Isolation of brucella spp from milk of brucellosis positive cows in São Paulo and Minas Gerais states

Isolamento de brucella spp do leite de vacas positivas para brucelose nos estados de São Paulo e Minas Gerais

Isolation of brucella spp from milk of brucellosis positive cows in São Paulo and Minas Gerais states^*

Isolamento de brucella spp do leite de vacas positivas para brucelose nos estados de São Paulo e Minas Gerais

Helio LANGONI¹; Sílvio Massaru ICHIHARA¹; Aristeu Vieira da SILVA¹; Renata Bonini PARDO¹; Flávia Bechelli TONIN¹; Lia Jeanne Pereira MENDONÇA¹; José Aparecido Dorta MACHADO²

CORRESPONDENCE TO:

Hélio Langoni

Núcleo de Pesquisa em Mastites

Departamento de Higiene Veterinária e Saúde Pública

Faculdade de Medicina Veterinária e Zootecnia da UNESP

Distrito de Rubião Júnior, s/n

18618-000 – Botucatu – São Paulo

e-mail: [e-mail]

SUMMARY

Brucellosis is a chronic zoonosis that plays an important role in Public Health. Considering the lack of data in Brazil regarding its presence in raw milk and non-pasteurized dairy products, we determined the presence of brucellae in milk from brucellosis positive animals. The slide agglutination test (SAT), tube agglutination test (TAT) and TAT treated with 2-mercaptoethanol were used to identify positive animals in studied herds. For 3 days, 300 ml milk samples/cow (75 ml/teat) were collected from all productive quarters of the positive animals. These were mixed and centrifuged. Part of the pellet and of the supernatant were inoculated in Farrel and Brodie-Sinton (BS) media supplemented with antimicrobial agents. The inoculated plates and tubes were incubated at 37ºC for 7 days, with 10 per cent CO₂ atmosphere. The suspected bacterial growth in BS media was immediately cultivated in agar Brucella media, under the same conditions. Colonies were submitted to identification procedures including Gram stain, CO₂ requirement, H₂S production, urease activity and growth in the presence of thionin and fuchsin. Of the 49 analysed samples, 15 (30.61%) contained Brucella abortus. The distribution was as follows: biotype 1 in one sample (2.04%), biotype 2 in eight (16.32%) and biotype 3 in six samples (12.25%).

UNITERMS: Milk; Brucella abortus; Cows; Brucellosis.

INTRODUCTION

Brucellosis occurs worldwide except in many European and Asian countries from which it has been erradicated²¹. As a herd disease, bovine brucellosis represents an economic problem; animals seldom eliminate infection without presenting symptoms and sequelae that directly affect the productive and reproductive aspects. The estimated economic loss exceeds 600 thousand dollars¹. It also has an enormous impact on public health because brucellosis can be transmitted to man either direct or by indirect contact with animal products⁵.

The prevalence of bovine brucellosis is variable in cattle but is generally higher among dairy cattle than range cattle due to the intensive farming practices to which these animals are submitted. In Brazil, the prevalence of the disease in bovine indicates that 2.49% of cattle seropositive and 2.04% show suspicious results².

Brucellic mastitis is chronic and often clinically unapparent. Because infected females excrete large numbers of viable brucellae in milk for months to years, apparently normal glands represent important sources of infection not only to other lactating cows but also to calves and humans who consume raw milk^12,15,21. This was demonstrated by Pedrix and Chirol¹⁸ who isolated Brucella spp. from milk samples collected during several lactations from serologically positive females that had recently aborted. Dafni et al.⁹ recovered B. melitensis from milk samples from brucellic animals and demonstrated that microorganisms can remain in latency, most commonly in udder tissues and in suprammamary lymph nodes. Zowghi et al.²³ recovered B. abortus from 26.32% of samples collected from positive animals and from 2.09% of 5,686 milk samples collected from serologically negative bovine females. B. abortus was found in 29.8% of milk samples obtained from cows with positive results to card test¹³.

Guercio et al.¹¹ observed a significant rise in prevalence of human brucellosis associated with the consumption of raw milk. Cooper⁷ extended this observation by showing that the main source of infection for the general population is not only contaminated raw milk but also non-pasteurized dairy products. Brucellae can survive food processing depending on maturation and acidification periods to which each product is submitted¹⁹.

Considering the importance of milk in the epidemiology of brucellosis as a zoonosis, we carried out this study to investigate the presence of brucellae organisms in milk from cows seropositive for brucellosis in São Paulo and Minas Gerais states.

MATERIAL AND METHOD

Milk samples were collected from Holstein, Gir and crossbred cows belonging to 10 dairy farms from São Paulo and Minas Gerais states with different milking management such as manual and mechanical, with milk yields ranging from 200 to 1,200 liters/day, all of the farms were deficient in milking hygiene procedures. These cows were in different lactation stages and were not vaccinated against brucellosis.

To identify animals with brucellosis, blood samples were initially collected from each bovine female, either from jugular or mammary vein with 40 x 20 needles, after careful local disinfection, and submitted to the slide-agglutination test (Huddleson test), tube-agglutination test (Wright-Bang test) and tube-agglutination test with 2-mercapthoethanol using B. abortus antigen¹⁷. Forty-nine animals with positive reactions to anyone of these diagnostics tests were submitted to clinical examination, which consisted in careful palpation of udder and suprammamary lymph nodes and inspection of milk secretion for presence of clots, flakes, discoloration and wateriness. Milk from each quarter was also examined using California Mastitis Test (CMT)²² for detection of subclinical mastitis.

In order to detect Brucella spp., milk samples of approximately 300 ml/cow (75 ml/teat) were collected from the first milking of the day during three consecutive days and were stored at 4ºC until laboratory procedures started. Before sampling, each teat was carefully washed, dried and the surface of the teat ends was sterilized by wiping with clean cotton dipped in 70º alcohol. Samples were collected in sterile vials.

For routine aerobic microbiological examination, 0.1 ml of each sample was inoculated in plates with 10% bovine blood agar and in plates with MacConkey agar, and incubated at 37ºC for 72 hours. Growth characteristics were observed at 24, 48 and 72 hours by macroscopic analysis of colonies. Microscopic study of smears treated with Gram stain followed. The isolated microorganisms were analysed by biochemical tests and identified according to Carter and Cole Jr⁶.

For brucellae investigation, the three milk samples collected from each animal was homogenized and centrifuged for 15 minutes at 6,000 rpm. A portion of the sediment was mixed with an equal volume of sterile saline and anphotericin B (4 µg/ml), nistatin (100 U/ml), nalidixic acid (5 µg/ml), vancomicin (20 µg/ml) and bacitracin (20 U/ml), were added. This mixture was left for 60 minutes at 4 to 8ºC to eliminate undesirable microorganisms. Two guinea pigs (Cavia porcellus) were inoculated intraperitoneally for each cow sample⁴ with 1.0 ml of the suspended material. These were observed daily. If symptoms or death were seen, a detailed necropsy was performed. A histopathological study of smears of samples from abnormal organs, specially lymph nodes efferent to inoculation point and spleen, was made. Tissue was stained with hematoxilin-eosin¹⁴ and by the Köster method as modified by Costa et al.⁸. A microbiological examination was also carried out on animals with no symptoms that were sacrificed at week three and six. Before sacrifice a blood sample was obtained to perform the described serological tests¹⁶.

The isolation of Brucella spp was performed on Farrel Medium plates¹⁰ inoculated with 0.1 ml of the sediment (SE) and with 0.1 ml of supernatant (SU) and incubated at 37ºC, for 7 days, under aerophlic condition (10% CO₂). In addition two tubes containing 4 ml of Brodie Sinton³ medium was inoculated with 0.5 ml of sediment and with 0.5 ml of supernatant and incubated as for Farrel Medium. Seven days later, to recover brucellae, 0.1 ml of this enriched culture was inoculated on to Brucella agar and reincubated.

Plates were observed daily for bacterial growth. Pinpoint, smooth, glistening and translucent colonies, resembling Brucella spp., were smeared and Gram stained. Morphological studies were performed by the modified Köster method⁸. Isolated organisms were submitted to the following tests to identify the biotype²⁰:

CO₂requirement for primary isolation: plates were streaked and incubated aerobically and under CO₂.

H₂S production: the isolate was grown on a trypticase soy agar with the test strip suspended over the slant. Blackening of the strip indicated a positive test.

Urease activity: a Christensen urea slope was inoculated with a loopful of a culture and incubated at room temperature. The test was regarded as negative if there was no reaction after 24 hours.

Growth in the presence of thionin and fuchsin: the test was carried out by incorporating the dyes thionin and basic fuchsin separately in trypticase soy agar at the concentration of 20 µg/ml (1:50,000) or 40 µg/ml (1:25,000). The medium was prepared by heating a 0.1 per cent solution of either dye in a boiling water bath for 20 minutes and then adding it to the required amount of autoclaved agar. The dye was mixed with the agar and poured into Petri dishes. A sterile swab was used to inoculate the dye media with a suspension of the test strain. The inoculated plates were incubated at 37ºC under 5-10 per cent CO₂ for 3-4 days and then examined for growth.

RESULTS AND DISCUSSION

Microbiological examination resulted in the recovery of several types of bacteria from 10% bovine blood agar and MacConkey agar. Seven (14.30%) milk samples with microbiological negative results, under routine conditions of aerobic culture, were positive for isolation of Brucella abortus biotype 3. This fact reinforces the necessity of using conditions to isolate species that are rarely involved in cases of mammary infections for accurate diagnosis.

Only three animals reacted positively to CMT, or had clinical mastitis (Tab. 1). The serological titers against Brucella abortus ranging between 1:100 UI and 1:1,600 UI, with 6.7% of 1:800 UI reactions, 13.3% of 1:200 UI, 26.7% of 1:100 UI and 1:1600 UI. Brucella abortus biotype 1, 2 and 3 were isolated in one or both differential media. When the isolation of Brucella from the sediment and the supernatant were compared, it was observed that the rates of isolation were higher when the sediment was inoculated on Farrel media than on Brucella Agar. In summary, Farrel media was better for the isolation of this microorganism with 40.0% and 46.6% positive using the supernatant and the sediment, respectively.

Table 1

Microbiological results isolated from supernatant (SU) and sediment (SE) from milk samples submitted to CMT of positive cows for brucellosis. Botucatu, 2000.

* serological titers of cows.

The microbiological results are in Tab. 1. The animals 3 and 4; 18 and 24; 30, 33, 34, 35 and 36; 39, 44 and 47 belonged, respectively, to same herd. The table shows the isolation of Brucella abortus biotype 1 in one sample (2.04%), biotype 2 in eight samples (16.32%) and biotype 3 in six samples (12.25%), making a total of 30.61% positive samples.

None of the sediment samples inoculated intraperitoneally resulted in the recovery of the agent from the organs examined. The guinea pig serum samples were negative to serum agglutination test. This probably occurred because of the small amount of brucellae in the samples. The amount being insufficient to cause the infection or the disease in the intraperitoneally inoculated animals. The histopathology of the spleen, lymphnodes and liver from all the animals inoculated showed no alterations. The cytology, using Köster modified method⁸, was normal, indicating that the animals were not infected after the intraperitoneal inoculation.

Considering that many people in our country consume raw milk, the isolation of Brucella spp. from 30.61% of the 49 samples studied shows the potential importance of the milk as a vehicle of this agent to men. A similar result was found by Huber et al.¹³ and Zowghi et al.²³ who isolated B. abortus from 29.8% and 26.32% respectively, from milk samples, and Zowghi et al.²³ also isolated this agent from serologic negative cows. Milk improperly pasteurized continues to be a potential vehicle of B. abortus for humans in Sao Paulo and Minas Gerais States.

RESUMO

A brucelose é uma zoonose crônica de importância para a Saúde Pública. Considerando o pequeno número de dados brasileiros sobre a sua presença em leite cru e derivados não-pasteurizados, estudamos a presença de brucelas em leite de animais sorologicamente positivos. A soroaglutinação rápida (SAR), a soroaglutinação lenta (SAL) e a soroaglutinação lenta com tratamento do soro com 2-mercaptoetanol foram utilizados para identificar os animais positivos nas propriedades estudadas. Amostras diárias de 300 ml de leite foram colhidas por três dias de todos os quartos mamários produtivos (75 ml/teto). As amostras eram misturadas e centrifugadas. Parte do sedimento e do sobrenadante foi inoculada em meios de Farrel e Brodie-Sinton (BS) suplementados com agentes antimicrobianos. As placas e tubos foram cultivados por sete dias a 37ºC, em microaerofilia. As colônias suspeitas no meio BS foram imediatamente repicadas para ágar-Brucella, e cultivadas sob a mesma condição. Os microrganismos isolados foram submetidos a procedimentos de identificação, incluindo a coloração de Gram, requerimento de CO₂, produção de H₂S, atividade da urease e crescimento na presença de tionina e fucsina. Das 49 amostras examinadas, isolou-se Brucella abortus de 15 (30,61%). Os biótipos isolados foram: biótipo 1 em uma amostra (2,04%), biótipo 2 em oito (16,32%) e biótipo 3 em seis amostras (12,25%)

UNITERMOS: Leite; Brucella abortus; Vacas; Brucelose.

Received: 17/03/2000

Accepted: 02/02/2001

• ¹
  - ACHA, P.N.; SZYFRES, B. Brucelosis. Publicaciones Científicas de la Organización Mundial de la Salud, n.503, p.14-36, 1986.
• ²
  - BRASIL. Ministério da Agricultura. Defesa Sanitária Animal. Brucelose bovina. Boletim da Defesa Sanitária Animal, v.18, n.1, p.41-8, 1984.
• ³
  - BRODIE, J.; SINTON, G.P. Fluid and solid media for isolation of Brucella abortus. Journal of Hygiene of Cambridge, v.7, n.3, p.359-67, 1975.
• ⁴
  - CARRILLO, C.G. Laboratory animal models for brucellosis studies. In: NIELSEN, K.; DUNCAN, J.R., eds. Animal Brucellosis Ontario : CRC Press, 1990. p.423-43.
• ⁵
  - CARRILLO, C.G. Situación de la brucelosis en las Américas. Comunicações Científicas da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, v.8, n.2, p.153-60, 1984.
• ⁶
  - CARTER, G.R.; COLE JR., J.R. Diagnostic procedures in veterinary bacteriology and mycology New York : Academic News, 1990. p.620.
• ⁷
  - COOPER, C.W. Risk factors in transmission of brucellosis from animals to humans in Saudi Arabia. Transactions of the Royal Society of Tropical Medicine and Hygiene, v.86, p.206-9, 1992.
• ⁸
  - COSTA, E.O.; OGASSAWARA, S.; CURY, R. Nova modificação na técnica de coloração diferencial de Brucella abortus Revista da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, v.11, p.107-10, 1974.
• ⁹
  - DAFNI, I.; HOYDA, B.; FEINHAKEN, D. Observations on Brucella melitensis infection in Israeli cattle herds. Israel Journal of Veterinary Medicine, v.46, n.1, p.13-9, 1991.
• ¹⁰
  - FARRELL, I.D. The development of a new selective medium for the isolation of Brucella abortus from contaminated sources. Research in Veterinary Science, v.16, n.3, p.280-6, 1974.
• ¹¹
  - GUERCIO, V. Aspetti epidemiologic della brucelosi umana ed animale in Sicilia. Giornalle de Mallatie Infettive i Parassitaire, v.37, n.6, p.375-81, 1985.
• ¹²
  - HARMON, B.G.; ADAMS, L.G.; FREY, M. Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages. American Journal of Veterinary Research, v.49, n.7, p.1092-7, 1988.
• ¹³
  - HUBER, J.D.; NICOLETTI, P. Comparison of the results of card, Rivanol, complement fixation, and milk ring tests with the isolation rate of Brucella abortus from cattle. American Journal of Veterinary Research, v.47, n.7, p.1529-31, 1986.
• ¹⁴
  - LUNA, L.G. Special techniques. In:___ Manual of histologic staining methods of the armed force Institute of Pathology New York : McGraw Hill, 1968. p.72-9.
• ¹⁵
  - LYRA, T.M.P. Epidemiologia da brucelose. Comunicações Científicas da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, v.8, n.2, p.177-90, 1984.
• ¹⁶
  - MAYFIELD, J.E. Detection of Brucella cells and cell components. In: NIELSEN, K.; DUNCAN, J.R., ed. Animal Brucellosis Ontario : CRC Press, 1990. p.107-20.
• ¹⁷
  - OLASCOAGA, R.C. Diagnóstico serológico de la brucelosis. Boletín Informativo del Centro Panamericano de Zoonosis, v.18, n.1, p.107-35, 1976.
• ¹⁸
  - PEDRIX, J.; CHIROL, C. Les vaches excentrices de Brucella dans le lait. Bulletin de la Societé Scientifique et Veterinaire et Medicine Companion Lyon, v.77, n.3, p.371-6, 1975.
• ¹⁹
  - PLOMMET, M. Survival of Brucella abortus in ripened soft cheese made from naturally infected cows milk. Le Lait, v.2, n.2, p.115-20, 1988.
• ²⁰
  - QUINN, P.J.; CARTER, M.E.; MARKEY, B.; CARTER, G.R. Brucella species In:___Clinical Veterinary Microbiology London : Wolfe Publishing, 1994. p.261-7.
• ²¹
  - RADOSTISTIS, O.M.; BLOOD, D.C.; GAY, C.C. Diseases caused by Brucella spp In: ___ Veterinary medicine: a textbook of the disease of cattle, sheep, pigs, goats and horses London : Baillière Tindall, 1994. p.787-812.
• ²²
  - SCHALM, O.W.; NOORLANDER, D.D. Experiments and observations leading of the California Mastitis Test. Journal of American Veterinary Medical Association, v.130, n.2, p.199-204, 1957.
• ²³
  - ZOWGHI, E.; EBADI, A.; MOHSENI, B. Isolation of Brucella organisms from the milk of seronegative cows. Archives Institute RAZI, v.42/43, n.1, p.35-8, 1992.

^* FAPESP – Fundação de Amparo a Pesquisa no Estado de São Paulo, Process n.96/08838-6.

FAPESP – Fundação de Amparo a Pesquisa no Estado de São Paulo, Process n.96/08838-6.

¹ Departamento de Higiene Veterinária e Saúde Pública da Faculdade de Medicina Veterinária e Zootecnia da UNESP, Botucatu – SP

² Médico-Veterinário Autônomo, Monte Sião – MG

Publication Dates

• Publication in this collection
  22 Aug 2001
• Date of issue
  Dec 2000

History

• Received
  17 Mar 2000
• Accepted
  02 Feb 2001