Occurrence of Leptospira Species from Pigs in Selected Local Government Areas of Kaduna State, Nigeria

Bridget Adah1*, Clara Kwanashie2, Haruna Kazeem2 and Samuel Mailafia1

1University of Abuja, Nigeria; 2Ahmadu bello university, Zaria, Nigeria.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | February 19, 2022; Accepted | May 10, 2022; Published | May 18, 2022

*Correspondence | Bridget Adah, University of Abuja, Nigeria; Email: bridget.adah@uniabuja.edu.ng

Citation | Adah, B., C. Kwanashie, H. Kazeem and S. Mailafia. 2022. Occurrence of Leptospira species from pigs in selected local government areas of Kaduna State, Nigeria. Veterinary Sciences: Research and Reviews, 8(1): 36-42.

DOI | https://dx.doi.org/10.17582/journal.vsrr/2022.8.1.36.42

Keywords | Occurrence, Isolation, Dark field microscopy, Leptospira, Pigs, Zoonoses

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Leptospirosis is an infectious and highly contagious commercially important disease of livestock which is widely distributed in the world (Bharti et al., 2003). It is caused by Leptospira organism within the phylum: Spirochaetes, order: Spirochaetales, family: Leptospiraceae, and Genus Leptospira (Scolamacchia et al., 2010). The genus comprises of saprophytic and pathogenic species with members also assembled into Serovars based on their antigenic relatedness with over 250 recognized serovars and each serovar acts differently within its maintenance host (Ngbede et al., 2013).

It is a disease that affects humans, mammals, birds, amphibians and reptiles (Langston and Hueter, 2003). Leptospirosis has been reported to be an indigenous disease in Nigeria with prevalence between 1.7% - 23% in cattle and 26% - 29% in human (Boey et al., 2019). The organism can survive in cold water environment; infections are most likely to occur in wet seasons than in dry seasons (Thiermann et al., 1985). The disease is an occupational hazard to swimmers through contact with rivers, streams or lakes which has been tainted with urine of rodents and wild animals (Bharti et al., 2003; Adah et al., 2018).

Definitive diagnosis of leptospirosis is usually provided by bacteriologic culture of the infecting organism (Toyokawa et al., 2011). In the selection of proper tissues or samples for isolation purposes, it is necessary to determine the stage of the disease. In cases of acute disease, isolation should focus on blood samples (Sukl et al., 2017). In the chronic form after seroconversion, the isolation should be ventured from the urine (Boey et al., 2019). Where there is a clinical disease or presence of an aborted foetus, isolation should be ventured from the kidney, liver and the aqueous humor (Faine, 1994). Due to production and reproductive losses which includes manifestations like agalactiae, decreased productivity and infertility, stillbirth, abortion, and low productivity due to the disease, leptospirosis is reported to be a major cause of economic/industrial losses in the livestock production (Agunloye et al., 2002). As a result of the public health and zoonotic importance attached to leptospirosis, there is need for creating awareness about the diseases worldwide (Alonghom et al., 2017; Burriel, 2010). The under reporting and paucity of information about the clinical confirmation and availability of required diagnostic tools has lead to the misdiagnosis of Leptospirosis for other interconnected diseases. Leptospirosis is often complicated in Nigeria due to negligence and unavailability of diagnostic tools and data, which is also seen in many other under developing countries worldwide (LERG, 2010).

The isolation of the organism is the preliminary bases by which all other antigen detection methods are evaluated (OIE, 2010). Cultural isolation of the organism is not only depending on the number of infectious organisms but also depends on the pathogenicity of the organisms (Kartrin et al., 2018). One vital superiority of bacteriologic culture and identification of Leptospira is the fact that, any serovar can be cultured, detected and subsequently identified (Collins et al., 2021). It can also aid in typing for various strains, required for epidemiological studies involving many serovars within particular species in different geographical locality (Vanina et al., 2018). This study is remarkable as it will supply information required for diagnostic and control strategies of the disease in Nigeria.

The aim of this study is to determine the occurrence of Leptospira organism from urine and blood of porcine species in Kaduna state, Nigeria and to establish the epidemiological implications and distribution of the disease in the study area.

Materials and Methods

Study area

The study was carried out in parts of Kaduna State (Zaria, Kaduna metropolis and Kafanchan), Nigeria. Zaria is located between latitude 11o04ʹN and longitude 7o42ʹE, covering an area of 300km2 and with a population of about 408,198 (Nimyel and Namadi, 2019). The vegetation is Northern Guinea Savannah zone, with rainfall ranging from 0.0 to 816.0 mm/month and temperature of 17oC to 33oC. Kafanchan is a town in Southern Kaduna located between latitude 9o34ʹN and longitude 8o18ʹE, with an estimated population of 83,092 (Nimyel and Namadi, 2019). Kaduna metropolis is located between latitude 10o31ʹN and longitude 7o26ʹE, covering an area of 46.053 km2 and with a population of about 736,000 (Nimyel and Namadi, 2019).

Study design

Cross-sectional study was designed using convenience sampling based on availability of pigs and willingness of pig farmers and abattoir owners to participate in this research. The sampling was carried out daily from June to August, 2012. Sampling covered areas of Zaria, Kaduna metropolis and Kafanchan which are the three senatorial zones and also constitute the nuclei for pig rearing in Kaduna state. In each farm, abattoir, household and market visited, samples were collected from 50% of the animals available to come about the sample size which was determined as recommended by Thrusfield (2007). The age, sex, breed, management practice, source of animals and location were recorded.

Sample collection and processing

Blood and urine samples were taken from 200 pigs in Kafanchan, Kaduna metropolis, and Zaria. About 10ml of blood was collected aseptically using 20ml syringe and 18 gauge needles, after proper restraining of animals, which was collected directly from the anterior vena cava of the heart of each pig using the hypodermic needle and syringe. About 5 ml each from the 10mls of blood collected was transferred into 2 clean test tubes with one containing heparin (anticoagulant) for culture and the one without heparin for serology, and both were labeled appropriately. The screw capped blood samples were transported immediately to the Ahmadu Bello University, Veterinary Microbiology Laboratory in a leak proof container with ice packs for processing (Miller et al., 1990).

Midstream urine samples were collected into sterile sample bottles after cleaning of the vulva in females and prepucial area of males using the clean catch urine sampling method. The pigs were encouraged to urinate using the massage method. Five mililiters of the collected urine collected directly unto a sterile screw cap bottle containing 0.5ml of filtered (0.4µm) 40% formaldehyde and transported to the laboratory placed in an ice packed container (4oC) as soon as possible for processing (Sakhaee et al., 2007).

Media preparation

Ellinghausen McCulough modified Johnson Harris (EMJH) semi solid medium (DifcoTM USA) was used for culture and isolation. The media was prepared following the manufacturers specification for each sample (urine and blood). 2.3 g of the EMJH basal media (DifcoTM USA), was weighed electronically and dissolved in 900ml of distilled water. It was then heated over a Bunsen burner flame to enable complete dissolution of the medium. The dissolved solution was sterilized via autoclave for 15mins. It was allowed to cool down to 40oC, and 100ml of the EMJH enrichment medium (DifcoTM USA) was added. 200µg/ml of 5 fluorouracil antibiotics was added to prevent growth of contaminants. The media was then dispensed into culture tubes at 5ml per culture tube (Sakhaee et al., 2007).

Identification

The identification of leptospiral organisms was done according to standard methods as described in the World Health Organization (WHO) leptospirosis guidelines (Abdollapour, 1995; Alonghom et al., 2017). For blood samples, the sample bottles containing heparin was inoculated with the suspected sample materials, into four 5ml tubes (containing Leptospira EMJH medium) with 1–2 drops of blood per tube and was incubated in the dark at 30oC. One drop of each urine sample was inoculated, immediately after sample collection into two 5ml sterile tubes of EMJH medium. Both tubes were incubated in the dark at 30oC based on standard methods (Isenberg, 1992).

All the cultured tubes were observed weekly for growth, which is evident by presence of turbidity, haze or a ring of growth. The culture was also examined microscopically, with dark field illumination at x400 magnification for Leptospira organisms, which appear as motile, tightly coiled spirochetes of about 1µm wide and 6–20µm long in size, with hooked end. All incubated culture media was kept for at least 5–8 weeks, after which negative growth tubes were discarded (Sakhaee et al., 2007: Ramesh et al., 2018).

Results and Discussion

Colonial and microscopic morphology

A total of 120 (12%) of the culture tubes incubated exhibited clear EMJH medium while two hundred and eighty-eight of the culture tubes showed cloudiness, haze, and turbidity of the EMJH medium. Nine of the culture tubes, which exhibited haze, turbidity and cloudiness showed presence of Leptospira species. The nine isolates were identified on dark field illumination at x400 magnification using dark field microscopy to be spiral, motile, and tightly coiled organisms, about 1µm wide and 7–18 µm long. The prevalence rate of Leptospira organisms in blood was 288 (3.5%) while the prevalence in urine was 1.0% with an overall prevalence rate of 4.5%.

The variation in prevalence rates obtained in our study was 3.5% from blood and 1.0 %, from urine as shown in Table 1. This shows that the organism can survive in urine, blood and perhaps other bodily fluids.

This research conducted at the bacteriology Laboratory of Ahmadu Bello University, Zaria, documents the occurrence of Leptospira organism in the study area as shown in Figure 1. Out of the 400 samples cultured, 9(4.5%) showed presence of Leptospira, on dark field illumination from pigs in Kaduna state (Figure 1). Our finding agrees with the previous reports of Agunloye et al. (2002) which reported similar prevalence of leptospirosis in Nigeria. This finding also concurs with similar studies from bovine kidneys in Plateau state which yielded the same prevalence rate of 4.5% (Ezeh et al., 1989).

 

A higher prevalence rate of 13.5% in humans has been reported in Enugu and its environs of eastern Nigeria by Onyemelukwe (1993). The sporadic nature of leptospirosis has been reported to be a major cause of morbidity and mortality in both animals and man (Bili et al., 2017). The differences in prevalence rates may be due to season of the year, with regards to time of sampling and also due to the difference in location, type of samples and sample size (Mohammad et al., 2020).

Our prevalence rate is lower than that obtained (4.5%) by Diallo et al. (1982) from Brown field rats (Arvicanthus niloticus) and this was attributed to the fact that rats serves as reservoir host for leptospira species (Diallo et al., 1982). The culture negative report obtained by (Diallo et al., 1982) from piggery sewage effluents collected at Kano and Kaduna may be as a result of the fastidious nature of the organism to grow (Bili et al., 2017).

In comparison with the isolation rate of Leptospira organisms from 3.5% blood and 1% urine, the percentage isolation from blood was higher than that obtained from urine, this might be due to the fact that more animals were sampled at the acute phase of the disease which permits serological responses as indicated in previous studies (Bili et al., 2017; Abena et al., 2017). Future studies involving leptospirosis should be focused on blood samples, while in chronic diseases; isolation should be from the urine (Udomsak et al., 2020).

Leptospirosis is a disease of economic and public health significance (Agunloye et al., 2001; Ngede et al., 2013), this research showed that the organism occurs more in pigs reared under extensive system than in semi intensive and intensive system as shown in Table 2. The extensive system of pig production in Nigeria allows pigs to roam about and scavenge for food. This may expose those pigs to various surfaces and niches for enzootic and occupational transmission of infection to animals or humans at risk (Ngbede et al., 2013: Mailafia et al., 2017).

 

Table 1: Occurrence of Leptospira species from blood and urine sample.

Sample type	No positive	Percentage positive (%)	No negative	Percentage negative (%)	Number of samples	Total percentage (%)
Blood	7	(3.5)	193	(96.5)	200	(100)
Urine	2	(1.0)	198	(99.0)	200	(100)
Total	9	(4.5)	391	195.5	400	(200)

 

Table 2: Zoographic studies of Leptospira positive cultures.

Culture tube no.	Sex	Age (Months)	Location	Breed	Source	Mgt. practice
7	Male	17	Zaria	Local	Farm	Intensive
9	Female	3	Zaria	Local	Farm	Intensive
10	Male	3	Zaria	Local	Farm	Intensive
15	Female	12	Zaria	Local	Household	Extensive
18	Female	2	Zaria	Local	Household	Semi-intensive
20	Male	2	Zaria	Local	Household	Semi-intensive
27	Male	17	Zaria	Local	Household	Extensive
28	Male	15	Zaria	Local	Household	Extensive
155	Male	15	Zaria	Local	Household	Extensive

 

In Nigeria, humans that come in contact with pigs or may be at risk of leptospirosis are the veterinarians, pig handlers, butchers, abattoir workers, coal miners, farmers, laboratory personnel’s, rodent control workers and pork meat vendors (Terpstra, 2006; Roman et al., 2018). This observation calls for urgent need to conduct more studies on porcine leptospirosis in Nigeria.

Conclusions and Recommendations

Findings of this research have demonstrated the use of dark field microscopy to determine the occurrence of Leptospira organisms in extensive, semi intensive and intensive system of pig production. The total prevalence rate was 4.5% and the prevalence was more (3.5%) in blood than urine (1.0%). This therefore calls for proper hygienic handling of porcine and porcine by products and good management practice. Public health awareness campaign and education, is very necessary as to enlighten the general public and clinicians on the control, economic and public health impact of porcine leptospirosis. This study has shown the occurrence of Leptospira organisms in pigs in Kaduna state at a prevalence of 4.5%. Further research on porcine leptospirosis, should be targeted at detecting IgG and the use of Polymerase Chain Reaction (PCR) in the diagnosis of leptospirosis. Veterinarians are encouraged to monitor and report cases of leptospirosis in Nigeria.

Acknowlegments

I will like to acknowledge all my co-authors for the value added to this manuscript and my special thanks goes to Mr. Dodo of the Department of Veterinary Microbiology, Ahmadu Bello University, Zaria for all the sacrifice made during this study.

Novelty Statement

In this study, leptospira specie was isolated from porcine blood and urine samples in Kaduna state Nigeria using cultural method of isolation while prior to this study, there was paucity of information on cultural isolation of Leptospira from porcine specie in Kaduna State, Nigeria using cultural method.

Author’s Contribution

BMJA: Contributed to the overall design, laboratory analysis, literature review and writing of the manuscript.

SM, KCN, and KHM: Contributed in the review and editing of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Abdollahpour, G.R., 1995. Isolation of Leptospira interrogans serovar grippotyphosa from a Heifer in New South Wales. Aust. Vet. J., 73: 109–110. https://doi.org/10.1111/j.1751-0813.1996.tb09990.x

Abena, P., Alexandra, V., Richard, P., and Yitade, G., 2017. Leptospirosis in the caribbean: Literature review. Panam Salud Publica, 10: 26633.

Adah, B.M.J., Kwanashie, C.N., Kazeem, H.M., and Mailafia, S., 2018. Prevalence of leptospira specie serovar brtislava in pigs from, Kaduna State, Nigeria using Competitive- ELISA. IOSR J. Agric. Vet. Sci., (10SR-JAVS) elssN., 11: 11-16.

Agunloye, C.A., Alabi, F.O., Odemuyiwa, S.O. and Olaleye, O.D., 2001. Leptospirosis in Nigeria: Seroepidemiological survey. Indian Vet. J., 78(5): 371-375.

Agunloye, C.A., Ajuwape, A.T.P., and Nottidge, H.O., 2002. Comparative study of the Prevalence of leptospirosis in vaccinated and unvaccinated dogs in Ibadan. Retrieved from Vet. Uni. Edu. Ng, on 26th January, 2010. At 8 pm.

Alonghom, K., Pattrarat, C., Kittikat, L., Waree, N., Vanaporn, W. and Nuvee, P., 2017. Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in nan province, a rural area in Thailand. Res. Vet. Sci., 3: 146-154.

Bharti, A.R., Nally, J.E., Ricaldi, J.N., Matthias, M.A., Diaz, M.M., and Lovett, M.A., 2003. Leptospirosis: A zoonotic disease of global importance. Lancet Infect. Dis., 3(12): 757-771. https://doi.org/10.1016/S1473-3099(03)00830-2

Bili, X., Le, S., Xia, F., Hailian, X., Yongzhang, Z., Jinhong, Q., Chengsong, C., Wei, Z., Yunfu, C., Yan, Z., Xiaokui, D., and Ping, H., 2017. A new model of self-resolving Leptospirosis in mice infected with a strain of Leptospirainterrogansserovar Autumnalis harboring LPS signaling only through TLR4. Emerg. Microbes Infect., 6: 36. https://doi.org/10.1038/emi.2017.16

Boey K., Shiokawa K., and Rajeev, S. 2019. Leptospira infection in rats: A literature review of global prevalence and distribution. PLOS Neglected Tropical Diseases., 13(8): e0007499.

Burriel, A.R., 2010. Leptospirosis: An important zoonotic disease. In: Current research. Technol. Educ. Topics Appl. Microbiol. Microbial Biotechnol., Mendez-Vilas A. (ed), Publ. Formatex Research Center Spain. 1: 687.

Collins L. F., Sheth A. N., Mehta C. C., Naggie S, Golub E. T., Anastos K., French A. L., Kassaye S., Taylor T., Fischl M. A., Adimora A. A., Kempf, M. C., Palella F. J., Tien P. G., and Ofotokun, I. 2021. The prevalence and burden of non - AIDS comorbidities among women living with or at risk for human immunodeficiency virus infection in the United States. Clinical Infectious Diseases., 72(8): 1301 - 1311. https://doi.org/10.3201/eid1712.110700

Diallo, A.A. and Dennis, S.M., 1982. Bacteriological survey of leptospirosis in Zaria, Nigeria. Trop. Geogr. Med., 34: 29–34.

Ezeh, A.O., Ellis, W.A., Kmety, E., Abiodun, A.A., and Addo, P.B., 1989. Bacteriological examination of bovine kidneys for leptospires in Plateau State, Nigeria. Revue Scientifique et technique (Int. Off. Epizoot.), 8(4): 1005–1008. https://doi.org/10.20506/rst.8.4.463

Faine, S., 1994. Leptospira and leptospirosis. CRC press, Boca Raton, Florida. pp. 353.

Isenberg, H.D., 1992. Clinical microbiology procedures handbook. Am. Soc. Microbiol. Washington D.C., pp. 56–89.

Kartrin, S.M., Astrid, T., Marlin, B., Mathias, H. and Lothar, K., 2018. Passive surveillance of leptospira infection in swine in Germany. Porcine Health Manag., 3: 018-0036-5.

Langston, C.E. and Heuter, K.J., 2003. Leptospirosis, A re-emerging zoonotic disease. Vet. Clin. N. Am. Small Anim. Pract., 33(4): 791-807. https://doi.org/10.1016/S0195-5616(03)00026-3

LERG, 2010. Lepto burden Epidemiology reference group. Accessed 23rd March, 2011 at 3:30 pm. pp. 693. http://www.who.int/zoonoses/diseases/lerg/en/index5.html.

Mailafia, S., Madubuike, S.A., Raji, A., Suleiman, M.M., Olabode, O.K., and Gods, P.R.O., 2017. Phenotypic Identification of Escherichia coli0157: H 7 isolated from cattle at Suleja Abattoir, Nigeria. Afr. J. Microbiol. Res., 10: 5897-8569.

Miller, D.A., Wilson, M.A., and Beran, G.W., 1990. The effect of storage time on isolation of Leptospira interrogans from bovine kidney. J. Vet. Diagn. Invest., 2: 63–65.

Mohammad, K., Ehsanollah, S., Fahimeh, B.A., Amir, A.B.S., Davoud, A., and Saber, E., 2020. Serological evidence of leptospirosis in Iran. A systematic review and meta-analysis. J. Microbiol. Pathol., 10: 1016.

Ngbede, E.O., Raji, M.A., Kwanashie, C.N., and Okolocha, E.C., 2013. Serosurvey of Leptospira species serovar Hardjo in cattle from Zaria, Nigeria. Rev. Med. Vet., 164(2): 85–89.

Nimyel, S.H., and Namadi, M.M., 2019. Determination of selected air quality parameters in Zaria and its environs, Kaduna State, Nigeria. J. Appl. Sci. Environ. Manag., 23(8): 1505–1510.

OIE, 2008. Office des epizootics. Leptospirosis, manual of diagnostic test and vaccines for terrestrial animals. pp. 251-264.

Onyemelukwe, N.F., 1993. A serological survey of leptospirosis in Enugu area of eastern Nigeria among people at occupational risk. J. Trop. Med. Hyg., 96(5): 301-304.

Ramesh, M., Bhagwan, D., Latika, P., Bhaskaran, U., Nithin, K., Rekha, U., Prasanna, M., and Vaman, K., 2018. Leptospirosis in coastal south India: A facility Based Study. Biol. Med. Res. Int., 17: 59-125.

Roman, T., Dominigue, G., Emilie, B., Marie-Estelle, S.G., Anna, R., Anthony, D.M.M., GregorioI, Mathieu, P. and Cyrille, G., 2018. Biodiversity of enviornmental leptospira: Improving identification and revisiting the diagnosis. Prontiers Microbiol., 10(33): 89-816.

Sakhaee, E., Abdollahpour, G.H.R., Bolourchi, M., Hasani, T.A.M., and Sattari, T.S., 2007. Serologic and Bacteriologic Diagnosis of Bovine Leptospirosis in Tehran Suburddiary farms. Iran. J. Vet. Res., 8(4): 325 – 332.

Scolamacchia, F., Handel, I.G., Fèvre, E.M., Morgan, K.L., Tanya, V. N., and Bronsvort, B.M.D., 2010. Serological Patterns of Brucellosis, Leptospirosis and Q - Fever in Bos indicus Cattle in Cameroon. PLoS One, 5(1): 8623. https://doi.org/10.1371/journal.pone.0008623

SukL, H.U., Nguyen, V.K., Huyen, N.X., Vuory, B.N., Hung, N.V., and Delia, G., 2017. Seroprevalence of specific Leptospira serovars in fattening pigs from 5 provinces in vietnam. BMC Vet. Res., 10: 1186/512917.

Terpstra, W.J., 2006. Historical perspectives in leptospirosis. Indian J. Med. Microbiol., 24(4): 316–320. https://doi.org/10.4103/0255-0857.29407

Thiermann, A.B., Handsaker, A.L., Mooseley, S.L., and Kingscote, B., 1985. New method for Classification of Leptospira isolates belonging to serogroup Pomona by restriction endonuclease analysis: Serovar Kennewicki. J. Clin. Microbiol., 21: 585-587.

Thrusfield, M., 2007. Veterinary epidemiology, 3rd Edition, Blackwell Science Limited, Oxford. www.blackwellpublishing.com

Toyokawa G., Cho H., Yukiko I., Masanori Y., Masashi T., Shinya H., Kazuhiro M., Noriaki S., Hirotoshi T., Tatsuhiko T., Helen I. F., John D. K., David E.N., Bruce A. J., Yoshihiko M. P., Yusuke, N. and Ryuji, H. 2011. The Histone Demethylase JMJD2B plays an essential role in human carcinogenesis through positive regulation of cyclin - dependent cyclase 6. Cancer Prevention Research., 4(12): 2051 - 2061.

Udomsak, N., Janijira, T., Prapaporn, S., Preeraya, S., Metawee, T., and Wirichada, P., 2020. Optimization of culture protocols to isolate Leptospira species from environmental water, field investigation and identification of factors associated with the presence of Leptospira species in the environment. Trop. Med. Infect. Dis., 5(2): 94. https://doi.org/10.3390/tropicalmed5020094