Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya.

A monoclonal antibody-based, enzyme immunoassay (antigen ELISA) for the detection of species-specific invariant antigens of Trypanosoma congolense, T. vivax or T. brucei in the serum of infected animals was evaluated as a means of diagnosis using bovine field sera from a trypanosomiasis endemic area, Nguruman, Kenya. Circulating trypanosome antigens were detected in 126 (96.2%) of 131 serum samples from animals with parasitologically confirmed diagnosis: 74.8% were positive for antigens of two or three trypanosome species, while 21.4% tested positive for one trypanosome species. When 70 sera from animals (at Nguruman), which had tested negative for trypanosomes by the buffy coat technique, were tested, 35 (50.0%) of them were shown to be antigen-ELISA positive: 24 (34.3%) showing infection with a single species and 11 (15.7%) with mixed infections. The predominant trypanosome species diagnosed in the two herds by antigen ELISA was T. vivax, which was detected in 133 (82.6%) of the 161 sera that tested positive for antigens, followed by T. congolense in 122 (75.8%) sera, with 109 (67.7%) showing evidence of mixed infections with two or three trypanosome species. In single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1.0%. Evidence for the specificity of the test was provided by analysis of field sera from 100 cattle, from a trypanosomiasis-free area, infected with other haemoparasites (anaplasmosis, babesiosis and theileriosis), which all tested negative in the assay.

A study of alpha-fetoprotein in primary liver cancer in Tanzania.

Alpha-fetoprotein (AFP) was detected, by Ouchterlony immunodiffusion technique, in 81.5% of patients with histologically confirmed diagnosis of hepatocellular carcinoma. The test gave negative results with 35 cases of acute viral hepatitis, 7 haemochromatosis, 6 micronodular cirrhosis and 2 cholangiocellular carcinoma. Curiously, one patient with postnecrotic cirrhosis, a well recognized sequela of viral hepatitis, whose liver cell regeneration also showed "atypical changes", was AFP positive. AFP was not detected in sera from the general population which comprised 1029 male blood donors, 144 antenatal and 106 maternity cases. The only exception was the case of a woman who aborted a 5-month old foetus. A follow-up serum sample taken 3 months later was, however, negative for AFP. The frequency of hepatitis B surface antigen (HBsAg) detection in patients with hepatocellular carcinoma (25.9%) was 4 to 5 times higher than that in the general population. This strong association between HBsAg and primary liver cancer in countries where liver tumours are often AFP secretors suggests a role for hepatitis B virus, not only in the aetiology of the cancer, but also in the reactivation of the gene encoding this foetal protein.

Identification of antigens for use in immunodiagnosis of Taenia saginata cysticercosis.

Fifteen metacestode antigens from Taenia saginata were defined by Laurell crossed immunoelectrophoresis and investigated for their potential use in immunodiagnosis of bovine cysticercosis. Several antigens cross reacted with those of some common cattle parasites. Three of the antigens, designated as numbers 4, 8 and 11, were selected on the basis of their restricted cross reactions and were isolated by affinity chromatography. These antigens showed high sensitivity and specificity values in the enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of bovine cysticercosis.

Comparison of human immune responses to purified Vero cell and human diploid cell rabies vaccines by using two different antibody titration methods.

Antibody responses to a conventional rabies preexposure regimen of a new purified Vero cell rabies vaccine (PVRV) and a human diploid cell vaccine (HDCV) were compared in 80 healthy Kenyan veterinary students. Forty-three of the students received the PVRV and 37 received the HDCV on days 0, 7, and 28. Antibody responses were monitored by using the rapid fluorescent-focus inhibition test (RFFIT) and an inhibition enzyme immunoassay (INH EIA) on days 0, 7, 28, and 49. Both vaccines elicited a rapid antibody response. A good correlation between the RFFIT titers and the INH EIA titers was obtained (r = 0.90). Our results also showed that the INH EIA was more reproducible and might therefore be a suitable substitute for the more expensive and less reproducible RFFIT. The geometric mean titers determined by both tests in the two groups of students were statistically similar during the test period. The RFFIT and the INH EIA gave comparable geometric mean titers, which differed significantly only on day 28 in the PVRV group. The effect of the new PVRV is comparable to that of the more expensive HDCV, as determined by the present test systems. The PVRV could therefore be the vaccine of choice, especially in tropical rabies-endemic areas, where the high cost of the HDCV has confined its use to a privileged few.

Legionella antibodies in human and animal populations in Kenya.

Using a microagglutination method, domestic and wild animal sera, together with human patient and healthy blood donor sera, have been analysed for titres against various Legionella species, comprising fourteen different serogroups. Generally, the level of moderately elevated titres, i.e. greater than or equal to 64, was low for all the aforementioned serum groups. Within the domesticated animals, cattle, pigs and dogs presented a much lower prevalence in Kenya than found elsewhere, whereas it was the other way round for goats. Human sera, either from patients or from healthy donors, did not react against L. pneumophila serogroups 1, 6, or 3, which in that sequence are the most common L. pneumophila serogroups in Europe, and in other geographic areas where legionellosis is common. High titres of greater than or equal to 256 against L. pneumophila serogroup 6 (two cattle) or against L. bozemanii strain Mi-15 (two cattle, one dog) indicate that although the epidemiological picture may be different from other parts of the world, Legionella infections exist in Kenya as well.

Two simple antigen-detection enzyme immunoassays for the diagnosis of Trypanosoma evansi infections in the dromedary camel (Camelus dromedarius).

A monoclonal antibody against a plasma membrane antigen of Trypanosoma rhodesiense was used in a microplate- and a tube-ELISA for the detection of T. evansi circulating antigen in camel sera from endemic areas in Mali and Kenya. The colour reactions could be read visually and the results obtained for both assays were identical. The sera from the control herd of 30 camels from a farm in a non-endemic area in Kenya were all negative for antigen, while 18 out of 20 sera from infected camels in an endemic area in Kenya and 16 out of 17 parasitaemic camels from a different endemic area in Mali were antigen-positive. More importantly, the antigen-detection assay gave positive results with 9 out of 20 and 13 out of 20 field sera from the two endemic areas in Kenya and Mali, respectively, collected from camels which had negative parasitological findings. The ease with which the tube-ELISA can be carried out makes it a potentially suitable tool for the diagnosis of T. evansi infections in the field.

Antigen-detection enzyme immunoassays for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infections in cattle.

Species-specific monoclonal antibodies against Trypanosoma vivax, T. congolense and T. brucei were used to develop antigen-capture enzyme-linked immunosorbent assays (antigen-ELISA) for the diagnosis of bovine trypanosomiasis. Each assay was subsequently used for the detection of species-specific circulating antigens in sera of cattle experimentally infected by tsetse transmission. In T. vivax and in T. congolense-infected animals, circulating antigens were detected as early as 10-12 days post-infection while for T. brucei infections, the antigens were detected 8-14 days after challenge. The appearance of the antigens in peripheral blood generally coincided with the onset of first parasitaemia. The antigen levels increased and were persistently present in circulation even on occasions where parasitaemia was not detectable by the buffy coat technique. Following treatment with Berenil (diminazene aceturate, Hoechst, W. Germany) at a dose of 7.0 mg/kg body weight, T. vivax and T. congolense antigens were cleared from circulation within 2 weeks. The rate of removal was slower but variable in T. brucei infections. In one group of 3 animals infected with T. congolense, however, the initial decline in "antigenaemia" was followed, several weeks later, by another rise in antigenaemia, possibly heralding a relapse of the infection. Parasitaemia was not demonstrable at this stage by the buffy coat technique. The circulating antigens were partly in the form of free antigens and partly as immune complexes. Not only are these antigen-detection ELISA assays likely to be of diagnostic importance, but they may also be useful as tools for evaluation of the efficacy of treatment.

Enteropathogenic Escherichia coli serotype O111:HNT isolated from preterm neonates in Nairobi, Kenya.

This investigation was initiated as a consequence of several cases of diarrhea in a nursery ward for preterm babies in Nairobi, Kenya. Ten lactose-positive colonies were isolated from the stools of each of 30 neonates, regardless of whether they had diarrhea; 229 strains were identified as Escherichia coli and 65 strains were identified as Klebsiella pneumoniae. Six strains were lost during laboratory handling. No other bacterial, viral, or parasitic enteropathogens were identified. Using synthetic alkaline phosphatase-labeled probes, the bacterial isolates were found to be negative for the presence of genes coding for heat-stable and heat-labile enterotoxins. Seventy-eight E. coli strains isolated from a total of 13 neonates possessed the E. coli enteropathogenic adhesion factor (EAF) gene, as demonstrated by the use of a cloned radiolabeled DNA fragment probe. These strains possessed similar plasmid profiles constituting a core plasmid profile, and while all adhered to HeLa cells, none produced Vero cell cytotoxins. The EAF gene was located on a 65-megadalton plasmid. Serotyping showed the strains to be of serogroup O111 and serotype H nontypable, a well known enteropathogenic type. Five neonates died during the outbreak, and the fatality rate was 30.7% (4 of 13) for neonates infected with EAF-positive E. coli strains compared with 7.7% (1 of 13) for neonates from whom only EAF-negative E. coli strains were isolated. K. pneumoniae only was isolated from five neonates.

Isolation of Campylobacter spp. and Yersinia enterocolitica from domestic animals and human patients in Kenya.

Rectal swabs or faecal samples from 992 domestic animals and 97 human patients in the Nairobi region were cultured for thermophilic Campylobacter species and Yersinia enterocolitica. The highest isolation rate of campylobacters was obtained from diarrhoeic pigs (55.1%), followed by healthy chicken (51.5%), diarrhoeic dogs (47.2%), healthy pigs (44.0%), healthy ducks (29.4%), healthy goats (6.3%), healthy cattle (5.8%), diarrhoeic humans (3.1%), and healthy sheep (2.0%). Only one strain of Y. enterocolitica was obtained. This isolate, which conformed to Nilehn's biotype 1, was recovered from one (0.7%) of the 150 healthy pigs examined. Out of 317 thermophilic campylobacters isolated, 163 (51.4%) were classified as C. jejuni, whereas 127 (40.1%) belonged to C. coli. The remaining 27 strains fell into three categories which did not conform to any defined species. Of the total number of isolates, 74.1% were resistant to metronidazole, 90.9% were resistant to triphenyltetrazolium chloride (TTC), and 50.2% reduced selenite. The results indicate that domestic animals may play a significant role in the epidemiology of human campylobacteriosis in the Nairobi region by serving as reservoirs. Y. enterocolitica seems to be rare among man and animals in this area.

An evaluation of the applicability of crossed immunoelectrophoresis in seroepidemiological studies of hydatid disease in livestock.

Hydatidosis is both an economic and public health problem all over the world. Although various tests have been used on serodiagnosis of the disease in laboratory infected sheep, there is a problem in detecting antibody to the parasite in sheep. The antibody response of people infected with E. granulosus differs from the antibody response of livestock. Crossed immunoelectrophoresis was used to search for antibodies in 44 goats known to have had the infection. Crossed immunoelectrophoresis was able to detect two antibodies produced by a goat naturally infected with hydatid disease. Three of 44 known cases were detected by the test method. None of the naturally infected animals' sera exhibited a pattern of arcs similar to the reference pattern. The test's sensitivity was poor.

Serological diagnosis of hydatid disease by enzyme linked immunosorbent assay (ELISA) using partially purified hydatid cyst fluid antigens.

Experimentally, two hydatid cyst fluid (HCF) antigens (antigens 4 and 5) were found to be the most immunogenic antigens in HCF. The two antigens were precipitated together from HCF. This was done by adding 2M phosphotungstic acid and 2M magnesium chloride solutions to clarified HCF while continuously stirring the mixture. The precipitate formed was suspended in physiological saline (P.S.). This antigens' solution was used to coat microtitre plates for indirect ELISA. Indirect ELISA was performed on 180 randomly selected bovine sera. The sensitivity of the test was found to be 98% while the specificity was 70%. The predictive value was 89%. Although the specificity of the test was relatively low, the test using these partially purified antigens was found to be useful because of its high sensitivity.