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Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia
Accepted Manuscript Title: Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia Author: Muza Kirjuˇsina Eduards Bakasejevs Patrizio Pezzotti Edoardo Pozio PII: DOI: Reference:
S0304-4017(16)30162-5 http://dx.doi.org/doi:10.1016/j.vetpar.2016.05.008 VETPAR 8008
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
23-12-2015 2-5-2016 7-5-2016
Please cite this article as: Kirjuˇsina, Muza, Bakasejevs, Eduards, Pezzotti, Patrizio, Pozio, Edoardo, Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia
Muza Kirjušina1, Eduards Bakasejevs1, Patrizio Pezzotti2, Edoardo Pozio2* 1
Institute of Live Science and Technology, Daugavpils University, Daugavpils, Latvia;
2
Istituto Superiore di Sanità, Rome, Italy
*Corresponding author. Tel.: +39 06 4990 2304; Fax +39 06 4990 3561. Email address: [email protected] (E. Pozio)
Highlights Estimation of Trichinella larva biomass in a naturally infected host population The number of larvae per gram of a muscle is representative of the total biomass Total larval burden and larvae/g coefficients were established for the pine marten
Abstract Parasites of the genus Trichinella are cosmopolitan nematodes infecting primarily wild animals, which represent the main reservoirs of these zoonotic pathogens. To investigate the transmission patterns of Trichinella spp. from wild to domestic animals and to humans and for the risk assessment of these parasites in a geographical area, it is important to know the number of possible transmission events deriving from carcasses of infected hosts. For this purpose, the evaluation of the larval biomass in reservoir hosts is needed. No data is available on how to estimate the biomass of Trichinella spp. larvae in muscles of naturally infected animals. The aim of this study was to evaluate the larval biomass in naturally infected pine martens (Martes martes) of Latvia, in which the prevalence of Trichinella britovi infection was over 50%. Single muscles or group of muscles (abdomen, back, diaphragm, intercostal muscles, muscles from the head, left and 1
right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail, and base and tip of the tongue) were collected from five skinned and eviscerated carcasses of T. britovi infected pine martens. Muscles were entirely removed from the bones and weighted. Each muscle or group of muscles was separately digested to detect the larvae per gram (LPG). Using linear regression, the larval burden in each muscle or group of muscles was evaluated to measure the possible prediction of the total animal larval burden (both as total number of larvae and as average LPG). All muscles were significantly predictive of the total burden with high “goodness of fit” (all adjusted R2 > 0.80; P ≤ 0.01), and the left shoulder provided the highest adjusted R2 (0.999). Then, to estimate the Trichinella britovi biomass in the pine marten population of Latvia, recent literature data on prevalence (56.2%, 95% CI: 47.8-64.3) and geometric mean LPG (1.26, 95% CI: 0.89-1.79) in the limb muscles of a sample representative of the whole Latvian pine marten population, were used. Using the predictive estimated relationship between LPG in the limb and that in the entire animals and by the estimated animal population and their mean total muscle weight, the Trichinella britovi biomass in the pine marten population of Latvia was estimated to be of 6,647,092 (95% CI: 3,840,030-11,100,000) larvae. The assessment of the biomass in nature can help to understand the epidemiological pattern of these pathogens, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology of this group of zoonotic nematodes.
Keywords: pine marten, Martes martes, Trichinella britovi, biomass, Latvia, epidemiology, sylvatic cycle
1.
Introduction Parasites of the genus Trichinella are cosmopolitan nematodes infecting mainly wild animals,
which represent the main reservoir hosts of these zoonotic pathogens (Pozio and Zarlenga, 2013). 2
Out of the 12 identified taxa in this genus, Trichinella britovi is the most prevalent species among wild carnivore mammals of Europe, western Asia, and North and Western Africa, but it infects also domestic and wild swine, which can be the source of infection for humans (Gottstein et al., 2009). The ecological niche of the infectious stage of these parasites is the striated muscle cell of their hosts, where the larva can survive from few weeks up to years waiting to be ingested from a new host (Gottstein et al., 2009). Studies on these zoonotic nematodes have estimated the larval burden, i.e. the number of larvae per g of muscle (LPG) in preferential muscles of important domestic and wild host species such as the domestic pig (Zimmermann and Schwarte, 1961; Olsen et al., 1964; Kapel and Gamble, 2000; Nöckler et al., 2005), horse (Pozio et al., 1999), wild boar (Kapel, 2001), and fox (Kapel et al., 2005), in order to establish the most suitable muscle/s for the detection of Trichinella spp. larvae to identify infected animals at the slaughterhouse, in the game abattoir, or for epidemiological surveys. The knowledge of the parasite biomass present in their hosts is useful to implement epidemiological studies on the transmission patterns of helminths in nature, and for monitoring and control programs. For gastrointestinal helminths, the biomass in a host can be approximately determined by the egg count in stool (Hollingsworth et al., 2013). No information is available on the biomass of Trichinella spp. larvae in striated muscles of naturally or experimentally infected animals. In the case of Trichinella, a transmission event from one to another host can occur, if the new host ingests at least one male larva and one female larva. On the basis of the number of larvae in a carcass, i.e. the biomass, the number of transmission events deriving from that carcass can be predicted. The integration of these data with epidemiological, biological and environmental data, will allow to assess the risk of exposure for domestic animals and for humans. The European pine marten (Martes martes) is a mustelid living from eastern to western and from northern to southern Europe (Wilson and Mittermeier, 2009). The aims of the present study were to count the total number of Trichinella spp. larvae in pine marten muscles, to establish if the
3
number of larvae in a selected muscle/s was significantly predictive of the total larval burden in the animal, and to evaluate the Trichinella spp. larval biomass in the pine marten population of Latvia. 2.
Materials and Methods
2.1
Animal and muscle collection According to the study design, the Trichinella larval biomass had to be investigated in five
carcasses of pine martens infected with different larval burdens of the same Trichinella species to obtain statistically significant data. These were selected among 13 skinned carcasses of pine martens killed by hunters in the Latgale region of Latvia, and forwarded to the Institute of Live Science and Technology, Daugavpils University, Daugavpils from July 2010 to June 2012 (Bakasejevs et al., 2012). Carcasses were eviscerated and a muscle sample of 25 g from the forelimb was tested to detect Trichinella spp. larvae by the magnetic stirrer digestion method according to the Commission Regulation 2075/2005 (European Commission, 2005). Out of 13 tested pine martens, 6 tested positive for Trichinella spp. larvae (Bakasejevs et al., 2012). Carcasses of three males and two females with 22.1, 69.8, 10.5, 37.4, and 2.9 (LPG in the forelimb, were selected for this study (Table 1). The five skinned and eviscerated carcasses were weighted; the head, tongue, diaphragm and rump with tail, were removed, and the carcass was cut in two parts along the median line. Then, single muscles (diaphragm, base and tip of the tongue) or group of muscles (abdomen, back, intercostal muscles, muscles from the head, left and right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail), were carefully removed, taking care not to leave striated muscles on the bones (Table 2). Each muscle or group of muscles were separately weighted by a precision scale (up to 10 mg). 2.2
Muscle digestion, larva collection and identification Each muscle or group of muscles were digested separately according to a published protocol
(European Commission, 2005). Trichinella spp. larvae were collected, washed, and counted in triplicate. When a high number of larvae was present in the sediment, larvae were diluted in tap 4
water and counted in triplicate. The number of larvae was expressed as total number of larvae and as LPG in the whole muscle or group of muscles. Twenty larvae per animal were fixed in 95% ethyl alcohol and then each single larva was identified at the species level by multiplex PCR according to a published protocol (Pozio and La Rosa, 2010). 2.3
Statistical analysis Several linear regression models for each single muscle (or group of muscles) were
performed, where the total larval burden (both as total number of larvae and as average LPG) was the dependent variable, while the larval burden in each muscle (or group of muscles) was the independent variable. To establish the ability of the larval burden in each muscle on predicting the total larval burden, we considered the estimated adjusted R2, which varies between 0 and 1, where 1 indicates a perfect fit. To predict the total larval burden, the estimated coefficient expresses the change in the total larval burden, per unit increase in the specific muscle considered. 2.4
Trichinella larva biomass calculation The total biomass of Trichinella spp. larvae in the pine marten population of Latvia was
estimated on the basis of estimates obtained in this study and external information as indicated in parentheses: 1. the estimated number of heads, i.e. 20,000-30,000, in the population (Ministry of Agriculture of Latvia) in the period 2010-2014; 2. the estimated prevalence and 95% confidence intervals (CI) of Trichinella britovi infection in this host species in the whole Latvian territory (see Deksne et al., 2016); 3. the estimated mean and 95% CI of LPG in the limb of each animal (see Deksne et al., 2016); 4. the estimated coefficient and 95% CI obtained by the previously described linear regression between the LPG in the limb and the LPG in the entire striated muscles of the animal; and 5. the estimated mean and 95% CI weight of striated muscles (Table 1). The expected total larval biomass and the corresponding 95% CI were estimated as the median value, and the 2.5th and the 97.5th percentiles of the distribution obtained by performing 100,000 simulations, where at each simulation the previous described parameters were randomly extracted on the basis of the reported variability. 5
3.
Results The weight of the five skinned and eviscerated carcasses ranged from 566 g to 1,249 g
(average 892.8 g), the mean weight of striated muscles was 488.7 g (95% CI: 339.4-636.3), the total number of larvae in the whole striated muscles per animal was 10,529 (range 640-32,294), and the average LPG ranged from 1.92 to 50.3 (Table 1). Larvae were identified as T. britovi (data not shown). No statistically significant difference was detected between the left and the right muscles (data not shown). The LPG ranking shows that the diaphragm of the five animals had the highest LPG (average 57; range 5.07-117.31), followed by the lower part of forelimb (average 38.26; range 3.89-86.25); whereas, the masseters and the other muscles of the head ranked at the last two positions for LPG. The group of muscles with the highest number of larvae was the neck (average 1,898 larvae, range 109-5,490 larvae) (Table 2; Supplementary Table 1). As shown in Fig. 1, all single muscles or group of muscles were significantly predictive of the total number of larvae (all adjusted R2 > 0.80, p<0.01) present in the whole striated muscles. Similar results were obtained using the LPG (Fig. 2), and the left shoulder LPG provided the highest adjusted R2 (0.999). In particular, it has been estimated that the average LPG in an animal was 0.80 (95% CI: 0.74-0.82, adjusted R2=0.997) times the LPG found in the limb (data not shown). Just by the digestion of a few grams from representative muscles or groups of muscles, it is possible to know the total number of larvae in a pine marten according to the following procedure: number of larvae/g x coefficient of the respective muscle or group of muscles (Table 2) x whole striated muscle weight (Table 1). An example on how the estimated total number of larvae and average LPG are derived from data of the pine marten N. 4, is shown in the supplementary Table 2 and supplementary Table 3, respectively. Finally, Table 3 shows the estimated T. britovi biomass in the pine marten population of Latvia. 6
4.
Discussion In this study on five infected carcasses of pine martens, we firstly investigated the relationship
between the number of T. britovi larvae found in each muscle, or group of them, and the total larval burden in the animal. The pine marten was selected for this study among other host species for its high prevalence of Trichinella spp. infection (Deksne et al., 2016) and for its low size in comparison to that of other hosts (e.g., foxes, wolves), which makes the muscle digestion easier and cheaper. Larvae found in each muscle had an elevated ability to predict the total larval burden in the animal (all adjusted R2 >0.80, p<0.05) both as the total number and the average LPG. Secondly, we provided an estimate of the biomass of T. britovi, which is the most widespread species among those of this genus circulating in Europe (Pozio et al., 2009), combining this data with those from Deksne et al. (2016). The pine marten was selected for its wildness, which makes this animal a good representative of the sylvatic cycle. The European pine marten has an opportunistic generalist diet feeding on carrions mainly during the winter time and on small rodents and insects during the spring and the summer (Pullianen and Ollinmaki, 1996; Brainerd and Rolstad, 2002; Posluszny et al., 2007). The variation of the estimated biomass was quite large, i.e. from 3,840,030 to 11,100,000, due to the uncertainty of the parameters used to calculate the biomass. There are some limitations and potential biases in this study, which should be underlined: 1. the whole striated muscles of only five infected pine martens were tested and they were selected among those with a high larval burden to better evaluate the larval burden differences among muscles; our method assumes that the relationship, we estimated between the larval burden (or the average LPG) with that in each specific muscle, also relies in animals with a lower larval burden; 2. we do not know if, over time, the decreasing larval burden in the muscles may or may not be uniform; and 3. the number of muscle larvae developing in a new host can vary and be independent from the number of ingested larvae due to the individual immune response.
7
The assessment of the Trichinella spp. biomass in nature can help to understand the transmission mechanisms of these pathogens in nature, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology and epidemiology of these group of zoonotic nematodes. This investigation was made easier by the detection of only one Trichinella species, T. britovi, in all the investigated pine martens of Latvia (Deksne et al., 2016). This is the first attempt to investigate the biomass of Trichinella parasites, and of T. britovi in particular. Further studies on other important reservoir hosts such as the red fox (Vulpes vulpes), the raccoon dog (Nyctereutes procyonoides), the wolf (Canis lupus), and the lynx (Lynx lynx), are needed.
Conflict of interest statement No financial or personal relationships are maintained with other people or organizations that could inappropriately influence or bias this paper.
Acknowledgements This study has been in part founded by the DG SANTE of the European Commission in the years 2012-2015.
References Bakasejevs, E., Daukšte, A., Zolovs, M., Zdanovska, A., 2012. Investigation of Trichinella in wildlife in Latgale region (Latvia). Acta Biol. Univ. Daugavp. 12, 1-5. Brainerd, S.M., Rolstad, J., 2002. Habitat selection by Eurasian pine martens Martes martes in managed forests of southern boreal Scandinavia. Wild. Biol. 8, 289-297. Deksne, G., Segliņa, Z., Jahundoviča, I., Esītea, Z., Bakasjevs, E., Bagrade, G., Keidāne, D., Interisano, M., Marucci, G., Tonanzi, D., Pozio, E., Kirjušina, M., 2016. 8
High prevalence of Trichinella spp. in sylvatic carnivore mammals of Latvia. Vet. Parasitol. (in press). DOI 10.1016/j.vetpar.2016.04.012. European Commission, 2005. Regulation (EC) No 2075/2005 of the European Parliament and of the Council of 5 December 2005 laying down specific rules on official controls for Trichinella in meat. Off. J. EC L 338, 60–82. Gottstein, B., Pozio, E., Nöckler, K., 2009. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin. Microbiol. Rev. 22, 127-145. Hollingsworth, T.D., Truscott, J.E., Anderson, R.M., 2013. Transmission dynamics of Ascaris lumbricoides – Theory and observation. In: Holland, C. (Ed.), Ascaris the neglected parasite, Academic press, London, pp. 231-262. Kapel, C.M., Gamble, H.R., 2000. Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs. Int. J. Parasitol. 30:215-221. Kapel, C.M., 2001. Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response. J. Parasitol. 87, 309-314. Kapel, C.M., Webster, P., Gamble, H.R., 2005. Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife. Vet. Parasitol. 132, 101-105. Ministry of Agriculture of Latvia. www.vmd.gov.lv/valsts-meza-dienests/statiskaslapas/medibas/valsts-meza-dienests/statiskas-lapas/skaitli-un-fakti?id=766#jump. Accessed 7 August, 2015. Nöckler, K., Serrano, F.J., Boireau, P., Kapel, C.M., Pozio, E., 2005. Experimental studies in pigs on Trichinella detection in different diagnostic matrices. Vet. Parasitol. 132, 85-90. Olsen, B.S., Villella, J.B., Gould, S.E., 1964. Distribution of Trichinella spiralis in muscles of experimentally infected swine. J. Parasitol. 50, 489-495. Pozio, E., Paterlini, F., Pedarra, C., Sacchi, L., Bugarini, R., Goffredo, E., Boni, P., 1999. Predilection sites of Trichinella spiralis larvae in naturally infected horses. J. Helminthol. 73, 233-237. 9
Pozio, E., Rinaldi, L., Marucci, G., Musella, V., Galati, F., Cringoli, G., Boireau, P., La Rosa, G., 2009. Hosts and habitats of Trichinella spiralis and Trichinella britovi in Europe. Int. J. Parasitol. 39, 71-79. Pozio, E., La Rosa, G., 2010. Trichinella. In: Liu, D. (Ed.), Molecular detection of Foodborne Pathogens. CRC Press, Taylor & Francis Group, Boca Raton, pp. 851-863. Pozio, E., Zarlenga, D.S., 2013. New pieces of the Trichinella puzzle. Int. J. Parasitol. 43, 983-997. Posluszny, M., Pilot, M., Goszczynski, J., Gralak, B., 2007. Diet of sympatric pine marten (Martes martes) and stone marten (Martes foina) identified by genotyping of DNA from faeces. Ann. Zool. Fennici 44, 269-284. Pullianen, E., Ollinmaki, P., 1996. A long-term study of the winter food niche of the pine marten Martes martes in northern boreal Finland. Acta Theriol. 41, 337-352. Wilson, D.E., Mittermeier, R.A., 2009. Handbook of the mammals of the world. Vol. 1. Carnivores. Lynx Edicions, Barcelona, 727 pp. Zimmermann, W.J., Schwarte, L.H., 1961. Distribution of Trichinella spiralis larvae in tissues of swine. Proc. Iowa Acad. Sci. 68, 553-557.
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Figure legend Fig. 1. Scatter plot, linear prediction with 95% CI and “goodness of fit” for each muscle or group of muscles between the larval burden in the muscle or group of muscles and the total larval burden in five Trichinella britovi naturally infected pine martens (Martes martes) of Latvia.
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Fig. 2. Scatter plot, linear prediction with 95% CI and “goodness of fit” for each muscle or group of muscles between the larval burden per g (LPG) of muscle or group of muscles and the average LPG burden in five Trichinella britovi naturally infected pine martens (Martes martes) of Latvia.
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Table 1: Main features of the five adult pine martens (Martes martes) infected with Trichinella britovi Animal code Skinned and eviscerated carcass weight (g) Striated muscle weight (g) Total number of larvae Average number of larvae/g in the whole striated muscles Number of larvae/g in the forelimb
1 1,249 684.69 8,885 12.97
2 1,190 641.80 32,294 50.30
3 846 461.04 4,120 8.90
4 566 322.8 6,705 20.77
5 613 333.33 640 1.92
22.1
69.8
10.5
37.4
2.9
13
Table 2: Average muscle weight, average number of larvae per muscle or group of muscles, and average number of larvae per gram (LPG) of five pine martens of Latvia, and coefficients for the total larval burden and total larva burden per g Average from five animals Total larval burden Total Muscle or group of muscles Weight coefficient number of LPG (g) (95% CI) larvae Diaphragm 5.0 285 57 39.03 (23.05-55.02) Lower part of left forelimb 12.9 604 47 18.54 (12.79-24.29) Lower part of right forelimb 11.0 407 37 26.67 (23.00-30.35) Right shoulder 9.3 290 31 38.56 (32.12-44.00) Lower part of right hind limb 16.2 471 29 22.69 (21.65-23.72) Left shoulder 14.0 412 29 24.88 (23.61-26.14) Upper part of right hind limb 39.4 1024 26 9.52 (8.33-10.71) Lower part of left hind limb 15.5 414 26 26.52 (22.57-30.47) Upper part of left hind limb 41.5 1086 26 8.77 (7.40-10.15) Tongue tip 1.2 28 24 408.12 (217.83-598.40) Upper part of right forelimb 21.7 472 22 22.23 (20.44-24.02) Upper part of left forelimb 19.5 400 20 25.37 (22.42-28.33) Neck 94 1898 20 5.77 (5.35-6.19) Back 76 1382 18 6.99 (5.82-8.15) Abdomen 17.8 295 16 33.43 (30.15-36.72) Tongue base 1.0 15 15 696.47 (643.53-749.40) Rump with tail 14.4 205 14 53.76 (46.68-60.84) Intercostal 49 645 13 16.54 (9.64-23.43) Masseter Head
8.1 20.3 Total 488.4
80 114 10,529
10 135.90 (102.04-169.76) 6 83.07 (69.95-96.18) 21
LPG coefficient (95% CI) 0.31 (0.13-0.50) 0.41 (0.18-0.65) 0.59 (0.52-0.67) 0.67 (0.59-0.75) 0.71 (0.67-0.76) 0.74 (0.71-0.77) 0.80 (0.63-0.96) 0.84 (0.73-0.94) 0.80 (0.60-0.99) 0.91 (0.51-1.30) 0.94 (0.81-1.07) 1.01 (0.86-1.16) 1.09 (0.97-1.22) 1.12 (0.78-1.46) 1.28 (1.09-1.47) 1.42 (1.25-1.59) 1.60 (1.39-1.82) 1.31 (0.54-2.09) 2.06 (1.71-2.41) 3.45 (2.53-4.38)
14
Table 3: Calculation of the Trichinella britovi biomass in pine marten population of Latvia. Estimates obtained through 100,000 simulations, where at each simulation a random number from each specific parameter and its reported variability is chosen. The reported estimates are the median (50th percentile), the 2.5th and the 97.5th percentile of the estimated distributions. Features Average (95% CI) a Estimated number of pine martens in Latvia 25,005 (20,048-29,875) Trichinella britovi prevalenceb 56.2% (49.0-66.4) b Geometric mean number of larvae/g in the limb 1.26 (0.89-1.79) c Mean weight (g) of pine marten muscles 488.3 (339.6-637.7) Mean number of larvae per animalc 477.4 (293.4-745.3) Estimated biomass 6,647,092 (3,840,030-11,100,000) a from www.vmd.gov.lv/valsts-meza-dienests/statiskas-lapas/medibas/valsts-mezadienests/statiskas-lapas/skaitli-un-fakti?id=766#jump; b from Deksne et al., 2016; c present work.
15
S0304-4017(16)30162-5 http://dx.doi.org/doi:10.1016/j.vetpar.2016.05.008 VETPAR 8008
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
23-12-2015 2-5-2016 7-5-2016
Please cite this article as: Kirjuˇsina, Muza, Bakasejevs, Eduards, Pezzotti, Patrizio, Pozio, Edoardo, Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Trichinella britovi biomass in naturally infected pine martens (Martes martes) of Latvia
Muza Kirjušina1, Eduards Bakasejevs1, Patrizio Pezzotti2, Edoardo Pozio2* 1
Institute of Live Science and Technology, Daugavpils University, Daugavpils, Latvia;
2
Istituto Superiore di Sanità, Rome, Italy
*Corresponding author. Tel.: +39 06 4990 2304; Fax +39 06 4990 3561. Email address: [email protected] (E. Pozio)
Highlights Estimation of Trichinella larva biomass in a naturally infected host population The number of larvae per gram of a muscle is representative of the total biomass Total larval burden and larvae/g coefficients were established for the pine marten
Abstract Parasites of the genus Trichinella are cosmopolitan nematodes infecting primarily wild animals, which represent the main reservoirs of these zoonotic pathogens. To investigate the transmission patterns of Trichinella spp. from wild to domestic animals and to humans and for the risk assessment of these parasites in a geographical area, it is important to know the number of possible transmission events deriving from carcasses of infected hosts. For this purpose, the evaluation of the larval biomass in reservoir hosts is needed. No data is available on how to estimate the biomass of Trichinella spp. larvae in muscles of naturally infected animals. The aim of this study was to evaluate the larval biomass in naturally infected pine martens (Martes martes) of Latvia, in which the prevalence of Trichinella britovi infection was over 50%. Single muscles or group of muscles (abdomen, back, diaphragm, intercostal muscles, muscles from the head, left and 1
right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail, and base and tip of the tongue) were collected from five skinned and eviscerated carcasses of T. britovi infected pine martens. Muscles were entirely removed from the bones and weighted. Each muscle or group of muscles was separately digested to detect the larvae per gram (LPG). Using linear regression, the larval burden in each muscle or group of muscles was evaluated to measure the possible prediction of the total animal larval burden (both as total number of larvae and as average LPG). All muscles were significantly predictive of the total burden with high “goodness of fit” (all adjusted R2 > 0.80; P ≤ 0.01), and the left shoulder provided the highest adjusted R2 (0.999). Then, to estimate the Trichinella britovi biomass in the pine marten population of Latvia, recent literature data on prevalence (56.2%, 95% CI: 47.8-64.3) and geometric mean LPG (1.26, 95% CI: 0.89-1.79) in the limb muscles of a sample representative of the whole Latvian pine marten population, were used. Using the predictive estimated relationship between LPG in the limb and that in the entire animals and by the estimated animal population and their mean total muscle weight, the Trichinella britovi biomass in the pine marten population of Latvia was estimated to be of 6,647,092 (95% CI: 3,840,030-11,100,000) larvae. The assessment of the biomass in nature can help to understand the epidemiological pattern of these pathogens, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology of this group of zoonotic nematodes.
Keywords: pine marten, Martes martes, Trichinella britovi, biomass, Latvia, epidemiology, sylvatic cycle
1.
Introduction Parasites of the genus Trichinella are cosmopolitan nematodes infecting mainly wild animals,
which represent the main reservoir hosts of these zoonotic pathogens (Pozio and Zarlenga, 2013). 2
Out of the 12 identified taxa in this genus, Trichinella britovi is the most prevalent species among wild carnivore mammals of Europe, western Asia, and North and Western Africa, but it infects also domestic and wild swine, which can be the source of infection for humans (Gottstein et al., 2009). The ecological niche of the infectious stage of these parasites is the striated muscle cell of their hosts, where the larva can survive from few weeks up to years waiting to be ingested from a new host (Gottstein et al., 2009). Studies on these zoonotic nematodes have estimated the larval burden, i.e. the number of larvae per g of muscle (LPG) in preferential muscles of important domestic and wild host species such as the domestic pig (Zimmermann and Schwarte, 1961; Olsen et al., 1964; Kapel and Gamble, 2000; Nöckler et al., 2005), horse (Pozio et al., 1999), wild boar (Kapel, 2001), and fox (Kapel et al., 2005), in order to establish the most suitable muscle/s for the detection of Trichinella spp. larvae to identify infected animals at the slaughterhouse, in the game abattoir, or for epidemiological surveys. The knowledge of the parasite biomass present in their hosts is useful to implement epidemiological studies on the transmission patterns of helminths in nature, and for monitoring and control programs. For gastrointestinal helminths, the biomass in a host can be approximately determined by the egg count in stool (Hollingsworth et al., 2013). No information is available on the biomass of Trichinella spp. larvae in striated muscles of naturally or experimentally infected animals. In the case of Trichinella, a transmission event from one to another host can occur, if the new host ingests at least one male larva and one female larva. On the basis of the number of larvae in a carcass, i.e. the biomass, the number of transmission events deriving from that carcass can be predicted. The integration of these data with epidemiological, biological and environmental data, will allow to assess the risk of exposure for domestic animals and for humans. The European pine marten (Martes martes) is a mustelid living from eastern to western and from northern to southern Europe (Wilson and Mittermeier, 2009). The aims of the present study were to count the total number of Trichinella spp. larvae in pine marten muscles, to establish if the
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number of larvae in a selected muscle/s was significantly predictive of the total larval burden in the animal, and to evaluate the Trichinella spp. larval biomass in the pine marten population of Latvia. 2.
Materials and Methods
2.1
Animal and muscle collection According to the study design, the Trichinella larval biomass had to be investigated in five
carcasses of pine martens infected with different larval burdens of the same Trichinella species to obtain statistically significant data. These were selected among 13 skinned carcasses of pine martens killed by hunters in the Latgale region of Latvia, and forwarded to the Institute of Live Science and Technology, Daugavpils University, Daugavpils from July 2010 to June 2012 (Bakasejevs et al., 2012). Carcasses were eviscerated and a muscle sample of 25 g from the forelimb was tested to detect Trichinella spp. larvae by the magnetic stirrer digestion method according to the Commission Regulation 2075/2005 (European Commission, 2005). Out of 13 tested pine martens, 6 tested positive for Trichinella spp. larvae (Bakasejevs et al., 2012). Carcasses of three males and two females with 22.1, 69.8, 10.5, 37.4, and 2.9 (LPG in the forelimb, were selected for this study (Table 1). The five skinned and eviscerated carcasses were weighted; the head, tongue, diaphragm and rump with tail, were removed, and the carcass was cut in two parts along the median line. Then, single muscles (diaphragm, base and tip of the tongue) or group of muscles (abdomen, back, intercostal muscles, muscles from the head, left and right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail), were carefully removed, taking care not to leave striated muscles on the bones (Table 2). Each muscle or group of muscles were separately weighted by a precision scale (up to 10 mg). 2.2
Muscle digestion, larva collection and identification Each muscle or group of muscles were digested separately according to a published protocol
(European Commission, 2005). Trichinella spp. larvae were collected, washed, and counted in triplicate. When a high number of larvae was present in the sediment, larvae were diluted in tap 4
water and counted in triplicate. The number of larvae was expressed as total number of larvae and as LPG in the whole muscle or group of muscles. Twenty larvae per animal were fixed in 95% ethyl alcohol and then each single larva was identified at the species level by multiplex PCR according to a published protocol (Pozio and La Rosa, 2010). 2.3
Statistical analysis Several linear regression models for each single muscle (or group of muscles) were
performed, where the total larval burden (both as total number of larvae and as average LPG) was the dependent variable, while the larval burden in each muscle (or group of muscles) was the independent variable. To establish the ability of the larval burden in each muscle on predicting the total larval burden, we considered the estimated adjusted R2, which varies between 0 and 1, where 1 indicates a perfect fit. To predict the total larval burden, the estimated coefficient expresses the change in the total larval burden, per unit increase in the specific muscle considered. 2.4
Trichinella larva biomass calculation The total biomass of Trichinella spp. larvae in the pine marten population of Latvia was
estimated on the basis of estimates obtained in this study and external information as indicated in parentheses: 1. the estimated number of heads, i.e. 20,000-30,000, in the population (Ministry of Agriculture of Latvia) in the period 2010-2014; 2. the estimated prevalence and 95% confidence intervals (CI) of Trichinella britovi infection in this host species in the whole Latvian territory (see Deksne et al., 2016); 3. the estimated mean and 95% CI of LPG in the limb of each animal (see Deksne et al., 2016); 4. the estimated coefficient and 95% CI obtained by the previously described linear regression between the LPG in the limb and the LPG in the entire striated muscles of the animal; and 5. the estimated mean and 95% CI weight of striated muscles (Table 1). The expected total larval biomass and the corresponding 95% CI were estimated as the median value, and the 2.5th and the 97.5th percentiles of the distribution obtained by performing 100,000 simulations, where at each simulation the previous described parameters were randomly extracted on the basis of the reported variability. 5
3.
Results The weight of the five skinned and eviscerated carcasses ranged from 566 g to 1,249 g
(average 892.8 g), the mean weight of striated muscles was 488.7 g (95% CI: 339.4-636.3), the total number of larvae in the whole striated muscles per animal was 10,529 (range 640-32,294), and the average LPG ranged from 1.92 to 50.3 (Table 1). Larvae were identified as T. britovi (data not shown). No statistically significant difference was detected between the left and the right muscles (data not shown). The LPG ranking shows that the diaphragm of the five animals had the highest LPG (average 57; range 5.07-117.31), followed by the lower part of forelimb (average 38.26; range 3.89-86.25); whereas, the masseters and the other muscles of the head ranked at the last two positions for LPG. The group of muscles with the highest number of larvae was the neck (average 1,898 larvae, range 109-5,490 larvae) (Table 2; Supplementary Table 1). As shown in Fig. 1, all single muscles or group of muscles were significantly predictive of the total number of larvae (all adjusted R2 > 0.80, p<0.01) present in the whole striated muscles. Similar results were obtained using the LPG (Fig. 2), and the left shoulder LPG provided the highest adjusted R2 (0.999). In particular, it has been estimated that the average LPG in an animal was 0.80 (95% CI: 0.74-0.82, adjusted R2=0.997) times the LPG found in the limb (data not shown). Just by the digestion of a few grams from representative muscles or groups of muscles, it is possible to know the total number of larvae in a pine marten according to the following procedure: number of larvae/g x coefficient of the respective muscle or group of muscles (Table 2) x whole striated muscle weight (Table 1). An example on how the estimated total number of larvae and average LPG are derived from data of the pine marten N. 4, is shown in the supplementary Table 2 and supplementary Table 3, respectively. Finally, Table 3 shows the estimated T. britovi biomass in the pine marten population of Latvia. 6
4.
Discussion In this study on five infected carcasses of pine martens, we firstly investigated the relationship
between the number of T. britovi larvae found in each muscle, or group of them, and the total larval burden in the animal. The pine marten was selected for this study among other host species for its high prevalence of Trichinella spp. infection (Deksne et al., 2016) and for its low size in comparison to that of other hosts (e.g., foxes, wolves), which makes the muscle digestion easier and cheaper. Larvae found in each muscle had an elevated ability to predict the total larval burden in the animal (all adjusted R2 >0.80, p<0.05) both as the total number and the average LPG. Secondly, we provided an estimate of the biomass of T. britovi, which is the most widespread species among those of this genus circulating in Europe (Pozio et al., 2009), combining this data with those from Deksne et al. (2016). The pine marten was selected for its wildness, which makes this animal a good representative of the sylvatic cycle. The European pine marten has an opportunistic generalist diet feeding on carrions mainly during the winter time and on small rodents and insects during the spring and the summer (Pullianen and Ollinmaki, 1996; Brainerd and Rolstad, 2002; Posluszny et al., 2007). The variation of the estimated biomass was quite large, i.e. from 3,840,030 to 11,100,000, due to the uncertainty of the parameters used to calculate the biomass. There are some limitations and potential biases in this study, which should be underlined: 1. the whole striated muscles of only five infected pine martens were tested and they were selected among those with a high larval burden to better evaluate the larval burden differences among muscles; our method assumes that the relationship, we estimated between the larval burden (or the average LPG) with that in each specific muscle, also relies in animals with a lower larval burden; 2. we do not know if, over time, the decreasing larval burden in the muscles may or may not be uniform; and 3. the number of muscle larvae developing in a new host can vary and be independent from the number of ingested larvae due to the individual immune response.
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The assessment of the Trichinella spp. biomass in nature can help to understand the transmission mechanisms of these pathogens in nature, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology and epidemiology of these group of zoonotic nematodes. This investigation was made easier by the detection of only one Trichinella species, T. britovi, in all the investigated pine martens of Latvia (Deksne et al., 2016). This is the first attempt to investigate the biomass of Trichinella parasites, and of T. britovi in particular. Further studies on other important reservoir hosts such as the red fox (Vulpes vulpes), the raccoon dog (Nyctereutes procyonoides), the wolf (Canis lupus), and the lynx (Lynx lynx), are needed.
Conflict of interest statement No financial or personal relationships are maintained with other people or organizations that could inappropriately influence or bias this paper.
Acknowledgements This study has been in part founded by the DG SANTE of the European Commission in the years 2012-2015.
References Bakasejevs, E., Daukšte, A., Zolovs, M., Zdanovska, A., 2012. Investigation of Trichinella in wildlife in Latgale region (Latvia). Acta Biol. Univ. Daugavp. 12, 1-5. Brainerd, S.M., Rolstad, J., 2002. Habitat selection by Eurasian pine martens Martes martes in managed forests of southern boreal Scandinavia. Wild. Biol. 8, 289-297. Deksne, G., Segliņa, Z., Jahundoviča, I., Esītea, Z., Bakasjevs, E., Bagrade, G., Keidāne, D., Interisano, M., Marucci, G., Tonanzi, D., Pozio, E., Kirjušina, M., 2016. 8
High prevalence of Trichinella spp. in sylvatic carnivore mammals of Latvia. Vet. Parasitol. (in press). DOI 10.1016/j.vetpar.2016.04.012. European Commission, 2005. Regulation (EC) No 2075/2005 of the European Parliament and of the Council of 5 December 2005 laying down specific rules on official controls for Trichinella in meat. Off. J. EC L 338, 60–82. Gottstein, B., Pozio, E., Nöckler, K., 2009. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin. Microbiol. Rev. 22, 127-145. Hollingsworth, T.D., Truscott, J.E., Anderson, R.M., 2013. Transmission dynamics of Ascaris lumbricoides – Theory and observation. In: Holland, C. (Ed.), Ascaris the neglected parasite, Academic press, London, pp. 231-262. Kapel, C.M., Gamble, H.R., 2000. Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs. Int. J. Parasitol. 30:215-221. Kapel, C.M., 2001. Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response. J. Parasitol. 87, 309-314. Kapel, C.M., Webster, P., Gamble, H.R., 2005. Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife. Vet. Parasitol. 132, 101-105. Ministry of Agriculture of Latvia. www.vmd.gov.lv/valsts-meza-dienests/statiskaslapas/medibas/valsts-meza-dienests/statiskas-lapas/skaitli-un-fakti?id=766#jump. Accessed 7 August, 2015. Nöckler, K., Serrano, F.J., Boireau, P., Kapel, C.M., Pozio, E., 2005. Experimental studies in pigs on Trichinella detection in different diagnostic matrices. Vet. Parasitol. 132, 85-90. Olsen, B.S., Villella, J.B., Gould, S.E., 1964. Distribution of Trichinella spiralis in muscles of experimentally infected swine. J. Parasitol. 50, 489-495. Pozio, E., Paterlini, F., Pedarra, C., Sacchi, L., Bugarini, R., Goffredo, E., Boni, P., 1999. Predilection sites of Trichinella spiralis larvae in naturally infected horses. J. Helminthol. 73, 233-237. 9
Pozio, E., Rinaldi, L., Marucci, G., Musella, V., Galati, F., Cringoli, G., Boireau, P., La Rosa, G., 2009. Hosts and habitats of Trichinella spiralis and Trichinella britovi in Europe. Int. J. Parasitol. 39, 71-79. Pozio, E., La Rosa, G., 2010. Trichinella. In: Liu, D. (Ed.), Molecular detection of Foodborne Pathogens. CRC Press, Taylor & Francis Group, Boca Raton, pp. 851-863. Pozio, E., Zarlenga, D.S., 2013. New pieces of the Trichinella puzzle. Int. J. Parasitol. 43, 983-997. Posluszny, M., Pilot, M., Goszczynski, J., Gralak, B., 2007. Diet of sympatric pine marten (Martes martes) and stone marten (Martes foina) identified by genotyping of DNA from faeces. Ann. Zool. Fennici 44, 269-284. Pullianen, E., Ollinmaki, P., 1996. A long-term study of the winter food niche of the pine marten Martes martes in northern boreal Finland. Acta Theriol. 41, 337-352. Wilson, D.E., Mittermeier, R.A., 2009. Handbook of the mammals of the world. Vol. 1. Carnivores. Lynx Edicions, Barcelona, 727 pp. Zimmermann, W.J., Schwarte, L.H., 1961. Distribution of Trichinella spiralis larvae in tissues of swine. Proc. Iowa Acad. Sci. 68, 553-557.
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Figure legend Fig. 1. Scatter plot, linear prediction with 95% CI and “goodness of fit” for each muscle or group of muscles between the larval burden in the muscle or group of muscles and the total larval burden in five Trichinella britovi naturally infected pine martens (Martes martes) of Latvia.
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Fig. 2. Scatter plot, linear prediction with 95% CI and “goodness of fit” for each muscle or group of muscles between the larval burden per g (LPG) of muscle or group of muscles and the average LPG burden in five Trichinella britovi naturally infected pine martens (Martes martes) of Latvia.
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Table 1: Main features of the five adult pine martens (Martes martes) infected with Trichinella britovi Animal code Skinned and eviscerated carcass weight (g) Striated muscle weight (g) Total number of larvae Average number of larvae/g in the whole striated muscles Number of larvae/g in the forelimb
1 1,249 684.69 8,885 12.97
2 1,190 641.80 32,294 50.30
3 846 461.04 4,120 8.90
4 566 322.8 6,705 20.77
5 613 333.33 640 1.92
22.1
69.8
10.5
37.4
2.9
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Table 2: Average muscle weight, average number of larvae per muscle or group of muscles, and average number of larvae per gram (LPG) of five pine martens of Latvia, and coefficients for the total larval burden and total larva burden per g Average from five animals Total larval burden Total Muscle or group of muscles Weight coefficient number of LPG (g) (95% CI) larvae Diaphragm 5.0 285 57 39.03 (23.05-55.02) Lower part of left forelimb 12.9 604 47 18.54 (12.79-24.29) Lower part of right forelimb 11.0 407 37 26.67 (23.00-30.35) Right shoulder 9.3 290 31 38.56 (32.12-44.00) Lower part of right hind limb 16.2 471 29 22.69 (21.65-23.72) Left shoulder 14.0 412 29 24.88 (23.61-26.14) Upper part of right hind limb 39.4 1024 26 9.52 (8.33-10.71) Lower part of left hind limb 15.5 414 26 26.52 (22.57-30.47) Upper part of left hind limb 41.5 1086 26 8.77 (7.40-10.15) Tongue tip 1.2 28 24 408.12 (217.83-598.40) Upper part of right forelimb 21.7 472 22 22.23 (20.44-24.02) Upper part of left forelimb 19.5 400 20 25.37 (22.42-28.33) Neck 94 1898 20 5.77 (5.35-6.19) Back 76 1382 18 6.99 (5.82-8.15) Abdomen 17.8 295 16 33.43 (30.15-36.72) Tongue base 1.0 15 15 696.47 (643.53-749.40) Rump with tail 14.4 205 14 53.76 (46.68-60.84) Intercostal 49 645 13 16.54 (9.64-23.43) Masseter Head
8.1 20.3 Total 488.4
80 114 10,529
10 135.90 (102.04-169.76) 6 83.07 (69.95-96.18) 21
LPG coefficient (95% CI) 0.31 (0.13-0.50) 0.41 (0.18-0.65) 0.59 (0.52-0.67) 0.67 (0.59-0.75) 0.71 (0.67-0.76) 0.74 (0.71-0.77) 0.80 (0.63-0.96) 0.84 (0.73-0.94) 0.80 (0.60-0.99) 0.91 (0.51-1.30) 0.94 (0.81-1.07) 1.01 (0.86-1.16) 1.09 (0.97-1.22) 1.12 (0.78-1.46) 1.28 (1.09-1.47) 1.42 (1.25-1.59) 1.60 (1.39-1.82) 1.31 (0.54-2.09) 2.06 (1.71-2.41) 3.45 (2.53-4.38)
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Table 3: Calculation of the Trichinella britovi biomass in pine marten population of Latvia. Estimates obtained through 100,000 simulations, where at each simulation a random number from each specific parameter and its reported variability is chosen. The reported estimates are the median (50th percentile), the 2.5th and the 97.5th percentile of the estimated distributions. Features Average (95% CI) a Estimated number of pine martens in Latvia 25,005 (20,048-29,875) Trichinella britovi prevalenceb 56.2% (49.0-66.4) b Geometric mean number of larvae/g in the limb 1.26 (0.89-1.79) c Mean weight (g) of pine marten muscles 488.3 (339.6-637.7) Mean number of larvae per animalc 477.4 (293.4-745.3) Estimated biomass 6,647,092 (3,840,030-11,100,000) a from www.vmd.gov.lv/valsts-meza-dienests/statiskas-lapas/medibas/valsts-mezadienests/statiskas-lapas/skaitli-un-fakti?id=766#jump; b from Deksne et al., 2016; c present work.
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