M.T. Rogan

Detection, screening and community epidemiology of taeniid cestode zoonoses: cystic echinococcosis, alveolar echinococcosis and neurocysticercosis.

Publisher Summary This chapter focuses on the public health importance of taeniid cestode zoonoses especially cystic echinococcosis, alveolar echinococcosis and T. solium cysticercosis. The chapter addresses the problem of neurocysticercosis and epilepsy in developing countries particularly Latin America and its public health consequences in the USA and elsewhere. E. multilocularis , which is the cause of one of the most pathogenic of all human parasitic infections, appears to be spreading in parts of western Europe, USA, and Japan. The remarkable development in the past 20 years or so of high resolution imaging technologies, such as computerized tomography (CT) scan and ultrasound have enabled precision detection of taeniid larval cystic infection in humans. Coupled with improvements in immunodiagnostic test sensitivity and antigen specificity, diagnosis of cestode zoonoses is now more accurate than ever before. Although further developments in immunodiagnosis are required, laboratory tests have provided improved capability for screening populations in both epidemiological and community studies. Parallel and recent development of genus specific coproantigen tests for human taeniasis and canine echinococcosis have also provided major new tools for epidemiological and surveillance programs.

Echinococcosis: disease, detection and transmission

Echinococcosis is one of the worlds most geographically widespread parasitic zoonoses, with transmission occurring in tropical, temperate and arctic biomes. Most human infections are due to Echinococcus granulosus transmitted between domestic dogs and livestock, but this cosmopolitan species also cycles between wild carnivores (principally canids) and wild ungulates. The other species with significant zoonotic potential is E. multilocularis that occurs naturally in fox definitive hosts and small mammal intermediate hosts. These two species cause human cystic or alveolar echinococcosis respectively, which may be considered serious public health problems in several regions including developed countries. This review provides an introductory overview to the Supplement and summarises the biology and epidemiology of these two related cestodes with an emphasis on applied aspects relating to detection, diagnosis and surveillance in animal and human populations, and includes aspects of transmission ecology, and also considers aspects of community epidemiology and potential for control.

IgG1 and IgG4 serum antibody responses in asymptomatic and clinically expressed cystic echinococcosis patients

IgG1 and IgG4 subclass antibody responses were investigated in two clinically different groups of hepatic cystic echinococcosis (CE) patients. One group consisted of surgically proven CE cases (clinically expressed hospitalized cases) and a second group comprised asymptomatic CE patients (first time community detected cases) diagnosed by portable ultrasound and serology in four different endemic communities. Fifty eight sera from surgically proven CE patients and 133 sera from asymptomatic ultrasound diagnosed CE cases were screened by ELISA using purified human hydatid cyst fluid antigen B for total specific IgG, IgG1 and IgG4 antibodies. Fifty sera from healthy individuals from within endemic regions were used as control negatives. Compared to control negatives total IgG antibody levels were elevated in both surgically proven (85%) and asymptomatic CE cases (77%) but there was no significant difference between the two groups. IgG1 subclass antibody levels were also elevated in both surgically proven (55%) and asymptomatic cases (58%) compared to endemic controls and similarly the difference in this response was not significant between these two groups. In contrast the prevalence of IgG4 antibodies in surgically confirmed chronic CE patients was greater (71%) than the respective IgG1 antibody levels (55%). The greatest difference in specific antibody response, between advanced surgically confirmed CE patients and ultrasound detected asymptomatic CE cases, was observed for IgG4 antibody levels which were detected in 71% of symptomatic compared to only 23% of asymptomatic patients (P < 0.0001). These observations confirm that IgG4 is an important diagnostic parameter for clinically expressed cystic echinococcosis in humans and suggest that a switch from a predominant IgG1 response to IgG4 might occur in CE patients as the disease progresses.

Cystic echinococcosis in semi-nomadic pastoral communities in north-west China.

In order to determine the prevalence of human cystic echinococcosis (CE) in semi-nomadic traditional pastoralist groups in north-west China, 2 large community studies were undertaken in Altai and Tacheng Prefectures in 1990/91 and 1995/96, respectively. The Kekergash community (Altai) comprised mainly ethnic Kazakhs, whereas the Narenhebuke community (Tacheng) comprised mainly Mongolians. Populations were screened for CE by abdominal ultrasound scan (US) and serological tests. The total prevalence of confirmed human CE was higher in Narenhebuke (2.7%, 49/1844) than in Kekergash (0.9%, 17/1861; P < 0.01). Within each community there was no significant difference of CE prevalence between the Kazakh and Mongolian groups, although Han Chinese exhibited twice the rate of CE (4.9%) in Narenhebuke compared to the dominant Mongolian population. For each community, human CE prevalence increased with age and there was a greater risk associated with the practice of home slaughter of livestock. Dogs were screened for Echinococcus granulosus infection and re-infection levels using a highly specific coproantigen test. The proportion of dogs with positive coproantigen tests was significantly higher in Narenhebuke (36.0%, 50/139) compared to Kekergash (17.8%, 16/90). In Narenhebuke the re-infection levels of dogs, as determined by coproantigen positivity, were higher in the winter quarters (49.4%, 39/79) compared to the summer quarters (18.3%, 11/60; P < 0.01). Furthermore, coproantigen re-test positivity was 25% at 3 months and 29.2% at 7 months. Highest dog coproantigen positivity was obtained over the winter period.

T‐cell activity associated with secondary infections and implanted cysts of Echinococcus granulosus in BALB/c mice

Selected cytokine profiles of lymphocytes were assessed in BALB/c mice infected with protoscoleces of Echinococcus granulosus. Late stages of the infection (three months +) were characterized by more dominant Th2 activity with elevated IL‐4 and IL‐10 and reduced IFN‐γ output by Con‐A and antigen stimulated splenocytes. Circumparasitic leucocytes produced mainly IL‐10 by five months post infection. A peak in IFN‐γ production in the first month of infection may suggest Th1 or Th0 activity at this time and this may be correlated with initial protoscolex death. In addition, cytokine profiles from mice implanted with intact hydatid cysts were also assessed. At two weeks post implantation all cysts were still viable and cytokine production was characterized mainly by elevated IL‐10 production. However, at four months post implantation, some of the cysts from two mice had been killed whilst all cysts in the remaining mouse remained viable. In the mice where dead cysts were present, elevated levels of IFN‐γ were detected from splenocytes and circumparasitic cells. Elevated IL‐4 was also evident with the splenocytes. In the mouse with viable cysts IFN‐γ production was reduced. Results indicate that IFN‐γ (Th1) activity may be correlated with killing of both protoscoleces and established cysts of E. granulosus.

Expression and immunological characterisation of Echinococcis granulosus recombinant antigen B for IgG4 subclass detection in human cystic echinococcosis

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.

In-vitro susceptibility of hydatid cysts of Echinococcus granulosus to nitric oxide and the effect of the laminated layer on nitric oxide production

Murine hydatid cysts of Echinococcus granulosus were incubated in vitro in the presence of nitric oxide produced from S‐nitroso‐N‐acetylpenicillamine (SNAP) or interferon‐γ activated peritoneal macrophages. In both situations, evidence of cyst damage and death was observed by microscopy in over 77% of cysts after 3 days, indicating that intact hydatid cysts could be susceptible to a Th1 driven macrophage attack. A crude extract of the laminated layer from cysts was found to be able to reduce the production of nitric oxide from activated macrophages in vitro and in vivo and this may have been due to phagocytosis of laminated layer fragments by the macrophages. The results indicate that, although cysts may be susceptible to the effects of nitric oxide, the laminated layer may be involved in downregulating nitric oxide production.

Echinococcus granulosus antigen B and seroreactivity in natural ovine hydatidosis

Hydatid cyst fluid from sheep and camels infected with Echinococcus granulosus, together with partially purified preparations of hydatid fluid antigen B and a recombinant antigen B product, were tested in an ELISA for their ability to detect IgG antibodies against E granulosus in the serum of naturally infected sheep. The antibody activity in sera from sheep naturally infected with Taenia hydatigena cysticercosis or Fasciola hepatica was also tested. All the antigen preparations from native hydatid cyst fluid were able to detect antibodies in the sera from a significant proportion of sheep with natural hydatid cyst infection, as identified by inspection at slaughter, although the seroreactivity was variable. The native antigen B preparation from camel hydatid cyst fluid gave the highest sensitivity in the ELISA (total 90 per cent), with 99 per cent specificity. In all cases, the recombinant antigen B was the least sensitive antigen (25 per cent) although it was highly specific (99 per cent).

Helminth species richness in wild wood mice, Apodemus sylvaticus, is enhanced by the presence of the intestinal nematode Heligmosomoides polygyrus

We analysed 3 independently collected datasets of fully censused helminth burdens in wood mice, Apodemus sylvaticus, testing the a priori hypothesis of Behnke et al. (2005) that the presence of the intestinal nematode Heligmosomoides polygyrus predisposes wood mice to carrying other species of helminths. In Portugal, mice carrying H. polygyrus showed a higher prevalence of other helminths but the magnitude of the effect was seasonal. In Egham, mice with H. polygyrus showed a higher prevalence of other helminth species, not confounded by other factors. In Malham Tarn, mice carrying H. polygyrus were more likely to be infected with other species, but only among older mice. Allowing for other factors, heavy residual H. polygyrus infections carried more species of other helminths in both the Portugal and Egham data; species richness in Malham was too low to conduct a similar analysis, but as H. polygyrus worm burdens increased, so the prevalence of other helminths also increased. Our results support those of Behnke et al. (2005), providing firm evidence that at the level of species richness a highly predictable element of co-infections in wood mice has now been defined: infection with H. polygyrus has detectable consequences for the susceptibility of wood mice to other intestinal helminth species.

Immunoblot evaluation of IgG and IgG-subclass antibody responses for immunodiagnosis of human alveolar echinococcosis.

Antigen binding of total-IgG and IgG-subclass antibodies from patients with alveolar or cystic echinococcosis (AE and CE) was assessed by immunoblotting. Antigen extracts were prepared from Echinococcus multilocularis protoscoleces (EmP) or from homogenized E. multilocularis metacestode tissue (EmCH). Antigens of approximately 44, 35, 21, 17.5 and 16.5 were recognized by total-IgG and IgG1- and IgG4-subclass antibodies in some of 50 human AE sera from China, Japan or France. The 44- and 35-kDa polypeptides, present in both EmP and EmCH extracts, were recognized by total-IgG antibodies in sera from 82% and 66% of the AE patients, respectively. However, over 30% cross-reactivity occurred between these two antigens and sera from CE and Taenia solium cysticercosis patients. The immunoblot specificities of the 27-, 21- and 17.5-kDa antigens in EmP for E. multilocularis infection were 73%, 88% and 93%, respectively. Recognition of the 17.5-kDa antigen in the EmP immunoblot was much higher for the Japanese AE cases (11/13; 85%) than for the French (9/19; 47%) or Chinese (9/18; 50%) AE cases. None of the CE cases from Uruguay or Libya, where human AE has not been reported, was seropositive for the 17.5-kDa antigen. Antibodies from three (7.3%) of the 41 Chinese CE cases recognized the 17.5-kDa antigen. Within the 13 Japanese AE sera, the combined detection by IgG1, IgG4 and total-IgG antibodies of the 27-, 21- and 17.5-kDa antigens in either EmP or EmCH immunoblots was greater than that by each class/subclass alone, increasing the overall sensitivity for AE patients. A combined ELISA/immunoblot approach, including IgG-subclass detection using E. multilocularis protocolex or cyst extracts, could be useful for the differential diagnosis of human alveolar echinococcosis. An algorithm for such an approach is given.