Comparison of Kato-Katz, PCR and coproantigen for the diagnosis of Taenia solium taeniasis.

Four methods were compared for the diagnosis of human taeniasis caused by Taenia solium. Fecal samples from persons living in a T. solium endemic region of Madagascar were examined for taeniid eggs by the Kato­Katz method. Subsequently, samples positive (n = 16) and negative (n = 200) for T. solium eggs were examined by (i) amplification of the fragment of small subunit of the mitochondrial ribosomal RNA (rrnS) gene using conventional polymerase chain reaction (PCR) and (ii) a nested PCR of a fragment of the T. solium Tso31 gene. Additionally, 12 egg-positive and all egg-negative samples were tested for coproantigen detection. A further 9 egg-positive fecal samples were examined using both PCRs. Of the 12 egg-positive samples tested by PCRs and coproantigen methods, 9 (75%) were positive by rrnS PCR, 3 (25%) using Tso31-nested PCR and 9 (75%) by coproantigen testing. None of the 200 egg-negative fecal samples was positive in either rrnS or Tso31-nested PCR. Twenty of the 25 egg-positive samples (80%) were positive in rrnS PCR, and DNA sequencing of PCR amplicons was obtained from 18 samples, all confirmed to be T. solium. Twelve of the 25 egg-positive samples (48%) were positive in the Tso31-nested PCR, all of which were also positive by rrnS PCR. It is suggested that species-specific diagnosis of T. solium taeniasis may be achieved by either coprological examination to detect eggs or coproantigen testing, followed by rrnS PCR and DNA sequencing to confirm the tapeworm species in egg-positive or coproantigen-positive samples.

Confirmation by necropsy of a high prevalence of porcine cysticercosis in a rural district of Madagascar.

Neurocysticercosis is recognized as an important health issue in the Malagasy population. To date, investigations into prevalence of infection with the causative agent, Taenia solium, in the parasite's natural animal intermediate hosts, have relied on serological methods which have been found to be non-specific. We determined the prevalence of porcine cysticercosis among pigs from a contiguous area of the Betafo and Mandoto administrative districts, Vakinankaratra Region, Madagascar. One hundred and four slaughter-weight pigs were examined by detailed necropsy examination including slicing of the heart, tongue, masseter muscles, diaphragm and carcase musculature. Thirty-seven animals (35.6%) were found infected with T. solium, representing one of the highest rates of infection ever reported, worldwide. These findings highlight the importance of T. solium in Madagascar and support the need for increased efforts to prevent the parasite's transmission to reduce its burden on the health of the Malagasy population.

Cytogenotoxicity Screening of Urban and Rural Marshes: An Integrated In Vivo Approach Coupling Fish and Plant-Based Tests Adapted for Low-Income Countries.

Effects of anthropogenic activities such as urbanization, population growth, and agriculture on water quality are major concerns particularly in low-income countries where water quality monitoring can be challenging. The purpose of the present study was to evaluate the cytogenotoxic potential of water from urban and rural Malagasy marshes, coupling a fish (Nile tilapia, Oreochromis niloticus) and a plant (Allium cepa) species as bioindicators. The fish and plants were exposed for 72 h to water sampled in the two locations investigated. Using the comet assay on fish erythrocytes, DNA strand breaks were assessed, while mitotic index and nucleolar alterations were estimated in cells of the plant root apex. Comet assays revealed significant DNA strand breaks to fish erythrocytes in both the marshes investigated while the mitotic index and nucleolar characteristics in the roots of A. cepa mainly highlighted potential cytotoxicity in the urban marsh. Our results demonstrate the advantages of coupling in vivo biological test systems to screen potential cytogenotoxicity of surface water in low-income countries where comprehensive data sets of aquatic contaminants are often lacking. Environ Toxicol Chem 2023;42:1266-1275. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Survival and moulting of Amblyomma variegatum nymphs under cold conditions of the Malagasy highlands.

Although Amblyomma variegatum is now regularly recorded up to 1600 m in altitude in the Malagasy highlands, where it was previously reported not to persist without a constant supply of ticks introduced from lower infested regions, some parts of the highlands remain tick-free. Studies were carried out to verify whether the cold climate prevailing in these areas in June-September could prevent the survival and moulting of nymphs, the tick life stage present in the environment at this period. Cohorts of engorged A. variegatum nymphs were released from June to August in six different sites (three in 2010, altitudes 1200-1415 m; three in 2011, altitudes 1585-1960 m) which were reported to be either tick-infested (two in 2010, one in 2011) or tick-free. The ticks were placed in cages driven into the soil and open at the bottom so that they could hide in the soil or root network. Of the 1975 nymphs released in 2010 and the 1494 released in 2011, 86% and 85% were recovered, respectively. Twenty to 23% of the recovered ticks were dead, and some of them were obviously predated; predation also likely contributed to the disappearance of the non-recovered ticks. When the rainy season started in October, 59% of the newly moulted adults were still alive in the cages. The moulting period lasted up to 20 weeks, depending on the site and release period. As verified in 2011, unfed nymphs could also survive the cold season. Various A. variegatum life stages are thus able to survive the adverse cold and/or dry seasons: unfed nymphs, engorged nymphs in developmental diapause, moulted adults in behavioural diapause as observed previously. Strong variation in mortality and recovery rates was observed between cages, highlighting the importance of the micro-environment and micro-climate for tick survival. The minimum temperature recorded in the field sites varied from 1.1 °C to 6.8 °C, but the tick-free sites were not the coldest ones; they were, however, those for which the temperature remained below 10 °C for the longest time over the study period. Recovery and mortality rates in the tick-free sites were similar to those of the tick-infested sites: the temperatures recorded during the study periods did not prevent ticks from surviving and moulting although it did delay the metamorphosis. Low temperature alone can therefore not explain the persistence of tick-free areas in the highlands. To further monitor survival, cohorts of engorged nymphs were also maintained in an incubator at 3.6 °C, 6.2 °C or 12.8 °C. More than 50% mortality was observed after 6 days at 3.6 °C, and after 15 days at 6.2 °C, whereas 18 days at 12.8 °C only delayed moulting. The collected survival, moulting and climatic data presented in this study should help to develop a predictive model to assess the distribution of A. variegatum according to climate characteristics.

Mining the genetic diversity of Ehrlichia ruminantium using map genes family.

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host. In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster. We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands). These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host-vector-pathogen interactions that could be crucial for developing broad-spectrum vaccines.

Amblyomma variegatum ticks and heartwater on three Caribbean Islands.

Amblyomma variegatum tick infestation, tick infection by Ehrlichia ruminantium (ER), and ER genetic diversity were studied in the Caribbean Islands of Guadeloupe, Marie-Galante, and Antigua between 2003 and 2005. Nested PCR for pCS20 was used to detect ER, while ER strains were characterized by sequencing or by restriction fragment length polymorphism (RFLP) profiles of map-1 PCR products. In 2003 in Guadeloupe, the prevalence of tick-infested herds was 35.6%. In Marie-Galante 79.1% of herds in 2003 and 73.8% in 2005 were infested, while only an average of 2.2% of the herds were infected in Antigua between this same period. In Marie-Galante, 19.1% of ticks were ER positive, and ER-infected ticks were found in 33.3% of the herds. In Antigua only 5.8% of the ticks were ER positive. High ER tick infection rate combined with a very high level of tick infestation highlight the risk of heartwater in Marie-Galante and Guadeloupe more than in Antigua. The three islands still represent a reservoir for tick and heartwater in the Caribbean. Nine different African and Caribbean map-1 ER genotypes were identified. This diversity was observed even in restricted areas, and identical map-1 genotypes were observed on all three islands. This high genetic diversity of ER strains suggests that there was a simultaneous introduction of several strains from African countries into the Caribbean region.

Renitelo cattle dermatophilosis and PCR-RFLP analysis of MHC gene.

Renitelo breed is a cattle breed created at Kianjasoa station (Madagascar) by a triple crossing Malagasy Zebu x Limousine x Afrikander. This breed besides many valuable advantages, such as rapid growth and drought power, presents a huge disadvantage which is sensitivity to skin disease, dermatophilosis, previously known as streptotrichosis. This disease caused by Dermatophilus congolensis is one of the major threats for the population of Renitelo cattle. An allele of MHC gene has been shown to be dramatically associated to hypersensitivity to the disease in other cattle breed. To bring further information to tick borne disease clinical survey, mainly dermatophilosis, we wanted to verify if such allele could be found in this breed. Renitelo cattle included in this study were chosen for the presence of dermatophilosis lesions in more or less severe form (N = 17). These animals were blood sampled and a genetic analysis on the MHC gene BoLA-DRB3 was performed, by PCR amplification using BOD 31 & BOD 32 primers. Amplified products were analyzed by RFLP using enzymes. Restriction band profiles were characterized according to previously defined patterns. Three cows out of the 17 cattle analyzed for MHC gene presented the hypersensitive allele FDA. Two out of the three hypersensitive cows were pure breed while one was half breed. All the cows presented dermatophilosis lesions at least during rainy season but one of them particularly suffered from severe lesions covering all its body and died of the illness. This study shows that hypersensitivity allele found in other bovine breeds can be found in Renitelo breed. This result seemed to suggest that this characterization could be utilized in breeding program for this breed.

Understanding the mechanisms of transmission of Ehrlichia ruminantium and its influence on the structure of pathogen populations in the field.

The understanding of the structure of Ehrlichia ruminantium stock population in the field was highlighted by experiments done in controlled conditions on the goat model. The mixture of strains observed in ticks seemed to be due to simultaneous infections rather than successive infections of the carrier. During a dual infection, the timing of Ehrlichia ruminantium circulation of the two stocks in hosts influenced their selection by ticks.

Nested PCR for detection and genotyping of Ehrlichia ruminantium: use in genetic diversity analysis.

Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium strains.