Prevalence and risk factors of Cryptosporidium species among domestic animals in rural communities in Northern South Africa.

To determine the prevalence and possible risk factors for the transmission of Cryptosporidium species among animals in rural Limpopo Province, South Africa. A total of 314 stool samples from 64 households were collected from animals in three villages situated in the Vhembe and Mopani Districts, South Africa and examined for Cryptosporidium, using the modified Ziehl Neelsen technique and confirmed by the real time PCR method. A questionnaire was developed to capture demographic data as well as other household information from the owners of the animals. Positive samples were further sequenced for the identification of the species present in the samples. The overall prevalence of Cryptosporidium among the animals was 31.2%. Of all the animal types tested goats (47.7%) appeared to be the most infected followed by cattle (26.8%) and chicken (7.4%). From the 64 households surveyed 43 (67.2%) had at least one or more infected animals. Adult animals were more infected (32%) compared to young animals (29%) but the difference was not statistically significant (p=0.793). The gender of the animal as well as the consistency of the stool did not affect the occurrence of Cryptosporidium; however, the level of education as well as the gender of the owners significantly affected the prevalence of Cryptosporidium among the animals they kept. C. parvum was the most commonly isolated organism while C. andersoni was identified in our region for the first time as well and occurred in both goats and cattle. This study showed a high prevalence of Cryptosporidium in domestic animals, which could constitute a health threat to both animals and humans in the region. The gender of the head of the Household and level of education were very significant factors in the prevalence of Cryptosporidium among the animals. Community education will be useful in helping reduce the impact of these infections.

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa.

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.

Serological diagnostic tools for the major tick-borne protozoan diseases of livestock.

Tick-borne protozoan diseases, babesiosis and theileriosis, are among the most important diseases affecting the productivity of livestock worldwide and resulting in high economic losses. A prerequisite for the control of these diseases is to study their epidemiology by mapping their distribution and seasonality. As clinical diagnostic and surveillance tools, serological tests such as the complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and the enzyme linked immunosorbent assay (ELISA) have been successfully used over decades. With the development in molecular biology, recombinantly expressed parasite molecules have emerged and substituted crude parasite antigen used in serology. A popular format of these tests is the antibody binding competitive inhibition and the indirect antibody detection ELISA. Under the precondition that these tests are correctly designed and validated, they provide a powerful tool for epidemiology, with greater advantages of affordability and amenability to standardization. This paper reviews the pathogenic tick-borne protozoan diseases and the respective diagnostic ELISA based serological tests currently available for serosurveillance.

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.

Estimation of the minimum number of Leishmania major amastigotes required for infecting Phlebotomus duboscqi (Diptera: Psychodidae).

BACKGROUND: Leishmaniasis is a vector-borne disease in which Leishmania parasites are transmitted by the bite of phlebotomine sand flies. Amastigotes are ingested by the sand fly vector with a blood meal taken from an infected host. This is followed by their differentiation into metacyclic promastigotes which are selectively released and permitted to migrate interiorly so as to make them available for transmission by bite. However, the actual number of amastigotes ingested by the sand fly in the blood meal is not known. OBJECTIVE: To investigate the minimum number of Leishmania major amastigotes required to cause an infection in Phlebotomus duboscqi following an infective blood meal. DESIGN: A laboratory based study. SETTING: Centre for Biotechnology Research and Development, Kenya Medical Research institute, Nairobi. RESULTS: Dissection of all fed sand flies at six days post-infective blood meal revealed that blood containing one amastigote per 0.3 microl in a total volume of 0.5 ml was able to cause an infection in the sand flies, but very few sand flies got infected (7.6% and 9.6% respectively). Concentrations of ten amastigotes per 0.3 microl in 0.5 ml gave infection rates of 35.4% and 26.3% respectively, suggesting that even when the concentration of amastigotes in a bloodmeal was high, not all sand flies feeding on it were able to pick up the parasites. CONCLUSIONS: These observations suggests that one amastigote is sufficient to cause an infection to a sand fly and as a result of multiplication in the gut and the existence of mechanisms that increase the number of infective bites delivered by a female sand fly they are able to sustain the transmission of leishmaniasis in an area.

Effects of anti-Leishmania monoclonal antibodies on the development of Leishmania major in Phlebotomus duboscqi (Diptera: Psychodidae).

BACKGROUND: Research in our laboratory has previously shown that immune-mediated transmission blocking may be applied to Leishmania infections and that the LPG molecule and anti-LPG monoclonal antibodies was found to be an excellent candidate against L. major infections. OBJECTIVE: To test the effect of monoclonal antibodies (MABs) raised against different species of Leishmania for their ability to inhibit development of Leishmania major in Phlebotomus duboscqi sand flies. DESIGN: A laboratory based study. SETTING: Centre for Biotechnology Research and Development, Kenya Medical Research Institute, Nairobi. RESULTS: Sand fly dissections on days two, four and six post-feeding showed that monoclonal antibodies against L. donovani (Ld2cb and Ld3A3) were the most effective at inhibiting L. major development than those raised against L. aethiopica, L. major or L. tropica. Ld2cb inhibited L. major development by 82% in sand flies fed on 1 x 10(6) amastigotes while Ld3A3 inhibited by 72%; 58% and 74% in those fed on 1 x 10(5) amastigotes respectively. Monoclonal antibodies against L. aethiopica (Lae 3c6) inhibited L. major development by 28% and 40% for sand flies fed on 1 x 10(6) and 1 x 10(5) amastigotes respectively. Anti-L. major monoclonal antibody (Lm5A5) inhibited L. major development by 16% in sand flies fed on 1 x 10(6) amastigotes and 25% in sand flies fed on 1 x 10(5) amastigotes. Anti-L. tropica antibody (Lt2c8) inhibited L. major development in P. duboscqi fed on 1 x 10(6) by 28 %and 33% in those fed on 1 x 10(5) amastigotes. Most of the parasites seen in sand flies which fed on L. donovani mABs (Ld2cb and Ld3A3) were nectomonads and a few haptomonads. In all the control groups, parasite development followed the normal developmental stages up to the metacyclic stage. In sand fly groups fed on monoclonal antibodies raised against L. aethiopica, L. major or L. tropica there was limited parasite development inhibition, and the promastigotes developed and migrated forward in a normal pattern as observed in the controls. CONCLUSIONS: These results suggests a possible role of humoral mechanisms in protection against leishmaniasis and potentially useful in reducing parasite development in the sand fly.

A survey of tick control methods used by resource-poor farmers in the Qwa-Qwa area of the eastern Free State Province, South Africa.

A survey conducted in five villages in a resource-poor farming community in Qwa-Qwa, using the rapid rural appraisal technique and a questionnaire survey, showed that a significant proportion of the farmers (84%) use traditional or alternative methods to control ectoparasites, while 16% use commercial acaricides (chi2 = 7.1; P < or = 0.05). Alternative control methods included the use of used engine oil, household disinfectant and paraffin. Killing of ticks was the main reason for control (40%), with disease control being second (20%). Other reasons given for controlling ticks were to prevent damage to teats, to provide animals with a clean appearance and to protect hides. Some 40% of farmers were aware of the effects of ticks on their animals. There is a need for farmer education that will provide information on integrated tick management and its advantages over absolute reliance on commercial acaricides.

Impact of waste disposal on health of a poor urban community in Zimbambwe.

OBJECTIVE: To assess excreta and waste disposal facilities available and their impact on sanitation related diseases in Epworth, an informal settlement on the outskirts of Harare. DESIGN: Descriptive cross-sectional survey. SETTING: This was a community based study of Epworth informal settlement. SUBJECTS: A total of 308 households were interviewed. Participating households were randomly selected from the three communities of Epworth. Secondary medical archival data on diarrhoeal disease prevalence was collected from local clinics and district health offices in the study areas. RESULTS: Only 7% of households were connected to the sewer system. The study revealed that in Zinyengere extension 13% had no toilet facilities, 48% had simple pits and 37% had Blair VIP latrines. In Overspill 2% had no toilet facilities, 28% had simple latrines and 36% had Blair VIP latrines while in New Gada 20% had no toilet facilities, 24% had simple pits and 23% had Blair VIP latrines. Although a significant percentage had latrines (83.2%), over 50% of the population were not satisfied with the toilet facilities they were using. All the respondents expressed dissatisfaction with their domestic waste disposal practices with 46.6% admitting to have indiscriminately dumped waste. According to the community, diarrhoeal diseases were the most prevalent diseases (50%) related to poor sanitation. Health statistics also indicated that diarrhoea was a major problem in this community. CONCLUSION: It is recommended that households and the local authorities concentrate on improving the provision of toilets, water and waste disposal facilities as a way of improving the health state of the community.

Cross-reactivity between antigens from Amblyomma cajennense and A. hebraeum (Acari: Ixodidae).

Laboratory animals exposed to feeding ticks develop resistance which is reflected by a decline in tick engorgement weight, egg-laying by adults and reduced egg viability. Serum antibodies from these hosts and their reaction with tick antigens have been detected by different methods, including precipitation techniques, immunofluorescent techniques, ELISA and Western blots. However, little is known about the effects of antibodies on ticks that engorge on resistant hosts, or which tissues of the tick body are possibly immunogenic. Some researchers, using immunohistochemistry, have detected host antibodies in the gut, salivary glands and haemolymph of ticks engorged on resistant animals. The same technique has helped considerably in determining antigenic sites or antibody targets in other arthropods. Consequently, immunohistochemistry techniques were used in this study to detect cross-reactivity between sera raised against Amblyomma cajennense (Fabricius, 1787) with Amblyomma hebraeum (Koch, 1844), and vice versa. The results show the existence of shared antigens between the 2 tick species. In general, our results point more to a 1-way cross-reactivity of A. hebraeum with A. cajennense than a reciprocal cross-reactivity, suggesting that A. hebraeum is more immunogenic than A. cajennense.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari: Ixodidae).

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Aomblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 microg extract in 0.03 ml of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo device before and 10 min, 1, 2, 4, 18, 24, 48, 72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60% increase in ear thickness) and a delayed reaction (18 h) (85% increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60% increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

Different approaches to the vaccination of free ranging village chickens against Newcastle disease in Qwa-Qwa, South Africa.

The aim of this study was to develop a strategy to control Newcastle disease (ND) in free ranging village chickens using the Nobilis ND Inkukhu vaccine (Intervet South Africa). The study was conducted at Thibella village in Qwa-Qwa, South Africa from April 2001 to October 2002. Three different routes of vaccination (administration via eye-drop, drinking water and feed) were investigated. The haemagglutination inhibition (HI) test was conducted monthly in order to measure the antibody response of village chickens after immunization against Newcastle disease. Using a South African isolate of velogenic ND virus, challenge trials were conducted to determine the efficacy of the vaccine. A questionnaire was provided to evaluate perceptions of farmers on vaccinations. The eye-drop vaccination route produced the highest HI titres ranging between 2.7 and 4.4, followed by the drinking water vaccination route with titres ranging between 2.3 and 4.0. The lowest titres were from the feed vaccination route which ranged between 1.6 and 3.0. Following the challenge, the entire control group died on the third and fourth day after infection. However, 70% of the chickens immunized by using either the eye-drop or drinking water route survived the challenge. Only 20% of the chickens from the group immunized through the feed route survived. Evidently both the eye-drop and drinking water routes were efficient in preventing disease. Necropsies showed that vaccinated chickens had mild lesions whilst control chickens had severe lesions compatible with Newcastle disease. The efficacy of the vaccine using either of the routes can be enhanced by administration of booster vaccinations at 3-month intervals during the first year of a vaccination campaign and then at 6-month intervals from the second year onwards. The majority of the owners indicated that they would prefer to vaccinate their flocks using the drinking water route.

A sero-epidemiological survey of blood parasites in cattle in the north-eastern Free State, South Africa.

A survey to determine the incidence of parasites in cattle (n = 386) was conducted in the north eastern Free State between August 1999 and July 2000. Giemsa-stained blood smears were negative for blood parasites. A total of 94% of the cattle were sero-positive for Babesia bigemina by indirect fluorescent antibody test while 87% were sero-positive for Anaplasma by enzyme-linked immunosorbent assay. The observation of negative blood smears but high incidence of positive serological results for Anaplasma and Babesia for the same group of cattle indicates that this area is endemic for these diseases but with a stable disease situation. All the animals were sero-negative for B. bovis and this is probably because the tick vector (Boophilus microplus) which transmits the disease is not present in the Free State Province. Two tick species belonging to the family ixodidae were found on cattle, namely Boophilus decoloratus and Rhipicephalus evertsi evertsi. In the present study significant differences in seasonal burdens of B. decoloratus occurred, with the highest infestations recorded from February to June. The presence of R. evertsi evertsi throughout the year without any or with small fluctuations in winter months was observed, with a peak from February to May.

Leishmania major-Phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes.

OBJECTIVES: To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut lysates have shared epitopes. DESIGN: A laboratory based study. SETTING: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. METHODS: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x 10(6) amastigotes in 20 microl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100 microl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100 microl of 1:100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37 degrees C and growth at 26 degrees C, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms. RESULTS: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5). CONCLUSIONS: These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105 kDa and 106 kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.

Parasitic helminths of veterinary importance in cattle, sheep and goats on communal farms in the northeastern Free State, South Africa.

The purpose of the study was to record and determine intensities, seasonal incidence and distribution of helminth parasites of veterinary importance that occur in cattle, sheep and goats in the northeastern Free State. The study was conducted at Harrismith and Kestell and in Qwa-Qwa from March 2000 to May 2001. Cattle of various breeds (including Bonsmara, Simmentaler and Friesian), Merino sheep and Angora goats were sampled. Faecal samples were analysed using the McMaster and Visser sieve techniques for egg counts and faecal cultures for 3rd-stage nematode larvae identification. Haemonchus and Oesophagostomum were the dominant nematode genera found to be infecting the animals. The socioeconomic status of the farmers in the study area was determined through a questionnaire survey aimed at recording their management strategies. It indicated that 81% of farmers take care of their livestock by feeding them with supplements. The low to moderate faecal egg counts from cattle showed that helminth infections in this region are still under control even though helminthosis seems to be a problem in small-stock, since EPG counts of more than > 1000 were found. Cattle farmers in this region are encouraged to continue with good animal husbandry practices that have ensured that helminth infections rates are kept low. Small-stock farmers are, however, encouraged to control helminth infections in their sheep and goats by anthelmintic treatment.

Diseases of free-ranging chickens in the Qwa-Qwa District of the northeastern Free State province of South Africa.

A total of 177 free-ranging chickens from 19 Qwa-Qwa villages were bled from wing veins over a period of 6 months (June-November 2000). Serological tests indicated that 5% of chickens tested had been exposed to Newcastle disease, 43% to infectious bronchitis and 63% to Mycoplasma gallisepticum infection. McMaster and Visser sieve techniques were used to determine helminth and coccidia from pooled fresh faecal samples. Helminths isolated in 37% of the villages investigated were Heterakis, Ascaridia and Capillaria species. Eimeria species were also isolated in 32% of the villages investigated. The red fowl mite (Dermanyssus gallinae) was isolated from some of the birds and their nests. Data from a questionnaire survey indicated that all farmers interviewed had never received any technical support and that their chickens had never been vaccinated against any avian diseases. Only 10.5% of the owners interviewed had scientific knowledge on poultry diseases. There is an urgent need for the government to support free-ranging poultry farmers by providing subsidised vaccinations and technical support in order to develop and stimulate economic development in impoverished rural areas of South Africa.

Leishmania donovani-derived lipophosphoglycan plus BCG induces a Th1 type immune response but does not protect Syrian golden hamsters (Mesocricetus auratus) and BALB/c mice against Leishmania donovani.

The efficacy of Leishmania donovani-derived lipophosphoglycan (LPG) plus Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine candidate against visceral leishmaniosis in susceptible BALB/c mouse and Syrian golden hamster (Mesocricetus auratus) models was investigated. Following a triple vaccination with a total dose of 150 microl BCG plus 60 microg or 30 microg of LPG for hamsters and BALB/c mice respectively, there were no noticeable side effects both locally and systemically; implying that the molecule was safe at this dosage level. Vaccinated animals demonstrated an activation of both the humoral as well as cell-mediated responses to LPG, which correlated with resistance against the disease. Protection by LPG plus BCG, was however, poor as the remaining immunized animals showed disease progression leading to severity of the disease as illustrated by emaciation, mass loss and heavy splenic parasitaemia in hamsters. These data nevertheless suggest that it may be rewarding to further evaluate the potential of LPG as a vaccine candidate in leishmaniosis using other adjuvants, which may enhance its immunogenicity.

Seasonal abundance of adult ixodid ticks infesting cattle belonging to resource-limited farmers in the north-eastern Free State Province of South Africa.

A study was undertaken to determine the seasonal abundance of the ticks infesting cattle owned by resource-limited farmers in the north-eastern Free State Province of South Africa. Infestations of Boophilus decoloratus Koch, 1844, Rhipicephalus evertsi evertsi Neumann, 1897, Rhipicephalus follis Donitz, 1910, Rhipicephalus gertrudae Feldman-Muhsam, 1960 and Rhipicephalus warburtoni Walker & Horak, 2000, were monitored on cattle of mixed breeds at monthly intervals from May 1998 to April 1999. High tick intensity on the cattle was observed between March and June, with a peak in May to June 1998 for B. decoloratus and R. evertsi evertsi. Few ticks from the other three species were recovered from the cattle. Small peaks in November to December 1998 for R. warburtoni, and in December 1998 for R. follis and R. gertrudae were observed. Following the winter (June to August), numbers of B. decoloratus declined markedly, while those of R. evertsi evertsi and other rhipicephalids increased. These results represent the first published information on ticks of veterinary importance infesting cattle in the north-eastern part of the Free State Province.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Leishmania major infections in Phlebotomus duboscqi fed on murine models immunized with L. major subcellular antigens and sandfly gut antigens.

The ability of antibodies in bloodmeals of mice and hamsters immunized with Leishmania major subcellular fractions and sandfly (Phlebotomus duboscqi) gut antigens to inhibit development of L. major in its vector P. duboscqi was examined. Antibodies from animals immunized with either L. major subcellular fractions alone or sandfly gut antigen alone were not very effective in inhibiting development of L. major in the sandfly. When P. duboscqi were fed on blood from animals immunized with both parasite flagella and sandfly gut antigen, development of L. major was significantly inhibited (P<0,05). Control sandflies fed on naive animals displayed a normal pattern of parasite development to the metacyclic stage. Electron microscopy studies showed that one of the mechanisms through which antisandfly gut antibody can cause inhibition of parasite development is by lysing sandfly gut epithelium. This study has demonstrated that it is possible to reduce transmission of leishmaniosis through immunization against both the parasite and its sandfly vector.