Risk factors and monitoring for water quality to determine best management practices for splash parks.

Splash parks have been associated with infectious disease outbreaks as a result of exposure to poor water quality. To be able to protect public health, risk factors were identified that determine poor water quality. Samples were taken at seven splash parks where operators were willing to participate in the study. Higher concentrations of Escherichia coli were measured in water of splash parks filled with rainwater or surface water as compared with sites filled with tap water, independent of routine inspection intervals and employed disinfection. Management practices to prevent fecal contamination and guarantee maintaining good water quality at splash parks should include selection of source water of acceptable quality.

Quantification of horizontal transmission of Salmonella enterica serovar Enteritidis bacteria in pair-housed groups of laying hens.

An important source of human salmonellosis is the consumption of table eggs contaminated with Salmonella enterica serovar Enteritidis. Optimization of the various surveillance programs currently implemented to reduce human exposure requires knowledge of the dynamics of S. Enteritidis infection within flocks. The aim of this study was to provide parameter estimates for a transmission model of S. Enteritidis in laying-type chicken flocks. An experiment was carried out with 60 pairs of laying hens. Per pair, one hen was inoculated with S. Enteritidis and the other was contact exposed. After inoculation, cloacal swab samples from all hens were collected over 18 days and tested for the presence of S. Enteritidis. On the basis of this test, it was determined if and when each contact-exposed hen became colonized. A transmission model including a latency period of 1 day and a slowly declining infectivity level was fitted. The mean initial transmission rate was estimated to be 0.47 (95% confidence interval [CI], 0.30 to 0.72) per day. The reproduction number R(0), the average number of hens infected by one colonized hen in a susceptible population, was estimated to be 2.8 (95% CI, 1.9 to 4.2). The generation time, the average time between colonization of a "primary" hen and colonization of contact-exposed hens, was estimated to be 7.0 days (95% CI, 5.0 to 11.6 days). Simulations using these parameters showed that a flock of 20,000 hens would reach a maximum colonization level of 92% within 80 days after colonization of the first hen. These results can be used, for example, to evaluate the effectiveness of control and surveillance programs and to optimize these programs in a cost-benefit analysis.

Prevalence of antibodies against Ascaris suum and its association with allergic manifestations in 4-year-old children in The Netherlands: the PIAMA birth cohort study.

The association between helminth infections and childhood atopic diseases remains controversial. The majority of studies have been carried out in tropical areas, whereas less information is available from western countries with low intensity of helminth infections. In the Netherlands, the infection of pigs with Ascaris suum is very common, particularly on pig farms with outdoor facilities. This helminth can also infect humans, causing visceral larva migrans. This study aims at determining the prevalence of antibodies against A. suum and its association with allergic symptoms and sensitisation in a population of 4-year-old children living in The Netherlands. Blood samples from 629 children from the prospective birth cohort Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study were examined for Ascaris antibodies. Data on allergic symptoms and sensitisation were collected using questionnaires and radioallergosorbent tests (RAST). A total of 45 out of 629 (7%) were found to be Ascaris-seropositive. In addition, a positive association between Ascaris seropositivity and wheeze in the last year, doctor-diagnosed asthma and food and aero-allergen sensitisation was found. These results support the hypothesis that low-level or transient infection with helminths enhances allergic reactivity.

Integrating the issues of global and public health into the veterinary education curriculum: a European perspective.

Veterinary public health is an essential field in public health activities, based upon veterinary skills, knowledge and resources and which aims to protect and improve human health and welfare. This discipline has evolved through three stages, beginning with the fight against animal diseases, moving on to include meat inspection and control of zoonoses and now encompassing a much broader health sciences education, with the goal of guaranteeing a safe and wholesome food supply, protecting human wellbeing and conserving the environment. Within the veterinary medicine curriculum, veterinary public health has undergone a similar development. At first, it was mainly concerned with slaughterhouse-based courses but in time it included the teaching of such subjects as epidemiology, the control of communicable (zoonotic) diseases and emergency preparedness. Veterinary medical faculties in Europe have adjusted their curricula over the past few years to reflect these changes in the subject and to meet the need for specialisation. It could be said that veterinary public health education has literally moved from the local abattoir to the global community. In this paper, the authors briefly discuss examples of veterinary medicine curricula at different universities. The veterinary public health curriculum of the Faculty of Veterinary Medicine, Utrecht, is then discussed in detail, as an example of the European perspective on integrating global and public health issues into the veterinary curriculum.

Fuchs' heterochromic iridocyclitis is not associated with ocular toxoplasmosis.

To analyze the association between Fuchs' heterochromic iridocyclitis (FHI) and toxoplasmosis, we performed ocular examinations and used various specific laboratory tests to establish a role for Toxoplasma gondii in the pathogenesis of FHI. Results were compared with those for other types of uveitis and healthy controls. Of the 88 patients with FHI, nine (10.2%) had toxoplasmosislike scars, but an association could not be proved by the indirect immunofluorescence antibody test or enzyme-linked immunosorbent assay, or by a test for cellular immunity to Toxoplasma antigen. Analysis of aqueous humor samples for Toxoplasma antibodies in patients with FHI also yielded negative results. On the basis of the negative results of these laboratory tests, we concluded that FHI is not associated with ocular toxoplasmosis.

Detection of toxoplasma antigen in tissues by means of enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for the detection of free toxoplasma antigens in patient sera was applied for demonstration of toxoplasmosis in tissues. Unfixed organs stored at room temperature remain antigen-positive for weeks, even when parasites are no longer demonstrable by means of direct immunofluorescence testing. Chronically infected brains, containing tenths of cysts do not react positive, whereas recently infected brains (proliferative stage) are positive, suggesting that with this ELISA method discrimination can be made between different stages of the infection.

Detection of specific immunoglobulins (IgG, IgM, IgA, IgE) and total IgE levels in human trichinosis by means of the enzyme-linked immunosorbent assay (ELISA).

Sera from 58 cases of confirmed human trichinosis were examined over an 11-month period. The enzyme-linked immunosorbent assay (ELISA) was used for the detection of class-specific serum antibodies (IgG, IgM, IgA, IgE) and for total serum IgE. The indirect immunofluorescent test served as reference technique. The ELISA proved to be more sensitive. Specific IgG antibodies were detected by ELISA in 100% of the clinical cases. Both IgG and IgM were demonstrable throughout the observation period of 11 months. Specific serum IgA was seen in 62% of the patients during the first 3 months, presumably the result of stimulation of the immune system by the adult worms in the intestine. Specific serum IgE was seen in a few cases only at the onset of the disease. Total serum IgE levels were elevated in 32% of the patients in the first month of the disease and in 17% after 11 months, compared to 10% of healthy individuals.

Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions.

Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E. coli (STEC) strains using different media under aerobic and anaerobic conditions. The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans. The main route of infection for humans is through the eating of badly cooked or handled beef. The major reservoir for STEC strains in cattle is the rumen. To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid. Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid. These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.

Serology in ocular toxoplasmosis.

The diagnostic value of toxoplasma serology in ocular disease was evaluated in the following groups of patients: (I) uveitis cases of various causes (n = 291); (II) consecutive posterior and panuveitis patients (n = 60); (III) patients with definite congenital and ocular toxoplasmosis (n = 8); (IV) cases of clinical ocular toxoplasmosis (n = 25); and control patients with uveitis of non-toxoplasma origin (n = 12). No relation was observed between the level of the dye test titres and the diagnosis of ocular toxoplasmosis (groups I and II). During the active stages of the disease no typical change of the titres occurred in several longitudinally studied patients with toxoplasmosis. In group III one case was discovered to be negative by the dye test despite active ocular disease; however, IgG antibodies against toxoplasma were detected by the ELISA technique. In group IV, which was investigated by the ELISA technique, 100% of the toxoplasmosis patients were positive for IgG versus 58% of the control patients. Circulating immune complexes containing IgG and toxoplasma antigen were detected in seven of 25 toxoplasmosis patients (28%) and in two of 12 control patients (16%). Our study shows that the definite diagnosis of ocular toxoplasmosis or its exclusion by serological means only is not yet feasible. The possible superiority of the ELISA test to the dye test warrants further investigation.

Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli.

Chicken meat contaminated with Campylobacter jejuni can be the source of human enteritis. To decrease the risk of human infection, Campylobacter should be controlled at farm levels. Orally given probiotic bacteria could prevent colonisation of chicken with pathogenic bacteria like Campylobacter. The aim of this study was to investigate the effect of different bacteria on Campylobacter growth. Our results demonstrated that bacteria isolated from conventional chicken had potential inhibitory activities against Campylobacter. Other bacteria not isolated from chickens but with known antagonistic capacities, e.g. Enterococcus (56 strains) and Escherichia coli (20 strains), did not show any negative effect on Campylobacter. Interestingly, one Lactobacillus (P93) strain isolated from the chicken gut showed bactericidal activity against all tested Campylobacter. The bactericidal effect was characterised as the production of organic acids in combination with probably production of an anti-Campylobacter protein. In a co-culture study of Campylobacter and Lactobacillus (P93), the culturability of Campylobacter was under the detection limit after 48 h of incubation. A chicken experiment is needed to further evaluate the effect of the promising probiotic bacteria against Campylobacter colonisation in chicken.

Suggestions for the construction, analysis and use of descriptive epidemiological models for the modernization of meat inspection.

There is consensus that scientifically validated, quantitative assessments of actual public health risks are a prerequisite for any sound modernization of current meat inspection procedures. This article outlines how such analyses could be conducted. Approaches that rely heavily upon extrapolations from theoretical dose-effect relationships are inadequate for the assessment of microbiological health risks associated with the production and consumption of meat. The use of highly structured and very elaborate descriptive epidemiological models covering the entire period from stable to table can be considered a promising solution. Health risks can be quantified by means of incidence rates and the influence of risk factors by means of odds ratios and (population) attributable fractions. A great advantage is that when it is not possible to quantify risks exactly, the descriptive models are detailed enough to be used in a hazard analysis critical control point (HACCP)-like approach and for writing validated codes of good manufacturing practice (GMP). There are, however, several conditions which have to be met before risk assessment can become the foundation of safety assurances for meat, such as active legislative support and the setting up of monitoring systems for zoonoses and other health hazards in animals and humans.

Identification and quantification of risk factors regarding Salmonella spp. on pork carcasses.

The main elements of a descriptive epidemiological model for Salmonella spp. in Dutch pig slaughterlines, and the subsequent quantification of risk factors regarding the contamination of carcasses, are described. There is a strong correlation between the number of live animals that carry Salmonella spp. in their faeces and the number of contaminated carcasses at the end of the slaughterline. Live animals that carry Salmonella spp. are 3-4 times more likely to end up as a positive carcass than Salmonella-free animals. Currently, about 70% of all carcass contamination results from the animals themselves being carriers, and 30% because other animals were carriers (i.e. cross contamination). Furthermore, it is estimated that in general between 5-30% of the carcasses produced may contain Salmonella spp. With respect to carcass contamination with Enterobacteriaceae and Salmonella spp., inadequately cleaned polishing machines (odds ratio, OR, 6) and 'inapt procedures during evisceration' (OR 11), i.e. faulty evisceration and hygiene practices, are the most important risk factors. An estimated 5-15% of all carcass contamination with Salmonella spp. occurs during polishing after singeing. The remainder is the result of current evisceration practices (55-90%) and, to a lesser extent, further processing (5-35%), i.e dressing, splitting and meat inspection. Less likely Salmonella spp. already present on the skin of the live animals survive scalding and singeing. However, because pigs are the only important source for the Salmonella contamination of the line and the carcasses produced, it can also be concluded that if Salmonella-free pigs were produced, consumers could be provided with virtually Salmonella-free pork. As long as Salmonella-positive animals enter abattoirs, there will always be transmission of Salmonella spp. to consumers, even if the process is carried out according to stringent codes of good manufacturing practices (GMP). EU regulations should, therefore, allow for the decontamination of caracasses with a safe substance, e.g. lactic acid, on the condition that the slaughterhouse strictly adhers to GMP principles.

Salmonella spp. on pork at cutting plants and at the retail level and the influence of particular risk factors.

This article describes the contamination of pork with Salmonella spp. in cutting plants and butchers' shops in The Netherlands and quantifies the influence of several risk factors. When contaminated carcasses are being processed, the main risk factors regarding cross contamination are inapt cleaning and disinfection (OR 12.8), manipulation of contaminated materials as such (OR 4.7) and (re)contaminated surfaces (OR 4.4). However, in the current situation, where contaminated carcasses are constantly being brought into cutting lines, interim cleaning and disinfection of surfaces and utensils during breaks and at the end of the working day will most likely prevent not more than about 10% of all cross contamination that takes place during a working day. Thus, as long as contaminated carcasses are being processed, about 90% of the cross contamination that occurs in cutting plants is practically unavoidable. It can therefore also be concluded that under these circumstances the implementation of codes of good manufacturing practices (GMP) and Hazard Analysis Critical Control Point (HACCP)-inspired production methods will only be marginally effective in the control of Salmonella spp. cross contamination in cutting lines. The same is more or less true for the processing of contaminated cuts or carcasses by butchers in shops and supermarkets. Furthermore, in contrast to the situation in cutting plants, it may be that up to 10% of butcher's shops or kitchens of restaurants become colonized for several weeks or months with their own endemic 'house flora' of Salmonella spp., which are originally introduced via the purchased contaminated products of animal origin. Though there are no hard data to substantiate this, it can be suspected that these shops and restaurants represent the more badly managed, i.e. poorly cleaned and disinfected, enterprises. However, several analytical limitations hinder an exact determination of the prevalence of Salmonella spp. contaminated pork and an exact quantification the influence of risk factors. The diagnostic value (i.e. the sensitivity, specificity, precision and predictive value) of the combination of swabbing of carcasses and cuts and the usually employed culturing methods, in particular, is largely unknown, and there are indications that it may be seriously questioned. Without a more thorough knowledge about the diagnostic value of current and future methods of sampling and identification, it is impossible to provide for more accurate estimations of the prevalence of Salmonella positive carcasses and cuts. Based on the research data, the incidence of contaminated cuts and retail-ready pork can not be estimated more precise than as somewhere between 5-40%. When compensating for the discussed methodological flaws, it must be assumed that currently the true prevalence of contaminated primal cuts and retail-ready pork in butchers' shops is about 25-30%, and that of minced pork and pork sausages about 50-55%. Lastly it is concluded that if carcasses were Salmonella-free, consumers could in principle be provided with virtually Salmonella-free pork. It is therefore recommended that the EU allows for a decontamination step in slaughterhouses with a substance that is generally recognized as safe, provided that the producers strictly adhere to GMP-principles.

Impact on human health of Salmonella spp. on pork in The Netherlands and the anticipated effects of some currently proposed control strategies.

The impact on human health of Salmonella spp. on pork in The Netherlands is described. Subsequently, the effects of some currently proposed control strategies in the Dutch pork production chain are evaluated and quantified with the aid of a simple mathematical model. The estimated average incidence of cases of salmonellosis in the Netherlands is about 450 cases per 100,000 person years at risk (pyar). Some special risk groups for which the risks could be quantified are (1) persons with underlying diseases, such as neoplasms or diabetes mellitus (1200 cases/100,000 pyar); (2) persons with achlorhydria or who excessively use antacids (1100 cases/100,000 pyar); (3) persons who have recently been treated with antibiotics that disturb the normal gut flora (1700 cases/100,000 pyar); (4) nurses (900 cases/100,000 pyar); (5) caterers (900 cases/100,000 pyar); (6) slaughterline personnel (1800 cases/100,000 pyar). Furthermore, it is estimated that 15% (5-25%) of all cases of salmonellosis in The Netherlands are associated with the consumption of pork. Currently, proposed control measures regarding Salmonella in pigs and on pork in The Netherlands are codes of good manufacturing practices (GMP) that, in fact, formalize recommendations that can be found in many handbooks about pig breeding and pig slaughtering. When evaluated by a mathematical model constructed for this purpose, the proposed GMP codes from farm to cutting/retail could, at best, reduce the current levels of Salmonella-positive pigs and pork by 50-60%. If pigs were bred according to the rather costly specific pathogen-free concept (SPF), the prevalence of contaminated carcasses and pork could in total be reduced by 95% or more. However, implementing GMP codes from the transport phase up to the cutting/retail phase coupled with a decontamination step at the end of the slaughterline would be just as effective as GMP in combination with breeding using the SPF-concept. It is therefore concluded that the most efficient and cost-effective way of reducing the 'Salmonella problem' entailed by the consumption of pork would be to decontaminate carcasses under the precondition that the entire production chain strictly adheres to GMP principles. Therefore, the EU should also allow for more possibilities regarding the decontamination of carcasses than is currently the case. It is also concluded that current EU regulations relying on hazard analysis of critical control points (HACCP)-inspired production in cutting plants will not be effective in reducing the prevalence of Salmonella spp. on pork. This is mainly because (1) there is currently an almost steady stream of Salmonella-positive carcasses that enter the cutting process; (2) when contaminated carcasses are being processed, further cross-contamination during working hours is unavoidable; (3) no steps in the cutting process are intentionally designed to effectively reduce the risks or consequences of cross contamination of cuts and retail-ready products.

Salmonella in slaughter pigs: the effect of logistic slaughter procedures of pigs on the prevalence of Salmonella in pork.

A substantial part of the finishing pigs in the Netherlands is infected with Salmonella. Infection of pigs with Salmonella can occur already on the farm. Pigs can also get infected or contaminated during transport, lairage or slaughter. The aim of this study was to evaluate the effect of separating pigs from Salmonella-infected farms from pigs from Salmonella-free farms during transport, lairage and slaughter on the prevalence of Salmonella on pork after slaughter. Two experiments were carried out. In the first experiment, farms were selected to participate, based on serology of the pigs (Dutch Salmonella ELISA). The pigs were slaughtered at the beginning of the day: firstly, sero-negative herds, secondly, sero-positive herds and thirdly, again sero-negative herds. The latter were slaughtered to investigate the effect of a contaminated slaughterline due to a previously slaughtered positive herd. In the second experiment, farms were selected to participate, based on both serology and bacteriology of the pigs on the farm. Two hundred pigs from Salmonella-free farms were slaughtered after 200 pigs from Salmonella-infected farms. Results showed that the prevalence of Salmonella in pork samples of sero-negative herds was lower than in samples of sero-positive herds. Results also showed that Salmonella contamination of carcasses after slaughter was partially caused by Salmonella-infected herds that were slaughtered before, and partially by residential flora of the slaughterhouse. It is concluded that separate slaughter of sero-negative pig herds can be useful to decrease the prevalence of Salmonella-contaminated pork after slaughter. To avoid cross-contamination by residential flora from trucks, lairage and slaughterline, cleaning and disinfection have to be improved.

Salmonella in slaughter pigs: prevalence, serotypes and critical control points during slaughter in two slaughterhouses.

The purpose of this study was to show the distribution of Salmonella in slaughtered pigs and the environment of the slaughterhouse. 1,114 samples of slaughtered pigs (six different samples for Salmonella isolation and one serum sample for ELISA on antibodies per pig) and 477 samples of the slaughterhouse environment were collected in two slaughterhouses on two sampling days per slaughterhouse. Salmonella was isolated from one or more samples of 47% of the pigs. The highest prevalence of Salmonella was observed in rectal content samples (25.6%), whereas the lowest prevalence of Salmonella was observed on the carcasses (1.4%). The prevalence of Salmonella in other samples was: 19.6% in tonsils, 9.3% on livers, 9.3% on tongues, and 9.3% in mesenterial lymphnodes. The prevalence of Salmonella in environmental samples was high in the drain water samples in both slaughterhouses (61%) and on the carcass splitter in one slaughterhouse (33%). Salmonella typhimurium was the most frequently isolated serotype in pig samples and environmental samples in both slaughterhouses: 43% of the Salmonella isolates from pigs and 33% of the Salmonella isolates from the environment was S. typhimurium. The results of this study show that Salmonella prevalences in pigs differ a lot, depending on which part of the pig is sampled. Not all different samples of the pig will become available for human consumption, but collecting more than one sample per pig showed that Salmonella can be found in almost the whole pig. The result of surface samples of carcass and liver gives information about hygiene during the slaughter process; the result of tonsils, lymphnodes and rectal contents, combined with the serological result, gives information about infection of the pig before the slaughter process (on the farm, during transport or in lairage). It can be concluded that results of Salmonella isolation of slaughter pigs should always be carefully interpreted, depending on the type of sample that has been collected.

Identification and quantification of risk factors in animal management and transport regarding Salmonella spp. in pigs.

This article discusses the main elements of a descriptive epidemiological model for Salmonella spp. in the pre-harvest stages of pork production, and the subsequent quantification of risk factors. About two thirds of all Dutch pig farms are more or less permanently infected. At infected finishing farms, the current probability that Salmonella-free pigs will become infected is about 85%. In the case that a certain pen is infected, the current probability that further pen-to-pen transmission will occur is about 90% and that (human) vectors will also spread the infection 60%. Between 5-30% of the animals may still excrete Salmonella spp. at the end of the finishing period, and this percentage can double during transport and lairage. In infected pigs at slaughter weight, the Salmonella spp. are foremost located in the digestive tract, its contents and the closely associated lymph nodes. Under the current circumstances, the lack of farm hygiene (odds ratio (OR) 39.7), (re)contaminated feed (OR 1.6), the use of broad spectrum antibiotics (OR 5.6), a positive Salmonella-status of animals before transport (roughly estimated OR 4.0), the lack of transport hygiene (roughly estimated OR 1.1) and transport stress (OR 1.9) are the most important risk factors regarding infections with Salmonella spp. Currently the role of on-farm contamination cycles with endemic ('house flora') Salmonella spp. is so important, that the role of other factors is difficult to ascertain. It is also concluded that the farm-phase forms the core of all current problems, and that better prevention and control can be achieved by, (i) very strict and consistent farm hygiene in combination with promotion of the colonization resistance of animals kept together with a prudent use of broad spectrum antibiotics; (ii) simultaneous execution of control programmes at breeding farms, multiplying farms and finishing farms; (iii) separate transport, lairage and slaughter of the animals thus produced.

Effect of acidified feed on susceptibility of broiler chickens to intestinal infection by Campylobacter and Salmonella.

Consumption of poultry meat is associated with human Campylobacter and Salmonella infections. One way to control the presence of these bacteria in broiler flocks is to make chickens less susceptible for colonisation. Acidification of feed may be a tool to reduce the Campylobacter and Salmonella carriage in broiler chickens. In the present experiments an acidified feed with high levels of organic acid, 5.7% lactic acid and 0.7% acetic acid, was applied. In an in vitro experiment the reduction or growth of Campylobacter and Salmonella was measured after addition of 10(7)cfu of these bacteria into a conventional broiler feed, acidified feed and fermented feed, whereas the numbers of Salmonella increased in non-acidified feed. The number of Campylobacter decreased 2-3 (10)log cfu. In the acidified and fermented feed a complete reduction of Campylobacter was observed within 20 min, and a total Salmonella reduction started after 1h, and was complete after 2h. Subsequently, an in vivo experiment with 100 individually housed broiler chickens showed that chickens fed acidified feed were less susceptible to an infection with Campylobacter than were chickens fed conventional feed. The size of reduction was however limited. The susceptibility for Salmonella colonisation was not affected by acidified feed. It is concluded that the role for acidified feed in the control of Campylobacter and Salmonella is limited.

Monitoring of transmission of Salmonella enterica serovars in pigs using bacteriological and serological detection methods.

The standard method to detect Salmonella positive pigs is bacteriological examination of the faeces, but in recent years the use of Salmonella-ELISA's have become available to screen pigs for serological evidence of infection. This study was conducted to monitor the transmission of five different Salmonella enterica serovars (S. Typhimurium, S. Brandenburg, S. Panama, S. Livingstone, and S. Goldcoast) in fattening pigs and to test the feasibility of Salmonella-ELISA, using seeder pigs as a mode of transmission. Serovar dependence in transmission was observed. The Salmonella-ELISA proved to be useful to detect S. Typhimurium and S. Brandenburg in herds but was of limited value to demonstrate S. Livingstone, S. Goldcoast, and S. Panama.