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Co-Infection with Capillaria hepatica and Bartonella elizabethae in a Brown Rat (Rattus norvegicus) from Nigeria
Kamani, J.,*1 Akanbi, B.O.,2 Baneth, G.,3 Morrick, D.,3 and Harrus, S.3
1 2
Parasitology Division, National Veterinary Research Institute, PMB 01Vom Plateau State, Nigeria. Central Diagnostic Division, National Veterinary Research Institute, PMB 01Vom Plateau State, Nigeria. 3 Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
* Corresponding author: Dr Joshua Kamani, Parasitology Division, National Veterinary Research Institute, PMB01 Vom, Plateau State, Nigeria. Tel: +2347035517715; +972552237079. Email: shapumani@yahoo.com.
AB ST RAC T
During a survey for Bartonella species in rodents in Vom, Plateau State, Nigeria, a brown rat (Rattus norvegicus) with multiple subcutaneous nodules was captured. Gross pathology examination revealed multifocal pale areas in the spleen and liver. Histopathologic examination of the liver revealed numerous multifocal well demarcated and walled-off granulomas compressing and destroying the hepatic cord and parenchyma. Granulomas contained Capillaria hepatica nematodes in different developmental stages with numerous typical ova. There was degeneration and necrosis of hepatocytes, bridging fibrosis and biliary hyperplasia and reduplication, surrounded by inflammatory foci composed of lymphocytes, plasma cells and macrophages. Tiny smooth bacterial colonies with metallic sheen were isolated from blood of the rat cultured on blood chocolate agar. DNA extracted from the blood and spleen of the rat was found positive for Bartonella spp. citrate synthase gene (gltA) by PCR. Sequence analysis shows 99% similarity to B. elizabethae. The identification of two zoonotic agents in a rat highlights the importance of rats in the transmission cycle of zoonotic diseases in Nigeria. Key words: Rattus norvegicus, Capillaria hepatica, Bartonella elizabethae, Nigeria.
Rodents especially rats (Rattus norvegicus, Rattus rattus, Cricetomyces gambianus) abound in domestic and peri-domestic settings in Nigeria, all year round (1). Their role in the transmission of zoonotic diseases is well documented (2-4). Their role in disease transmission in Nigeria is even greater as rats are being hunted and consumed as a source of protein by some local ethnic groups. In addition, the act of trapping rats, bringing them to the homestead and preparing them for human consumption without proper protective clothing, litters the environment with rat feces and expose humans especially children, who play around contaminated playground, to zoonotic agents. Capillaria hepatica is a worldwide distributed zoonotic nematode
INTRODUCTION
of mammals, primarily infesting the liver of rats (5, 6). Consumption of C. hepatica unembryonated eggs may cause pseudoparasitism in man, with no apparent clinical signs or symptoms of disease. True infection, with liver localization of the parasite and the development of hepatic lesions, requires the ingestion of infective eggs. The contamination of playgrounds by infective eggs can explain the high susceptibility of children to capillariasis (7). Rattus norvegicus have also been found to carry various zoonotic Bartonella species (2, 8-10). In the United States and Portugal, 18% of rodents tested were Bartonella bacteremic, and some isolates from R. norvegicus were closely related to B. elizabethae (9). The latter species was associated with endocarditis in humans (9, 11).
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
230
Kamani, J.
Case Reports
Since rodents are the principal hosts of C. hepatica and also reservoir of Bartonella spp., contact between humans and rodents can be a potential health hazard. There is paucity of information of bartonellosis in man and animals in Nigeria. We present in this report a co-infection of zoonotic agents; C. hepatica and B. elizabethae in a Rattus norvegicus in Nigeria. CASE REPORT
During a survey for bartonellosis in rodents from Vom, Nigeria, one of the rats (Rattus norvegicus) trapped was noticed to be emaciated with poor body condition. Diffuse solitary nodules were present on its body and hard solid masses could be palpated in its abdomen. The rat was humanely euthanized according to the guidelines of the Animal Welfare and Ethics Committee of the National Veterinary Research Institute (NVRI), Vom, Nigeria. Approximately 2ml of blood was collected in sterile ethyelene diamine tetra acetic acid (EDTA) tube and stored at -20oC until further analyzed. The heart, liver, skin and spleen were harvested. Each tissue was divided into two parts. One part was preserved in buffered formalin while the other was placed in a sterile tube and kept at -20oC until further analyzed. Ticks were removed from its body and pooled into a universal bottle containing absolute ethanol. The ticks were identified as Rhipicephalus sanguineus. Tissues in buffered formalin were processed at the Central Diagnostic Division, NVRI Vom. Five µm paraffin-wax sections of organs were dewaxed and stained with hematoxylin-eosin (H&E), mounted on charged microscope slides and observed under a Carl Zeiss light microscope for histopathological changes. The other samples were transported to The Koret school of Veterinary Medicine, Israel, for further analysis. For bacterial culture, 200μl of thawed whole blood sample was plated onto blood chocolate agar. The plate was incubated at 35oC in 5% CO2 and checked every other day for growth of Bartonella species. Suspected colonies were randomly selected and separately sub-cultured onto different fresh agar plates. The resulting pure cultures were stained using Gram stain. DNA was extracted from the spleen, liver, heart and ticks using Illustra tissue and cell genomicPrep miniSpin
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
kit (GE Healthcare, UK, Limited) and from blood using the BiOsticTM Bacteremia DNA Isolation Kit (MO Bio Laboratories, Inc., Carlsbad, CA, USA) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the primers, BhCS871p: (5’-GGGGACCAGCTCATGGTGG-3’) and BhCS1137n: (5’-AATGCAAAAAGAACAGTAAACA-3’) for amplification of a 379 bp fragment of the citrate synthase gene (gltA) of Bartonella spp. Positive and negative controls were included in each PCR run. PCR was performed in 25 μl total volume as follows: 3μl of DNA template, 1μl of 10mM each primer, 1μl Mgcl2, 20μl of ultra pure PCR water. PCR was performed using Syntezza PCR-Ready High Specificity (Syntezza Bioscience, Israel). Amplification was performed using a programmable conventional thermocycler (Biometra, Goettingen, Germany) using the following program parameters: an initial denaturing at 95°C for five minutes, and 35 cycles at 95°C for one minute, 56°C for one minute, and 72°C for one minute. The PCR products were analyzed for the presence of amplicons of the correct size by electrophoresis of 6μl of the products in 1.5% agarose gels containing ethidium bromide. Amplicons of the proper size were identified by comparison to the positive control lane on the agarose gel and comparison to a 50bp DNA molecular ladder. DNA amplicons from positive samples were purified (EXOSAP-IT, USB, Cleveland, Ohio, USA) and sequenced using forward primers at the Center for Genomics Technologies, Hebrew University of Jerusalem. DNA sequences obtained were evaluated with MEGA 5 software and compared for similarity to sequences in GenBank, using the BLAST program hosted by NCBI, National Institutes of Health, Bethesda, MD USA http:/ www.ncbi.nlm.nih.gov/BLAST. Sequences of Bartonella spp. derived from Rattus norvegicus in this study were deposited in GenBank under the following accession numbers JX065633 and JX065634. Approximately 80% of the liver was studded with multifocal to coalescing pale granulomas. Abscesses were seen under the skin with marked infiltration of polymorphonuclear cells. There were no gross or histopathologic lesions in the heart, lungs and kidneys. However, in the liver there was compression and destruction of the hepatic cord and parenchyma by numerous multifocal well demarcated and walled-off granulomas containing nematode parasites in different developmental
Co-infection of Zoonotic Pathogens in a Rat
231
Case Reports
stages and ovoid typical C. hepatica ova of up to 20-30µm in diameter. Multifocally cross sections of whole larva and slender adult stages of the nematode parasites were noticed. The larvae and adult stages had thick curticle, narrow rim of clear space, body wall, hypodermis and a coelomyrian striated muscle (Figures 1 & 2). Well developed digestive and reproductive organs (esophagus and a gravid uterus) containing numerous ova with polar eminence at each end, consistent with Capillaria hepatica, were seen. Degeneration and necrosis of hepatocytes, bridging fibrosis and biliary hyperplasia and reduplication, surrounded by inflammatory foci composed of lymphocytes, plasma cells and macrophages were present (Figures 1 & 2).
Tiny smooth bacterial colonies with metallic sheen were isolated from blood of the rat cultured on blood chocolate agar. The colonies were Gram negative cocci to rod shaped bacteria. Bartonella gltA DNA with 99% sequence homology with GenBank deposited B. elizabethae was identified from the blood and spleen of the Rattus norvegicus rat. Phylogenic analysis performed using the maximum likelihood method showed that the sequences from this study formed a distinct clade with B. elizabethae accession number GU056192 from the Genbank (Figure 3). DISCUSSION
Figure 1: Liver histopathology (×10); Multifocal cross sections, whole larva and slender adult stage of the nematode parasites, Capillaria hepatica. (H&E)
Pathologies consistent with C. hepatica were evident on gross and histopathology of the liver. However, the rat appeared to be emaciated and in poor body condition contrary to the report by Ceruti et al. (7) in which C. hepatica infected rats, appeared to be in a good body condition. The pathologies observed in this rat could be as a result of synergistic effects between C. hepatica and B. elizabethae since pathologies due to bartonellae infections alone are not commonly observed in rodents (12). Bartonella DNA has been isolated from rodent spleens in Israel (13) which is consistent with our finding and may be due to pooling of infected erythrocytes in the spleen. Rattus norvegicus have been shown to harbor several Bartonella species, including B. tribocorum and B. elizabethae which were implicated as a cause of human diseases (9, 11, 14). It has been shown that rodent-associated Bartonella species could act as human pathogens and suggested that the predominantly urban rat R. norvegicus could be a reservoir for human infection among inner-city residents living in poor social conditions (9).
Figure 2: Liver histopathology (×10); Within individual fibrous connective tissue walled-off peri portal areas are numerous Capillaria hepatica ovoid ova of up to 20-30μm, with polar eminence at each end. Note the degeneration and necrosis of hepatocytes with bridging fibrosis. (H&E)
Figure 3: The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. Stability of inferred phylogeny was assessed by using bootstrap analysis of 1000 randomly generated trees. Sequences from this study are in dotted lines.
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
232
Kamani, J.
Case Reports
In conclusion, the finding of these zoonotic agents in a rat captured in the vicinity of human habitation coupled with the fact that people in this area consume rat meat highlights the serious danger posed by rodents to human health. Physicians and medical workers should consider bartonellosis in the diagnosis of cardiac conditions in Nigeria. Further investigation is needed to determine the role of Bartonella spp. in general and Bartonella elizabethae in particular in human endocarditis cases in Nigeria.
ACKNOWLED GEMENTS The authors acknowledge Daniel Yarsura and Osnat Eyal (Koret Shool of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot Israel) for technical assistance.
CONFLICT OF I NT EREST There was no conflict of interest in this study.
1. Njoku, I.: Studies on the seasonal variations and prevalence of helminth fauna of the black rat, Rattus rattus (l) (Rodentia: muridae) from different microhabitats in Nsukka, Nigeria. Anim. Res. Internat. 6: 1063 – 1071, 2009. 2. Kosoy, M. Y., Regnery, R. L., Tzianabos, T., Marston, E., Jones, D.C., Green, D., Maupin, G.O., Olson, J. G., and Childs, J.E.: Distribution, diversity, and host specificity of Bartonella in rodents from the southeastern United States. Am. J. Trop. Med. Hyg. 57: 578–588, 1997. 3. Bai, Y., Kosoy, M.Y., Maupin, G.O., Tsuchiya, K.R., and Gage, K.L.: Genetic and ecological characteristics of Bartonella communities in rodents in southern China. Am. J. Trop. Med. Hyg. 66: 622–627, 2002. 4. Kaiser, P. O., Riess, T., O’Rourke, F., Linke, D. and Kempf, V. A. J.: Bartonella spp.: Throwing light on uncommon human infections. International J. Med. Microbiol. 301: 7–15, 2011.
REFERENCES
5. Conlogue, G., Foreyt, W., Adess, M., and Levine, H.: Capillaria hepatica (Bancroft) in select rat populations of Hartford, Connecticut, with possible public health implications. J. Parasitol. 65: 105-108, 1979. 6. Namue, C., and Wongsawad, C.: A survey of helminth infection in rats (Rattus spp.) from Chiang Mai Moat. Southeast Asian J. Trop. Med. Pub. Hlth. 28: 179-183, 1997. 7. Ceruti, R., Sonzogni, O., Origgi, F., Vezzoli, F., Cammarata, S., Giusti, A. M., and Scanziani, E.: Capillaria hepatica infection in commensal brown rats (Rattus norvegicus) from the urban area of Milan, Italy. J. Vet. Med. B. 48: 235-240, 2001. 8. Birtles, R. J., Canales, J., Ventosilla, P., Alvarez, E., Guerra, H., Llanos-Cuentas, A., Raoult, D., Doshi, N., and Harrison, T. G.: Survey of Bartonella species infecting intradomicillary animals in the Huayllacalla´n Valley, Ancash, Peru, a region endemic for human bartonellosis. Am. J. Trop. Med. Hyg.60: 799–805, 1999. 9. Ellis, B. A, Regnery, R. L., Beati, L., Bacellar, F., Rood, M., Glass, G. G., Marston, E., Ksiazek, T.G., Jones, D., and Childs, J. E.: Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease? J. Infect. Dis. 180: 220–224, 1999. 10. Gundi, V. A., Davoust, B., Khamis, A., Boni, M., Raoult, D., and La Scola, B.: Isolation of Bartonella rattimassiliensis sp. nov. and Bartonella phoceensis sp. nov. from European Rattus norvegicus. J. Clin. Microbiol. 42: 3816–3818, 2004. 11. Daly, J. S., Worthington, M. G, Brenner, D. J., Moss, C. W., Hollis, D.G., Weyant, R.S., Steigerwalt, A.G., Weaver, R. E., Daneshvar, M. I. and O’Connor, S. P.: Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J. Clin. Microbiol. 31: 872–881, 1993. 12. Bai, Y., Calisher, C. H., Kosoy, M. Y., Root, J.F., and Doty, J.B.: Persistent Infection or successive reinfection of deer mice with Bartonella vinsonii subsp. arupensis. App. Environ. Microbiol.77: 1728–1731, 2011. 13. Morick, D., Baneth, G., Avidor, B., Kosoy, M. Y., Mumcuoglu, K. Y., Mintz, D., Eyal, O., Goethe, R., Mietze, A., Shpigel, N. and Harrus, S.: Detection of Bartonella spp. in commensal rodents in Israel using HRM real-time PCR. Vet. Microbiol. 139: 293–297, 2009. 14. Comer, J. A., Diaz, T., Vlahov, D., Monterroso, E. and Childs, J.E.: Evidence of rodent-associated Bartonella and Rickettsia infections among intravenous drug users from central and east Harlem, New York City. Am. J. Trop. Med. Hyg.65: 855–60, 2001.
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
Co-infection of Zoonotic Pathogens in a Rat
233
Co-Infection with Capillaria hepatica and Bartonella elizabethae in a Brown Rat (Rattus norvegicus) from Nigeria
Kamani, J.,*1 Akanbi, B.O.,2 Baneth, G.,3 Morrick, D.,3 and Harrus, S.3
1 2
Parasitology Division, National Veterinary Research Institute, PMB 01Vom Plateau State, Nigeria. Central Diagnostic Division, National Veterinary Research Institute, PMB 01Vom Plateau State, Nigeria. 3 Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
* Corresponding author: Dr Joshua Kamani, Parasitology Division, National Veterinary Research Institute, PMB01 Vom, Plateau State, Nigeria. Tel: +2347035517715; +972552237079. Email: shapumani@yahoo.com.
AB ST RAC T
During a survey for Bartonella species in rodents in Vom, Plateau State, Nigeria, a brown rat (Rattus norvegicus) with multiple subcutaneous nodules was captured. Gross pathology examination revealed multifocal pale areas in the spleen and liver. Histopathologic examination of the liver revealed numerous multifocal well demarcated and walled-off granulomas compressing and destroying the hepatic cord and parenchyma. Granulomas contained Capillaria hepatica nematodes in different developmental stages with numerous typical ova. There was degeneration and necrosis of hepatocytes, bridging fibrosis and biliary hyperplasia and reduplication, surrounded by inflammatory foci composed of lymphocytes, plasma cells and macrophages. Tiny smooth bacterial colonies with metallic sheen were isolated from blood of the rat cultured on blood chocolate agar. DNA extracted from the blood and spleen of the rat was found positive for Bartonella spp. citrate synthase gene (gltA) by PCR. Sequence analysis shows 99% similarity to B. elizabethae. The identification of two zoonotic agents in a rat highlights the importance of rats in the transmission cycle of zoonotic diseases in Nigeria. Key words: Rattus norvegicus, Capillaria hepatica, Bartonella elizabethae, Nigeria.
Rodents especially rats (Rattus norvegicus, Rattus rattus, Cricetomyces gambianus) abound in domestic and peri-domestic settings in Nigeria, all year round (1). Their role in the transmission of zoonotic diseases is well documented (2-4). Their role in disease transmission in Nigeria is even greater as rats are being hunted and consumed as a source of protein by some local ethnic groups. In addition, the act of trapping rats, bringing them to the homestead and preparing them for human consumption without proper protective clothing, litters the environment with rat feces and expose humans especially children, who play around contaminated playground, to zoonotic agents. Capillaria hepatica is a worldwide distributed zoonotic nematode
INTRODUCTION
of mammals, primarily infesting the liver of rats (5, 6). Consumption of C. hepatica unembryonated eggs may cause pseudoparasitism in man, with no apparent clinical signs or symptoms of disease. True infection, with liver localization of the parasite and the development of hepatic lesions, requires the ingestion of infective eggs. The contamination of playgrounds by infective eggs can explain the high susceptibility of children to capillariasis (7). Rattus norvegicus have also been found to carry various zoonotic Bartonella species (2, 8-10). In the United States and Portugal, 18% of rodents tested were Bartonella bacteremic, and some isolates from R. norvegicus were closely related to B. elizabethae (9). The latter species was associated with endocarditis in humans (9, 11).
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
230
Kamani, J.
Case Reports
Since rodents are the principal hosts of C. hepatica and also reservoir of Bartonella spp., contact between humans and rodents can be a potential health hazard. There is paucity of information of bartonellosis in man and animals in Nigeria. We present in this report a co-infection of zoonotic agents; C. hepatica and B. elizabethae in a Rattus norvegicus in Nigeria. CASE REPORT
During a survey for bartonellosis in rodents from Vom, Nigeria, one of the rats (Rattus norvegicus) trapped was noticed to be emaciated with poor body condition. Diffuse solitary nodules were present on its body and hard solid masses could be palpated in its abdomen. The rat was humanely euthanized according to the guidelines of the Animal Welfare and Ethics Committee of the National Veterinary Research Institute (NVRI), Vom, Nigeria. Approximately 2ml of blood was collected in sterile ethyelene diamine tetra acetic acid (EDTA) tube and stored at -20oC until further analyzed. The heart, liver, skin and spleen were harvested. Each tissue was divided into two parts. One part was preserved in buffered formalin while the other was placed in a sterile tube and kept at -20oC until further analyzed. Ticks were removed from its body and pooled into a universal bottle containing absolute ethanol. The ticks were identified as Rhipicephalus sanguineus. Tissues in buffered formalin were processed at the Central Diagnostic Division, NVRI Vom. Five µm paraffin-wax sections of organs were dewaxed and stained with hematoxylin-eosin (H&E), mounted on charged microscope slides and observed under a Carl Zeiss light microscope for histopathological changes. The other samples were transported to The Koret school of Veterinary Medicine, Israel, for further analysis. For bacterial culture, 200μl of thawed whole blood sample was plated onto blood chocolate agar. The plate was incubated at 35oC in 5% CO2 and checked every other day for growth of Bartonella species. Suspected colonies were randomly selected and separately sub-cultured onto different fresh agar plates. The resulting pure cultures were stained using Gram stain. DNA was extracted from the spleen, liver, heart and ticks using Illustra tissue and cell genomicPrep miniSpin
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
kit (GE Healthcare, UK, Limited) and from blood using the BiOsticTM Bacteremia DNA Isolation Kit (MO Bio Laboratories, Inc., Carlsbad, CA, USA) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the primers, BhCS871p: (5’-GGGGACCAGCTCATGGTGG-3’) and BhCS1137n: (5’-AATGCAAAAAGAACAGTAAACA-3’) for amplification of a 379 bp fragment of the citrate synthase gene (gltA) of Bartonella spp. Positive and negative controls were included in each PCR run. PCR was performed in 25 μl total volume as follows: 3μl of DNA template, 1μl of 10mM each primer, 1μl Mgcl2, 20μl of ultra pure PCR water. PCR was performed using Syntezza PCR-Ready High Specificity (Syntezza Bioscience, Israel). Amplification was performed using a programmable conventional thermocycler (Biometra, Goettingen, Germany) using the following program parameters: an initial denaturing at 95°C for five minutes, and 35 cycles at 95°C for one minute, 56°C for one minute, and 72°C for one minute. The PCR products were analyzed for the presence of amplicons of the correct size by electrophoresis of 6μl of the products in 1.5% agarose gels containing ethidium bromide. Amplicons of the proper size were identified by comparison to the positive control lane on the agarose gel and comparison to a 50bp DNA molecular ladder. DNA amplicons from positive samples were purified (EXOSAP-IT, USB, Cleveland, Ohio, USA) and sequenced using forward primers at the Center for Genomics Technologies, Hebrew University of Jerusalem. DNA sequences obtained were evaluated with MEGA 5 software and compared for similarity to sequences in GenBank, using the BLAST program hosted by NCBI, National Institutes of Health, Bethesda, MD USA http:/ www.ncbi.nlm.nih.gov/BLAST. Sequences of Bartonella spp. derived from Rattus norvegicus in this study were deposited in GenBank under the following accession numbers JX065633 and JX065634. Approximately 80% of the liver was studded with multifocal to coalescing pale granulomas. Abscesses were seen under the skin with marked infiltration of polymorphonuclear cells. There were no gross or histopathologic lesions in the heart, lungs and kidneys. However, in the liver there was compression and destruction of the hepatic cord and parenchyma by numerous multifocal well demarcated and walled-off granulomas containing nematode parasites in different developmental
Co-infection of Zoonotic Pathogens in a Rat
231
Case Reports
stages and ovoid typical C. hepatica ova of up to 20-30µm in diameter. Multifocally cross sections of whole larva and slender adult stages of the nematode parasites were noticed. The larvae and adult stages had thick curticle, narrow rim of clear space, body wall, hypodermis and a coelomyrian striated muscle (Figures 1 & 2). Well developed digestive and reproductive organs (esophagus and a gravid uterus) containing numerous ova with polar eminence at each end, consistent with Capillaria hepatica, were seen. Degeneration and necrosis of hepatocytes, bridging fibrosis and biliary hyperplasia and reduplication, surrounded by inflammatory foci composed of lymphocytes, plasma cells and macrophages were present (Figures 1 & 2).
Tiny smooth bacterial colonies with metallic sheen were isolated from blood of the rat cultured on blood chocolate agar. The colonies were Gram negative cocci to rod shaped bacteria. Bartonella gltA DNA with 99% sequence homology with GenBank deposited B. elizabethae was identified from the blood and spleen of the Rattus norvegicus rat. Phylogenic analysis performed using the maximum likelihood method showed that the sequences from this study formed a distinct clade with B. elizabethae accession number GU056192 from the Genbank (Figure 3). DISCUSSION
Figure 1: Liver histopathology (×10); Multifocal cross sections, whole larva and slender adult stage of the nematode parasites, Capillaria hepatica. (H&E)
Pathologies consistent with C. hepatica were evident on gross and histopathology of the liver. However, the rat appeared to be emaciated and in poor body condition contrary to the report by Ceruti et al. (7) in which C. hepatica infected rats, appeared to be in a good body condition. The pathologies observed in this rat could be as a result of synergistic effects between C. hepatica and B. elizabethae since pathologies due to bartonellae infections alone are not commonly observed in rodents (12). Bartonella DNA has been isolated from rodent spleens in Israel (13) which is consistent with our finding and may be due to pooling of infected erythrocytes in the spleen. Rattus norvegicus have been shown to harbor several Bartonella species, including B. tribocorum and B. elizabethae which were implicated as a cause of human diseases (9, 11, 14). It has been shown that rodent-associated Bartonella species could act as human pathogens and suggested that the predominantly urban rat R. norvegicus could be a reservoir for human infection among inner-city residents living in poor social conditions (9).
Figure 2: Liver histopathology (×10); Within individual fibrous connective tissue walled-off peri portal areas are numerous Capillaria hepatica ovoid ova of up to 20-30μm, with polar eminence at each end. Note the degeneration and necrosis of hepatocytes with bridging fibrosis. (H&E)
Figure 3: The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. Stability of inferred phylogeny was assessed by using bootstrap analysis of 1000 randomly generated trees. Sequences from this study are in dotted lines.
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
232
Kamani, J.
Case Reports
In conclusion, the finding of these zoonotic agents in a rat captured in the vicinity of human habitation coupled with the fact that people in this area consume rat meat highlights the serious danger posed by rodents to human health. Physicians and medical workers should consider bartonellosis in the diagnosis of cardiac conditions in Nigeria. Further investigation is needed to determine the role of Bartonella spp. in general and Bartonella elizabethae in particular in human endocarditis cases in Nigeria.
ACKNOWLED GEMENTS The authors acknowledge Daniel Yarsura and Osnat Eyal (Koret Shool of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot Israel) for technical assistance.
CONFLICT OF I NT EREST There was no conflict of interest in this study.
1. Njoku, I.: Studies on the seasonal variations and prevalence of helminth fauna of the black rat, Rattus rattus (l) (Rodentia: muridae) from different microhabitats in Nsukka, Nigeria. Anim. Res. Internat. 6: 1063 – 1071, 2009. 2. Kosoy, M. Y., Regnery, R. L., Tzianabos, T., Marston, E., Jones, D.C., Green, D., Maupin, G.O., Olson, J. G., and Childs, J.E.: Distribution, diversity, and host specificity of Bartonella in rodents from the southeastern United States. Am. J. Trop. Med. Hyg. 57: 578–588, 1997. 3. Bai, Y., Kosoy, M.Y., Maupin, G.O., Tsuchiya, K.R., and Gage, K.L.: Genetic and ecological characteristics of Bartonella communities in rodents in southern China. Am. J. Trop. Med. Hyg. 66: 622–627, 2002. 4. Kaiser, P. O., Riess, T., O’Rourke, F., Linke, D. and Kempf, V. A. J.: Bartonella spp.: Throwing light on uncommon human infections. International J. Med. Microbiol. 301: 7–15, 2011.
REFERENCES
5. Conlogue, G., Foreyt, W., Adess, M., and Levine, H.: Capillaria hepatica (Bancroft) in select rat populations of Hartford, Connecticut, with possible public health implications. J. Parasitol. 65: 105-108, 1979. 6. Namue, C., and Wongsawad, C.: A survey of helminth infection in rats (Rattus spp.) from Chiang Mai Moat. Southeast Asian J. Trop. Med. Pub. Hlth. 28: 179-183, 1997. 7. Ceruti, R., Sonzogni, O., Origgi, F., Vezzoli, F., Cammarata, S., Giusti, A. M., and Scanziani, E.: Capillaria hepatica infection in commensal brown rats (Rattus norvegicus) from the urban area of Milan, Italy. J. Vet. Med. B. 48: 235-240, 2001. 8. Birtles, R. J., Canales, J., Ventosilla, P., Alvarez, E., Guerra, H., Llanos-Cuentas, A., Raoult, D., Doshi, N., and Harrison, T. G.: Survey of Bartonella species infecting intradomicillary animals in the Huayllacalla´n Valley, Ancash, Peru, a region endemic for human bartonellosis. Am. J. Trop. Med. Hyg.60: 799–805, 1999. 9. Ellis, B. A, Regnery, R. L., Beati, L., Bacellar, F., Rood, M., Glass, G. G., Marston, E., Ksiazek, T.G., Jones, D., and Childs, J. E.: Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease? J. Infect. Dis. 180: 220–224, 1999. 10. Gundi, V. A., Davoust, B., Khamis, A., Boni, M., Raoult, D., and La Scola, B.: Isolation of Bartonella rattimassiliensis sp. nov. and Bartonella phoceensis sp. nov. from European Rattus norvegicus. J. Clin. Microbiol. 42: 3816–3818, 2004. 11. Daly, J. S., Worthington, M. G, Brenner, D. J., Moss, C. W., Hollis, D.G., Weyant, R.S., Steigerwalt, A.G., Weaver, R. E., Daneshvar, M. I. and O’Connor, S. P.: Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J. Clin. Microbiol. 31: 872–881, 1993. 12. Bai, Y., Calisher, C. H., Kosoy, M. Y., Root, J.F., and Doty, J.B.: Persistent Infection or successive reinfection of deer mice with Bartonella vinsonii subsp. arupensis. App. Environ. Microbiol.77: 1728–1731, 2011. 13. Morick, D., Baneth, G., Avidor, B., Kosoy, M. Y., Mumcuoglu, K. Y., Mintz, D., Eyal, O., Goethe, R., Mietze, A., Shpigel, N. and Harrus, S.: Detection of Bartonella spp. in commensal rodents in Israel using HRM real-time PCR. Vet. Microbiol. 139: 293–297, 2009. 14. Comer, J. A., Diaz, T., Vlahov, D., Monterroso, E. and Childs, J.E.: Evidence of rodent-associated Bartonella and Rickettsia infections among intravenous drug users from central and east Harlem, New York City. Am. J. Trop. Med. Hyg.65: 855–60, 2001.
Israel Journal of Veterinary Medicine Vol. 68 (4) December 2013
Co-infection of Zoonotic Pathogens in a Rat
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