Parasite-specific antibody and cellular immune responses in human infected with Necator americanus and Oesophagostomum bifurcum.

In this study parasite-specific antibody, cellular reactivity and Thl-type or Th2-type cytokine responses were investigated in humans concurrently infected with Necator americanus and Oesophagostomum bifurcum. The prospects for O. bifurcum-specific serodiagnosis based on IgG4 and IgE were evaluated. IgG4 showed low specificity for O. bifurcum due to antigen cross-reactivity with N. americanus, while IgE specifically distinguished between hookworm and O. bifurcum, and, in doubly infected patients, levels of O. bifurcum-specific as well as N. americanus-specific IgE were significantly elevated compared to those with N. americanus mono-infections. Cellular immunity was not strictly dominated by a Thl- or Th2- type reactivity. In co-infected patients cellular unresponsiveness to parasite antigens was observed, while cellular production of tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) was greater in those doubly infected. Th2-type cytokines (interleukin-5 and interleukin-10) were produced in equal amounts by peripheral blood mononuclear cells from individuals with mono- and coinfections. Such mixed Thl-type and Th2-type immune responsiveness associated with persisting gastrointestinal parasitic nematodes may reflect a state of infection at which parasite-induced inflammatory and enteropathogenic responses co-exist, and furthermore, helminth coinfection will not only suppress parasite-specific cellular responsiveness but may also direct cytokine production towards a "permissive Th1-type cytokine profile" that favours parasite persistence.

Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples.

Until recently infection of humans with Oesophagostomum bifurcum was regarded as a rare zoonosis. But in northern Togo and Ghana its prevalence is 50% or more in certain villages. Diagnosis is hampered by the fact that the eggs of O. bifurcum are morphologically identical to those of the hookworm Necator americanus. Stools have to be cultured for 7 days to allow eggs to hatch to the characteristic third-stage (L3) larvae. We evaluated the applicability of specific polymerase chain reactions (PCRs) to amplify DNA from faecal samples as an alternative method for the differential diagnosis of the two infections. Oesophagostomum bifurcum-PCR was positive in 57 of 61 faecal samples known to contain O. bifurcum L3 larvae in coproculture. Necator americanus PCR was positive in 137 of 146 faecal samples known to contain N. americanus L3 larvae in coproculture. PCR also detected 26 additional O. bifurcum cases in 72 samples from O. bifurcum endemic villages in which no O. bifurcum larvae were found and 45 N. americanus cases in 78 samples in which no N. americanus larvae were found in coproculture. No O. bifurcum DNA was detected in 91 stool samples from individuals from two non-endemic villages. These results prove the usefulness of specific PCR assays as epidemiological tools to estimate the prevalence of O. bifurcum and N. americanus infections in human populations.

Prenatal immune priming with helminth infections: parasite-specific cellular reactivity and Th1 and Th2 cytokine responses in neonates.

The present investigation aimed to determine to what extent maternal helminth infection primes parasite-specific cellular responsiveness in neonates. Umbilical cord mononuclear blood cells (UCBC) and peripheral blood mononuclear cells (PBMC) from mothers proliferated in response to mitogenic stimulation with concanavalin A, as well as to bacterial Streptococcus pyogenes-derived (streptolysin O) and helminth-specific antigens of Necator americanus and Onchocerca volvulus. Cellular responses to Echinococcus multilocularis (Em) and Oesophagostomum bifurcum (Oes), helminth parasites not endemic in the study area, were absent (for Em) or very low (for Oes due to antigenic cross-reactivity). Cellular responsiveness to mitogen and antigens was higher in mothers than in their neonates. Several Th1-type (IL-2, IL-12, and IFN-gamma) and Th2-type (IL-5 and IL-10) cytokines were produced by UCBC from neonates and PBMC from mothers. Low levels of IFN-gamma were elicited by UCBC in response to helminth and bacterial antigens, while secretion of IL-2 was pronounced and similarly high in neonates and their mothers. Amounts of IL-5 produced by UCBC in response to bacterial SL-O and mitogenic stimulation (PHA) were low, but equivalent levels of IL-5 were induced by intestinal helminth and filaria-derived antigens in neonates and mothers. A pronounced production of IL-10 and IL-12 by UCBC was observed--spontaneous IL-10 and IL-12 secretion by UCBC was higher in neonates than by PBMC from mothers. Net amounts of IL-10 elicited by helminth antigens were similar, while net IL-12 in response to mitogen, and bacterial and helminth antigens was significantly higher in mothers than their offspring. Our results indicate that human maternal helminth infection does sensitize in utero for parasite-specific cellular responsiveness in offspring, and also activates specific production of several cytokines, and such children do not present a dominant expression of immunity of either Th1 or Th2.

The role of pigs as transport hosts of the human helminths Oesophagostomum bifurcum and Necator americanus.

We conducted a study in an endemic area of both Oesophagostomum bifurcum and Necator americanus in northern Ghana to examine the possibility of pigs acting as transport hosts for these two human helminth species, due to the commonly observed coprophagic habits of pigs. Under controlled conditions four parasite-free pigs consumed fresh faeces from people heavily infected with both helminths, and faeces were subsequently collected from the rectum of the pigs from 5 to 50 h post-feeding. Four to five per cent of the O. bifurcum and N. americanus eggs fed to the pigs were viable and retrieved as third-stage larvae after coproculture of the pigs' faeces. We discuss the possible impact of the coprophagic habits of pigs as potential parasite transport hosts during different seasons in this area of West Africa.

The capacity of the third-stage larvae of Oesophagostomum bifurcum to survive adverse conditions.

Human infections with the intestinal nematode Oesophagostomum bifurcum are commonly found in the Sudan savannah of northern Togo and Ghana. Apparently, the long and hot dry season in this region does not prevent transmission, which is believed to take place through ingestion of the infective, third-stage larvae (L3). Oesophagostomum L3 cultured from human stools, unlike the larvae of Necator americanus, were shown to survive desiccation. In addition, 93% of the O. bifurcum L3 frozen for 24 h at -15 degrees C regained motility when brought back into ambient temperatures. The L3 also survived the acidity of an artificial mixture made to resemble the gastric juices of humans. Desiccated larvae could even be rehydrated in this mixture, indicating the possibility of dust-borne infections. The sturdiness of the L3 is likely to contribute to the high transmission intensity in northern Togo and Ghana.

Diagnosis of Oesophagostomum bifurcum and hookworm infection in humans: day-to-day and within-specimen variation of larval counts.

Oesophagostomum bifurcum, as well as hookworm infections are hyperendemic among humans in northern Togo and Ghana. For parasite-specific diagnosis a coproculture is obligatory, because only the infective larvae, and not the eggs, can be distinguished morphologically. The sensitivity of duplicate coprocultures from a single stool sample was found to be above 90% in comparison to a gold standard of 10 coprocultures made from a single stool specimen. Prevalence of infection with O. bifurcum and hookworm further increased with the number of coprocultures made from each individual stool. Notwithstanding the high sensitivity, intensity of infection per individual varied considerably from day-to-day and the number of larvae found in different samples out of 1 stool also varied highly, both showing a heterogeneous distribution. Surprisingly, daily fluctuation and within-specimen variation could not be differentiated from each other, probably because of the variation created by the coproculture technique. To estimate the intensity of infection, it is sufficient to make repeated coprocultures from only 1 individual stool sample. Laborious collection of stool samples on subsequent days does not give better estimates of the individual infection status.

Geographic distribution and epidemiology of Oesophagostomum bifurcum and hookworm infections in humans in Togo.

In contrast to the rest of the world, infections with Oesophagostomum bifurcum are commonly found in humans in northern Togo and Ghana. In addition, infections with hookworm are endemic in this region. In the present study, a detailed map of the geographic distribution of O. bifurcum and hookworm infections in northern Togo was made. There were a number of foci with high prevalence of infection with O. bifurcum. All the villages examined were infected with hookworm, and the distribution was quite patchy. Women were infected with O. bifurcum more often than men, while infections with hookworm were more prevalent in men than in women. The prevalence and intensity of infection with both parasites were clearly age-dependent. We estimate that more than a 100,000 people in Togo are infected with O. bifurcum and more than 230,000 are infected with hookworm.

Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.

Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia.

In higher vertebrates, two opposite behavioral coping strategies can be distinguished that are associated by a typical neuroendocrine pattern. Little is known about the individual variation in the stress response in lower vertebrates such as teleosts. In the present study, rainbow trout were fitted with an indwelling aortic catheter for repeated blood sampling and exposed to severe hypoxia and subsequent recovery and their behavior was characterized semiquantitatively during hypoxia. Blood levels of catecholamines, cortisol, glucose, FFA, lactate, and electrolytes were measured. About 60% of the fish survived the experiment whereas the others died during the recovery period. Behavioral strategy appeared to be highly related to survival since nonsurviving fish displayed strenuous avoidance behavior involving burst type activity whereas surviving fish did not panic and remained quiet. These behavioral differences were associated with marked differences in plasma catecholamine levels, which were 4- to 5-fold higher in nonsurviving fish as compared to survivors whereas the cortisol response tends to be lower in nonsurviving fish. Plasma lactate levels in nonsurvivors were 4- to 5-fold higher as compared to survivors while a severe hyperkalemia developed during recovery indicating the loss of intracellular homeostasis. The individual differences in behavioral concepts and neuroendocrine activation observed in rainbow trout during stress show great similarity with the active and passive coping strategies distinguished in higher vertebrates and may be determinant for survival during hypoxia.