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Brucella infection in fresh water fish: Evidence for natural infection of Nile catfish, Clarias gariepinus, with Brucella melitensis
Veterinary Microbiology 141 (2010) 321–325
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Brucella infection in fresh water fish: Evidence for natural infection of Nile catfish, Clarias gariepinus, with Brucella melitensis Wael F. El-Tras a, Ahmed A. Tayel b, Mahmoud M. Eltholth a,c,*, Javier Guitian c a
Department of Hygiene and Preventive Medicine (Zoonoses), Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt Genetic Engineering and Biotechnology Research Institute, Menufiya University, Egypt c Department of Veterinary Clinical Sciences, Royal Veterinary College, London, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2009 Received in revised form 17 September 2009 Accepted 22 September 2009
Brucellosis is endemic among ruminants in the Nile Delta region of Egypt, where recent reports suggest that the incidence of human infection is increasing. In this region the practice of throwing animal waste into Nile canals is common. As a result, water can be contaminated with potential zoonotic pathogens such as B. melitensis that could infect fish. This study aimed at isolating and characterizing B. melitensis from Nile catfish. Serum samples from 120 catfish captured from Nile canals and 120 farmed catfish were tested for the presence of antibodies against Brucella spp. by using the Rose Bengal Test (RBT) and the Rivanol test (Riv T). Skin swabs from all fish and samples from internal organs (liver, kidney and spleen) from all serologically positive fish were cultured to identify B. melitensis biovar 3 isolates. Polymerase Chain Reaction (PCR) was used to confirm the results. 9.2% and 8.3% of serum samples from Nile catfish were positive by RBT and Riv T, respectively. None of the samples from farmed catfish were seropositive. B. melitensis biovar 3 was isolated from 5.8%, 4.2%, 5.8% and 13.3% of liver, kidney and spleen samples and skin swabs, respectively. To our knowledge this is the first report of isolation of B. melitensis biovar 3 from fresh water fish. Our results suggest that Nile catfish are naturally infected with B. melitensis biovar 3 and this may play a role in the epidemiology of brucellosis. The public should be aware of the consequences of disposing of animal waste into the canals and public health authorities should consider the potential role of catfish as a source of infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Brucellosis Catfish Zoonosis
1. Introduction Brucellosis is a zoonotic disease of mammals, caused by species of the genus Brucella (Moreno et al., 2002). For several decades it has been recognized as a significant public health problem in the Middle East and recent reports suggest that its incidence is increasing in both, ruminants and humans (Benkirane, 2006; Refai, 2002) and
* Corresponding author at: Department of Veterinary Clinical Sciences, Veterinary Epidemiology and Public Health Group, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield AL9 7TA, UK. E-mail address: [email protected] (M.M. Eltholth). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.017
that currently applied control measures may not be capable of reducing the levels of infection in ruminants (Hegazy et al., 2009). In Egypt, Brucella melitensis biovar 3 is considered to be the predominant species of Brucella isolated from humans and animals (Refai, 2002). Ruminant brucellosis can cause abortion, weak offspring, infertility, loss of milk production, and has been responsible for major economic losses (Radostits et al., 2000). The main routes for human infection are consumption of contaminated milk and raw dairy products and direct contact with infected ruminants (Kiel and Khan, 1993; Bilal et al., 1991; Namiduru et al., 2003; Almuneef et al., 2004; Jennings et al., 2007). International guidelines for the prevention of human brucellosis such as those issued by the World
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Health Organization emphasize public education to avoid consumption of unpasteurized milk and dairy products, personal hygiene for at risk occupational groups (farmers, butchers, veterinarians) and precautions during handling and disposal of placenta, discharges and fetuses from aborted animals (Corbel, 2006). Despite the preeminent role of ruminants as a source of human infection, other sources cannot be ruled out since it would be feasible that humans become infected via direct contact or consumption of tissues from infected non-ruminant species. Several Brucella species have been isolated from domestic animals, wildlife and marine mammals (Corbel and Brinley-Morgan, 1984; Godfroid, 2002). Gelev and Gelev (1988) isolated a pathogenic bacterium antigenically related with classical Brucella (S form) from trout in Bulgaria, but 2 years later this pathogen was identified as Hafnia alvei (Gelev et al., 1990). Salem and Mohsen (1997) experimentally inoculated 15 Nile catfish with B. melitensis biovar 3 and then sacrificed 3 fish weekly for 5 weeks. Fourteen catfish were seropositive and 9 were positive for bacteriological examination in which Brucella organisms were isolated from liver and spleen. Although this study did not prove that the infecting strain was virulent and capable of producing disease in catfish, it demonstrates that Nile catfish may be experimentally infected by B. melitensis biovar 3 and points at the possibility of catfish becoming potential vectors of transmission in natural systems. The aim of this work was to investigate natural infection of catfish with Brucella organisms via isolation and characterization of this pathogen from wild and farmed catfish captured from the Nile Delta region of Egypt. 2. Materials and methods 2.1. Fish samples Between February and May 2008 a total of 120 live catfish were collected from 17 sites in small tributaries of Nile canals in the governorates of Kafrelsheikh, Menufiya, Gharbiya and Dekahliya in the Nile Delta region, Egypt. Fish were collected at each site with the help of local fishermen using traditional fishing methods. Another 120 live catfish were collected from 7 fish farms from Kafrelsheikh, Elbehera and Dekahliya governorates and unlikely to be exposed to water contaminated by carcasses and other contaminated animal materials. Only adult fish that had reached market weight (between 600 and 800 g) were collected. Skin swabs were obtained immediately after catching the fish by gentle rubbing of fish surface with a dry sterile cotton–wool swab stick. Fish were transported alive and individually separated in 30 l sterile portable tanks to the diagnostic laboratory of the Department of Hygiene and Preventive Medicine, College of Veterinary Medicine, Kafrelsheikh University for bacteriological examination. Blood samples were obtained from the caudal blood vessels with a sterile syringe after anaesthetizing the fish with 0.4 g/l tricaine methanesulfonate (MS-222, Crescent Research Chemicals, Phoenix, AZ, USA) and 0.8 g/l NaHCO3. Blood samples were allowed to clot overnight at 4 8C, then
centrifuged for 10 min at 5000 g to obtain the serum. Serum samples were kept in polyethylene Eppendorf test tubes at 20 8C until serological examination. Necropsy was carried out immediately after blood sampling and specimens of internal organs (liver, kidney and spleen) were obtained under aseptic technique, macerated separately in sterile phosphate buffered saline (pH 7.2) and inoculated onto Brucella medium base (CMO 169, OXOID, UK). Once the results of the serological tests were available, samples from seronegative fish were excluded from subsequent tests. 2.2. Serological examination 2.2.1. Rose Bengal Test Equal amounts of serum sample and Rose Bengal Antigen (30 ml each) were mixed on a white enamel plate. The mixture was agitated gently on a rocker for 4 min. Any visible agglutination was considered to be positive (Alton et al., 1988; OIE, 2004). Positive and negative control sera and Rose Bengal Antigen (Brucella abortus strain 99) were obtained from the Veterinary Serum and Vaccine Research Institute, Cairo, Egypt. 2.2.2. Rivanol test The Rivanol Brucella Antigen and Rivanol Solution were obtained from the Veterinary Serum and Vaccine Research Institute, Cairo, Egypt. The test was carried out by mixing 400 ml of serum sample with equal volume of rivanol solution and shaken in a test tube, then allowed to stand for 5–60 min, and then centrifuged for 5 min, at 1000 g. 30 ml of rivanol antigen was mixed with 80 ml, 40 ml, 20 ml and 10 ml of the supernatant to obtain 1:25, 1:50, 1:100 and 1:200 dilutions. Plates were rotated and kept for 6 min under cover to prevent evaporation, 6 min later the plates were rotated again. Complete agglutination at 1:25 was considered positive (Quinn et al., 1994). 2.3. Bacteriological analysis Primary isolation was carried out by inoculating samples on Brucella medium base (CMO 169, OXOID, UK) and Brucella selective supplement (SR 0083, OXOID, UK). All inoculated plates were incubated under 5–10% CO2 at 37 8C for up to 2 weeks. The initial identification of Brucella organisms was by colony morphology, Gram stain and modified Ziel–Neelsen’s stain. To identify Brucella isolate biotypes, the following biochemical tests were applied; aerobic growth without CO2, H2S production, urease production, growth on thionin and basic fuschin, agglutination with Brucella anti-sera A and M and lysis by Tbilisi (Tb) phage (Alton et al., 1988; OIE, 2004; Quinn et al., 1994). 2.4. Bacterial DNA extraction All isolated bacterial strains which were identified as B. melitensis biovar 3 by bacteriological analysis were subjected to DNA extraction and subsequent polymerase chain reaction (PCR) amplification for confirmation as Brucella. Bacterial DNA was extracted by Genomic DNA
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Table 1 Results of culture and PCR methods for identification of B. melitensis biotypes from catfish samples. PCR method
Culture method
Total number of samplesa
Type of samplesb
Source of Catfish
%
No.
%
No.
5.0 4.2 5.0 13.3
6 5 6 16
5.8 4.2 5.8 15.8
7 5 7 19
11 11 120 120
Liver Kidney Spleen Skin swab
Nile canals
0
120
Skin swab
Fish farms
0 a b
0
0
Only liver, kidney and spleen samples from seropositive fish were tested. Skin swabs were tested for all fish, seropositive and seronegative. Organs from seropositive catfish and skin swabs from all fish.
purification kit (Puregene Genetra, MN, USA) according to the manufacturer instructions. 2.5. Primers used for PCR amplification Brucella-specific primer pairs were designed to amplify a fragment of the Brucella omp2 gene (Bardenstein et al., 2002). These primers were synthesized at AGERI (Cairo, Egypt) on an ABI 392 DNA/RNA synthesizer (Applied Biosystems). The sequence of the forward primer was P1 50 -TGGAGGTCAGAAATGAAC-30 and for reverse was P2 50 GAGTGCGAAACGAGCGC-30 2.6. Polymerase chain reaction (PCR) PCR amplification was performed according to Mullis and Faloona (1987). Following hot start treatment at 95 8C for 3 min, PCR was performed with an Eppendorf Thermocycler (Eppendorf, Hamburg, Germany) as follows: 35 cycles of PCR, with 1 cycle consisting of 20 s at 95 8C for DNA denaturation, 1 min at 50 8C for DNA annealing, and 1 min at 72 8C for polymerase-mediated primer extension. The last cycle included incubation of the sample at 72 8C for 7 min. PCR products were digested by using the PstI restriction enzyme (Boehringer GmbH, Mannheim, Germany). Ten microliters of the amplified product was analyzed by electrophoresis in ethidium bromide stained 1.5% agarose gel in TEA buffer. The molecular weights were estimated using a PCR marker with molecular size of 50– 2000 base pair (bp), (Sigma–Aldrich Product No. P9577) then photographed using a Polaroid camera. PCR was carried out in the Genetic Engineering and Biotechnology Research Institute, El Sadat City, Menufiya University, Egypt. 3. Results Among Nile catfish, 11 (9.2%) were seropositive against Brucella spp. by RBT and 10 (8.3%) by Riv T. The frequency of agreement for the two tests was 99.1%; 10 samples (8.3%) were positive by both tests, 109 samples (90.8%) were negative by both tests. One positive sample by RBT was negative using Riv T. All samples from farmed catfish were negative for both tests. Positive PCR results were obtained for 16 (13.3%) skin swabs and 6 (5.0%), 5 (4.2%) and 6 (5.0%) liver, kidney and spleen samples respectively. No Brucella spp. were isolated from skin swabs from the fish farms. All seropositive catfish samples and those with infected internal organs were found to have positive skin
swabs but not all fish with positive skin swabs were seropositive; in one of the sites (Bletag-Gharbia), all catfish with positive skin swabs were seronegative. The bacteriological and PCR results are summarized in Table 1. All catfish with isolates from internal organs had congested (dark red-colored) liver and spleen. Out of the 16 catfish with isolates from skin swabs, 5 had multiple focal surface lesions which were not pathologically investigated further. Skin lesions were only observed in seropositive fish with positive skin swabs. Following PCR product digestion with PstI, the appearance of the omp2 gene fragment with molecular weights of 282, 238 and 44 bp (Fig. 1) corresponded to the profile previously reported for the B. melitensis reference strain 16 M and isolates of the vaccine strain Rev1 examined by Bardenstein et al. (2002). 4. Discussion Although brucellosis has been reported in humans, domestic animals, wild animals and marine animals (Corbel and Brinley-Morgan, 1984; Godfroid, 2002), to our knowledge this is the first report that confirms the presence of Brucella in fresh water fish in a natural system. Freshwater fish have been known as a symptomatic carrier and vector for several potential zoonotic bacterial pathogens such as Aeromonas spp. (Palumbo et al., 1989), Vibrio spp. (Lehane and Rawlin, 2000), some species of the family Enterobacteriacae such as Edwardsiella spp. (Meyer and Bullock, 1973), Mycobacterium spp. (Ho et al., 2006), Streptococcus iniae (Weinstein et al., 1997) and Erysiplothrix rhusiopathiae (Gorby and Peacock, 1988). In relation to Brucella, to our knowledge, the only available evidence of fish infection is the study by Salem and Mohsen (1997), which suggested that Nile catfish is susceptible. Our results demonstrate that Nile catfish can be naturally infected by B. melitensis biovar 3 and suggest that catfish should be considered as a possible reservoir for B. melitensis biovar 3 and may have a role in the epidemiology of the disease in the Nile Delta region. Although the goal of our study was not to estimate the frequency of Brucella infection in catfish in the region, the detection of B. melitensis biovar 3 from 13.3% of skin swabs and from around 5%, of internal organs sampled from fish from four different sites suggests the level of exposure in the Nile canals to B. melitensis biovar 3 is considerable. The finding of a higher number of positive samples by bacterial culture than by PCR may reflect poor quality DNA extractions from some samples or the existence of some, to date unreported, diversity encom-
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(Lowry and Smith, 2007), our results suggest that in the studied region, contact with Nile catfish may be a source of human infection. It seems plausible that humans could become infected with B. melitensis via similar routes to other pathogens such as Edwardsiella tarda, the causative agent of emphysematous putrefactive disease in catfish (Vandepitte et al., 1983); humans could be infected through puncture wounds during handing of fish or by contamination of existing cuts and abrasions. B. melitensis should then be considered among the pathogens potentially transmitted to humans by fish and the risk of transmission may be considerable in the Nile delta region.
Fig. 1. PCR detection of B. melitensis from Nile catfish samples (Lanes 1– 10), by comparison of characteristic 282 and 44 bp bands to a 50–2000 bp PCR size marker (Lane M).
passing the binding sites for the primers used in this study. The major difference between the two bodies of water is that water in fish farms is less likely to be contaminated as a result of direct access of livestock or disposal of carcases and contaminated materials (aborted feti, fetal membranes and placenta). It is known from the field experience of the authors of this work that these practices are not uncommon in the Nile Delta region and previous studies have shown that fish can be infected with bacteria in polluted aquatic environments (Geldreich and Clarke, 1966; Guzman et al., 2004; Pal and Dasgupta, 1992). The negative results obtained for catfish captured from fish farms in the same area may be attributed to the above reasons. Salem and Mohsen (1997) observed skin lesions in catfish experimentally inoculated with 105 cells of B. melitensis biovar 3. The skin lesions observed in the present study may also be attributable to infection with B. melitensis biovar 3. Catfish infection with B. melitensis biovar 3 could be the result of skin penetration or ingestion of contaminated materials in the water canal. Following subcutaneous inoculation of Nile catfish with B. melitensis, Salem and Mohsen (1997) detected serological response and isolated the pathogen from internal organs from seropositive fish. The evidence presented here strongly suggests, for the first time, a link between brucellosis in freshwater fish and brucellosis in other species. B. melitensis biovar 3 is the main cause of brucellosis in ruminants and humans in Egypt (Refai, 2002). It seems plausible that Nile catfish are naturally infected by contaminated materials from infected ruminants that reach the water canals. It has always been assumed that the reason for the high incidence of human brucellosis in the region is the contact with exposed and infected ruminants and the consumption of some of their products such as raw milk and raw milk products (Kiel and Khan, 1993; Bilal et al., 1991; Namiduru et al., 2003; Almuneef et al., 2004; Jennings et al., 2007). Our results indicate that direct contact with infected catfish may also be a source of human infection. Given the zoonotic potential of B. melitensis (Seleem et al., in press) and the potential of fish to act as healthy carriers that transmit pathogens to humans by different routes
References Almuneef, M.A., Memish, Z.A., Balkhy, H.H., Alotaibi, B., Algoda, S., Abbas, M., Alsubaie, S., 2004. Importance of screening household members of acute brucellosis cases in endemic areas. Epidemiol. Infect. 132, 533– 540. Alton, G.G., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the Brucella Laboratory. INRA, Paris. Bardenstein, S.M., Mandelboim, T.A., Ficht, M.B., Banai, M., 2002. Identification of the Brucella melitensis vaccine strain Rev. 1 in animals and humans in Israel by PCR analysis of the Pst I site polymorphism of its omp2 gene. J. Clin. Microbiol. 40, 1475–1480. Benkirane, A., 2006. Ovine and caprine brucellosis: World distribution and control/eradication strategies in West Asia/North Africa region. Small Rumin. Res. 62, 19–25. Bilal, N., Ghazi, J., Raymond, A.B., Olfat, F.M., Nariman, M.E., 1991. Brucellosis in Asia region of Saudi arabia. Saudi Med. J. 12, 37–41. Corbel, M.J. 2006. Brucellosis in humans and animals: Produced by the World Health Organization in collaboration with the Food and Agriculture Organization of the United Nations and World Organisation for Animal Health. Corbel, M.J., Brinley-Morgan, W.J., 1984. Genus Brucella. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, pp. 377–388. Geldreich, E.E., Clarke, N.A., 1966. Bacterial pollution indicators in the intestinal tract of freshwater fish. Appl. Microbiol. 14, 429–437. Gelev, I., Gelev, E., 1988. A new species of fish-pathogenic bacterium antigenically related to classical Brucellae. Zbl. Bakt. Hyg. 269, 1–6. Gelev, I., Gelev, E., Steigerwalt, A.G., Carter, G.P., Brenner, D.J., 1990. Identification of the bacterium associated with haemorrhagic septicaemia in rainbow trout as Hafnia alvei. Res. Microbiol. 141, 573–576. Godfroid, J., 2002. Brucellosis in wildlife. Rev. Sci. Tech. Off. Int. Epiz. 21, 277–286. Gorby, G., Peacock, J., 1988. Erysipelothrix rhusiopathiae endocarditis: microbiologic, epidemiologic, and clinical features of an occupational disease. Rev. Infect. Dis. 10, 317–325. Guzman, M.C., Bistoni, Mde.L., Tamagnini, L.M., Gonzalez, R.D., 2004. Recovery of Escherichia coli in fresh water fish, Jenynsia multidentata and Bryconamericus iheringi. Water Res. 38, 2367–2373. Hegazy, Y.M., Ridler, A.L., Guitian, F.J., 2009. Assessment and simulation of the implementation of brucellosis control program in an endemic area of the Middle East. Epidemiol. Infect. 137, 1436–1448. Ho, M.H., Ho, C.K., Chong, L.Y., 2006. Atypical mycobacterial cutaneous infections in Hong Kong: 10-year retrospective study. Hong Kong Med. J. 12, 21–26. Jennings, G.J., Hajjeh, R.A., Girgis, F.Y., Fadeel, M.A., Maksoud, M.A., Wasfy, M.O., Sayed, N.E., Srikantiah, P., Luby, S.P., Earhart, K., Mahoney, F.J., 2007. Brucellosis as a cause of acute febrile illness in Egypt. Trans. R. Soc. Trop. Med. Hyg. 101, 707–713. Kiel, F.W., Khan, M.Y., 1993. Brucellosis among hospital employees in Saudi Arabia. Infect. Control Hosp. Epidemiol. 14, 268–272. Lehane, L., Rawlin, G.T., 2000. Topically acquired bacterial zoonoses from fish: a review. Med. J. Aust. 173, 256–259. Lowry, T., Smith, S.A., 2007. Aquatic zoonoses associated with food, bait, ornamental, and tropical fish. J. Am. Vet. Med. Assoc. 231, 876–880. Meyer, F.P., Bullock, G.L., 1973. Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus punctatus). Appl. Microbiol. 25, 155–156. Moreno, E., Cloeckaert, A., Moriyo´n, I., 2002. Brucella evolution and taxonomy. Vet. Microbiol. 90, 209–227. Mullis, K.B., Faloona, F.A., 1987. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335– 350.
W.F. El-Tras et al. / Veterinary Microbiology 141 (2010) 321–325 Namiduru, M., Gungor, K., Dikensoy, O., Baydar, I., Ekinci, E., Karaoglan, I., Bekir, N.A., 2003. Epidemiological, clinical and laboratory features of brucellosis: a prospective evaluation of 120 adult patients. Int. J. Clin. Pract. 57, 20–24. OIE, 2004. Bovine brucellosis. In: Manual of the Diagnostic Tests and Vaccines for Terrestial Animals, Office International Des Epizooties, Paris, France, pp. 409–438. Pal, D., Dasgupta, C.H., 1992. Microbial pollution in water and its effect on fish. J. Aquat. Anim. Health. 4, 32–39. Palumbo, S.A., Bencivengo, M.M., Del Corral, F., Williams, A.C., Buchanan, R.L., 1989. Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin. J. Clin. Microbiol. 27, 854–859. Quinn, P.J., Markey, B.K., Carter, G.R., 1994. Clinical Veterinary Microbiology. Mosby, London, pp. 262–267.
325
Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Veterinary Medicine, 9th ed. ELBS Bailliere Tindall, London, UK, pp. 870– 871. Refai, M., 2002. Incidence and control of brucellosis in the Near East region. Vet. Microbiol. 90, 81–110. Salem, S.F., Mohsen, A., 1997. Brucellosis in fish. Vet. Med-Czech 42, 5–7. Seleem, M.N., Boyle, S.M., Sriranganathan, N. Brucellosis: A re-emerging zoonosis. Vet. Microbiol., in press. Vandepitte, J., Lemmens, P., de Swert, L., 1983. Human edwardsiellosis traced to ornamental fish. J. Clin. Microbiol. 17, 165–167. Weinstein, M.R., Litt, M., Kertesz, D.A., Wyper, P., Rose, D., Coulter, M., McGeer, A., Facklam, R., Ostach, C., Willey, B.M., Borczyk, A., Low, D.E., 1997. Invasive infections due to a fish pathogen, Streptococcus iniae. S. iniae study group. N Engl. J. Med. 337, 589–594.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Brucella infection in fresh water fish: Evidence for natural infection of Nile catfish, Clarias gariepinus, with Brucella melitensis Wael F. El-Tras a, Ahmed A. Tayel b, Mahmoud M. Eltholth a,c,*, Javier Guitian c a
Department of Hygiene and Preventive Medicine (Zoonoses), Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt Genetic Engineering and Biotechnology Research Institute, Menufiya University, Egypt c Department of Veterinary Clinical Sciences, Royal Veterinary College, London, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2009 Received in revised form 17 September 2009 Accepted 22 September 2009
Brucellosis is endemic among ruminants in the Nile Delta region of Egypt, where recent reports suggest that the incidence of human infection is increasing. In this region the practice of throwing animal waste into Nile canals is common. As a result, water can be contaminated with potential zoonotic pathogens such as B. melitensis that could infect fish. This study aimed at isolating and characterizing B. melitensis from Nile catfish. Serum samples from 120 catfish captured from Nile canals and 120 farmed catfish were tested for the presence of antibodies against Brucella spp. by using the Rose Bengal Test (RBT) and the Rivanol test (Riv T). Skin swabs from all fish and samples from internal organs (liver, kidney and spleen) from all serologically positive fish were cultured to identify B. melitensis biovar 3 isolates. Polymerase Chain Reaction (PCR) was used to confirm the results. 9.2% and 8.3% of serum samples from Nile catfish were positive by RBT and Riv T, respectively. None of the samples from farmed catfish were seropositive. B. melitensis biovar 3 was isolated from 5.8%, 4.2%, 5.8% and 13.3% of liver, kidney and spleen samples and skin swabs, respectively. To our knowledge this is the first report of isolation of B. melitensis biovar 3 from fresh water fish. Our results suggest that Nile catfish are naturally infected with B. melitensis biovar 3 and this may play a role in the epidemiology of brucellosis. The public should be aware of the consequences of disposing of animal waste into the canals and public health authorities should consider the potential role of catfish as a source of infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Brucellosis Catfish Zoonosis
1. Introduction Brucellosis is a zoonotic disease of mammals, caused by species of the genus Brucella (Moreno et al., 2002). For several decades it has been recognized as a significant public health problem in the Middle East and recent reports suggest that its incidence is increasing in both, ruminants and humans (Benkirane, 2006; Refai, 2002) and
* Corresponding author at: Department of Veterinary Clinical Sciences, Veterinary Epidemiology and Public Health Group, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield AL9 7TA, UK. E-mail address: [email protected] (M.M. Eltholth). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.017
that currently applied control measures may not be capable of reducing the levels of infection in ruminants (Hegazy et al., 2009). In Egypt, Brucella melitensis biovar 3 is considered to be the predominant species of Brucella isolated from humans and animals (Refai, 2002). Ruminant brucellosis can cause abortion, weak offspring, infertility, loss of milk production, and has been responsible for major economic losses (Radostits et al., 2000). The main routes for human infection are consumption of contaminated milk and raw dairy products and direct contact with infected ruminants (Kiel and Khan, 1993; Bilal et al., 1991; Namiduru et al., 2003; Almuneef et al., 2004; Jennings et al., 2007). International guidelines for the prevention of human brucellosis such as those issued by the World
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Health Organization emphasize public education to avoid consumption of unpasteurized milk and dairy products, personal hygiene for at risk occupational groups (farmers, butchers, veterinarians) and precautions during handling and disposal of placenta, discharges and fetuses from aborted animals (Corbel, 2006). Despite the preeminent role of ruminants as a source of human infection, other sources cannot be ruled out since it would be feasible that humans become infected via direct contact or consumption of tissues from infected non-ruminant species. Several Brucella species have been isolated from domestic animals, wildlife and marine mammals (Corbel and Brinley-Morgan, 1984; Godfroid, 2002). Gelev and Gelev (1988) isolated a pathogenic bacterium antigenically related with classical Brucella (S form) from trout in Bulgaria, but 2 years later this pathogen was identified as Hafnia alvei (Gelev et al., 1990). Salem and Mohsen (1997) experimentally inoculated 15 Nile catfish with B. melitensis biovar 3 and then sacrificed 3 fish weekly for 5 weeks. Fourteen catfish were seropositive and 9 were positive for bacteriological examination in which Brucella organisms were isolated from liver and spleen. Although this study did not prove that the infecting strain was virulent and capable of producing disease in catfish, it demonstrates that Nile catfish may be experimentally infected by B. melitensis biovar 3 and points at the possibility of catfish becoming potential vectors of transmission in natural systems. The aim of this work was to investigate natural infection of catfish with Brucella organisms via isolation and characterization of this pathogen from wild and farmed catfish captured from the Nile Delta region of Egypt. 2. Materials and methods 2.1. Fish samples Between February and May 2008 a total of 120 live catfish were collected from 17 sites in small tributaries of Nile canals in the governorates of Kafrelsheikh, Menufiya, Gharbiya and Dekahliya in the Nile Delta region, Egypt. Fish were collected at each site with the help of local fishermen using traditional fishing methods. Another 120 live catfish were collected from 7 fish farms from Kafrelsheikh, Elbehera and Dekahliya governorates and unlikely to be exposed to water contaminated by carcasses and other contaminated animal materials. Only adult fish that had reached market weight (between 600 and 800 g) were collected. Skin swabs were obtained immediately after catching the fish by gentle rubbing of fish surface with a dry sterile cotton–wool swab stick. Fish were transported alive and individually separated in 30 l sterile portable tanks to the diagnostic laboratory of the Department of Hygiene and Preventive Medicine, College of Veterinary Medicine, Kafrelsheikh University for bacteriological examination. Blood samples were obtained from the caudal blood vessels with a sterile syringe after anaesthetizing the fish with 0.4 g/l tricaine methanesulfonate (MS-222, Crescent Research Chemicals, Phoenix, AZ, USA) and 0.8 g/l NaHCO3. Blood samples were allowed to clot overnight at 4 8C, then
centrifuged for 10 min at 5000 g to obtain the serum. Serum samples were kept in polyethylene Eppendorf test tubes at 20 8C until serological examination. Necropsy was carried out immediately after blood sampling and specimens of internal organs (liver, kidney and spleen) were obtained under aseptic technique, macerated separately in sterile phosphate buffered saline (pH 7.2) and inoculated onto Brucella medium base (CMO 169, OXOID, UK). Once the results of the serological tests were available, samples from seronegative fish were excluded from subsequent tests. 2.2. Serological examination 2.2.1. Rose Bengal Test Equal amounts of serum sample and Rose Bengal Antigen (30 ml each) were mixed on a white enamel plate. The mixture was agitated gently on a rocker for 4 min. Any visible agglutination was considered to be positive (Alton et al., 1988; OIE, 2004). Positive and negative control sera and Rose Bengal Antigen (Brucella abortus strain 99) were obtained from the Veterinary Serum and Vaccine Research Institute, Cairo, Egypt. 2.2.2. Rivanol test The Rivanol Brucella Antigen and Rivanol Solution were obtained from the Veterinary Serum and Vaccine Research Institute, Cairo, Egypt. The test was carried out by mixing 400 ml of serum sample with equal volume of rivanol solution and shaken in a test tube, then allowed to stand for 5–60 min, and then centrifuged for 5 min, at 1000 g. 30 ml of rivanol antigen was mixed with 80 ml, 40 ml, 20 ml and 10 ml of the supernatant to obtain 1:25, 1:50, 1:100 and 1:200 dilutions. Plates were rotated and kept for 6 min under cover to prevent evaporation, 6 min later the plates were rotated again. Complete agglutination at 1:25 was considered positive (Quinn et al., 1994). 2.3. Bacteriological analysis Primary isolation was carried out by inoculating samples on Brucella medium base (CMO 169, OXOID, UK) and Brucella selective supplement (SR 0083, OXOID, UK). All inoculated plates were incubated under 5–10% CO2 at 37 8C for up to 2 weeks. The initial identification of Brucella organisms was by colony morphology, Gram stain and modified Ziel–Neelsen’s stain. To identify Brucella isolate biotypes, the following biochemical tests were applied; aerobic growth without CO2, H2S production, urease production, growth on thionin and basic fuschin, agglutination with Brucella anti-sera A and M and lysis by Tbilisi (Tb) phage (Alton et al., 1988; OIE, 2004; Quinn et al., 1994). 2.4. Bacterial DNA extraction All isolated bacterial strains which were identified as B. melitensis biovar 3 by bacteriological analysis were subjected to DNA extraction and subsequent polymerase chain reaction (PCR) amplification for confirmation as Brucella. Bacterial DNA was extracted by Genomic DNA
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Table 1 Results of culture and PCR methods for identification of B. melitensis biotypes from catfish samples. PCR method
Culture method
Total number of samplesa
Type of samplesb
Source of Catfish
%
No.
%
No.
5.0 4.2 5.0 13.3
6 5 6 16
5.8 4.2 5.8 15.8
7 5 7 19
11 11 120 120
Liver Kidney Spleen Skin swab
Nile canals
0
120
Skin swab
Fish farms
0 a b
0
0
Only liver, kidney and spleen samples from seropositive fish were tested. Skin swabs were tested for all fish, seropositive and seronegative. Organs from seropositive catfish and skin swabs from all fish.
purification kit (Puregene Genetra, MN, USA) according to the manufacturer instructions. 2.5. Primers used for PCR amplification Brucella-specific primer pairs were designed to amplify a fragment of the Brucella omp2 gene (Bardenstein et al., 2002). These primers were synthesized at AGERI (Cairo, Egypt) on an ABI 392 DNA/RNA synthesizer (Applied Biosystems). The sequence of the forward primer was P1 50 -TGGAGGTCAGAAATGAAC-30 and for reverse was P2 50 GAGTGCGAAACGAGCGC-30 2.6. Polymerase chain reaction (PCR) PCR amplification was performed according to Mullis and Faloona (1987). Following hot start treatment at 95 8C for 3 min, PCR was performed with an Eppendorf Thermocycler (Eppendorf, Hamburg, Germany) as follows: 35 cycles of PCR, with 1 cycle consisting of 20 s at 95 8C for DNA denaturation, 1 min at 50 8C for DNA annealing, and 1 min at 72 8C for polymerase-mediated primer extension. The last cycle included incubation of the sample at 72 8C for 7 min. PCR products were digested by using the PstI restriction enzyme (Boehringer GmbH, Mannheim, Germany). Ten microliters of the amplified product was analyzed by electrophoresis in ethidium bromide stained 1.5% agarose gel in TEA buffer. The molecular weights were estimated using a PCR marker with molecular size of 50– 2000 base pair (bp), (Sigma–Aldrich Product No. P9577) then photographed using a Polaroid camera. PCR was carried out in the Genetic Engineering and Biotechnology Research Institute, El Sadat City, Menufiya University, Egypt. 3. Results Among Nile catfish, 11 (9.2%) were seropositive against Brucella spp. by RBT and 10 (8.3%) by Riv T. The frequency of agreement for the two tests was 99.1%; 10 samples (8.3%) were positive by both tests, 109 samples (90.8%) were negative by both tests. One positive sample by RBT was negative using Riv T. All samples from farmed catfish were negative for both tests. Positive PCR results were obtained for 16 (13.3%) skin swabs and 6 (5.0%), 5 (4.2%) and 6 (5.0%) liver, kidney and spleen samples respectively. No Brucella spp. were isolated from skin swabs from the fish farms. All seropositive catfish samples and those with infected internal organs were found to have positive skin
swabs but not all fish with positive skin swabs were seropositive; in one of the sites (Bletag-Gharbia), all catfish with positive skin swabs were seronegative. The bacteriological and PCR results are summarized in Table 1. All catfish with isolates from internal organs had congested (dark red-colored) liver and spleen. Out of the 16 catfish with isolates from skin swabs, 5 had multiple focal surface lesions which were not pathologically investigated further. Skin lesions were only observed in seropositive fish with positive skin swabs. Following PCR product digestion with PstI, the appearance of the omp2 gene fragment with molecular weights of 282, 238 and 44 bp (Fig. 1) corresponded to the profile previously reported for the B. melitensis reference strain 16 M and isolates of the vaccine strain Rev1 examined by Bardenstein et al. (2002). 4. Discussion Although brucellosis has been reported in humans, domestic animals, wild animals and marine animals (Corbel and Brinley-Morgan, 1984; Godfroid, 2002), to our knowledge this is the first report that confirms the presence of Brucella in fresh water fish in a natural system. Freshwater fish have been known as a symptomatic carrier and vector for several potential zoonotic bacterial pathogens such as Aeromonas spp. (Palumbo et al., 1989), Vibrio spp. (Lehane and Rawlin, 2000), some species of the family Enterobacteriacae such as Edwardsiella spp. (Meyer and Bullock, 1973), Mycobacterium spp. (Ho et al., 2006), Streptococcus iniae (Weinstein et al., 1997) and Erysiplothrix rhusiopathiae (Gorby and Peacock, 1988). In relation to Brucella, to our knowledge, the only available evidence of fish infection is the study by Salem and Mohsen (1997), which suggested that Nile catfish is susceptible. Our results demonstrate that Nile catfish can be naturally infected by B. melitensis biovar 3 and suggest that catfish should be considered as a possible reservoir for B. melitensis biovar 3 and may have a role in the epidemiology of the disease in the Nile Delta region. Although the goal of our study was not to estimate the frequency of Brucella infection in catfish in the region, the detection of B. melitensis biovar 3 from 13.3% of skin swabs and from around 5%, of internal organs sampled from fish from four different sites suggests the level of exposure in the Nile canals to B. melitensis biovar 3 is considerable. The finding of a higher number of positive samples by bacterial culture than by PCR may reflect poor quality DNA extractions from some samples or the existence of some, to date unreported, diversity encom-
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(Lowry and Smith, 2007), our results suggest that in the studied region, contact with Nile catfish may be a source of human infection. It seems plausible that humans could become infected with B. melitensis via similar routes to other pathogens such as Edwardsiella tarda, the causative agent of emphysematous putrefactive disease in catfish (Vandepitte et al., 1983); humans could be infected through puncture wounds during handing of fish or by contamination of existing cuts and abrasions. B. melitensis should then be considered among the pathogens potentially transmitted to humans by fish and the risk of transmission may be considerable in the Nile delta region.
Fig. 1. PCR detection of B. melitensis from Nile catfish samples (Lanes 1– 10), by comparison of characteristic 282 and 44 bp bands to a 50–2000 bp PCR size marker (Lane M).
passing the binding sites for the primers used in this study. The major difference between the two bodies of water is that water in fish farms is less likely to be contaminated as a result of direct access of livestock or disposal of carcases and contaminated materials (aborted feti, fetal membranes and placenta). It is known from the field experience of the authors of this work that these practices are not uncommon in the Nile Delta region and previous studies have shown that fish can be infected with bacteria in polluted aquatic environments (Geldreich and Clarke, 1966; Guzman et al., 2004; Pal and Dasgupta, 1992). The negative results obtained for catfish captured from fish farms in the same area may be attributed to the above reasons. Salem and Mohsen (1997) observed skin lesions in catfish experimentally inoculated with 105 cells of B. melitensis biovar 3. The skin lesions observed in the present study may also be attributable to infection with B. melitensis biovar 3. Catfish infection with B. melitensis biovar 3 could be the result of skin penetration or ingestion of contaminated materials in the water canal. Following subcutaneous inoculation of Nile catfish with B. melitensis, Salem and Mohsen (1997) detected serological response and isolated the pathogen from internal organs from seropositive fish. The evidence presented here strongly suggests, for the first time, a link between brucellosis in freshwater fish and brucellosis in other species. B. melitensis biovar 3 is the main cause of brucellosis in ruminants and humans in Egypt (Refai, 2002). It seems plausible that Nile catfish are naturally infected by contaminated materials from infected ruminants that reach the water canals. It has always been assumed that the reason for the high incidence of human brucellosis in the region is the contact with exposed and infected ruminants and the consumption of some of their products such as raw milk and raw milk products (Kiel and Khan, 1993; Bilal et al., 1991; Namiduru et al., 2003; Almuneef et al., 2004; Jennings et al., 2007). Our results indicate that direct contact with infected catfish may also be a source of human infection. Given the zoonotic potential of B. melitensis (Seleem et al., in press) and the potential of fish to act as healthy carriers that transmit pathogens to humans by different routes
References Almuneef, M.A., Memish, Z.A., Balkhy, H.H., Alotaibi, B., Algoda, S., Abbas, M., Alsubaie, S., 2004. Importance of screening household members of acute brucellosis cases in endemic areas. Epidemiol. Infect. 132, 533– 540. Alton, G.G., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the Brucella Laboratory. INRA, Paris. Bardenstein, S.M., Mandelboim, T.A., Ficht, M.B., Banai, M., 2002. Identification of the Brucella melitensis vaccine strain Rev. 1 in animals and humans in Israel by PCR analysis of the Pst I site polymorphism of its omp2 gene. J. Clin. Microbiol. 40, 1475–1480. Benkirane, A., 2006. Ovine and caprine brucellosis: World distribution and control/eradication strategies in West Asia/North Africa region. Small Rumin. Res. 62, 19–25. Bilal, N., Ghazi, J., Raymond, A.B., Olfat, F.M., Nariman, M.E., 1991. Brucellosis in Asia region of Saudi arabia. Saudi Med. J. 12, 37–41. Corbel, M.J. 2006. Brucellosis in humans and animals: Produced by the World Health Organization in collaboration with the Food and Agriculture Organization of the United Nations and World Organisation for Animal Health. Corbel, M.J., Brinley-Morgan, W.J., 1984. Genus Brucella. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, pp. 377–388. Geldreich, E.E., Clarke, N.A., 1966. Bacterial pollution indicators in the intestinal tract of freshwater fish. Appl. Microbiol. 14, 429–437. Gelev, I., Gelev, E., 1988. A new species of fish-pathogenic bacterium antigenically related to classical Brucellae. Zbl. Bakt. Hyg. 269, 1–6. Gelev, I., Gelev, E., Steigerwalt, A.G., Carter, G.P., Brenner, D.J., 1990. Identification of the bacterium associated with haemorrhagic septicaemia in rainbow trout as Hafnia alvei. Res. Microbiol. 141, 573–576. Godfroid, J., 2002. Brucellosis in wildlife. Rev. Sci. Tech. Off. Int. Epiz. 21, 277–286. Gorby, G., Peacock, J., 1988. Erysipelothrix rhusiopathiae endocarditis: microbiologic, epidemiologic, and clinical features of an occupational disease. Rev. Infect. Dis. 10, 317–325. Guzman, M.C., Bistoni, Mde.L., Tamagnini, L.M., Gonzalez, R.D., 2004. Recovery of Escherichia coli in fresh water fish, Jenynsia multidentata and Bryconamericus iheringi. Water Res. 38, 2367–2373. Hegazy, Y.M., Ridler, A.L., Guitian, F.J., 2009. Assessment and simulation of the implementation of brucellosis control program in an endemic area of the Middle East. Epidemiol. Infect. 137, 1436–1448. Ho, M.H., Ho, C.K., Chong, L.Y., 2006. Atypical mycobacterial cutaneous infections in Hong Kong: 10-year retrospective study. Hong Kong Med. J. 12, 21–26. Jennings, G.J., Hajjeh, R.A., Girgis, F.Y., Fadeel, M.A., Maksoud, M.A., Wasfy, M.O., Sayed, N.E., Srikantiah, P., Luby, S.P., Earhart, K., Mahoney, F.J., 2007. Brucellosis as a cause of acute febrile illness in Egypt. Trans. R. Soc. Trop. Med. Hyg. 101, 707–713. Kiel, F.W., Khan, M.Y., 1993. Brucellosis among hospital employees in Saudi Arabia. Infect. Control Hosp. Epidemiol. 14, 268–272. Lehane, L., Rawlin, G.T., 2000. Topically acquired bacterial zoonoses from fish: a review. Med. J. Aust. 173, 256–259. Lowry, T., Smith, S.A., 2007. Aquatic zoonoses associated with food, bait, ornamental, and tropical fish. J. Am. Vet. Med. Assoc. 231, 876–880. Meyer, F.P., Bullock, G.L., 1973. Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus punctatus). Appl. Microbiol. 25, 155–156. Moreno, E., Cloeckaert, A., Moriyo´n, I., 2002. Brucella evolution and taxonomy. Vet. Microbiol. 90, 209–227. Mullis, K.B., Faloona, F.A., 1987. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335– 350.
W.F. El-Tras et al. / Veterinary Microbiology 141 (2010) 321–325 Namiduru, M., Gungor, K., Dikensoy, O., Baydar, I., Ekinci, E., Karaoglan, I., Bekir, N.A., 2003. Epidemiological, clinical and laboratory features of brucellosis: a prospective evaluation of 120 adult patients. Int. J. Clin. Pract. 57, 20–24. OIE, 2004. Bovine brucellosis. In: Manual of the Diagnostic Tests and Vaccines for Terrestial Animals, Office International Des Epizooties, Paris, France, pp. 409–438. Pal, D., Dasgupta, C.H., 1992. Microbial pollution in water and its effect on fish. J. Aquat. Anim. Health. 4, 32–39. Palumbo, S.A., Bencivengo, M.M., Del Corral, F., Williams, A.C., Buchanan, R.L., 1989. Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin. J. Clin. Microbiol. 27, 854–859. Quinn, P.J., Markey, B.K., Carter, G.R., 1994. Clinical Veterinary Microbiology. Mosby, London, pp. 262–267.
325
Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Veterinary Medicine, 9th ed. ELBS Bailliere Tindall, London, UK, pp. 870– 871. Refai, M., 2002. Incidence and control of brucellosis in the Near East region. Vet. Microbiol. 90, 81–110. Salem, S.F., Mohsen, A., 1997. Brucellosis in fish. Vet. Med-Czech 42, 5–7. Seleem, M.N., Boyle, S.M., Sriranganathan, N. Brucellosis: A re-emerging zoonosis. Vet. Microbiol., in press. Vandepitte, J., Lemmens, P., de Swert, L., 1983. Human edwardsiellosis traced to ornamental fish. J. Clin. Microbiol. 17, 165–167. Weinstein, M.R., Litt, M., Kertesz, D.A., Wyper, P., Rose, D., Coulter, M., McGeer, A., Facklam, R., Ostach, C., Willey, B.M., Borczyk, A., Low, D.E., 1997. Invasive infections due to a fish pathogen, Streptococcus iniae. S. iniae study group. N Engl. J. Med. 337, 589–594.