteriology assays were not always correlated. Out of 79 isolation-positive samples, 11, 17, 22, and 28 samples were negative by PCR following the Santos, PK, GT, and Boom protocols, respectively (Table 1). There were also samples that were negative by the bacteriological assay but positive by PCR, as quantified in the following list: Santos, 5; Boom, 15; PK, 19; and GT, 23 samples. Cortez et al. (2001) detected 4 PCR-positive samples in 54 samples classified as negative by classical bacteriological isolation, whereas Fekete et al. (1990) found 2 PCR-positive samples in 52 isolation-negative samples. Buyukcangaz et al. (2011) found a sensitivity and specificity of 83% and 94%, respectively, when comparing PCR and bacteriological methods. The authors suggested that the culture-negative but PCR-positive samples were caused by cross-contamination between samples and nonviable or unculturable Brucella organisms (Buyukcangaz et al. 2011). PCR detects living and dead organisms, but cultures detect only live organisms (Abdalla and Hamid 2012).

PCR specificities were not calculated because they depend exclusively on the primers used (Fekete et al. 1990). The same PCR conditions were used for DNA extracted by all 4 methods. In the reaction, the primers B4 and B5 designed by Baily et al. (1992) for B. abortus and B. melitensis detection were used. These primers showed high sensitivity, high specificity, and low genetic similarity with other bacteria in previous studies (Baily et al. 1992, Matar et al. 1996, Richtzenhain et al. 2002, Elfaki et al. 2005, Queipo-Ortuño et al. 2005, O'Leary et al. 2006, Baddour and Alkhalifa 2008). However, the primer pair can also amplify DNA from Ochrobactrum spp., a human pathogen (Cieslak et al. 1992, Navarro et al. 1999).

Cotyledon was the best organ for detection of B. abortus due to the high relative sensitivity (97%) of the PCR assay (Table 2). B. abortus multiplication is stimulated by the erythritol polyalcohol, which can be found in the gravidic uterus (Carter and Chengappa 1991). This factor could explain the better detection of B. abortus in this organ. Alexander et al. (1981) verified that a gram of cotyledon from a B. abortus-infected cow has 1.4×10¹³ Brucella. The high bacterial load makes the cotyledon the ideal organ for B. abortus detection because the sensitivity of the PCR assay depends on the number the microorganisms in the sample (Fekete et al. 1990).

A high cumulative relative sensitivity was observed in the supramammary lymph node (75%) and udder (79%) (Table 2). The increased level of detection could also be attributed to erythritol in the mammary tissues (Carter and Chengappa 1991); other authors described similar results. Bishop et al. (1994) isolated B. abortus in the supramammary lymph node from more than 90% of infected animals, whereas Leal-Klevezas et al. (1995) found B. abortus in this organ in more than 80% of infected animals. O'Leary et al. (2006) concluded B. abortus can be isolated from lymph nodes.

Hemoglobin, porphyrins, and other molecules with a heme moiety could act as inhibitors of DNA amplification (Navarro et al. 1999, Baddour and Alkhalifa 2008). Thus, insufficient washing of blood-rich organs could limit Brucella detection. To eliminate these inhibitors, spleen samples were washed several times. However, compared with the bacteriological assay, the lowest cumulative relative sensitivity was found in spleen (57%, Table 2). The number of positive cases in spleen was statistically lower than those found in other organs, with the exception of prescapular lymph node. Few growth-phase B. abortus were found in spleen of infected cows (Bosseray 1983). However, Matrone et al. (2009) demonstrated that in aborted fetuses, there is a higher probability of B. abortus detection in the spleen and lung than in the liver and bronchial lymph node. This discrepancy can be explained partially by the amount of host genomic DNA, which affects the sensitivity of the assay as observed previously (Mukherjee et al. 2007). The sensitivity of these assays could be improved by increasing the volume of template DNA in the PCR and/or by concentrating the DNA in a smaller volume elution (Kattar et al. 2007).

The most sensitive detection of B. abortus in infected cow organs was obtained by extracting bacterial DNA with the Santos and PK methods, and a greater number of positive samples originated from cotyledon, suggesting that these methods and this organ should be used for B. abortus detection. However, organ pools containing supramammary lymph node, prescapular lymph node, and udder can also be tested. Because of the disadvantages of classical bacteriological methods, the best direct diagnosis of B. abortus in organs of infected cows includes PCR with DNA extracted by the Santos or PK protocols. Because current PCR methods have differences in sensitivity and specificity among many testing laboratories, the extraction and amplification protocols should be standardized for consistent diagnostic results.

Footnotes

Acknowledgments

The authors thank FAPESP, CAPES, and CNPq for financial support.

Author Disclosure Statement

No competing financial interests exist.

References

Abdalla

, Hamid

. Comparison of conventional and non-conventional techniques for the diagnosis of bovine brucellosis in Sudan. Trop Anim Health Prod, 2012; 44:1151–1155.

Alexander

, Schnurrenberger

, Brown

. Number of Brucella abortus in the placenta, umbilicus and fetal fluid of two naturally infected cows. Vet Rec, 1981; 108:500.

Alişkan

. The value of culture and serological methods in the diagnosis of human brucellosis. Mikrobiyol Bul, 2008; 42:185–195.

Araj

. Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents, 2010; 36,Suppl 1:S12–S17.

Baddour

, Alkhalifa

. Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood. Can J Microbiol, 2008; 54:352–357.

Baily

, Krahn

, Drasar

, Soker

. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg, 1992; 95:271–275.

Bishop

, Bosman

, Herr

. Bovine brucellosis. Coetzer

JAN

, Thomson

, Tustin

. Infectious diseases of livestock. Austin: Texas A&M University Press, College Station, 1994; 1053–1066.

Boom

, Sol

, Salimans

, Jansen

et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol, 1990; 28:495–503.

Bosseray

. Kinetics of placental colonization of mice inoculated intravenously with Brucella abortus at day 15 of pregnancy. Br J Exp Pathol, 1983; 64:612–616.

10.

Brasil. Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Programa Nacional de Controle e Erradicação da Brucelose e da Tuberculose (PNCEBT). Instrução Normativa do Serviço Nacional Animal número 6. Regulamento PNCEBT. January 8, 2004[cited 2012 June 07]www.agricultura.gov.br/. 2012 November 22.

11.

Bricker

, Halling

. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol, 1994; 32:2660–2666.

12.

Buyukcangaz

, Sen

, Carli

, Kahya

. Comparison of direct culture versus PCR for the detection of Brucella in aborted fetuses of cattle and sheep in Turkey. Vet Rec, 2011; 23;168:430.

13.

Carter

, Chengappa

. Brucella. Carter

, Chengappa

. Essentials of Veterinary Bacteriology and Mycology, 4th. Philadelphia: Lea & Febiger, 1991; 196–200.

14.

Christopher

, Umapathy

, Ravikumar

. Brucellosis: Review on the recent trends in pathogenicity and laboratory diagnosis. J Lab Physicians, 2010; 2:55–60.

15.

Chomkzynski

. Reagent for the single step simultaneous isolation of RNA, DNA and proteins from cells and tissue samples. BioTechniques, 1993; 15:532–534536–537.

16.

Cieslak

, Robb

, Drabick

, Fischer

. Catheter-associated sepsis caused by Ochrobactrum anthropi: Report of a case and review of related nonfermentative bacteria. Clin Infect Dis, 1992; 14:902–907.

17.

Corbel

, Elberg

, Cosivi

. Brucellosis in humans and animals. Geneva: WHO Press, 2006.

18.

Cortez

, Scarcelli

, Soares

, Heinemann

et al. Detection of Brucella DNA from aborted bovine foetuses by polymerase chain reaction. Aust Vet J, 2001; 79:500–501.

19.

Dauphin

, Hutchins

, Bost

, Bowen

. Evaluation of automated and manual commercial DNA extraction methods for recovery of Brucella DNA from suspensions and spiked swabs. J Clin Microbiol, 2009; 47:3920–3926.

20.

Elfaki

, Uz-Zaman

, Al-Hokail

, Nakeeb

. Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagn Microbiol Infect Dis, 2005; 53:1–7.

21.

Ewalt

, Bricker

. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol, 2000; 38:3085–3086.

22.

Fekete

, Bantle

, Halling

, Sanborn

. Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J Appl Bacteriol, 1990; 69:216–227.

23.

Foster

, Osterman

, Godfroid

, Jacques

et al. Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int J Syst Evol Microbiol, 2007; 57:2688–2693.

24.

Godfroid

, Nielsen

, Saegerman

. Diagnosis of brucellosis in livestock and wildlife. Croat Med J, 2010; 51:296–305.

25.

Hamdy

ME.

, Amin

. Detection of Brucella species in the milk of infected cattle, sheep, goats and camels by PCR. Vet J, 2002; 163:299–305.

26.

Kattar

, Zalloua

, Araj

, Samaha-Kfoury

et al. Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis. Diagn Microbiol Infect Dis, 2007; 59:23–32.

27.

Leal-Klevezas

, Martínez-Vázquez

, López-Merino

, Martínez-Soriano

. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol, 1995; 33:3087–3090.

28.

Mantur

, Amarnath

, Shinde

. Review of clinical and laboratory features of human brucellosis. Indian J Med Microbiol, 2007; 25:188–202.

29.

Matar

, Khneisser

, Abdelnoor

. Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31 kilodalton Brucella antigen DNA. J Clin Microbiol, 1996; 34:477–478.

30.

Matrone

, Keid

, Rocha

VCM

, Vejarano

et al. Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308. Braz J Microbiol, 2009; 40:480–489.

31.

Morata

, Queipo-Ortuño

, de Dios Colmenero

. Strategy for optimizing DNA amplification in a peripheral blood pcr assay used for diagnosis of human Brucellosis. J Clin Microbiol, 1998; 36:2443–2446.

32.

Morgan

. Brucellosis in animals: diagnosis and control. Proc R Soc Med, 1969; 62:1050–1052.

33.

Mukherjee

, Jain

, Patel

, Nair

. Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals. J Med Microbiol, 2007; 56,Pt 10:1309–1316.

34.

Mullis

, Faloona

. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol, 1987; 155:335–350.

35.

Navarro

, Fernandez

, Escribano

, Solera

. PCR assay for diagnosis of human brucellosis. J Clin Microbiol, 1999; 37:1654–1655.

36.

Newby

, Hadfield

, Roberto

. Real-time PCR detection of Brucella abortus: A comparative study of SYBR green I,5′-exonuclease, and hybridization probe assays. Appl Environ Microbiol, 2003; 69:4753–4759.

37.

Ocampo-Sosa

, Agüero-Balbín

, García-Lobo

. Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3. Vet Microbiol, 2005; 110:41–51.

38.

O'Leary

, Sheahan

, Sweeney

. Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Res Vet Sci, 2006; 81:170–176.

39.

Paulin

, Ferreira Neto

. O Combate à brucelose bovina. Situação brasileira. Jaboticabal: Funep, 2003.

40.

Queipo-Ortuño

, Colmenero

, Baeza

et al. Comparison between light cycler real time polymerase chain reaction (PCR) assay with serum and PCR, enzyme linked immunosorbant assay with whole blood samples for the diagnosis of human brucellosis. Clin Infect Dis, 2005; 40:260–264.

41.

Redkar

, Rose

, Bricker

, DelVecchio

. Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol Cell Probes, 2001; 15:43–52.

42.

Ribeiro

. Comparação de protocolos de extração de DNA para detecção de Mycobacterium bovis através da PCR em homogeneizado de órgãos. [dissertation] São Paulo (SP): University of São Paulo, 2006.

43.

Richtzenhain

, Cortez

, Heinemann

, Soares

et al. A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses. Vet Microbiol, 2002; 87:139–147.

44.

Santos

, Goes Filho

, Nery

, Duppre

et al. Evaluation of PCR mediated DNA amplification in non-invasive biological specimens for subclinical detection of Mycobacterium leprae. FEMS Immunol Med Microbiol, 1995; 11:113–120.

45.

Scholz

, Hubalek

, Sedlácek

, Vergnaud

et al. Brucella microti sp. nov., isolated from the common vole Microtus arvalis. Int J Syst Evol Microbiol, 2008; 58:375–382.

46.

Scholz

, Nöckler

, Göllner

, Bahn

et al. Brucella inopinata sp. nov., isolated from a breast implant infection. Int J Syst Evol Microbiol, 2010; 60,Pt 4:801–808.

47.

Sreevatsan

, Bookout

, Ringpis

, Perumaalla

et al. Multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle. J Clin Microbiol, 2000; 38:2602–2610.

48.

Vinodh

, Raj

, Govindarajan

, Thiagarajan

. Detection of Leptospira and Brucella genomes in bovine semen using polymerase chain reaction. Trop Anim Health Prod, 2008; 40:323–329.

49.

Wilson

. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol, 1997; 63:3741–3751.