Molecular identification of Trypanosoma brucei gambiense in naturally infected pigs, dogs and small ruminants confirms domestic animals as potential reservoirs for sleeping sickness in Chad.

Human African trypanosomiasis (HAT) has been targeted for zero transmission to humans by 2030. Animal reservoirs of gambiense-HAT could jeopardize these elimination goals. This study was undertaken to identify potential host reservoirs for Trypanosoma brucei gambiense by detecting its natural infections in domestic animals of Chadian HAT foci. Blood samples were collected from 267 goats, 181 sheep, 154 dogs, and 67 pigs. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) were performed to search for trypanosomes. DNA was extracted from the buffy coat, and trypanosomes of the subgenus Trypanozoon as well as T. b. gambiense were identified by PCR. Of 669 blood samples, 19.4% were positive by RDT and 9.0% by CTC. PCR revealed 150 animals (22.4%) with trypanosomes belonging to Trypanozoon, including 18 (12%) T. b. gambiense. This trypanosome was found in all investigated animal species and all HAT foci. Between animal species or villages, no significant differences were observed in the number of animals harboring T. b. gambiense DNA. Pigs, dogs, sheep and goats appeared to be potential reservoir hosts for T. b. gambiense in Chad. The identification of T. b. gambiense in all animal species of all HAT foci suggests that these animals should be considered when designing new control strategies for sustainable elimination of HAT. Investigations aiming to decrypt their specific role in each epidemiological setting are important to achieve zero transmission of HAT.

TITLE: L'identification moléculaire de Trypanosoma brucei gambiense chez les porcs, les chiens et les petits ruminants naturellement infectés confirme les animaux domestiques comme réservoirs potentiels de la maladie du sommeil au Tchad. ABSTRACT: La trypanosomiase humaine africaine (THA) a été ciblée pour une interruption de sa transmission en 2030. Le réservoir animal de la THA à Trypanosoma brucei gambiense pourrait compromettre ces objectifs d'élimination. Cette étude a été entreprise pour identifier des potentiels hôtes réservoirs de Trypanosoma brucei gambiense en détectant ses infections naturelles chez des animaux domestiques des foyers tchadiens de la THA. Des échantillons de sang ont été prélevés chez 267 chèvres, 181 moutons, 154 chiens et 67 porcs. Le test de diagnostic rapide (TDR) et la centrifugation en tube capillaire (CTC) ont été utilisés pour mettre en évidence les trypanosomes. L'ADN a été extrait des couches leucocytaires et les trypanosomes du sous-genre Trypanozoon ainsi que T. b. gambiense ont été identifiés par PCR. Sur les 669 échantillons de sang analysés, 19,4& % étaient positifs au TDR et 9,0& % à la CTC. La PCR a révélé 150 (22,4& %) animaux avec des trypanosomes du sous-genre Trypanozoon donc 18 (12& %) portant l'ADN de T. b. gambiense. Ce dernier a été identifié chez toutes les espèces animales de tous les foyers de la THA. Entre les espèces animales ou les villages, aucune différence significative n'a été observée entre le nombre d'animaux ayant l'ADN de T. b. gambiense. Les porcs, les chiens, les moutons et les chèvres sont apparus comme des hôtes réservoirs potentiels de T. b. gambiense au Tchad. L'identification de T. b. gambiense, chez toutes les espèces animales étudiées dans tous les foyers, suggère de considérer ces animaux dans la conception des nouvelles stratégies de lutte visant une élimination durable de la THA. Des investigations visant à décrypter leur rôle spécifique dans chaque contexte épidémiologique sont nécessaires pour parvenir à une transmission nulle de la THA.

Genetic diversity and population structure of goliath frogs (Conraua goliath) from Cameroon.

The goliath frog (Conraua goliath) is an Endangered species exclusively found in Cameroon and Equatorial Guinea. Climate change, deforestation and overhunting are principal causes driving this species to extinction. Therefore, a better understanding of the genetic diversity and population structure of this species is necessary to improve conservation efforts. Here we used two mitochondrial genes (Cytochrome Oxidase subunit 1 (COI) and 16S) extracted from 54 C. goliath individuals from six localities in Cameroon to examine their genetic diversity. The result shows a low DNA substitution between the sequences. There were four 16S and two COI haplotypes in total. Overall, genetic diversity was very low for all the genes with nucleotide diversity of 0.00106 and 0.00007 for 16S and COI respectively. The Tajima D and Fu Fs statistics were negative. The TCS haplotype network revealed a predominant and ancestral haplotype (H1) for these genes which is distributed in the 6 populations. Pairwise genetic differentiation (FST) generated between these populations using 16S revealed very high differentiation between populations from Nkam and Mungo Administrative Divisions in Cameroon. In contrast, we observed low differentiation among the geographically clustered Mungo and Nkam populations. Overall, human activities and perhaps climate change can appear to have depleted genetic diversity in the scattered populations that remain of this amphibian. To sustain the Goliath frog, we suggest to the Cameroonian government to implement more effective strategies to conserve and manage remnant populations of this iconic species through more effort against poaching which contribute to reduce the genetic diversity.

Microfilariae infestation of goliath frogs (Conraua goliath) from Cameroon.

The goliath frog (Conraua goliath) is endemic to Equatorial Guinea and Cameroon. It is an endangered species but little information is known about its parasites. To understand the impact of blood parasites on this species, we microscopically examined blood smears from 78 goliath frogs in February and November 2016 (dry and wet seasons) from six localities in Littoral Region of Cameroon, and we sequenced mitochondrial DNA from positive samples. Microfilariae were found in 33/78 (42.3%) goliath frogs at six locations. No other haemoparasite species was detected. Morphological characteristics of microfilariae were also described, and specimens from each frog species were similar. DNA sequencing data from the mitochondrial Cytochrome Oxidases sub unit I (COI) gene revealed a close relationship with Icosiella neglecta, a microfilaria documented in other European, Asian, and African frogs. However, sequences were sufficiently genetically distant (0.118) that they may define a new species of Icosiella. The infection burden of microfilariae varied by site, with season (65% in dry season to 23% in rainy season), and by sex, (male frogs had significantly higher parasite burdens than females (p < 0.0001)). However, this may have been confounded by size as the microfilaria intensity increased with frog weight (p < 0.0001), and males were larger than females. Microfilaria infection intensity varied from 1 to 120 per 50 µl of blood. Microfilaria induced a significant increase (p < 0.05) in the number of white blood cells (WBC) counted compared to uninfected frogs, but there was no statistically significant variation in red blood cell (RBC) count, plasma cholesterol level (p = 0.210) or plasma glucose level (p = 0.100).

Distribution of Plasmodium falciparum gametocytes and malaria-attributable fraction of fever episodes along an altitudinal transect in Western Cameroon.

BACKGROUND: Highland areas are hypoendemic zones of malaria and are therefore prone to epidemics, due to lack of protective immunity. So far, Cameroon has not succeeded in implementing a convenient and effective method to detect, prevent and forecast malaria epidemic in these peculiar zones. This monitoring and evaluation study aims to assess the operational feasibility of using the human malaria infectious reservoir (HMIR) and the malaria-attributable fraction of fever episodes (MAFE) as indicators, in designing a malaria epidemic early warning system (MEWS). METHODS: Longitudinal parasitological surveys were conducted in sentinel health centres installed in three localities, located along an altitudinal transect in Western Cameroon: Santchou (750 m), Dschang (1,400 m) and Djuttitsa (1,965 m). The syndromes of outpatients with malaria-like complaints were recorded and their blood samples examined. The HMIR and the MAFE were estimated and their spatial-temporal variations described. RESULTS: The prevalence of asexual Plasmodium infection in outpatients decreased with increasing altitude; meanwhile the HMIR remained fairly constant, indicating that scarcity of malaria disease in highlands is likely due to absence of vectors and not parasites. In lowland, children carried the heaviest malaria burden in the form of febrile episodes, and asexual parasites decreased with age, after an initial peak in the 0-5 year's age group; however, they were similar for all age groups in highland. The HMIR did not show any variation with age in the plain; but some discrepancies were observed in the highland with extreme age groups, and migration of populations between lowland and highland was suspected to be the cause. Plasmodium infection was perennial in the lowland and seasonal uphill, with malaria disease occurring here mostly during the short dry season. The MAFE was high and did not change with altitude. CONCLUSION: It is obvious that a malaria outbreak will cause the sudden rise of HMIR and MAFE in highland, prior to the malaria season; the discrepancy with lowland would then help detecting an incipient malaria epidemic. It is recommended that in designing the MEWS, the National Malaria Control Programme should include these parameters and put special emphasis on: altitude, age groups and seasons.

The evolution of HIV-1 group M genetic variability in Southern Cameroon is characterized by several emerging recombinant forms of CRF02_AG and viruses with drug resistance mutations.

The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by circulating recombinant forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naïve patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naïve patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naïve patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1.

In vitro anthelminthic efficacy of Dichrocephala integrifolia (Asteraceae) extracts on the gastro-intestinal nematode parasite of mice: Heligmosomoides bakeri (Nematoda, Heligmosomatidae).

OBJECTIVE: To evaluate the ovicidal and larvicidal activities of aqueous and ethanolic extracts of leaves of Dichrocephala integrifolia (D. integrifolia) against the eggs (fresh and embryonnated), the first and second larval stages of Heligmosomoides bakeri. In order to verify if this medicinal plant possesses active compounds capable of inhibiting the embryonation and hatching of eggs or to induce the mortality of larvae (L1 and L2). METHODS: dried extracts were diluted in distilled FIV water to obtain five different concentrations: 625, 1,250, 2,500, 3,750 and 5,000 µg/mL. Fresh eggs obtained from artificially infected mice feces were exposed to these different concentrations for 48 h. Time of contact for embryonated eggs was 6 h while L1 and L2 larvae were exposed for 24 h. Distilled water (placebo) and 1.5% DMSO were used as negative controls. RESULTS: Distilled water, and 1.5% DMSO had no effect on embryonation, hatching and larval survival. Aqueous extracts of D. integrifolia showed a weak activity against all stages of the parasite at all concentrations tested. On the contrary, the ethanolic extract of D. integrifolia inhibited the embryonation of 87.5% of fresh eggs, the hatching of 81.1% of embryonated eggs and induced the mortality of 98.1% and 98% of L1 and L2 larvae respectively at 5,000 µg/mL. CONCLUSIONS: The results of the present study indicate that the ethanolic extracts of D. integrifolia contained compounds with ovicidal and larvicidal properties. In spite of these results, in vivo tests, studies on toxicity and mechanism of action of active compounds are also needed to validate the utilisation of this medicinal plant by population of Dschang-Cameroon to treat gastro-intestinal parasites.

A preliminary analysis of some epidemiological factors involved in porcine cysticercosis in Bafut and Santa subdivisions, North West Region of Cameroon

OBJECTIVE@#To examine the magnitude and some risk factors of porcine cysticercosis in Bafut and Santa, two sub-divisions noted for pig farming in the North West Region of Cameroon.@*METHODS@#A total of 499 pigs in 300 households were examined by tongue inspection and serologically by Ag-ELISA. Information was sought on the age and sex of the animals, prevailing husbandry systems, types of feed, the state of each pen and the state of toilets. Futhermore, a questionnaire was administered to the farmers to determine their awareness on taeniasis/cysticercosis and related factors.@*RESULTS@#The prevalence of the disease was significantly higher in Santa (10.2%) than in Bafut (4.2%), although there was a higher level of awareness in both localities (62.3%). Age of pigs, traditional rearing systems (roaming, tethered, earth floor pen, raised floor pen), faecal disposal in the environment and poor sanitation significantly influenced the seroprevalence of porcine cysticercosis in both localities. Most farmers (79.7%) used a combination of concentrate, grass and kitchen waste to feed pigs. Financial loss from porcine cysticercosis was estimated at 346 900 CFA representing 2% of total income.@*CONCLUSIONS@#Control measures advanced here include sensitization campaigns, periodic examination and treatment of infected pigs by veterinarians, improved husbandry systems, proper use of standard latrines regularly inspected by sanitary officers, and sound hygienic and sanitary practices.

In vitro activities of acetonic extracts from leaves of three forage legumes (Calliandra calotyrsus, Gliricidia sepium and Leucaena diversifolia) on Haemonchus contortus.

OBJECTIVE: To assess ovicidal activity of three acetonic extracts from the leaves of three forage legume, Calliandra calotyrsus (C. calotyrsus), Gliricidia sepium (G. sepium) and Leucaena diversifolia (L. diversifolia) in vitro on Haemonchus contortus (H. contortus). METHODS: Eggs were exposed for 24 hours to five different concentrations (0.075, 0.15, 0.3, 0.6 and 1.25 mg/mL) of acetonic extracts at room temperature (24 °C). Distilled water and 0.4% Tween were used in the bioassay as negative controls. RESULTS: The later did not affect embryonation and egg hatching of H. contortus. Conversively, significant effects were obtained with the acetonic extracts of leaves of all three plants and the maximum activity was observed with the highest concentration (1.25 mg/mL). The acetonic extract of G. sepium was found to be more active (2.9% and 0.0% for embryonation and egg hatching, respectively) than the other substances 16.5% and 33.5%, respectively for C. calothyrsus, 33.7% and 33.3%, respectively for L. diversifolia. CONCLUSIONS: These results suggest that the three forage legumes do possess ovicidal properties and further studies on larvae should be carried out.

A comparative study of the ovicidal and larvicidal activities of aqueous and ethanolic extracts of pawpaw seeds Carica papaya (Caricaceae) on Heligmosomoides bakeri

OBJECTIVE@#To assess the ovicidal and larvicidal activities of aqueous and ethanolic extracts of pawpaw seeds Carica papaya (Caricaceae) on the eggs and first stage larvae (L(1)) of Heligmosomoides bakeri.@*METHODS@#Eggs of this parasite were obtained from experimentally infested mice (Mus musculus) and larvae were from eggs after incubation at 25∘C for about 72 hours. The eggs and larvae were exposed to ten different concentrations (0.125, 0.25, 0.375, 0.5, 0.75, 1.0, 1.25, 1.75, 2.25 and 2.75 mg/mL) of both aqueous and ethanolic extracts respectively for 72 hours. Distilled water and 0.05% ethanol used as placebo and negative control, respectively.@*RESULTS@#Placebo and negative control group all showed average 92% embryonnation, 98% egg hatching and 2% larval mortality, and did not affect development and larval survival. The extracts inhibited embryonic development, egg hatching and larval survival. In general, the ovicidal and larvicidal activities increased with increasing concentration of different extracts. The aqueous extract was found to be more potent on eggs than on larvae. At 2.75 mg/mL, only 8% of eggs embryonnated and 50% hatched to L(1) vs 57% embryonic development and 79% hatching occurred in the ethanolic extract. However, this later extract was more efficient in preventing larval development producing 96% mortality as against 68% with the aqueous extract.@*CONCLUSIONS@#These results shows the ovicidal and larvicidal properties of aqueous and ethanolic pawpaw seeds extracts.

Bionomics of Anopheline species and malaria transmission dynamics along an altitudinal transect in Western Cameroon.

BACKGROUND: Highland areas of Africa are mostly malaria hypoendemic, due to climate which is not appropriate for anophelines development and their reproductive fitness. In view of designing a malaria control strategy in Western Cameroon highlands, baseline data on anopheline species bionomics were collected. METHODS: Longitudinal entomological surveys were conducted in three localities at different altitudinal levels. Mosquitoes were captured when landing on human volunteers and by pyrethrum spray catches. Sampled Anopheles were tested for the presence of Plasmodium circumsporozoite proteins and their blood meal origin with ELISA. Entomological parameters of malaria epidemiology were assessed using Mac Donald's formula. RESULTS: Anopheline species diversity and density decreased globally from lowland to highland. The most aggressive species along the altitudinal transect was Anopheles gambiae s.s. of S molecular form, followed in the lowland and on the plateau by An. funestus, but uphill by An. hancocki. An. gambiae and An. ziemanni exhibited similar seasonal biting patterns at the different levels, whereas different features were observed for An. funestus. Only indoor resting species could be captured uphill; it is therefore likely that endophilic behaviour is necessary for anophelines to climb above a certain threshold. Of the ten species collected along the transect, only An. gambiae and An. funestus were responsible for malaria transmission, with entomological inoculation rates (EIR) of 90.5, 62.8 and zero infective bites/human/year in the lowland, on the plateau and uphill respectively. The duration of gonotrophic cycle was consistently one day shorter for An. gambiae as compared to An. funestus at equal altitude. Altitudinal climate variations had no effect on the survivorship and the subsequent life expectancy of the adult stage of these malaria vectors, but most probably on aquatic stages. On the contrary increasing altitude significantly extended the duration of gonotrophic cycle and reduced: the EIR, their preference to human blood and consequently the malaria stability index. CONCLUSION: Malaria epidemiological rooting in the outskirts of Western Cameroon highlands evolves with increasing altitude, gradually from stable to unstable settings. This suggests a potential risk of malaria epidemic in highlands, and the need for a continuous epidemiological surveillance.