A brief overview of food hygiene legislation.

Following several animal disease outbreaks and food contaminant scandals in Europe in recent years, the European Commission adopted the White Paper on Food Safety in 2000. This White Paper contains a number of recommendations aimed to increase food safety, improve the traceability of food products and regain consumer confidence in the food industry. To this effect a package of new European legislation on food and feed has been prepared with the following characteristics: responsibility of food safety lies with the food business operator, while the competent authority of the Member State verifies correct implementation of the new rules. Production should be based on good hygienic practice and HACCP principles and products are subject to microbiological criteria and temperature limits. The legislation deals with all food and covers the entire food chain ("from stable to table"). The general framework of the new food hygiene legislation is explained. The General Food Law (Regulation (EC) No 178/2002) is discussed in more detail as well as the Regulations concerning food hygiene. The characteristics and requirements of each one of the three Hygiene Regulations is presented (Regulation (EC) No 852/2004, Regulation (EC) No 853/2004 and Regulation (EC) No 854/2004) with a particular emphasis on the changes in the new (horizontal) legislation as compared to the old (vertical) Directives. Implementing measures of the Hygiene Regulations have been published in the form of four Commission Regulations in December 2005. The implementing measures deal with technical issues often in great detail and became applicable at the same time as the Hygiene Regulations with effect of 1 January 2006. The major issues as laid down in the four Commission Regulations are presented. Finally, various guidance documents are mentioned. These documents are available on the Internet site (http//ec.europa. eu/food/food/biosafety/hygienelegislation/guide_en.htm) of DG SANCO and explain in plain language some of the topics of the Hygiene Regulations.

Parasite kinetics and cellular responses in goats infected and superinfected with Trypanosoma congolense transmitted by Glossina morsitans centralis.

Trypanosoma congolense infected tsetse were fed on the flanks of goats at sites drained by the prefemoral lymph node. The efferent lymphatic of this lymph node was surgically cannulated and the lymph was collected daily and examined for appearance of parasites, lymph flow and cells. Trypanosomes were detected in the lymph 4 days after infection, which was 2 days prior to the appearance of the local skin reaction or the presence of parasites in the blood. Once the animal became parasitaemic, trypanosomes were found to recirculate in the lymphatic system, appearing in the lymph of the contralateral lymph node 11 days after infection. In goats infected with T. congolense and superinfected 12 or 13 days later with a different tsetse-transmitted T. congolense serodeme, parasites belonging to the second serodeme were apparently delayed in their development in the skin and appeared up to 7 days later in the efferent lymph when compared to control animals. This delay in development might have implications for field situations where superinfections frequently occur; it might result in limiting the number of serodemes of T. congolense an animal can be infected with at any one time.

Dose and stage dependency for the development of local skin reactions caused by Trypanosoma congolense in goats.

Intradermal inoculation of metacyclic forms of Trypanosoma congolense propagated in vitro caused skin reactions in goats similar to the local skin reaction (chancre) induced by the bite of an infected tsetse fly. The onset, size and duration of these local skin reactions were dose-dependent. Whereas one cultured metacyclic T. congolense was sufficient to cause a local skin reaction in a goat, over 10(7) bloodstream forms of T. congolense were necessary to elicit a detectable skin reaction and while T. congolense parasites present in lymph did not cause local skin reactions, trypanosomes collected from oedematous fluid of the chancre did. - Using non-dividing irradiated bloodstream forms it was estimated that 10(8) T. congolense were required to induce a detectable local skin reaction. - Intradermal needle inoculation of procyclic forms (uncoated trypomastigotes) of T. congolense propagated in vitro induced an intense inflammatory response which was similar to that found in the early phases of the reaction elicited by metacyclic trypanosomes. This suggests that the uncoated trypomastigotes which are known to be present in the saliva of infected tsetse may play a role in the initial development of the chancre. - The data obtained for the local skin reaction suggest the presence of an intracutaneous dividing stage of T. congolense which is intermediate between the metacyclic and bloodstream forms.

Trypanosomiasis in cattle in Gambia: incidence, prevalence and tsetse challenge.

The incidence of trypanosome infections, measured by a Berenil Index in experimental herds of 10 Zebu and 10 N'Dama cattle, was compared with tsetse challenge and with the prevalence of parasitaemia in local N'Dama at three villages in Gambia. Tsetse challenge was more strongly correlated with the incidence of parasitaemia in the Zebu than in the N'Dama. There was a strong correlation between prevalence and incidence of infection in the N'Dama. There was no correlation, however, between prevalence of infection in cattle and tsetse challenge unless the data were offset by 3-5 months. The Berenil Index in the Zebu increased at about twice the rate as in the N'Dama under corresponding levels of challenge. It is concluded that whereas incidence of infection in susceptible animals is best measured independently, it can, under stable conditions, be inferred from an assessment of tsetse challenge.

Interaction between physiological status in N'Dama cows and trypanosome infections and its effect on health and productivity of cattle in Gambia.

Data collected for three years on incidence of trypanosome infections, degree of anaemia as assessed by packed red cell volume (PCV) and live weights of four groups of cows of varying physiological status were analysed. The animals were not harbouring trypanosomes during a period of two to three months before exposure to periods of increasing density of tsetse flies (Glossina morsitans submorsitans) while grazing in savannah woodlands. The groups of cows were formed on the following basis: pregnant and lactating (lactating-pregnant) (Group 1, n = 143); pregnant and not lactating (dry-pregnant) (Group 2, n = 69); non-pregnant and lactating (lactating-open) (Group 3, n = 160); non-pregnant and not lactating (dry-open) (Group 4, n = 49). Monthly trypanosome prevalence was highest (17.5%) in the cows with the highest physiological stress (Group 1), followed by Group 3 (11.1%) and Group 2 (10.0%) with the lowest prevalence found in the least stressed cows, Group 4 (1.6%). Average PCV values for dry-pregnant cows (Group 2; 27.0%) and dry-open cows (Group 4; 26.2%), whether infected or not, were higher than those lactating (Group 1; 25.3% and Group 3; 23.6%). A body weight gain of 4.3 kg between the month of October and the following June was recorded for dry-pregnant cows (Group 2) whereas a weight loss of 16 kg occurred in the lactating-pregnant and lactating-open cows (Groups 1 and 3), with more severe losses recorded in infected than uninfected cows. Dry-open cows (Group 4) maintained their weight during the observation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and cultivation in vitro to the infective, metacyclic stage of Trypanosoma (Nannomonas) simiae from Glossina morsitans submorsitans.

Two separate trypanosome isolations were made from a single Nannomonas-infected Glossina morsitans submorsitans from The Gambia. Inoculation of a piglet with the infected hypopharynx produced an infection with Trypanosoma simiae. DNA was isolated from the bloodstream forms to prepare a probe specific for this species. Trypanosomes isolated from the fly midgut were frozen in liquid nitrogen and then cultivated in vitro. Amplification of this population and elimination of a yeast contaminant were achieved by two passages through laboratory G. m. morsitans. Further cultivation in vitro resulted in the production of epimastigotes and, later, metacyclic forms. Two pigs inoculated with cultivated metacyclic forms developed infections with atypical, relapsing parasitaemias and extended survival time. Neither the metacyclic forms, nor bloodstream forms derived from them, infected calves. The identity of various stages of the in vitro cultivated, procyclic-derived stock was confirmed morphologically and with the T. simiae-specific DNA probe.

Influence of fever and flurbiprofen on trypanosome growth.

The administration of flurbiprofen, a potent non-steroidal anti-inflammatory drug (NSAID), to goats infected with trypanosomes resulted in high elevated parasitaemia and suppression of fever. In contrast to goats, rats infected with trypanosomes do not show febrile reactions. Therefore, the role of body temperature was investigated with yeast-induced fever in Trypanosoma evansi and T. brucei infected rats. These investigations did not support the hypothesis that a high body temperature causes a drop in parasitaemia. In goats infected with trypanosomes, it is also unlikely that fever has an inhibitory influence on the parasitaemia. In these animals, rises in parasitaemia could be provoked by doses of flurbiprofen as low as 1/20 of the normal doses and these doses did not or only partly suppressed fever. No effect on parasite growth could be obtained when flurbiprofen was added in concentrations up to 32 micrograms/ml directly to T. brucei cultures. Moreover, no growth promoting factor(s) could be identified in vitro in serum from flurbiprofen-treated goats.

Flurbiprofen and immunosuppression of Trypanosoma brucei infection in the goat.

Goats infected with Trypanosoma brucei and treated with the non-steroidal anti-inflammatory agent flurbiprofen, showed a marked increase in parasitaemia, followed in one of the four goats by death. The in vitro response to mitogens of peripheral blood lymphocytes and separated T- and B-lymphocytes from healthy goats treated with flurbiprofen was normal when compared with non-treated animals. T. brucei-infected goats, not treated with flurbiprofen, showed a marked immunosuppression which was mainly localized in the B-enriched lymphocyte fraction. A combination of T. brucei infection and treatment with flurbiprofen led to even more suppression, because the T-lymphocyte function was also suppressed. It is concluded that flurbiprofen first causes a rise in the parasitaemia and that this high parasitaemia is responsible for the observed immunosuppression.

Effects of trypanosome and helminth infections on health and production parameters of village N'Dama cattle in The Gambia.

The effects of trypanosome and helminth infections on health and production parameters in 2000 village N'Dama cattle were assessed periodically. Blood examination showed Trypanosoma congolense and Trypanosoma vivax to be prevalent, while strongylid-type eggs were those most frequently encountered in faecal samples. A distinct seasonal fluctuation was detected for both blood levels of trypanosomes and helminth egg output. Strongylid burden and trypanosome infection had significant negative effects on packed red cell volume levels and body weights mainly in animals of 2-3 years old. Clear indications of an increased susceptibility to trypanosomosis were found in animals affected by helminths. Similarly, animals infected with trypanosomes were more frequently infested with strongyles and egg counts were higher than in cattle in which no trypanosomes were detected.

The interaction of Trypanosoma congolense and Haemonchus contortus infections in trypanotolerant N'Dama cattle.

The interactions between Trypanosoma congolense and Haemonchus contortus infections were studied in N'Dama calves. A total of 38 N'Dama bulls was divided into four groups and each group infected either with H. contortus 1 week after infection with T. congolense or with T. congolense 4 weeks after infection with H. contortus, or with either infection singly. Parasitological (faecal egg counts, parasitaemia), haematological (packed cell volume, white blood cell counts, albumin) and clinical parameters (body weight change, mortality rate) were compared among the various groups. The results showed a reduced prepatent period and a markedly increased pathogenicity of H. contortus infections in animals with a concurrent T. congolense infection. The most harmful combination was a H. contortus infection 1 week after the T. congolense infection which resulted in a progressive and severe anaemia, accompanied by hypoalbuminaemia, increased weight loss and high mortality. The anaemia induced by dual infections showed a low responsiveness to chemotherapy and in several cases supportive treatment did not help recovery. The results also showed that animals with a concurrent T. congolense and H. contortus infection ran a higher risk of succumbing during the infection, and also during 10 weeks following treatment. Although infections with T. congolense alone produced no clinical signs, they were found to significantly reduce the ability of infected animals to mount a normal response to a subsequent H. contortus infection. It was concluded that the increased H. contortus egg excretion observed in animals infected with both parasites might significantly increase the risk of nematode infections and that the reduced prepatent period might necessitate more frequent anthelmintic treatments. These interactions should, therefore, be considered wherever attempts are made to control these two diseases.

Study of the effect of gamma-irradiation on bovine serum samples on the ability of monoclonal antibodies to detect invariant antigens of Trypanosoma congolense, T. vivax and T. brucei in enzyme-linked immunosorbent assays.

Samples of bovine serum from uninfected and African trypanosomes-infected animals were tested before and after gamma-irradiation, using three sandwich enzyme-linked immunosorbent assays (ELISA). Each test system utilized a different monoclonal antibody, reputedly allowing the specific detection of conserved-invariant cytoplasmic antigens of trypanonosomes, T. congolense, T. vivax, and T. brucei, respectively. Results have identified two groups of samples. The first contained samples where there were unequivocal ELISA results indicating positivity and negativity, for non-irradiated samples. In this group, irradiation had no effect on the diagnostic sensitivity of the assays. All samples shown to be positive before irradiation remained positive and those shown to be negative, remained negative. There was, however, a statistically significant reduction in signal in each of the ELISAs following irradiation. The second group contained samples identified before irradiation as flanking the diagnostic negative/positive threshold of OD > or =0.05. These showed a negative bias after irradiation of the order of OD -0.01, which was shown to be statistically significant by paired t-statistics. Without correction of the given diagnostic negative/positive threshold, bovine sera with OD values around the threshold were expected to deliver more false negative test results upon irradiation. This was confirmed when serological data were compared with parasitological findings; where three times more false negative test results were found from irradiated serum samples. Consequently, for this group of irradiated bovine samples tested by ELISA, the re-adjustment of the diagnostic negative/positive threshold of the ELISAs using defined irradiated serum samples is recommended; otherwise, the frequency of false negative results might be increased.

Susceptibility of African buffalo and Boran cattle to Trypanosoma congolense transmitted by Glossina morsitans centralis.

Four African buffalo (Syncerus caffer) and four Boran cattle (Bos indicus) were each exposed to the bites of 10 tsetse flies infected with Trypanosoma congolense. Although both groups of animals became infected, the buffalo showed no clinical signs of trypanosomiasis while the cattle suffered from the disease characterized by pronounced skin reactions, high parasitaemia and severe anaemia. The prepatent periods in the buffalo varied from 18 to 27 days in comparison with 11 to 14 days in the cattle. In the buffalo, skin reactions were only detectable by histological examination of skin biopsies, the peak of parasitaemia was at least a hundredfold below that in cattle and after 54 days parasites were no longer detected. In contrast, the cattle had a continuous high parasitaemia until they were treated with a trypanocidal drug 60 days after infection. Neutralizing antibody to metacyclic trypanosomes appeared in the buffalo during the prepatent period, 15-20 days after infection, whereas in cattle neutralizing antibody was not detected until 10 days after the first peak of the parasitaemia, 25-30 days after infection.

Interference in the establishment of tsetse-transmitted Trypanosoma congolense, T. brucei or T. vivax superinfections in goats already infected with T. congolense or T. vivax.

An interference phenomenon that delays superinfection with a trypanosome species different from that used for the initial infection has been found to occur in goats. Following tsetse transmission of Trypanosoma brucei to goats already infected with T. congolense, there was a delay in chancre development, as well as in the appearance of T. brucei and anti-T. brucei antibodies in the blood when compared to previously uninfected goats. However, there was no delay in the establishment of a tsetse-transmitted superinfection with T. vivax in goats already infected with either T. congolense or in animals already infected with a different serodeme of T. vivax.

Effect of gamma-irradiation on serum samples on the diagnostic performance of ELISA methods for the detection of trypanosomal antibodies.

The study investigated the effect of gamma-irradiation on bovine serum samples on the ability of enzyme-linked immunosorbent assay (ELISA) methods to detect trypanosomal antibodies. The serum samples were analysed using two standardised indirect ELISA systems. Higher measurement values were observed for most gamma-irradiated antibody positive and negative test samples. Using cut-off points, determined from the analysis of a non-irradiated trypanosomal antibody-negative population, the gamma-irradiated sera data showed that there was an increased risk of misclassifying samples as false positive or cross-reactive due to increased analytical sensitivity and decreased analytical specificity. The intraplate precision and agreement between tested and expected values of measurements were not altered throughout. The impact on the assays' diagnostic performance was estimated by analysing diagnostic sensitivity, diagnostic specificity and related parameters. The data demonstrated that although there was a bias of higher measurement values after gamma-irradiation, this could be compensated after readjustment of the cut-off points to obtain best separation of antibody-positive and -negative samples. Thus, for each assay, no significant difference of the diagnostic proficiency was found before and after gamma-irradiation. The practical implications are discussed of a serum sterilisation procedure using (60)Co gamma-rays for routine sample testing, assay validation and trypanosomosis monitoring and tsetse-fly control and eradication programmes.

Charting methods to monitor the operational performance of ELISA method for the detection of antibodies against trypanosomes.

Four indirect enzyme-linked immunosorbent assays (ELISAs) for the detection of antibody against trypanosomes using antigen-precoated plates (Trypanosoma congolense and T. vivax) were used in 15 veterinary diagnostic laboratories in Africa and Europe. The study provided data allowing an evaluation of charting methods with respect to the operational performance of each ELISA. Data from standardised internal quality control (IQC) samples were plotted on charts and used as the assay performance indicators with reference to expected upper and lower control limits. Based on unprocessed (optical density) and normalised absorbance values (calculated as a percentage positivity of a control), dispersion of values from the expected data range was estimated plotting the location and deviation of the values. In addition, assay precision was estimated plotting the distribution of coefficients of variation<10% of the IQCs. Binding ratios of controls were calculated to estimate the assay proficiency with respect to the accuracy of assessing that the IQC samples tested positive or negative in the test proper. The graphical analysis of dispersion of absorbance values in combination with assay precision and proficiency criteria was considered fully satisfactory to evaluate the operational performance of the ELISAs and provided useful decision criteria for plate acceptance and rejection. The establishment of standardised and transparent IQC data charting methods for the indirect ELISAs provided an increased measure of confidence to national laboratories with respect to their reports on disease occurrence. Moreover, the relative assay performances between all laboratories were examined using summary data charts with reference to the performance criteria described. The IQC data were also examined using modified Youden plot analysis demonstrating that indirect ELISA methods can be successfully applied at diagnostic laboratories in the tropics for monitoring trypanosomosis control programmes.

Evaluation of antigen-coating procedures of enzyme-linked immunosorbent assay method for detection of trypanosomal antibodies.

Research was undertaken to improve the antigen-coating step of indirect enzyme-linked immunosorbent assay (ELISA) method through the use of polystyrene 96-well plates precoated with antigenically stabile crude trypanosomal antigens. The plates were precoated with antigens, air dried and sealed before being packed in plastic bags with silica gel desiccant packets. Such plates stored at +4 and +37 degrees C provided an assay performance, which was superior to that of plates freshly coated with antigens from a frozen stock. Antigen-precoated plates consistently proved stable after storage up to +50 degrees C for at least 1 year. The accuracy of the assay was not affected, i.e. trypanosomal antibody-positive sera were clearly discriminated from trypanosomal antibody-negative negative sera. In contrast, lyophilized trypanosomal antigens lacked stability on storage at +37 degrees C for longer than 1 month. It was concluded that the routine use of antigen precoated polystyrene plates for the enzyme immunoassay technique will contribute to improved assay robustness at an acceptable diagnostic proficiency. The modified coating procedure will also provide an improved quality assurance and standardization procedure for the assay, which is required to allow the reliable detection of trypanosomal antibodies and comparison of data from different laboratories.

Detection of Trypanosoma congolense antibodies with indirect ELISAs using antigen-precoated microtitre plates.

The study reports the performance of four indirect enzyme-linked immunosorbent assays (ELISAs) for antibody (AB) detection using microtitre plates which were precoated with native or heat/detergent denatured antigens (AGs) from Trypanosoma congolense (T.c.) and T. vivax (T.v.), and stored for between 1 to 206 days at +37 degrees C. Bovine serum samples were obtained by sequential bleeding of 3-months old T.c.-infected bulls and their uninfected cohorts, as well as by a single bleeding of uninfected adult cattle. The first day of AB detection, and observations on samples after this (defined as estimated ELISA sensitivity), depended on the cut-off value in the specific ELISAs. Cut-off values from pre- and early post-infection samples of individual animals demonstrated a seroconversion in all ELISAs on average after 10-15 days post-infection (dpi). The AB detection was delayed in the T.c. native and denatured AG-based ELISAs using cut-off points from uninfected cohort cattle (16.5 dpi, 19.3 dpi) and the adult cattle population (22.1 dpi, 25.0 dpi). The T.v. AG-based ELISAs however lacked crossreactiviy to T.c. ABs. The estimated sensitivity of each T.c. AG-based ELISA was above 96% throughout, but significantly lower for the T.c. native AG-based ELISA (91.1%) when the adult cattle derived cut-off point was used (p<0.01). The sensitivity of the phase contrast buffy coat technique was similar to the T.c. AG-based ELISAs, but significantly lower when the T.c. denatured AG-based ELISA was used at the adult cattle derived cut-off point (p<0.05). The implications of the results and future research aspects on ELISAs to detect trypanosomal ABs and AGs are discussed.

Infectious agents associated with diarrhoea of calves in the canton of Tilarán, Costa Rica.

A case-control study of calves under 3 months of age was carried out by weekly visits to 15 farms in the canton of Tilarán, Costa Rica. Most farms were dedicated to beef or dual-purpose (DP) production. Faecal samples were collected over a 6-month period from a total of 194 calves with clinical signs and from 186 animals without clinical signs of diarrhoea as assessed by a scoring system. The samples were investigated for the presence of viruses, bacteria and parasites. Torovirus was detected for the first time in Costa Rica and was present in 14% of calves with diarrhoea and in 6% of the controls. Coronavirus and Rotavirus were less frequently encountered in either one of the groups (in 9 and 7% of scouring calves and in 1 and 2% of controls, respectively). Escherichia coli was detected in 94% of all the faecal samples, but isolates from only three samples from calves with diarrhoea contained the K99 antigen. Similarly, Salmonella was found only in scouring calves. Cryptosporidium oocysts were detected in animals with signs of diarrhoea, while other coccidia oocysts, Strongylida and Strongyloides eggs were frequently found in animals both with and without diarrhoea. A conditional logistic regression (CLR) analysis to compare healthy and scouring calves showed a significant difference with regard to the presence of Torovirus, Rotavirus and Coronavirus.

Development of Trypanosoma congolense, T vivax and T brucei in the skin reaction induced in goats by infected Glossina morsitans centralis: a light and electron microscopical study.

The development and distribution of Trypanosoma congolense, T vivax and T brucei in the skin of goats was examined after the animals were bitten by infected Glossina morsitans centralis. Following the tsetse bite, the trypanosomes in the skin multiplied, reaching maximum numbers when the skin reaction (chancre) of the host attained its maximum size. In goats infected with T vivax and T brucei, trypanosomes were observed circulating in the blood before the peak of the chancre, while in T congolense-infected goats microscopically detectable parasites were found in blood only during the decline of the chancre. In contrast to T vivax, large numbers of T congolense and T brucei parasites were found in the skin following tsetse-transmitted infection. Ultrastructural differences were observed in T congolense and T brucei indicating an intracutaneous transformation from metacyclic to blood stream forms. T congolense forms in the skin reactions had a well developed secretory reticulum, small mitochondria and lacked large lipid inclusions compared to metacyclic and blood stream forms. The intracutaneous forms of T brucei had smaller mitochondria, the glycosomes were of more uniform size and the rough endoplasmic reticulum was less developed than in metacyclic or blood stream forms.