ABSTRACT
The prevalence, morphometry and molecular characterisation of Fasciola species from slaughtered cattle and sheep, and snails from Maiduguri, Gombe and Jos were investigated. Prevalence of Fasciola spp. was studied by determination of eggs of the parasite in both faeces and bile, while morphometric description was done using standard keys and descriptions. For molecular characterisation, the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA), 28S rDNA and NADH dehydrogenase subunit 4 (NAD4) respectively were amplified from individual Fasciola isolated from bile duct by polymerase chain reaction (PCR). All collected snails were subjected to morphological identification using standard keys. The DNA of Fasciola spp. was similarly characterised in Lymnaea natalensis by the use of 28S rDNA, while the Lymnaea (Radix) natalensis was characterised by the use of 18S rDNA. Representative amplicons of both Fasciola spp and Lymnaea natalensis were sequenced, and NCBI databases were used for sequence homology analysis using BLAST and ClustalW programs, while phylogenetic analysis was done in ApE and Molecular Evolutionary Genetics Analysis (MEGA). Combined location prevalence rate was 27.52% for cattle and 11.97% for sheep. For cattle, sex, age or breed had no significant (p≥0.05) impact on prevalence rate, while for sheep, only age had an impact, as more adult than young sheep were infected (12.62% compared to 2.63%). Jos, had a significantly (p<0.05) higher prevalence of 35.43% followed by Gombe (26.99%), while Maiduguri had the lowest (19.63%). In cattle, there was a negative association between the number of positive animals and egg per gram of faeces and bile (EPG), with Maiduguri having a mean EPG of 65.85±13.2 followed by Gombe 45.48± 10.8 and Jos the least (14.4± 1.34). For sheep, Jos had significantly (p<0.0.010) higher prevalence rate (24.35%) than both Maiduguri (6.16%) and Gombe (5.52%). Actual mean EPGs were 19.71, 36.34 and 14.47 for the respective locations. For both cattle and sheep, mean EPG were significantly (p<0.05) higher by the bile sedimentation method than by faecal analysis. For cattle, values were 41.12±5.8 versus 15.72± 3.8, while for sheep values were 50.88±15 versus 8.36±1.9 for bile and faeces respectively. Month of sampling had a significant (p≤0.05) influence on infection rate with most animals infected in January – Febuary, being the months with highest infections. Morphological differences were observed in linear measurements and ratios. Three useful morphological parameters (BL, CL, CW) and one ratio (BW/BL) showed significant (p<0.05) variations among samples from the locations, and may therefore be relevant for phenotypic differentiation of species. The molecular identification using ITS-1, 28S rDNA and NAD4 and the sequencing revealed the presence of both F.hepatica and F.gigantica in the study areas. Analaysis of the overall genetic sequence data showed that 64.7% of the sequences of Fasciola isolates were F. gigantica, while 35.3% were F. hepatica. Of the F.hepatica isolates, 66.6% were from Jos. The phylogenetic tree constructed based upon the ITS-1 sequences revealed a close relationship (95-98%) with isolates of F. gigantica from Bukina Faso and South Africa, while the F. hepatica isolate had 84% identity with that from Iran. The 18S rRNA of Lymnaea natalensis was identified molecularly at 450bp. The sequences had between 99-100% similarities among themselves and 98-100% with other deposited reference Lymnaea natalensis sequences. All our Lymnaea (Radix) natalensis sequences formed a clade different from the clade formed by other reference sequences. Lymnaea (Radix) natalensis sequences from Nigeria seem to clade separately from deposited sequences from GenBank. Conclusively, the prevalence of Fasciola spp indicates a high morbidity in the sampled animals. The study had shown that, a well defined relationship exists between egg counts in bile and faeces of cattle, but not sheep in this study This study also confirmed the presence of F.gigantica and strongly suggests the existence of F.hepatica for the first time, to the best of our knowledge, using molecular tools. The findings of the two species of Fasciola (F. hepatica and F.gigantica) may have implications for livestock and human infections and vaccine types to be developed in the control of fasciolosis in the study locations and Nigeria.The findings also confirmed the existence of Lymnaea (Radix) natalensis and its role as intermediate host of Fasciola spp in the study areas. In addition, experimental infection of different breeds of cattle and sheep with Fasciola spp, complete sequence of the ITS-1, 28S rDNA, NAD4 of Fasciola spp and 18S rDNA of Lymnaea (Radix) natalensis and investigation of snails such as Melanoides tuberculata and Biomphalaria pfeifferei as potential intermediate hosts of Fasciola spp are recommended for further studies.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
The disease, fasciolosis is a parasitic disease of domestic ruminants caused by two liver fluke species: Fasciola hepatica and Fasciola gigantica (Trematoda). Fasciola hepatica has a cosmopolitan distribution, mainly in temperate zones, while Fasciola gigantica is found in tropical regions of Africa and Asia (Mulugeta et al., 2011). Infection with Fasciola is of veterinary and medical importance, particularly in areas of high-density cattle and sheep production (Taha et al., 2014). Fasciola spp. occur commonly in the bile duct and liver of sheep, goat, cattle and buffalo, mule, pig and may rarely be found in unusual hosts such as man and horse (Bhatia et al., 2006). The economic importance of fasciolosis is mostly due to mortality and high losses from reduced feed efficiency, weight gains, milk production, reproductive performance, carcass quality and work output in draught animals, and condemnation of livers at slaughter (Vassilev and Jooste, 1991). An estimate of more than US$200 million dollars globally, is lost every year in livestock products due to the effects of fasciolosis (Moazeni et al., 2005). Fasciolosis also has the widest geographic spread of any emerging vector-borne zoonotic disease.World Health Organisation (WHO) estimated that at least 2.4 million people are infected in more than 70 countries worldwide, with several million at risk. Although, the infection in humans was described as accidental, and characterised by jaundice caused by obstruction of the biliary tree (Moghadami and Mardani, 2008), there have been increased reports of the occurrence of the infection in man of recent. No continent is free from fascioliasis, and it is likely that where animal cases are reported, human cases also exist (WHO, 2016). Varying prevalence of the infection has been reported in animals and man (Mulugeta et al., 2011; Biu et al., 2013; Jean-Richard et al., 2014). In animals, the infection is more important in cattle where high prevalence rates have been reported in comparison to other domestic animals (Jean-Richard et al., 2014). In Nigeria, several investigators have reported the disease in cattle and small ruminants; for example Ardo and Aliyara (2014) reported 0.32% and 0.23% in sheep and goats respectively in Yola, while Magaji et al., (2014) reported 27.68% prevalence among slaughtered cattle at Sokoto central abattoir. Similarly, previous reports from Zaria, Nigeria, ranked fasciolosis among top pathological conditions encountered in slaughtered animals at abattoirs (Raji et al., 2010; Alawa et al., 2011). Adult Fasciola live in bile ducts producing eggs that are excreted with the faeces. The life cycle of the disease involves hatching of the eggs in 14 days at a temperature of 22-26oC (Bhatia et al., 2006). Hatching occurs in moist conditions only after the first larval stage, the miracidium, has formed and when ambient temperatures rise above 5-6oC (Radostits et al., 2006). Miracidia must find and invade the tissues of a suitable snail host within 24-30 hours.Cercariae which emerge from snails about 6-8 weeks after infection, encyst on pasture as metacercariae, the infective form for ruminants and other susceptible hosts. Following ingestion of metacercariae by grazing animals, the parasites (newly excysted juveniles, NEJ) emerge from their cysts in the intestine, traverse the intestinal wall, which takes just a few hours, before migrating through the liver capsule and into the parenchyma (Molina-Hernández et al., 2015). Here, their feeding and migratory activities cause tissue perforation and haemorrhage, leading to extensive tissue damage. After about 7–8 weeks, the parasites migrate into the bile ducts, mature and produce 20,000–24,000 eggs per fluke per day which are released onto the pasture with the faeces (Urquhart et al., 1987). Clinically, fasciolosis presents with distended abdomen, ascites, anaemia, stiff gait, loss of appetite, black scouring and bottle jaw, similar to signs seen in amphistosmosis and haemonchosis. Three types of clinical presentations; acute, subacute and chronic forms have been reported (Bhatia et al., 2006). These presentations may however depend on the state of the animal at the time of infection especially cattle. Cattle may overtime develop a partially protective immune response to F. hepatica in regions where the infection occurs. The interaction of such factors as age of the host, innate resistance, previous exposure and present level of parasite exposure determine the degree of parasite establishment and the pathologic impact of the infection. Older cattle with previous exposures have a greater resistance to infection than young parasite-naive calves (Kaplan, 2001). The snails belonging to the genera; Lymnaea, Amphipepla, Simlimnaea, Galba, Fossaria, Stagniciola and Pseudosuccinea serve as intermediate hosts, with specific species restricted to different geographical regions of the world (Smyth 1994; Bhatia et al., 2006). In Nigeria, the most important snail is Lymnaeaa natalensis. These intermediate hosts are found in shallow water and are capable of migrating for long distances over mud and wet pasture, thereby increasing the chances of exposure of susceptible animals (Bhatia et al., 2006)
1.2 Statement of Research Problem
Fasciola hepatica infects more than 300 million cattle and 250 million sheep worldwide and together with F. gigantica, cause significant economic losses to global agriculture; through lost productivity such as reduction in milk and meat yields (Mas-Coma et al., 2005). Previous report by Talukder et al. (2010) estimated a conservative amount of over US$ 3.2 billion per annum, as losses due to fasciolosis in production animals worldwide, which was slightly higher than the US$ 3 billion estimated recently by Elelu and Eisler (2017). According to a World Health Organization (WHO) report in 2007, fasciolosis was limited in the past to specific and typical geographical areas (endemiotopes), but is now widespread throughout the world. Until recently, human cases occurred occasionally but are now increasingly reported from Europe, the Americas and Oceania (where only F. hepatica is transmitted) and from Africa and Asia (where the two species overlap)(WHO, 2016). The epideomiology of fasciolosis is linked to a myriad of factors. The presence of streams, wetlands and pastures on farms were significantly associated with the presence F. hepatica infection in cattle herds previously (Olsen et al., 2015). The metacercariae of Fasciola spp. take about six days to reach the liver or only about 48 hours in exceptional cases (Bhatia et al., 2006).The acute phase of fasciolosis characterised by severe haemorrhage caused by the migrating juvenile flukes in the hepatic tissue rupturing the blood vessels. The liver parenchyma particularly the ventral lobe associated with gall bladder is severely damaged, assuming an uneven surface covered with blood clots (Gupta, 2014). Most of the pathologies described in fasciolosis are often due to the migration of the immature stages of the parasite and are manifested as early as 2 weeks after the infection (Guobadia and Fagbemi, 1997). Fasciola gigantica has been previously characterised from African countries of Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric parameters (Amor et al., 2011). For sometimes now, the identification of Fasciola spp. based on traditional morphological features has been used for speciation (Ai et al., 2011). This is however limited, especially in regions where the two common species are known to overlap. Similarly, the emergence of an intermediate/hybrid Fasciola in some endemic foci has opened a new dimension in the study of this important disease. This hybrid has long been reported from Asian countries of Japan, Korea, China and Vietnam (Itagaki and Tsutsumi, 1998; Agatsuma et al., 2000; Lin et al., 2007; Itagaki et al., 2009) and most recently from Pakistan (Mufti et al., 2014). The hybrid form was also confirmed in Egypt by the aid of morphological features (Periago et al., 2008) and molecularly using mitochondrial and ribosomal gene markers (Amer et al., 2011). Understanding genetic structure and status of genetic variation of the parasite populations has important implications on epidemiology and effective control of fasciolosis (Rokni et al., 2010). Equally, molecular study will provide information on the taxonomic status of Fasciola isolates against the traditional reliance on morphological characterisation.
1.3 Justification of the Study
Among all the livestock, ruminants, comprising sheep, goats and cattle, constitute the largest group reared by farm families in the country‟s agricultural system. Nigeria has an estimated population of 34.5 million goats, 22.1 million sheep and 13.9 million cattle (Lawal-Adebowale, 2012). However, about 90 per cent of the country‟s cattle population and 70 per cent of the sheep and goat populations are concentrated in the northern part of the country (Lawal-Adebowale, 2012).
Most studies aimed at determination of prevalence of Fasciola spp. conducted in Nigeria (Nwosu and Srivastava, 1993; Omowaye et al., 2012; Adang et al., 2015) and Southern Lake Chad region (Jean-Richard, 2014), have been based on examination of liver at post-mortem. Hence, comparatively, fewer reports exist on the detection of Fasciola by coprology, the traditional detection method in the laboratory, than by post-mortem examination in the abattoir. The exact prevalence of fasciolosis is most likely underestimated due to the lack of comprehensive epidemiological surveys performed in potentially endemic areas. Furthermore, in some areas where reports exist, there is a time lag of up to a decade or more, thereby making such reports obsolete and probably not in tune with current realities.
The faecal egg count provides an estimate of the degree of infection with Fasciola spp. in the hosts. However; it is deficient in estimation due to intermittent release of bile into the intestine. Also, the inability to sample bile for eggs in living animals, makes bile a bad candidate for sampling in clinical applications (Radostits et al., 2006). Thus, estimating both faecal and bile egg counts will provide important information on the quantum of infection and exact relationship between eggs in faeces and bile (Radostits et al., 2006). The characterisation of Fasciola spp. by the use of morphological keys has been applied in different parts of the world including Africa. Such include the study by Biu et al. (2013) in Nigeria, Chauke et al. (2014) in Zimbabwe and Shaldoum et al. (2015) in Egypt. Despite the inability of morphological keys to accurately differentiate between the two Fasciola species especially regarding immature/juvenile flukes (Ashrafi et al., 2006), available evidences have shown that it is still important when careful morphometry is carried out. In fact, for research activities in Africa and Asia, where both species overlap, Mas-Coma et al. (2009a) recommended a minimum morphometrical study of adult flukes parameters such as body length (BL), maximum body width (BW), BL/BW ratio, distance between the ventral sucker and the end of the body (VS-P) and the distance between the ventral sucker and the union of the vitelline gland (Vit-P) distance and eggs for inclusion within genetic characterisation studies. Where available, species variations among isolates from different geographical locations presumably provide an idea of ecological adaptation as with other helminthes such as Haemonchus contortus (Rahman and Hamid, 2007; Kumsa et al., 2008). Molecular identification of parasites including Fasciola is important as it helps with information on the epidemiology, genetic variation and diagnosis (Liu et al., 2014). For Fasciola spp, this often employs the use of genetic markers such as internal transcribed spacers (ITS 1&2) and 5.8S of the nuclear ribosomal DNA, 28S ribosomal ribonucleic acid (rRNA), 18S rRNA, mitochondrial NADH dehydrogenase I (NDI) and Cytochrome C Oxidase I (COI) genes. Because of its highly repeated and conserved regions, the nuclear ribosomal DNA is especially designed for molecular studies (Chilton, 2004; Choe et al., 2011). These markers have successfully been used to differentiate between the two common species including the intermediate form. Despite the widespread use of moleular techniques for genetic characterisation and for determining inter- and intra-species variability, there is dearth of information on their application in Nigeria. Most importantly, there is need to use molecular techniques to determine the available Fasciola species infecting cattle, sheep and goats, which are the most important animal hosts in Nigeria. The need to characterise the isolates of cattle and sheep origins from different geographical regions of Nigeria cannot be overemphasized in view of the available evidences on the restriction of the two important species of Fasciola to specific climatic zones. These climatic zones are represented by some regions in Nigeria. Similarly, the emergence of a hybrid/intermediate form of Fasciola in some endemic regions including Africa underscores the need to determine the isolates present in Nigeria, particularly the Northern part of the country where about 90% and over 70% of the cattle and small ruminants are reared (Lawal-Adebowale, 2012). Because of the larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes detected in Chinese aspermic Fasciola populations than in aspermic populations from other neighboring countries, Ichikawa-Seki et al.(2017a) proposed that aspermic Fasciola flukes should be termed as „hybrid‟ Fasciola. The snail intermediate hosts of Fasciola spp. belong to the genus Lymnaea. Lymnaea truncatula and Lymnaea natalensis are the major vector hosts in temperate and tropical regions respectively (Bhatia et al., 2006; Radostits et al., 2006). In the previous report of Schillhorn van Veen (1980) as cited in Brown (2005), L. natalensis was described as the main intermediate host if not the only one in large areas of Africa for Fasciola, particularly west Africa. The previous report of Dar et al.(2010) on the experimental transmission of Fasciola hepatica by Lymnaea natalensis in Egypt and the finding of the sporocysts of Fasciola gigantica in Achatina fulica, an edible snail not known for the transmission of parasite in Nigeria (Igbinosa et al.,2016), may need further investigation of these snails. Morphological characterisations have successfully been applied to Lymnaeaid snails from France (Hurtrez-Boussès et al., 2005), Brazil (Carvalho et al., 2004) and Nigeria (Falade and Otarigho, 2015). Similar characterisation molecularly using genetic markers such as ITS-1, ITS-2, 16S ribosomal (Correa et al., 2010) and 18S ribosomal gene (Howell et al., 2012) have either confirmed Lymnaea natalensis or differentiated among the various species of Lymnaea.
1.4 Aim of the Study
The research was to determine the prevalence and characterise Fasciola spp from snails, and slaughtered cattle and sheep from Maiduguri, Gombe and Jos abattoirs.
1.5 Objectives of the Study
The objectives were to:
- determine the prevalence of Fasciola in bile and faeces of slaughtered cattle and sheep from Maiduguri, Gombe and Jos abattoirs.
- characterise the Fasciola isolates morphometrically using body length (BL), body width (BW), body length to width ratio (BL/BW); Cone Length (CL).
morphologically identify snail samples from Maiduguri, Gombe and Jos.
- molecularly characterise Fasciola from the slaughtered cattle, sheep and also sampled snail tissues using PCR.
- molecularly characterise Lymnaea species from the study areas
- sequence the DNA amplicons and determine their evolutionary relationship with reference sequences in the GenBank.
1.6 Research Questions
- What is the prevalence of Fasciola in bile and faeces of slaughtered cattle and sheep from
- Can morphological features (body length (BL), body width (BW), body length to width ratio (BL/BW); Cone Length (CL)) characterise Fasciola spp from the study areas
- Can morphological keys identify snails from the study locations?
- Can PCR characterise Fasciola spp from the slaughtered cattle, sheep and the sampled snail tissue?
- Can PCR characterise Lymnaea species from Maiduguri, Gombe and Jos?
- Can DNA amplicons be sequenced and evolutionary relationships determined?
