The wild boar (
The magnetic stirrer digestion is the gold standard method for the direct detection of
Trichinelloscopy and artificial digestion were previously used for the identification of
Romania is located in Eastern Europe, in the north of the Balkan Peninsula. The country has a temperate-continental climate of transitional type, with four clearly defined seasons (Trusca & Alecu, 2005). The animals originated from Bihor County (Fig. 1), a high-altitude area covered by forest. During the hunting season, in 2018, 84 plasma and diaphragm samples were collected from wild boars for research purposes. The blood samples, from each animal, were collected by jugural venipuncture into EDTA tubes. Plasma was obtained after the centrifugation of blood samples at 2500 RPM for 15 min and frozen at -15 °C until further use.
For artificial digestion, 5 g of diaphragm tissues were used from each animal. Four digestion pools of 100 g and one digestion pool of 20 g were made. For each pool of 100g, 2 l of heated water, 10 g of pepsin powder (1: 10,000 NF) and 16 ml of hydrochloric acid 25 % were added. The artificial digestion method was performed on all the samples according to Gamble and others (2000), in order to detect and collect the larvae for species identification.
The artificial digestion method was carried out as stated in the European community regulation EU 2015/1375.
Eighty four samples of plasma were tested for the presence of anti-
Samples with a cut off value (E/P- positivity index calculation formula) equal or lower than 30 % were considered negative, while values that ranged from 30 % to 40 % were doubtful. Moreover, positive samples were associated with E/P values that were equal or higher than 40 % (IDEXX ELISA technical guide, 2007).
Twenty six plasma samples were tested with the Western blot technique. The samples were chosen randomly from the total of 84 samples previously tested in ELISA. Antigens were separated on a 10 % SDS-PAGE and transferred onto a nitrocellulose membrane. Plasma, diluted 1/100 in TBS, with an addition of 5 % milk was incubated for 1 hour at room temperature under gentle shaking. After 3 washes in TBS-tween, secondary antibodies diluted 1/15 000 in TBS, with 5 % milk were incubated in the same conditions, followed by 3 repetitions of the washing step. The obtained bands were identified by chemiluminescence with GeldocTM reader (Bio-Rad, Bulletin 6376).
The prevalence of
All applicable national and institutional guidelines for the care and use of animals were followed.
Among the 84 animals included in the study, 45 (53.6 %) were females (38 adults, 7 juveniles) and 39 (46.4 %) were males (30 adults, 9 juveniles). All the samples were negative when artificial digestion was conducted. In ELISA 54 samples (64.2 %; 95 % CI: 53.7 – 74.2) were positive and 6 samples (7.1 %; 95 % CI: 1.4 – 12.5) were doubtful. Western blot was performed on 26 random samples, from which only 6 samples (23.0 %; 95 % CI: 5.6 – 40.3) gave a positive result. All the positive samples had three bands which were situated in the margin of 46 – 56.8.kD. Furthermore, ELISA and Western blot showed different results in the 26 samples tested. Thus, from the six samples that were positive in Western blot, only five were positive in ELISA. In addition, 11 (42.3 %; 95 % CI: 31.1 – 78.8) of the samples that were negative in Western blot, were positive in ELISA, as shown in Table 1. A Kappa value of 0.18 was found between ELISA and Western blot results, which demonstrated a slight agreement between the two methods used. Also, a Kappa value of 0.0 was observed in case of Western blot and artificial digestion, but also in case of ELISA and artificial digestion, respectively showing no inter-rated agreement between the methods chosen for diagnosis. Moreover, the seroprevalence of
The comparative results for artificial digestion, ELISA, and Western blot.
Nr. Ctr. | Gender | Artificial digestion | ELISA (E/P) | Western blot (bands) |
---|---|---|---|---|
1 | Male | N | D (34.55%) | N |
3 | Male | N | D (38.66%) | N |
4 | Male | N | P (66.42%) | N |
6 | Female | N | P (41.85%) | N |
8 | Female | N | D (32.18%) | N |
11 | Female | N | N (29.51%) | N |
15 | Male | N | P (47.33%) | N |
18 | Female | N | P (52.52%) | N |
25 | Female | N | P (40.79%) | N |
30 | Female | N | D (30.58%) | P (47.4; 51.8; 55.6 kD) |
32 | Male | N | P (157.72%) | P (46.4; 50.7; 58.5 kD) |
39 | Female | N | P (62.04%) | N |
68 | Male | N | P (53.37%) | N |
79 | Male | N | N (27.20%) | N |
81 | Male | N | N (26.78%) | N |
83 | Male | N | P (171.46%) | P (48.7; 52.3; 57.4 kD) |
92 | Female | N | P (63.20%) | P (47.4; 51.8; 55.6 kD) |
104 | Male | N | P (57.03%) | P (47.0; 51.8; 56.7 kD) |
115 | Female | N | D (39.11%) | N |
154 | Female | N | N (25.70%) | N |
156 | Male | N | P (65.00%) | N |
157 | Male | N | P (49.52%) | P (46.0; 50.2; 55.7 kD) |
158 | Female | N | P (42.48%) | N |
166 | Female | N | D (37.12%) | N |
169 | Female | N | P (56.14%) | N |
176 | Female | N | P (50.10%) | N |
In case of artificial digestion, the lack of other regional muscles samples that are recommended in EU regulation can be responsible for the negative results. Kapel (2001) suggested that diaphragm and tongue muscles were the predilection sites in wild boars, independent of
Although artificial digestion was negative, ELISA and Western blot showed high rates of positivity. Indirect ELISA is commonly used for the surveillance of
Taken together, these results indicate the circulation of
In Romania, the human population can acquire trichinellosis mainly by consuming raw pork and wild boar meat. The meat from these animals are usually prepared accordingly to local habits and traditions. Commonly, the wild boar meat products (dried raw salami, raw dried sausage, smoked pastrami e.g.) are not subjected to thermal processing, exposing the population to parasitic infestations. In 1993, controlled pig meat was mixed with infested wild boar meat, causing an extensive outbreak in Romania (Cironeanu & Ispas, 2002). Moreover, in January 2007, in the city of Timisoara, 5 humans acquired trichinellosis after consuming sausages with infected wild boar meat mixed with pork (Neghina, unpublished data).
Epidemiological actions that limit the spread of the infestation to humans and animals should be widely available and correctly applied by pig breeders, hunters, and consumers to prevent future outbreaks. These measures should include hygiene conditions in backyard pig breeding and raising, testing of wild boar meat from hunters and the consumption of only controlled wild boar meat by the local population (Neghina, 2010). However, in countries with high percentages of
The EU Regulation 2015/1375 highlights the importance, in case of wild boar meat, of using a sufficient amount of samples from the predilection sites, meaning foreleg, tongue or diaphragm muscles. In this regard and giving the recommendation of using at least 10 g of muscle tissue from each animal, is essential to ensure a correct training for the personnel that is responsible for the sample collection and inspection.
Serological evidences confirmed the exposure of wild boars from Bihor County, to