Molecular analysis of the congopain gene family.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken

Intestinal parasitic infections in children presenting with diarrhoea in outpatient and inpatient settings in an informal settlement of Nairobi, Kenya

BACKGROUND: The distribution of and factors associated with intestinal parasitic infections are poorly defined in high risk vulnerable populations such as urban slums in tropical sub-Saharan Africa. METHODS: In a cross sectional study, children aged 5 years and below who presented with diarrhoea were recruited from selected outpatient clinics in Mukuru informal settlement, and from Mbagathi District hospital, Nairobi, over a period of two years (2010–2011). Stool samples were examined for the presence of parasites using direct, formal-ether concentration method and the Modified Ziehl Neelsen staining technique. RESULTS: Overall, 541/2112 (25.6%) were positive for at least one intestinal parasite, with the common parasites being; Entamoeba histolytica, 225 (36.7%),Cryptosporidium spp. 187, (30.5%), Giardia lamblia, 98 (16%).The prevalence of intestinal parasites infection was higher among children from outpatient clinics 432/1577(27.4%) than among those admitted in hospital 109/535 (20.1%) p < 0.001. Infections with E. histolytica, and G. lamblia were higher among outpatients than inpatients (13.8% vs 1.3% p < 0.001 and 5.8% vs 1.3% p < 0.049) respectively, while infection with Cryptosporidium spp. was higher among inpatients than outpatients (15.3% vs 6.7%) respectively p < 0.001. Other parasites isolated among outpatients included Isospora belli, 19 (1.2%), Ascaris lumbricoides, 26 (1.6%), and Hymenolepis nana 12 (0.8%), with the remainder detected in less than ten samples each. HIV-infected participants were more likely to be infected with any parasite than uninfected participants, Adjusted Odds Ratio (AOR), 2.04, 95% CI, 1.55-2.67, p < 0.001), and with Cryptosporidium spp. (AOR, 2.96, 95% CI 2.07-4.21, p < 0.001).The inpatients were less likely to be infected with E. histolytica than outpatients (AOR, 0.11, 95% CI, 0.51- 0.24, p < 0.001), but more likely for inpatients to be infected with Cryptosporidium spp. than outpatients (AOR, 1.91, 95% CI, 1.33-2.73, p < 0.001). Mixed parasitic infections were seen in 65 (12.0%) of the 541 infected stool samples. CONCLUSION: Intestinal parasitic infections are common in urban informal settlements’ environment. Routine examinations of stool samples and treatment could benefit both the HIV infected and uninfected children in outpatient and inpatient settings

The potential role of roaming dogs in establishing a geographically novel life cycle of taeniids (Echinococcus spp. and Taenia spp.) in a non-endemic area

Cystic Echinococcosis (CE) is endemic in humans and livestock in many pastoral communities in Kenya. The distribution of the disease is enhanced by several factors, including livestock trade, which has allowed for the spread of CE to non-endemic areas such as western Kenya. Dogs' roaming behaviour, with consequent contamination of the environment with intestinal parasites, could then lead to parasite establishment. This study examined dogs' infection levels with taeniid eggs and their potential role in contaminating the environment with intestinal parasites. Methodology: We selected sixteen ruminant slaughterhouses in Busia and Bungoma Counties, and around each slaughterhouse we identified ten homesteads owning free-roaming dogs. We administered a questionnaire on dog management practices to the homestead owner and collected a faecal sample from the dog's rectum. In homesteads around 8 of the 16 slaughterhouses, we collared dogs with a GPS tracker to assess their movement patterns. The faecal samples were examined microscopically following zinc-chloride sieving-floatation technique for the presence of taeniid eggs and other canine intestinal parasites. Polymerase Chain Reaction – Restriction Fragment Length Polymorphism of NADH dehydrogenase subunit 1 gene and sequencing were used to confirm taeniid eggs identified during microscopy. Additionally, the Coproantigen-ELISA was used to detect the presence of taeniid antigen in a sub-set of the faecal samples. Results: Helminths detected in the 155 dogs sampled included hookworms (n = 92; 59.4%), ascarids (n = 15; 9.7%), and taeniids (n = 1; 0.6%). Through Copro-PCR, 13 eggs extracted from the sample of the only taeniid infected dog were sequenced and identified as E. canadensis (G6/7) [n = 1], Taenia multiceps [n = 1], and Taenia serialis [n = 6]; the remaining were indeterminate. Of the 77 faecal samples tested for E. granulosus sensu lato (s. l.) with the Copro-ELISA, 64 (83.1%) were negative, 12 (15.6%) were positive, while 1 (1.3%) was suspicious. The dogs travelled a median of 13.5 km daily, and 28 dogs visited the slaughterhouses during the 5-day recording period. Conclusion: The results indicate a relatively high carriage of zoonotic parasites by free-roaming domestic dogs in western Kenya, which poses a risk to human and livestock populations. We report for the first time a domestic lifecycle of Echinococcus canadensis and Taenia multiceps in western Kenya, as well as a presumptive sylvatic cycle of coenurosis by T. serialis. We recommend an extensive and ongoing Copro-antigen survey of dog faeces, broader assessment of dog parasites with zoonotic potential, adherence to slaughterhouse management practices, and dog-ownership programmes to highlight the importance of deworming and restricted dog movements

Cystic echinococcosis in donkeys in eastern Africa

Cystic echinococcosis (CE) is endemic in humans and domestic animals in eastern Africa. All the species of the Echinococcus granulosus sensu lato complex have been reported in this region except for E. equinus, possibly due to the small number of studies involving equids. This study reports the frequency of different Echinococcus species in donkeys from eastern Africa. A total of 5961 donkeys were examined during meat inspection in 3 slaughterhouses in Kenya. Identification of Echinococcus spp. was achieved through polymerase chain reaction-restriction fragment-length polymorphism and sequencing of the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 gene. The prevalence of CE was 5.7% (337/5961). The 263 genotyped cysts belonged to E. equinus (n = 163), E. granulosus sensu stricto (n = 70), E. canadensis (G6/7) (n = 26) and E. ortleppi (n = 4). One donkey harboured a metacestode of Spirometra theileri. All E. equinus cases, except 2, originated from southern Ethiopia, whereas the other species were more evenly distributed across the study area. Most of the cysts belonging to E. equinus were fertile (111/163), while those of the other species were non-fertile. This is the first report of Echinococcus spp. in donkeys from sub-Saharan Africa and the first confirmation of E. equinus in East Africa. The frequent fertility of E. equinus cysts in donkeys affirms their suitability as intermediate hosts of this species, while low frequency and cyst fertility suggest a marginal role of donkeys in the transmission of E. granulosus s. s., E. canadensis (G6/7) and E. ortleppi

Prevalence of cryptosporidiosis in dairy cattle, cattle-keeping families, their non-cattle-keeping neighbours and HIV-positive individuals in Dagoretti Division, Nairobi, Kenya

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).This paper reports a study estimating the prevalence of cryptosporidiosis, an emerging zoonosis, in people and cattle in Dagoretti, Nairobi. A repeated cross-sectional survey was carried out among randomly selected cattle keepers in Dagoretti, their dairy cattle and their non-cattle-keeping neighbours in the dry and wet seasons of 2006. A survey was also carried out among a group of people living with human immunodeficiency virus (HIV). Faecal samples were examined for Cryptosporidium oocysts using the modified Ziehl–Neelsen method; 16 % of the samples were also examined using immunofluorescence antibody (IFA) technique. Quality control consisted of blind reviews of slides, examining split samples and confirming slide results with IFA. We found that members of dairy households had a dry season cryptosporidiosis prevalence of 4 % and wet season prevalence of 0.3 %, and non-dairy households, a prevalence of 5 and 0 %, respectively. The cattle dry season prevalence was 15 %, and the wet season prevalence, 11 %. The prevalence in people living with HIV was 5 %. The laboratory quality control system showed some inconsistency within and between different tests, indicating challenges in obtaining consistent results under difficult field and working conditions. In conclusion, this is the first reported study to simultaneously survey livestock, livestock keepers and their neighbours for cryptosporidiosis. We failed to find evidence that zoonotic cryptosporidiosis is important overall in this community. This study also draws attention to the importance of quality control and its reporting in surveys in developing countries

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya

This paper is part of a special supplement on assessing and managing urban zoonoses and food-borne disease in two African cities (Nairobi, Kenya and Ibadan, Nigeria).A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl–Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twentyfive samples (20 %) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68 %) as Cryptosporidium parvum-like, four samples (16 %) as Cryptosporidium ryanae, three samples (12 %) as Cryptosporidium andersoni and one sample (4 %) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans

Genetic Diversity of Cryptosporidium in Children in an Urban Informal Settlement of Nairobi, Kenya.

INTRODUCTION:Globally Cryptosporidium and Giardia species are the most common non-bacterial causes of diarrhoea in children and HIV infected individuals, yet data on their role in paediatric diarrhoea in Kenya remains scant. This study investigated the occurrence of Cryptosporidium species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi. METHODS:This was a prospective cross-sectional study in which faecal specimen positive for Cryptosporidium spp. by microscopy from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to the paediatric ward at the Mbagathi District Hospital were characterized. The analysis was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene for subtyping. RESULTS:C. hominis was the most common species of Cryptosporidium identified in125/151(82.8%) of the children. Other species identified were C. parvum 18/151(11.9%), while C. felis and C. meleagridis were identified in 4 and 2 children, respectively. Wide genetic variation was observed within C. hominis, with identification of 5 subtype families; Ia, Ib, Id, Ie and If and 21 subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and HIV status or patient type. CONCLUSION:C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, whereas genetic diversity was low within C. parvum with only one subtype family IIc identified

Prevalence and Intensity of Gastrointestinal Parasites in Donkeys in Selected Abattoirs in Kenya

The aim of this study was to determine the prevalence and intensity of gastrointestinal parasites in donkeys slaughtered in Kinamba, Mogotio, and Lodwar slaughterhouses and their association with several host factors. A survey was done between July and September 2017 in three slaughterhouses. Faecal samples were collected per rectum from all the study donkeys for faecal egg counts (EPG) and morphological identification of the eggs. At slaughter, the gastrointestinal tracts were opened and examined visually, and all helminth parasites collected were subjected to morphological identification. Prevalence and intensity were calculated based on the helminth identified and EPG. A total of 282 donkeys were sampled. A majority of the donkeys (89%) were in poor body condition. Ten helminth parasite species were identified in 85.5% donkeys. They were Strongylus vulgaris (52.8%), Parascaris equorum (20.2%), Strongylus edentatus (12.1%), Anaplocephala perfoliata (10.3%), Setaria equina (3.5%) Anaplocephala magna (2.5%), Cylicocyclus auriculatus (2.1%), Cyathostomum species (1.8%), Strongylus equinus (0.4%), and Triodontophorus serratus (0.4%). A significant percentage (55.3%) had no eggs in their feces, 39% had low infection, 5% had medium, and only 0.7% were heavily infected. Prevalence rates via use of the EPG showed Strongyles (44.7%), Parascaris equorum (5.3%), Oxyuris equi (11%), Triodontophorus tenuicolis (0.7%), Habronema species (0.7%), and cestodes eggs (0.4%). No significant differences were observed between fecal Strongylus egg count and age, sex, and pregnancy status. However, donkeys with poor body condition shed more Strongylus eggs in feces. Helminth infections are prevalent in donkeys in Kenya; however, this is not reflected in coprological analyses. These helminth parasites may contribute to poor body condition, ill health, and poor productivity of donkeys

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya

A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl–Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twenty-five samples (20 %) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68 %) as Cryptosporidium parvum-like, four samples (16 %) as Cryptosporidium ryanae, three samples (12 %) as Cryptosporidium andersoni and one sample (4 %) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans