First report of the visceral leishmaniasis vector Phlebotomus martini (Diptera: Psychodidae) in Tanzania.

Phlebotomus martini is a known vector of visceral leishmaniasis caused by Leishmania donovani in sub-Saharan Africa. The disease is known to be endemic in areas of north and south Sudan, Kenya, Ethiopia, Uganda, and Somalia but has not been reported from Tanzania. In this report we present the first documented collection of P. martini and P. vansomerenae in Tanzania. Sand flies were collected using standard dry-ice baited CDC light traps (John W. Hock Company, Gainesville, FL) from five sampling sites in the Arusha and Kilimanjaro regions from 14 to 20 July 2010. Phlebotomus martini was collected from all sites and represented 6.6% of the total identified sand flies. Phlebotomus martini ranged from 4.5 to 9.4% of the total identified catch from the four sites in the Kilimanjaro region and 17.9% of the total identified catch at the one collection site in the Arusha region. In addition, one male specimen of the sibling species, Phlebotomus vansomerenae, was found at Chemka Springs in the Kilimanjaro region. These data indicate the presence of an established population(s) of P. martini in northern Tanzania that could support L. donovani transmission in an area with no prior case history of visceral leishmaniasis.

Factors influencing individual and community participation in the control of tsetse flies and human African trypanosomiasis in Urambo District, Tanzania.

This study was carried out to assess the knowledge and level of individual and community participation in the control of Human African trypanosomiasis in Urambo District, western Tanzania. Semi structured questionnaires were used to collect information from individuals at house hold level. Retrospective data of HAT was sought from the medical officers in-charge of health facilities. The results indicate that, 191 (90.5%, n = 211) individuals knew tsetse flies and 187 (88.6%, n = 211) knew HAT. All nine key informants reported that, the communities were aware of HAT while seven key informants reported that, the communities were aware of health risks associated with tsetse bites in human. There was poor knowledge about the role played by animals in the transmission of HAT (26.7%, n = 187). Majority of those who knew HAT (n = 187) were willing to contribute labour (70.1%) and money (64.2%) to tsetse and HAT control whereas amongst those who knew tsetse flies, 66.5% and 60.7% were willing to contribute labour and money, respectively. Amongst those who knew any HAT control technique (n = 108), 78.7% and 82.4% were willing to contribute money and labour respectively. A total of 454 cases of HAT were reported in the area from 1999 to 2006. It is concluded that, the factors influencing individual and community participation include the knowledge of tsetse, HAT and control measures.

Factors Influencing Individual and Community Participation in the Control of Tetse Flies and Human African Trypanosomiasis in Urambo District; Tanzania

This study was carried out to assess the knowledge and level of individual and community participation in the control of Human African trypanosomiasis in Urambo District; western Tanzania. Semi structured questionnaires were used to collect information from individuals at house hold level. Retrospective data of HAT was sought from the medicalofficers in-charge of health facilities. The results indicate that; 191 (90.5 ; n = 211) individuals knew tsetse flies and 187(88.6; n=211) knew HAT. All nine key informants reported that; the communities were aware of HAT while seven key informants reported that; the communities were aware of health risks associated with tsetse bites in human. There was poor knowledge about the role played by animals in the transmission of HAT (26.7; n=187). Majority of those who knew HAT (n = 187) were willing to contribute labour (70.1) and money (64.2) to tsetse and HAT control whereas amongst those who knew tsetse flies; 66.5 and 60.7 were willing to contribute labour and money; respectively. Amongst those who knew any HAT control technique (n = 108); 78.7 and 82.4were willing to contribute money and labour respectively. A total of 454 cases of HAT were reported in the area from 1999 to 2006. It is concluded that; the factors influencing individual and community participation include the knowledge of tsetse; HAT and control measures

Immunization of young chicks using graded dose of wild strain of Eimeria tenella.

A wild strain of Eimeria tenella was isolated and utilized for immunization studies. Its optimal sporulation was attained at room temperature 24-25 degrees C after 24-48 h. Two groups of chicks were immunized by dosing a graded dose of five oocysts/chick/day for 6 days followed by 50 oocysts/chick/day for 7 days. A third group was not immunized and served as a negative control. Immunized chicks gained mass at the same rate as unimmunized ones, but when challenged with 200,000 oocysts/chick, mass gains declined in the unimmunized group. The growth rate of immunized chicks was not affected by challenge (P > 0.05). Upon challenge, unimmunized chicks produced significantly more oocysts than immunized chicks (P < 0.005). Immunized chicks withstood a challenged with 200,000 oocysts/chick without developing any clinical signs whereas the unimmunized chicks developed typical clinical signs of coccidiosis. Unimmunized chicks had significantly more severe lesions in the caecum than any other group (P > 0.005) and also produced significantly more oocysts than any other group (P > 0.005).

Theileria parva: in vitro studies on the effects of holding temperature, pH and medium on sporozoite infectivity.

The effects of holding temperature, pH and medium on the infectivity of Theileria parva sporozoites were investigated using an in vitro infectivity assay. The sporozoite infectivity lasted for 72 h at a holding temperature of 4 C but for only 24 h at 24 degrees C. Sporozoite infectivity was found to be sensitive to pH variations and sporozoites were most infective between pH 7 and pH 8. There was a significant loss in infectivity at pH 5 and infectivity was almost totally abolished at pH 9. Theileria parva sporozoites are usually held and manipulated in Eagle's minimum essential medium (MEM) with Earles' salts. In this study. Leibovitz-15 supplemented with 15% fetal bovine serum gave a significantly better infectivity than Eagle's MEM (3.8 log units versus 1.0 log units) or any other medium. The importance of proper management of the T. parva sporozoite environment in the laboratory or field is emphasized by the findings in these studies and might also explain some of the failures of vaccination when the pH of the holding medium was allowed to deteriorate.

Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats.

Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1.

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs.

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Application of PCR and DNA probes in the characterisation of trypanosomes in the blood of cattle in farms in Morogoro, Tanzania.

Polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) probes were used to characterise trypanosomes from cattle in Morogoro region of Tanzania. Blood samples collected from 390 beef and dairy cattle in selected farms in Morogoro region were examined for presence of trypanosomes using the buffy coat technique (BCT) and blood smears (BSs). Fifty-two animals were found infected: 40 with Trypanosoma congolense, 10 with T. vivax and two with both T. congolense and T. vivax. DNA extracted from all the parasitologically positive and 62 randomly selected parasitologically negative samples were subjected to PCR amplification using primers specific for different trypanosome species. Using a set of seven specific-pairs of primers on the parasitologically positive samples, we detected only T. congolense, either the Savannah- or the Kilifi-type, as single or mixed infections. With the PCR, trypanosome DNA could be detected in 27 (43%) out of 62 samples that were parasitologically negative. DNA hybridisation using probes specific for Savannah- or Kilifi-types T. congolense, or T. vivax, confirmed the presence of these parasites in cattle kept on some farms in Morogoro region of Tanzania. From these studies, it is clear that there is a need to undertake molecular epidemiological studies to determine the distribution of trypanosome species and subspecies, and to assess the economic impact of these parasites in the productivity of livestock in Tanzania. In particular, it would be desirable to verify the assumed association between the different presentations of trypanosomosis on one hand and genotypes of T. congolense on the other.

Use of an in vitro infectivity assay in comparison with histological techniques in the study of Theileria parva sporozoite maturation.

Adult male and female Rhipicephalus appendiculatus ticks infected with Theileria parva (Muguga 3087) were fed on rabbits and the development of infection was monitored daily using light microscopy and an in vitro titration technique able to quantify the infectivity of sporozoite suspensions. The salivary glands stained with methyl green pyronine showed presence of infection in some unfed ticks. The intensity of staining was shown to increase with the number of days the ticks had fed. The in vitro technique, on the other hand, could detect infection only in ticks which had fed for 3-5 days. Feeding of ticks on rabbits for 4 days produced significantly more sporozoites than any other lengths of feeding (P = 0.001). The in vitro assay was also able to demonstrate differences between male and female R. appendiculatus in production of infective sporozoites. Female ticks produced significantly more sporozoites than male ticks (P = 0.002).