ABSTRACT

Culicoides are tiny blood sucking flies of the family ceratopogonidae, and are most abundant of hematophagous insect with over 1500 species approximaly known worldwide. Culicoides milnei is the principal vector for the transmission of Mansonella perstans, which is one of the filarial nematodes causing Masonellosis in the human population. There have been little or knowledge in either maintaining this wild species (Culicoides milnei) in captivity in the field or rearing them in the laboratory. This work was designed to optimized Field maintenance and Laboratory Rearing Conditions for the Improvement of Survival of Engorged Culicoides milnei for the Large Scale Production of Infective larvae of Mansonella perstans. A total of 6100 culicoides milnei were collected from 2 donors with microfilaria load of (16450mf/ml) (750mf/ml) respectively. The engorged flies were fed daily with 15% sucrose solution while mortality and survival was visually monitor under different rearing conditions until dissection. Data was exported in to SPSS in to excel sheet for analysis. Kruskal wallis test and Mann whitney test was used to test for different variable. It was observed that flies maintain and reared up 12days had the highest survival (79.1%) with a significant difference (P < 0.001). as compare to 13 and 14days. Flies that were reared from donor 2 had a survival rate of (77.8%) with a significant difference of (P < 0.001)  For the various rearing conditions, flies maintain and reared inside cold box (on ice park) had a survival rate of (70.1%) with a significant different of (P = 0.0143). Flies in which tubes were moist with distilled had (69.1%) survival with a significant difference of (P = 0.0146).  Lastly, flies maintained and reared under the temperature 24ᵒC/75% RH had (71.8%) survival with a significant difference (P < 0.001). For the standard conditions to maintain and reared the engorged flies  to survive for the large scale  production of infective larvae in the field and laboratory the flies, should be place inside cold (ice park), tubes moist with distilled water and reared under temperature of 24ᵒC/75%RH.  

CHAPTER ONE

INTRODUCTION

1.0 Background of the study

Culicoides are tiny blood sucking flies of the family ceratopogonidae and are most abundant of hematophagous insects, which occur throughout most of the inhabited world, transmitting some disease-causing pathogens of humans and domestic or wild animals (WHO, 2009). Approximately, 1500 Culicoides species have been identified worldwide (Sheffer, 2011) with the notable exceptions of New Zealand, Iceland and the Hawaiian Islands, they also range from sea level to over 4000 m in elevation (Mellor et al., 2000, Borkent, 2012). The distribution, abundance and seasonal occurrence of these holometabolous flies is determined by the availability of moisture-rich habitats that are essential for the development of immature stages (Mellor et al., 2000). In areas where Culicoides are very abundant, they constitute a biting nuisance (Wanji et al., 2019) to humans, domestic and wild animals.

Their bites are sometimes associated with allergic skin reactions, which in some individuals may result in urticaria. Culicoides are diverse both in species and in the pathogens they transmit. They are involved in the transmission of animal and human viruses (Snyman et al., 2021; Zuzana et al., 2021),   such as African Horse, sickness virus (AHSV) and Blue Tongue Virus (BTV) as well as the filarial worms of the genus Mansonella, (Mansonella perstans. M streptocerca, M Ozzardi and Mansonella spp DEUX) the causative agents of Masonellosis. These holometabolous flies are associated with aquatic or semiaquatic habitats, e.g. mud or moist soil around streams, ponds and marshes.  They are also associated with the dung of various animals such as cattle, bufalo and equines, and breed in rotting fruit, cacti, banana stems and leaf detritus (Hopkins 1952; Meiswinkel et al., 2004) .Therefore the  presence of animals, especially livestock such as cattle, horses and sheep also play an important role in the abundance/distribution of Culicoides spp.

The first definite association of flies of the family Ceratopogonidae with transmission of filarial infection was that made by sharp (1927, 1928) who reported on the development of Acanthocheilonema perstans in Culiciodes grahamii and Culicoides austeni (milnei) in Cameroon. However, Henrard and peel, (1949) and Chardome and peel (1949), strongly suggest that sharp was actually working with Dipetalonema streptocerca instead of Acanthocheilonema perstans. Seven Culicoides species were reported by Glick, (1990) to be found in Cameroon of which, Nicholas and Hopekins (1952) incriminated Culiciodes grahammi and Culicoides milnei as vector transmitting Mansonella perstans in south West Cameroon.  These studies were carry out long ago and the vector as well as entomological indices of disease transmission might have changed.        

Culicoides control programs are generally implemented to reduce biting nuisance of livestock and/or humans and arbovirus transmission. Control measures can be applied against both larvae and adults. In the framework of Integrated Pest/Vector Management (IPM/IVM) and One Health criteria, habitat manipulation and larvicidal treatments with chemical and/or bio-rational insecticides have been the mostly used strategies against Culicoides larvae. (Carpenter et al., 2008).

Mansonella perstans is a vector-borne human filarial nematode, transmitted by tiny blood-sucking female flies called midges (Bregani et al., 2007). Mansonellosis is one of several filarial nematode infections for which humans are the definitive host.  It is for this reason that it is put in the same category as several parasitic infections of importance to global public health, including onchocerciasis, lymphatic Filariasis, Dracunculiasis, and Loiasis. While some of these major filarial infections have gained international attention such as Onchocerciasis (river blindness) (Gardon et al., 2002), Dracunculiasis (Guinea worm) (Barry, 2018; Cairncross, 2002) and lymphatic Filariasis have been the focus of global eradication efforts while Mansonellosis has been neglected.

Mansonella perstans is a little known but widespread human filarial parasite in many parts of Sub-Saharan Africa (Cairncross, 2002). 33 countries in Sub-Saharan Africa, with about 580 million inhabitants (World Bank, 2008), appear to be endemic for transmission and infection prevalence are often very high in endemic areas, even among children. Nelson (1965) considered M. perstans to be one of the most prevalent parasites of man in tropical Africa. In spite of this, very few studies (Wanji et al., 2019) have been carried out on its epidemiology and the associated health consequences in endemic populations, and no simple and effective drug therapy for treatment and control of the infection has been identify, despite the number of broad spectrum activity of anthelminthic drugs, increased resistance calls for new anthelminthic drugs.

Chemotherapy is the main approach in the treatment of M. perstans infections and 3 antifilarial drugs, diethylcarbamazine (DEC) and ivermectin (IVM), as well as various anthelmintic benzimidazoles (mebendazole, levamizole, albendazole and thiabendazole) have been documented as the most commonly prescribed drug for M. perstans (Simonsen et al., 2011). It can thus be classified as one of the most neglected among the Neglected Tropical Diseases. Major reasons for this lack of attention are that M. perstans infections prevail in poor rural populations and that it has not been linked with a clear and distinct clinical picture with only mild symptoms like subcutaneous lesions, fever, headaches, arthralgia, and neurologic manifestation (CDC, 2009). Although symptoms may often not be immediately visible, distinct, there could be more hidden effects of infection. M perstans might potentially interfere with the host’s regulatory mechanism and influence the outcome of other infection such as Malaria, Tuberculosis and HIV, which often thrive in the same environment (Babu et al., 2009), has clearly shown that helminthes attenuate the TH1 immune response required for the control of M. tuberculosis infection (Simonsen et al., 2010).

 Much of the information available on M. perstans infections has in fact been obtained as a side-product from studies of other filarial parasites – considered more important from a public health perspective – such as Loa loa, Onchocerca volvulus and Wuchereria bancrofti, in which individuals were found to have M. perstans co-infection. Among the three types of human Mansonellosis, the one caused by M perstans is usually regarded as the most important to treat because it pathology is not well known. (Asgeirsson et al., 2017). There exist genetically distinct strain of M. perstans (strain that lack Wolbachia) which make it difficult to formulate treatment guideline. In M. perstans endemic areas, very few studies (Wanji et al.,2016 ;Simonsen et al.,2011) have tried to incriminate the species of vectors implicated in the transmission of the parasite especially in the forest areas of Cameroon. Given the limited information on Culicoides species in the Littoral Region of Cameroon, The aim of this study was to optimize field and laboratory rearing conditions for the improvement of the survival of engorged Culicoides milnei, for large-scale production of infective larvae of Mansonella perstans.

1.1 Statement of the problem.

Biting midges (Ceratopogonidae) are identify globally as vector of some diseases of medical and veterinary importance.  It is estimated that 114 million individuals are infected with Mansonella perstans in west, East and central Africa and in some neo-tropical regions of central and south America and that 600 million people live in high risk areas in 33-African countries. Their bites are painful and a nuisance to the human population where these Culicoides species are abundant and they are a hindrance to tourism, as tourists cannot visit biting midges infested sites. Although Mansonellosis infection may not have high clinical consequences to humans, however the presence of the parasite in human have been shown to have some immune modulatory consequences (Ritter, 2018; Glick, 1990). In the animal husbandry, they are known to transmit several diseases (African Horse sickness virus (AHSV), Blue Tongue Virus (BTV) and Schmallenberg virus (SBV), equine encephalitis, Ephemeral fever, Epizootic Hemorrhagic Disease virus (EHDV), and akabane) to the livestock populations in Europe, Africa, Asia, south and central Americas (Du Toit, 1944; Mellor et al., 2004; Lysyk, 2006 and Carpenter et al., 2013). Again several reports ( Dyce Sharp, 1928, Hopkin, 1952, Hopkins and Nicholas 1952, Nicholas and Kershaw 1954 and Wanji et al., 2019) have shown that Culicoides milnei may be  the principal vector in the transmission of Mansonella perstans, which is one of the most wide spread filarial nematode causing Mansonellosis in the human population especially in sub –Saharan Africa. There have been little or no knowledge on the standard field maintenance and laboratory rearing conditions of engorged Culicoides milnei for the mass production of Mansonella perstans infective larvae. . The aim of this study was to find out the optimum maintenance/rearing conditions for the improvement on the survival of the engorged Culicoides milnei for the large-scale production of infective larvae of Mansonella perstans.

1.2 Rationale

Over the years, more than 1,400 Culicoides species (Ceratopogonidae) (Boorman, 1994, Scheffer, 2011) have been described worldwide with exceptions from the Antarctica and New Zeeland (Borkent 2012, Mellor et al., 2004, Meiswinkel et al, 2004, Venter 2012). The medical and veterinary importance of these hematophagous insects cannot be over emphasis as they are responsible for the transmission of several diseases to both animals (Africa horse sickness virus, bluetongue virus, equine encephalitis) and human (Mansonellosis). There have been several attempts in the world to successfully rear other Culicoides species in the laboratory such as Culicoides stellifer and Culicoides sonorensis Wirth and Jones (1979), but very few reports have mentioned rearing of Culicoides species in the laboratory in Cameroon such as C. grahamii. C. fulvithrox, C. innornatipennis, C. milnei (Wanji et al., 2019) again none of these studies were aimed at developing standard field maintenance or laboratory rearing conditions for the mass production of infective larvae of Mansonella perstans. This study is of great significance to optimizing field maintenance and laboratory rearing conditions of Culicoides milnei for the large-scale production of the infective larvae of Mansonella perstans, which could be used for future drug screening, animal model development and genotyping.

1.3 Hypothesis

Optimizing field maintenance and laboratory rearing conditions of engorged Culicoides milnei may play a key role in their long-term survival of up to 14 days a period of development from microfilariae to infective larvae

1.4 General Objective

 To optimize field maintenance and laboratory rearing conditions for the improvement of the survival of engorged Culicoides milnei, for large-scale production of infective larvae of Mansonella perstans.

1.5 Specific Objectives

1. To Collect engorged  Culicoides milnei using drop trap techniques
2. To monitor the survival of the vector under different rearing conditions within a period   of 1-14 days post infection.
3. To know the standard rearing conditions for the survival of the vector for the production of infective larvae after dissection.

1.6 Research questions

1. What is the best method of collecting engorged culicoides milnei?
2. What is survival rate of the vector maintained and reared within the period of 1-14 days post infection?
3. What is the standard rearing conditions for the survival of the vector for the production of infective larvae after dissection?