View of Comparative Bacteriological Study of Two Wild Boar Populations in Sierra Morena (Jaén, Spain)

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Comparative Bacteriological Study of Two Wild Boar Populations in Sierra Morena (Jaén, Spain)

Antonio NOTARIO

¹⁾

, Javier MORO

²⁾

, Olimpio FUENTES

³⁾

, Luis CASTRESANA

¹⁾

Eduardo RESPALDIZA

⁴⁾

, Ovidiu IONESCU

⁵⁾

, Andras NAHLIK

⁶⁾

1)Universidad Politéchnica de Madrid, Ciudad Universitaria 28040, Madrid, Spain; [email protected]

2)Autonomous Body, Spanish National Parks, 7-11 Centro de Lugar Nuevo, Spain; [email protected]

3) Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria INIA, Crta. de la Coruña, 28040, Madrid, Spain; [email protected]

4) Madrid Regional Veterinary Laboratory, Ctra. Guadalix de la Sierra, Colmenar Viejo, 28770, Madrid, Spain; [email protected]

5)Transilvania University, 1 Sirul Beethoven St., 500123 Brasov, Romania; [email protected]

6)University of West Hungary, Bajcsy-Zsilinszky u. 4, 9400 Sopron, Hungary; nahlik@ emk.nyme.hu

Abstract

The aim of this study was to investigate the presence of various bacterial species in the wild boar populations of Lugar Nuevo and Selladores-Contadero woodlands from Sierra Morena (Spain). Bacteriological analyses were carried out on a total of 229 wild boar individuals hunted in the period 2000-2003 in eleven experimental plots which are representative for the different biotopes of the area.

The following species were detected: Brucella ovis, Clostridium sp., Corynebacterium sp., Streptococcus pneumoniae, Escherichia coli, Mycobacterium bovis, Mycobacterium avium subsp. paratuberculosis, Mannheimia haemolytica, Pasteurella multocida, Pseudomonas aeruginosa, Chlamydophila psittaci, Staphylococcus aureus and Staphylococcus sp. and the bacterial prevalence was estimated for each of them. The results provide useful indications of the health status of wild boar in both locations and highlight the potential of the wild boar populations to act as biological reservoirs of certain microorganisms that can be passed onto other vertebrate wild animals and humans.

Keywords: wild boar, post-mortem, bacteriology, infection prevalence Introduction

Several studies on specific aspects of the bacteriology of wild boar have been recently published by the outstand- ing authors Acevedo-Whitehouse et al. (2005), Gortázar et al. (2003, 2005), Vicente et al. (2002, 2005, 2006), Se- gales et al. (2005).

According to Gortazar (2007), there are a number of diseases to be considered in the wild boar. It is the case for tuberculosis by strains of bovine and caprine origin in- cluded in the Mycobacterium tuberculosis Complex, wide- spread in game preserves and hunting-designated areas of Southern Spain and probably linked to a high density of ungulates in this territory. Brucellosis is also common as a result of the role of reservoirs of Brucella spp played by wild animals, which convey the potential infection to large domestic animals with significant implications in public health. Salmonellosis, a well known zoonosis has to be taken into account too, especially in those outbreaks with high mortality rates that can be wrongly diagnosed as either African swine fever or Hog cholera, both viral diseases. Analogous interest should be focused on other transmissible agents such as Escherichia coli and Campy- lobacter spp., present in the digestive tract of wild boars,

Chlamydophila psittaci and Erysipelothrix rhusiopathiae, aetiological agent responsible of Swine erysipelas.

This study, subsidized by the Autonomous Body, Span- ish National Parks, and carried out from 2000 to 2003, was designed to provide a thorough knowledge of the various bacterial species isolated from wild boar populations in Lugar Nuevo and Selladores-Contadero.

Materials and methods

The woodlands of Lugar Nuevo (9,233.55 hectares) and Selladores-Contadero (10,455.89 hectares), 60 km away from each other and managed by the autonomous body, Spanish National Parks, lie in the northwest of the province of Jaén, in a peripheral area of the southern side of Sierra Morena. The latter is a mountain range that forms the link between the Castilian Plateau and the Riv- er Guadalquivir Valley. Slopes are moderate to high and sunny. Most soils showed neutral or slightly acid pH values and medium texture. The climate can be classified as of Mediterranean type with mild winters, extremely dry and hot summers and a moderate rainfall regime (approx.

600 mm), characterized by an uneven distribution of rain throughout the year. The indigenous flora consists of the

Received 12 October 2010; accepted 28 November 2010

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A division of woodlands in eleven plots (seven in the woodlands of Lugar Nuevo and four in those of Selladores- Contadero) following patterns of homogeneity of habitat and behavioural basis was made so as to make proper investigation feasible and statistically valid. Tab. 1, 2 and 3 depict some of the studied parameters (i.e. total surface, perimeter, lowest and highest points above the sea level, vegetation type).

Duration of sanitary on-spot checks and sampling Collection of 229 samples (142 from Lugar Nuevo and 87 from Selladores-Contadero) took place from 2000 to 2003. Animals were randomly selected and immediate post-mortem examinations were done at local sanitary units/facilities equipped for that specific purpose. Speci- mens were aseptically submitted for analysis within 2-5 hours after death.

standard vegetation of Mediterranean forests and reforested pinewoods.

Land plot division

Due to the difficulties associated with the size of the covered area under investigation, a subsequent division of land plots with similar or equivalent features in their habi- tats was performed, so that valid results could be obtained in different seasons.

Tab. 1. Area and altitude of the studied plots (LN=Lugar Nuevo; SC=Selladores- Contadero)

Land plot

identification Area (hectares)

Highest point above

sea level (metres)

Lowest point above sea level

(metres) Coscojal

(P0LN) ^455,6 ¹⁹⁷ ⁷⁰⁶

Fuencubierta y

Solana (P1LN) ^1800,31 ²³⁵ ⁶⁴⁸

Estrecho

(P2LN) ^1138,09 ²⁴¹ ⁶⁵²

Valdelipe (P3LN) ^1445,24 ¹⁹⁹ ⁶³¹ Navalpacho

(P4LN) ^1372,73 ²¹⁴ ⁵⁵⁹

Centenera-Piedra

bermeja (P5LN) ^1654,95 ¹⁹⁹ ⁶⁷⁴

Cabrera

(P6LN) ^1366,64 ¹⁹⁹ ⁶⁷⁴

Selladores

(P1SC) ^2700,39 ³⁷² ⁹⁹⁰

Navalcardo-

Vallejones (P2SC) ^2595,45 ⁴⁴⁵ ⁷⁶⁷ Contadero

(P3SC) ^3062,92 ³³⁷ ⁷⁷⁹

Lentisquillo

(P4SC) 2097,13 423 780

Tab. 3. Vegetation characteristics of the Selladores- Contadero area

Vegetation type Area (ha)

P1SC P2SC P3SC P4SC Standard Mediterranean

mountain area vegetation

736,27 916,79 310,09 324,49

Meadow/pasture including groves

of holm-oaks

0,00 77,52 0,00 0,00

Reforested pinewood

P. pinea, P. pinaster 927,15 1470,26 1427,81 1369,55

Cultivated area ^0,00 ^0,00 ^3,91 ^0,00 Thicket ^369,36 ^7,04 ^102,66 ^94,20 Grassland ^54,44 ^12,44 ^170,21 ^29,38 Mastic Tree forest&

Olive woods ^748,29 ^40,85 ^808,69 ^21,10 Other ^0,58 ^15,57 174,68 242,59

Total 2836,08 2540,47 2998,05 2081,31

Tab. 2. Vegetation characteristics of the Lugar Nuevo area

Vegetation type Area (ha)

P0LN P1LN P2LN P3LN P4LN P5LN P6LN

Standard Mediterranean mountain area vegetation ^0,59 ^94,68 ^106,99 ^180,90 ^445,12 ^1161,40 ^146,33 Meadow/pasture including groves of holm-oaks ^50,55 ^272,18 ^255,31 ^48,81 ^155,36 ^20,64 ^34,22

Reforested pinewood P. pinea, P. pinaster ^394,97 ^728,66 ^726,43 ^1129,4 ^479,33 ^382,57 ^1158,7

Riparian vegetation ^9,49 ^0,13 ^24,68 ^3,81 ^77,02 ^26,02 ^0,00

Thicket ^0,00 ^183,36 ^0,78 ^11,51 ^11,51 ^0,00 ^0,00

Grassland ^0,00 ^25,77 ^0,00 ^63,18 ^27,18 ^47,62 ^27,32

Mastic Tree forest and Olive woods ^0,00 ^495,52 ^6,79 ^0,00 ^171,12 ^0,00 ^0,00

Other ^0,00 ^0,00 ^17,11 ^7,61 ^6,09 ^16,65 ^0,03

Total 455,6 1800,3 1138,1 1445,2 1372,7 1654,9 1366,6

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Sample collection and processing

A system of protocols containing relevant information was used. Details reflected a vast range of indicators, such as:1. Identification chart-Sample number, plot identifi- cation, sex, estimated age, corpse weight, carcass weight, height to the withers, head-rump length, thoracic circum- ference.

2. Macroscopic study-External abnormalities and anato- mopathological findings in offals and tissues of carcasses (lymph nodes, abscesses, presence of endoparasites).

3. Sampling for bacteriological tests - Representative portions of lung, spleen, liver, intestines, lymph nodes as well as abscesses.

4. Laboratory diagnosis

a. Direct microscopy. Diverse techniques were used alone or in conjunction with cultural methods to stain smears from lesions and specimens. This is the case for the Gram stain, acid-fast stains such as Thiam-Hok and Kiny- oun besides those like either the Giemsa stain or the Stamp stain capable of revealing Brucella spp. and Chlamydophila psittaci.

b. Isolation and identification of causative agents. Inocu- lation of liquid media (Peptone Broth, Brain Heart Infu- sion Broth) with specimens (lung, liver, spleen, intestinal mucosa and/or lymph nodes) was carried out in those samples positive to reactions specified in section 4a. The isolation of strains was performed through plate streak- ing of selective agars (Columbia Agar enriched with 5%

of sheep blood, Endo-Columbia Agar to which 5% sheep blood was incorporated, Triple Sugar Iron Agar, and Mac- Conkey Agar) from those cultures in liquid media showing turbidity as a way of demonstrating growth. For identification of species CTA medium with cystine and trypticase as well as commercial strips (API 20 E, API Staph, API ID 32 Staph, API 20 A, API 20 Strep and API CORYNE) were used. Preparation of Lowenstein-Jensen medium re- quired supplementation with either tiophen-2-carbonic acid hydrazide (TCH) or mycobactin, as specified by Fu- entes Pérez (1988). The choice of ready-to-use media, was based on the results obtained in the Gram reaction. All products, including the miniaturized identification sys- tems were supplied by Bio-Mérieux.

Results and discussion

The results of the 229 analyzed samples are summa- rized in Tab. 4 to 11.

While in 67% of the sampled population at least one microorganism was isolated, in 33% of the population a lack of presence of viable strains was evident (Tab. 4).

The following species were detected: Brucella ovis, Clostridium spp., Corynebacterium spp., Streptococcus pneumoniae, Escherichia coli, Mycobacterium bovis, My- cobacterium avium subsp. paratuberculosis, Mannheimia

haemolytica, Pasteurella multocida, Pseudomonas aerugi- nosa, Chlamydophila psittaci (PLT), Staphylococcus aureus and Staphylococcus spp.

A wider variety of bacterial pathogens is clearly ob- served during May, June and July. (Tab. 6).

“Fuencubierta-Solana” and “Valdelipe” in Lugar Nue- vo and “Selladores” in Selladores-Contadero were the land plots data depicting populations with a higher infection rate (Tab. 7 and 8).

Chlamydophila psittaci and Mycobacterium avium sub- sp. paratuberculosis (MAP) affect wild boars of all ages and conditions.

Tab. 4. Sample distribution, reflecting positive and negative cases, for each location

Monte Number of

samples Negative

samples Positive samples

Lugar Nuevo 142 45 97

Selladores-Contadero 87 30 57

Total 229 75 154

Tab. 5. Bacterial species detected in the studied areas

Bacterial species

Positive cases on its own

Positive cases associated with

other species Total Lugar Nuevo

Mycobacterium avium subsp.

Paratuberculosis (MAP) 10 17 27

Staphylococcus spp. 1 0 1

Mycobacterium bovis 0 1 1

Streptococcus pneumoniae 2 6 8

Mannheimia haemolytica 2 13 15

Pasteurella multocida 0 5 5

Pseudomonas aeruginosa 1 3 4

Brucella ovis 0 1 1

Corynebacterium spp. 2 5 7

Escherichia coli 3 3 6

PLT 18 52 70

Staphylococcus aureus 0 0 0

Clostridium spp. 0 0 0

Selladores-Contadero Mycobacterium avium subsp.

Paratuberculosis (MAP) 4 11 15

Staphylococcus spp. 0 1 1

Mycobacterium bovis 0 0 0

Streptococcus pneumoniae 0 5 5

Mannheimia haemolytica 2 5 7

Pasteurella multocida 0 1 1

Pseudomonas aeruginosa 0 2 2

Brucella ovis 0 0 0

Corynebacterium spp. 0 0 0

Escherichia coli 3 1 4

PLT 15 28 43

Staphylococcus aureus 0 1 1

Clostridium spp. 1 0 1

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Mycobacterium avium subsp. paratuberculosis (MAP) is present in both woodlands with an equivalent prevalence of 19%. Mycobacterium bovis is not relevant as solely detected in one sample. Staphylococcus sp. and S. aureus show a low prevalence with a slightly higher value in Selladores- Contadero. Brucella ovis and Mycobacterium bovis are not significant having only been isolated in one case in Lugar Nuevo.

Streptococcus pneumoniae is present in both woodlands with a similar prevalence rate of 6%, whilst Mannheimia

haemolytica shows a slightly higher prevalence in Lugar Nuevo.

Pasteurella multocida has a significantly lower preva- lence rate with higher values in Lugar Nuevo. Pseudomo- nas aeruginosa shows equivalent prevalence rates of 2.67%

in both locations.

In the case of Corynebacterium spp. remarkable differ- ences can be seen between prevalence rates in Lugar Nue- vo (4.83%) and absence in Selladores-Contadero (0%).

Clostridium spp. is only present in Selladores-Contadero.

Tab. 6. Presence of different bacterial species along the year (both locations)

Bacterial species Month _Total

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Mycobacterium avium subsp.

Paratuberculosis (MAP) 3 2 4 4 1 2 8 5 4 5 4 0 42

Staphylococcus spp. 0 0 0 0 0 1 1 0 0 0 0 0 2

Mycobacterium bovis 0 0 0 0 0 0 0 1 0 0 0 0 1

Streptococcus pneumoniae 0 1 1 1 5 1 1 1 1 1 0 0 13

Mannheimia haemolytica 1 1 1 2 3 6 2 2 1 1 1 1 22

Pasteurella multocida 0 0 1 0 0 0 1 0 1 2 1 0 6

Pseudomonas aeruginosa 1 0 0 0 1 2 1 1 0 0 0 0 6

Brucella ovis 0 0 0 0 0 0 0 0 0 0 1 0 1

Corynebacterium spp. 0 1 0 0 1 1 1 1 0 1 0 1 7

Escherichia coli 2 0 1 0 1 2 1 1 0 1 1 0 10

Staphylococcus aureus 0 0 0 0 0 0 1 0 0 0 0 0 1

Clostridium spp 0 0 0 0 0 0 0 0 0 0 1 0 1

PLT ⁸ ⁷ ¹¹ ¹³ ¹¹ ¹³ ⁹ ⁶ ¹¹ ⁸ ¹⁰ ⁶ ¹¹³

Total number of isolations

15 12 19 20 23 28 26 18 18 19 19 8 225

Nº of negative samples

10 5 6 7 6 3 7 6 6 7 9 3 75

Total number of analyzed samples

24 14 17 20 20 22 25 18 18 19 21 11 229

Monthly infection prevalence

* 58 64 65 65 70 86 72 67 67 63 57 73 67

*Monthly infection prevalence rate, calculated by dividing the number of positive cases by the total number of investigated animals. It is not a biohazard indicator but a monthly prevalence rate

Tab. 7. Plot distribution and level of presence (%) of bacterial species in different locations of Lugar Nuevo

Bacterial species Number of positive cases Level of

presence(%) P0 LN P1 LN P2 LN P3 LN P4 LN P5 LN P6 LN Total

Mycobacterium avium subsp. Paratuberculosis (MAP) 0 7 7 3 4 5 1 27 18,62

Staphylococcus spp. 0 0 0 1 0 0 0 1 0,69

Mycobacterium bovis 0 1 0 0 0 0 0 1 0,69

Streptococcus pneumoniae 1 0 2 1 2 2 0 8 5,52

Mannheimia haemolytica 1 4 3 2 3 1 1 15 10,34

Pasteurella multocida 0 1 0 3 0 1 0 5 3,54

Pseudomonas aeruginosa 0 2 0 1 0 1 0 4 2,76

Brucella ovis 0 0 1 0 0 0 0 1 0,69

Corynebacterium spp. 0 1 1 2 1 0 2 7 4,83

Escherichia coli 0 3 0 0 2 1 0 6 4,14

PLT 3 15 7 15 9 13 8 70 48,27

Total number of isolations 5 34 21 28 21 24 12 145 100

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Tab. 8. Plot distribution and level of presence (%) of bacterial species in different locations of Selladores-Contadero

Bacterial species Number of positive cases Level of

presence (%)

P1 SC P2 SC P3 SC P4 SC Total

Mycobacterium avium subsp. Paratuberculosis (MAP) 4 0 5 6 15 18,75

Staphylococcus spp. 0 0 1 0 1 1,25

Mycobacterium bovis 0 0 0 0 0 0

Streptococcus pneumoniae 2 1 1 1 5 6,25

Mannheimia haemolytica 5 0 1 1 7 8,75

Pasteurella multocida 0 1 0 0 1 1,25

Pseudomonas aeruginosa 1 0 1 0 2 2,50

Brucella ovis 0 0 0 0 0 0

Corynebacterium spp. 0 0 0 0 0 0

Escherichia coli 0 2 1 1 4 5,00

Staphylococcus aureus 0 1 0 0 1 1,25

Clostridium spp 0 1 0 0 1 1,25

PLT 15 9 10 9 43 53,75

Total number of isolations 27 15 20 18 80 100

Tab. 9. Summary of bacterial prevalence rates (%) in the different age cohorts (both locations) Months

Bacterial species 0-12 13-24 25-36 37-48 49-60 61-72 73

Mycobacterium avium subsp. Paratuberculosis (MAP) 14,29 18,03 12,50 27,27 11,54 33,33 33,33

Staphylococcus spp. 1,79 0,00 0,00 3,03 0,00 0,00 0,00

Mycobacterium bovis 0,00 0,00 3,13 0,00 0,00 0,00 0,00

Streptococcus pneumoniae 3,57 8,20 9,38 3,03 3,85 11,11 0,00

Mannheimia haemolytica 12,50 13,11 12,50 0,00 3,85 0,00 16,67

Pasteurella multocida 5,36 0,00 0,00 6,06 0,00 0,00 8,33

Pseudomonas aeruginosa 3,57 3,28 3,13 3,03 0,00 0,00 0,00

Brucella ovis 0,00 0,00 0,00 0,00 3,85 0,00 0,00

Corynebacterium spp. 3,57 3,28 3,13 0,00 3,85 0,00 8,33

Escherichia coli 5,36 3,28 12,50 3,03 0,00 0,00 0,00

Staphylococcus aureus 0,00 0,00 0,00 0,00 0,00 0,00 8,33

Clostridium spp 0,00 0,00 0,00 3,03 0,00 0,00 0,00

PLT 42,86 59,02 46,88 42,42 53,85 44,44 50,00

Negative samples 32,12 27,87 34,38 30,30 42,31 55,56 25,00

Tab. 10. Bacterial prevalence rate (percentage related to total) in the wild boar population of Lugar Nuevo

Bacterial species P0 LN P1 LN P2 LN P3 LN P4 LN P5 LN P6 LN Total Prevalence (%)

Mycobacterium avium subsp. Paratuberculosis (MAP) 0 7 7 3 4 5 1 27 19,01

Staphylococcus spp. 0 0 0 1 0 0 0 1 0,70

Mycobacterium bovis 0 1 0 0 0 0 0 1 0,70

Streptococcus pneumoniae 1 0 2 1 2 2 0 8 5,63

Mannheimia haemolytica 1 4 3 2 3 1 1 15 10,56

Pasteurella multocida 0 1 0 3 0 1 0 5 3,52

Pseudomonas aeruginosa 0 2 0 1 0 1 0 4 2,82

Brucella ovis 0 0 1 0 0 0 0 1 0,70

Corynebacterium spp. 0 1 1 2 1 0 2 7 4,93

Escherichia coli 0 3 0 0 2 1 0 6 4,23

PLT 3 15 7 15 9 13 8 70 49,30

Total number of isolations 5 34 21 28 21 24 12 145

Negative samples 2 7 8 7 8 7 6 45

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Escherichia coli shows an analogous prevalence rate in both woodlands. Mannheimia haemolytica and Escherichia coli prevalence dramatically increases in young animals (up to 3 years). Chlamydophila psittaci is widely distributed and has a higher prevalence in Selladores-Contadero.

Conclusions

From this study it can be stated that bacterial species present in domestic swine are similarly distributed in boar populations from Lugar Nuevo and Selladores-Contadero, acting as biological reservoirs of certain microorganisms that can be passed onto other vertebrate wild animals and humans. So, Mycobacterium avium subsp. paratuberculosis (MAP) which is not relevant in adult boars, may well be pointed out as major pathogen in certain ungulates such as red deer (Cervus elaphus), causing clinical conditions closely resembling that of tuberculosis by Mycobacterium bovis.

Acknowledgements

We would like to express our gratitude to Professor Respaldiza Cardeñosa, Member of the Royal College of Veterinary Surgeons of Spain, for his continued guidance, time, constructive advice and willingness to share his in- sight and wisdom throughout all these years as well as to the Autonomous Body, Spanish National Parks, part of the Department of Environment Preservation for supporting and subsidizing the present study.

References

Acevedo-Whitehouse, K., J. Vicente, C. Gortázar, U. Höfle , I. G.

Fernández de Mera and W. Amos (2005). Genetic resistance to bovine tuberculosis in the Iberian wild boar. Molecular Ecology 14(10):3209-3217.

Tab. 11. Bacterial prevalence rate (percentage relative to total) in the wild boar population of Selladores-Contadero

Bacterial species P1 SC P2 SC P3 SC P4 SC Total Prevalence(%)

Mycobacterium avium subsp. Paratuberculosis (MAP) 4 0 5 6 15 17,24

Staphylococcus spp. 0 0 1 0 1 1,15

Mycobacterium bovis 0 0 0 0 0 0

Streptococcus pneumoniae 2 1 1 1 5 5,75

Mannheimia haemolytica 5 0 0 1 7 8,05

Pasteurella multocida 0 1 0 0 1 1,15

Pseudomonas aeruginosa 1 0 1 0 2 2,30