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13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen in Raw Ovine Milk Produced in Central Italy

13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen in Raw Ovine Milk Produced in Central Italy

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A. R. Attili et al.




Keywords Ovine Milk Mycobacterium avium subsp. paratuberculosis Public


13.1 Introduction

Paratuberculosis, otherwise known as Johne’s disease (1895), is a contagious,

chronic, and incurable granulomatous disease caused by Mycobacterium avium

subsp. paratuberculosis (MAP). MAP can infect a wide range of domestic and wild

ruminants and a series of reared animals (Pavlik et al. 2000). Transmission is

achieved by congenital and fecal-oral routes, including by food intake and/or

contaminated water. Infected colostrum and milk can be a source of infection for

young animals. In sheep, the three described forms are paucibacillary, multibacillary, and asymptomatic.

Paratuberculosis causes chronic enteritis, with animals showing lower ponderal

index values, ruffled hairs, dehydration, decrease in milk production, hypofertility,

and in the late stages, intermittent diarrhea and progressive weight loss. Paratuberculosis causes significant economic losses due to decreased production and

premature culling of infected animals (Smith et al. 2009). A potential role of MAP

in the etiology of Crohn’s disease in humans has been suggested. In 1913, Dalziel

reported the clinical and histopathological similarities among animal paratuberculosis, intestinal tuberculosis, and chronic granulomatous enteritis in humans.

Recent studies have also suggested the involvement of MAP in the pathogenesis of

human type I diabetes: 70 % positivity was observed in Sardinia, Italy, and in

England, while 40 % positive correlation was recorded in Lombardia, Italy (Rosu

et al. 2009). MAP can survive pasteurization treatments if present in milk at a high

concentration, with consequent risk to public health (Ellingson et al. 2005).

Food safety problems and the risk of transmission of zoonotic agents have

increased with the growing use of automated raw milk systems, mainly in cattle, as

authorized by Regulation (EC) 853/2004, together with increased consumption of

cheeses made from raw milk. The aim of this work was to estimate the prevalence

of MAP infection and the degree of safety of raw ovine milk and its role as a

source of MAP infection for humans in the Marche Region (Italy), where the

production of raw milk cheeses is high.

13.2 Materials and Methods

The survey was carried out between October 2009 and July 2010. A total of 697

ewes, older than 2 years, were randomly selected from 17 farms located in the

Marche region which produced raw milk cheese. The animals were Comisana,

Massese, Fabriano, Sarda, Sopravvissana, and mixed breed ewes at the beginning,


Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen


end, or peak of lactation. From each animal, after teat disinfection with chlorhexidine, approximately 100 ml of milk were collected. Samples were kept at 4 °C

during transport to the laboratory and frozen at -20 °C until microbiological


All milk samples were tested for the detection of anti-MAP antibodies using a

commercial immunosorbent assay kit (ELISA Paratuberculosis screening kit;

Institut PourquierÒ, France) following the protocol that the manufacturer recommends for bovine milk. Briefly, serum was diluted 1:2 and pre-incubated with

Mycobacterium phlei, limiting the possibility of cross-reactions with heterologous

mycobacteria and significantly improving the specificity of the ELISA. The test was

validated when the optical density (OD450 nm) of the positive control was C0.350

and the ratio of OD means of the positive and negative controls was C3. The milk

samples were recorded as positive if they scored C40 %, questionable if between

30 and 40 % and negative if \30 %. All samples were subjected to BC and to ZN

staining. The isolation of MAP was performed on Herrold’s egg yolk medium

(HEYM) containing mycobactin J (2 lg/ml) following the protocol previously

developed by Gao et al. (2005). A 50-ml aliquot of milk was centrifuged at

3,000 rpm for 30 min, and the serum was removed. After the addition of 20 ml of

benzalkonium chloride (0.3 %), samples were left at room temperature for 2 h with

stirring at 30-min intervals and then subjected to a final centrifugation at 2,500 rpm

for 10 min. The supernatant was removed, and the sediment resuspended in 0.5 ml

of antibiotic solution (50 mg amphotericin B, 100 mg nalidixic acid, 100 mg

vancomycin, and BHI broth 18.5 g/L, Sigma-Aldrich, Italy). After incubation at

37 °C for 24 h, 0.2 ml of the suspension was spread on HEYM and placed at 37 °C

for 16 weeks. Periodic inspections allowed for the identification of suspected

colonies and ZN staining highlighted the typical clusters of acid-fast bacilli. Statistical analysis was conducted using STATA software version 5 (STATA Corporation, College Station, TX, USA) and differences assessed using the Chi-square

test. P values\0.05 were considered significant. The degree of agreement between

tests was estimated using the kappa coefficient (k) statistic, and the k-value interpreted as reported by Holton et al. (1998).

13.3 Results

ELISA was able to identify 12/17 (70 %) MAP-infected ovine dairy farms. MAP

antibodies were evidenced in 37 (5.3 %) milk samples with a mean optical density

of 1.5 ± 0.8 (SD). In particular, 7.8 % (n = 270) and 3.7 % (n = 427,

P = 0.021) of ewes tested positive at early/late and peak lactation, respectively.

MAP infection was confirmed in 24.3 and 21.6 % of ELISA-positive milk samples

(n = 37) by ZN staining and BC, respectively (Table 13.1).

A fair (k = 0.43) and slight (k = 0.33) agreement was observed between

ELISA–ZN and ELISA–BC, respectively. When the higher antibody-level milk


A. R. Attili et al.

Table 13.1 Mycobacterium avium subsp. paratuberculosis in the milk of ovine dairy flocks

reared in the Marche region (Central Italy), using three diagnostic tests




ZN staining








samples were considered, an almost perfect agreement (k = 0.90) was attained

between milk ELISA–BC or –ZN.

13.4 Discussion

In Italy, the normative (Intesa Stato Regioni 25/1/2007) defines the criteria of

acceptability of raw milk sold directly to consumers or by automated distributors.

It individualizes parameters that must be respected for the most important bacterial

agents of food-borne illness, but these do not include emergent pathogens of public

health significance such as MAP (Intesa Governo, Regioni e Provincie Autonome

di Trento e Bolzano N.5/CSR 2007; Autori Vari 2006).

Many authors have suggested the transmission of various pathogens to the

consumer through the consumption of raw milk. Those that play a particularly

important role are E. coli O157: H7, thermophilic Campylobacter, Salmonella

spp., Listeria monocytogenes, Staphylococcus aureus, and Yersinia enterocolitica.

The danger of these pathogens, MAP and other zoonotic agents should lead to an

attitude of caution in consuming raw milk.

Our results indicate that almost two-thirds of the sampled farms were infected

and that MAP is excreted in the milk. Eight (47.0 %) and twelve (70.6 %) of the 17

dairy sheep farms in the Marche region were positive using BC or ZN staining

combined with ELISA, respectively.

The indirect ELISA, performed on milk, was significantly more sensitive when

carried out on sheep at the beginning/end of lactation than when performed on

animals at the peak of lactation (P \ 0.021). This is in agreement with previous

works of Williams and Millar (1979) and Nielsen et al. (2002), and could be

attributed by the decrease in IgG concentration during peak lactation, causing a

dilution effect due to increased milk production. The 21.6 % of raw milk samples

that were positive for MAP on ELISA is lower than that reported by Gao et al.

(2009), who isolated the organism in 34.6 % (44/133) of samples, but is higher

than that obtained in other studies, which recorded rates of isolation of 10.0 %

(Ayele et al. 2005), 8.3 % (Streeter et al. 1995) and 11.7 % (Singh et al. 2007).

This study also demonstrated that an immunoenzymatic technique validated

from bovine milk can also be applied to the sheep (Ngu Ngwa et al. 2010).

In conclusion, the data obtained from this study suggest that ovine paratuberculosis is widespread in the Marche region and that MAP, eliminated through the

milk of infected sheep, represents a potential risk of infection to humans via


Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen


consumption of raw milk and its products. A precautionary approach to legislation

should therefore be taken by the competent authority for the protection of public



Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I (2005) Mycobacterium avium subspecies

paratuberculosis cultured from locally and commercially pasteurized cow’s milk in the Czech

Republic. Appl Environ Microbiol 71:1210–1214

Ellingson JL, Anderson JL, Koziczkowski JJ, Radcliff RP, Sloan SJ, Allen SE, Sullivan MN

(2005) Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized

whole milk by two culture methods and PCR. J Food Prot 68(5):966–972

Gao A, Odumeru J, Raymond M, Mutharia L (2005) Development of improved method for

isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age

of milk, centrifugation, and decontamination. Can J Vet Res 69:81–87

Gao A, Odumeru J, Raymond M, Hendrick S, Duffield T, Mutharia L (2009) Comparison of milk

culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on

samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis. Can J

Vet Res 73(1):58–64

Holton LL, Scott EM, Nolan AM, Reid J, Welsh E, Flaherty D (1998) Comparison of three

methods used for assessment of pain in dogs. J Am Vet Med Assoc 212(1):61–66

Intesa Governo, Regioni e Provincie Autonome di Trento e Bolzano in materia di vendita di latte

crudo per l’alimentazione umana. Repertorio Atti N.5/CSR del 25/01/2007

Ngu Ngwa V, Attili AR, Preziuso P, Valente C, Cuteri V (2010). A comparative study of serum

and milk ELISAs for the diagnosis of Ovine Paratuberculosis (Ovine Johne’s Disease: OJD).

The Acts of 18th International Congress of FeMeSPRum: 32–37

Nielsen SS, Grohn YT, Enevoldsen C (2002) Variation of the milk antibody response to

paratuberculosis in naturally infected dairy cows. J Dairy Sci 85:2795–2802

Pavlik I, Bartl J, Dvorska L, Svastova P, Du Maine R, Machackova M, Ayele WY, Horvathova A

(2000) Epidemiology of paratuberculosis in wild ruminants studied by restriction fragment

length polymorphism in the Czech Republic during the period 1995–1998. Vet Microbiol


Rosu V, Ahmed N, Paccagnini D, Gerlach G, Fadda G, Hasnain SE, Zanetti S, Sechi LA (2009)

Specific immunoassays confirm association of Mycobacterium avium subsp. paratuberculosis

with type-1 but not type-2 diabetes mellitus. PLoS One 4(2):e4386

Singh SV, Singh AV, Singh R, Sandhu KS, Singh PK, Sohal JS, Gupta VK, Vihan VS (2007)

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and

fecal-PCR for the diagnosis of bovine Johne’s disease (BJD) in India. Comp Immunol

Microbiol Infect Dis 30:175–186

Smith RL, Grohn YT, Pradhan AK, Whitlock RH, Van kessel JS, Smith JM, Wolfgang DR,

Schukken YH (2009) A longitudinal study on the impact of Johne’s disease status on milk

production in individual cows. J Dairy Sci 92(6):2653–2661

Streeter RN, Hoffsis GF, Bechnielsen S, Shulaw WP, Rings M (1995) Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am J Vet

Res 56:1322–1324

Vari A (2006) Parere in materia di Rischi per la Salute degli animali derivanti da

un’alimentazione con prodotti lattiero caseari pronti all’uso senza ulteriore trattamento.

EFSA J 347:1–21

Williams MR, Millar P (1979) Changes in serum immunoglobulin levels in Jerseys and Friesians

near calving. Res Vet Sci 26:81–84

Chapter 14

Canine Filariosis in Sardinia:

Epidemiological Findings in the Ogliastra


A. Scala, C. Solinas, A. P. Pipia, G. Sanna, A. Varcasia and G. Tosciri

Abstract From May 2010 to April 2011, 242 family dogs from 14 municipalities

of the Ogliastra region (Sardinia) were examined for microfilaremia using the

modified knott method. Overall, 32.6 % microfilaremia were recorded. The species found were Acanthocheilonema reconditum (28.1 %), Dirofilaria immitis

(11.6 %), and Dirofilaria repens (11.2 %). The most important risk factors for

microfilaremia were the age of dogs ([6 years) and only for D. repens, the differences in the use of the dog (pet versus hunting and guard). A negative correlation was also found between the altitude and the prevalence of D. immitis and

D. repens. In light of these epidemiological results, it would therefore be desirable

to implement pharmacological prophylaxis to prevent filariosis in dogs in the

monitored region.

Keywords Dog

Á Filariosis Á Modified knott method Á Ogliastra (Sardinia)

14.1 Introduction

Dog heartworms are currently one of the most important parasitological diseases,

due to the health impact in animals (i.e., Dirofilaria immitis), the possible zoonotic

implications (especially Dirofilaria repens), and recent epidemiological changes.

In particular, concerning the latter point, several authors have recently shown an

expansion of the potential vectors of Dirofilaria spp., such as Aedes albopictus (the

tiger mosquito) (Pietrobelli 2010).

Moreover, climatic and environmental changes, as well as the increase in the

possible handling of dogs for activities related to tourism, hunting, etc. (Pietrobelli

A. Scala Á C. Solinas Á A. P. Pipia Á G. Sanna Á A. Varcasia (&) Á G. Tosciri

Dipartimento di Medicina Veterinaria, Sassari, Italy

e-mail: varcasia@uniss.it

C. Boiti et al. (eds.), Trends in Veterinary Sciences,

DOI: 10.1007/978-3-642-36488-4_14, Ó Springer-Verlag Berlin Heidelberg 2013



A. Scala et al.

2010), make the epidemiological picture of canine heartworm extremely variable.

For all of these reasons, it should be monitored continuously. In this context, we

have carried out an investigation to provide adequate answers to veterinarians on

prophylactic and therapeutic measures against these parasites.

The first survey carried out in Sardinia on canine heartworm by Arru et al.

(1968) showed the presence of microfilariae in 11.2 % of examined hosts. Dirofilaria repens was then the most widespread species (7.1 %), followed by Dirofilaria immitis (1.6 %), and Acanthocheilonema (syn. Dipetalonema) reconditum

(1.2 %), and 1.2 % were mixed infections.

Subsequently, other investigations were carried out by Tarantini et al. (1983)

and Garippa et al. (1993), and more recently by Scala et al. (2004). The latter study

confirmed some data, such as the rate of microfilaremia (12.3 %) and the spreading

of D. immitis (3.8 %), but also an increase in A. reconditum (7.4 %) and a decrease

in the prevalence of D. repens (Scala et al. 2004). This latest survey also enabled

the identification of some areas of the island with a higher risk of infection from

D. immitis, such as the Oristano and Sulcis-Iglesiente regions, where the prevalence of microfilariae of this species was 17.1 and 7.4 %, respectively.

Unfortunately, these regions have been recently confirmed to be at particular

risk for the occurrence of filariasis by D. immitis (Genchi et al. 2010); in the

district of Oristano, the first infection from D. immitis in cats in Sardinia was also

reported (Scala et al. 2009).

In the same Oristano region, the seroprevalence of 14 % for D. immitis in a cat

population (Scala et al. 2010) was highlighted. The presence of filariosis in cats

therefore confirms the high parasitic pressure for D. immitis in this area (Dillon


In this survey, to complete the epidemiological picture of the different areas of

Sardinia, we report the results of an epidemiological survey carried out on canine

heartworm in dogs from Ogliastra, the central-eastern region of Sardinia.

14.2 Materials and Methods

From May 2010 to April 2011, 242 blood samples collected from dogs (129 males

and 113 females) were examined with the modified knott technique. Examined

dogs, ranging in age from 6 months to 19 years (mean = 3.9 years, SD = 2.98),

were from 14 municipalities of the Ogliastra region. The sampling was carried out

with the help of the owners by collecting data on age, sex, type of hair (shaved,

short, or long), and the use of the dog. A clinical examination to ascertain the

presence or absence of symptoms of canine filariosis by D. immitis was also

performed. The identification of microfilariae was performed according to the

morphometric keys as described by Euzeby (1981).

Data were stratified by sex, age, living environment, season, and dog category,

and then processed statistically using the software EPI INFO version 6.04.


Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region


14.3 Results

Microfilariae were found in 32.6 % of the examined dogs (79), belonging to the

species A. reconditum, D. immitis, and D. repens, identified in 28.1 % (68), 11.6 %

(28), and 11.2 % (27) of examined animals, respectively. Multiple infections were

observed in 24 dogs (9.9 %) [5 D. immitis ? D. repens (2.1 %); 8 D. immitis ? A. reconditum (3.3 %); and 11 D. immitis ? D. repens ? A. reconditum

(4.5 %)].

Microfilaremia was detected in 36.4 % of males (47/129) and in 28.3 % of

females (32/113), with odds ratio (OR) values of 1.45 (0.81 \ OR \ 2.59).

Results of prevalence were processed by stratifying the animals by gender for each

Filariidae and are reported in Table 14.1.

No significant differences were found for the prevalence of Filariidae species in

either sex (v2 = 1.804; P = 0.179), although there was a slight tendency to

significance for A. reconditum, where higher prevalence was found in females

(v2 = 2.72; P = 0.099) with OR values of 1.62 (0.88 \ OR \ 2.98).

Prevalence of microfilaremia stratified by host age is shown in Table 14.2.

OR values for the various age classes of dogs with microfilaremia and for each

Filariidae are reported in Table 14.3.

The prevalence in dogs stratified according to category is shown in Table 14.4.

The stratification of the data into three categories according to the type of fur

did not show statistically significant differences (P [ 0.05). Prevalence of

Table 14.1 Filariidae

prevalence stratified by

animal gender

A. reconditum

D. immitis

D. repens

Males (129)

n (%)

Females (113)

n (%)

42 (32.6)

16 (12.4)

16 (12.4)

26 (23.0)

12 (10.6)

11 (9.7)

Table 14.2 Microfilaremia prevalence in dogs stratified by age

Classes of age


Microfilariae + D. immitis + D. repens + A. reconditum


n (%)

n (%)

n (%)

+ n (%)

\1 year

1 year

2 years

3–5 years

6–10 years

[10 years

v2 trend

Level of significance









2 (33.3 %)

9 (23.7 %)

18 (29.5 %)

31 (35.6 %)

15 (35.7 %)

4 (50 %)


P = 0.016


4 (10.5 %)

9 (14.8 %)

8 (9.2 %)

6 (14.3 %)

1 (12.5 %)


P = 0.727


4 (10.5 %)

7 (11.5 %)

10 (11.5 %)

6 (14.3 %)



P = 0.239

2 (33.3 %)

9 (23.7 %)

16 (26.2 %)

24 (27.6 %)

13 (31 %)

4 (50 %)


P = 0.189


A. Scala et al.

Table 14.3 Odds ratio values for each age class of dogs and for each Filariidae

Classes of age (Years)


D. immitis

D. repens

A. reconditum























Table 14.4 Prevalence stratified according to dog category




D. immitis


+n (%)

+ n (%)





Level of







7 (38.9 %)

67 (32.5 %)

5 (27.8 %)


P = 0.773

5 (27.8 %)

20 (9.7 %)

3 (16.7 %)


P = 0.056













D. repens

+ n (%)

A. reconditum

+ n (%)

5 (27.8 %)

19 (9.2 %)

3 (16.7 %)


P = 0.042

3 (16.7 %)

61 (29.6 %)

4 (22.2 %)


P = 0.427

D. immitis and D. repens found in 14 municipalities was negatively correlated with

the altitude of the same municipalities (D. immitis -0.542, P = 0.045; D. repens

-0.593, P = 0.026) (Pearson correlation).

14.4 Conclusions

Our results show the presence of the three Filariidae previously described in

Sardinia in the Ogliastra region: A. reconditum, D. immitis, and D. repens.

Additionally, in this region, A. reconditum plays a predominant role. This species

differs from the others of the genus Filaria for its intermediate hosts, and therefore

for its mode of transmission. This finding could explain the lack of a negative

correlation between the altitude of the municipalities of the positive dogs and

prevalence rates.

The presence of A. reconditum requires the implementation of an accurate

differential diagnosis with D. immitis microfilariae that have the same length. The

prevalence of Filariidae found in Ogliastra, especially for D. immitis, requires the

implementation of chemoprophylaxis. It is obvious that, as for other coastal areas

of Sardinia, such as Oristano and Sulcis-Iglesiente, which are already reported as

risk areas for D. immitis, the Ogliastra region should be considered an area in

which this parasite is an important health problem for dogs.


Canine Filariosis in Sardinia: Epidemiological Findings in the Ogliastra Region



Arru E, Nuvole A, Mann P (1968) La filariosi del cane in Sardegna. Riv Parasitol 30:49–58

Dillon AR (1986) Feline heartworm disease. In: Otto GF (ed) Proceeding Heartworm Symposium

American Heartworm Society, Washington, DC, p 149–154

Euzeby J (1981) Diagnostic Experimental des Helminthoses animals. Livre 1 Edition Information

Tecniques des services Vétérinaires, Paris

Garippa G, Sanna ML, Delogu E, Arru E (1993) Ulteriori indagini sulla filariosi del cane in

Sardegna. Atti Soc It Sci Vet 48:1417–1420

Genchi M, Scala A, Piazza C, Melis R, Viglietti A (2010) Can San Pietro island (Sardinia) be a

possible cause of filarial infection throuhghout Europe? Parassitol 52(1–2):318

Pietrobelli M (2010) Dirofilariosi canina: un esempio di federalismo parassitario? La Sett Vet

698(suppl 5–6):7–18

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aggiornamenti epidemiologici (1998–2003). Atti Soc It Sci Vet 58:120–121

Scala A, Pazzola M, Giobbe M, Sanna G, Briguglio P, Lutzu M, Carta A, Genchi M (2009) Is a

heartworm preventive treatment also advisable in Sardinia? 2hd Eur Dir Days 197

Scala A, Briguglio P, Sanna G, Pipia AP, Varcasia A, Garippa G, Genchi M, Genchi C (2010)

Feline hearthworm (Dirofilaria immitis) infection in Sardinia Parassitol 52(1-2):257

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Parasitol 25:361–364

Chapter 15

Comparison of Serum and Meat Juice

for Detection of Anti-Toxoplasma gondii

Antibodies in Hunted Wild Boars

(Sus scrofa)

D. Ranucci, F. Veronesi, I. Di Matteo, R. Branciari, D. Miraglia,

C. Marini and D. Piergili Fioretti

Abstract Samples of serum and diaphragm meat juice obtained from wild boars

were tested for anti-Toxoplasma gondii antibodies by means of an indirect

immunofluorescent antibody test (IFAT). The results were compared to evaluate

the concordance between the two matrices. A total of 160 blood and muscle juice

samples were tested. Antibodies against T. gondii were detected in 28 (17.5 %)

blood samples and in 22 meat juice samples (13.75 %). The results showed an

‘‘almost perfect’’ concordance between serum and meat juice analyses

(K value = 0.86). These preliminary data show that diaphragm meat juice could

be an alternative to serum when using IFAT for T. gondii monitoring purposes in

wild boars.

Keywords Wild boar

Á Toxoplasma gondii Á Meat juice Á IFAT

15.1 Introduction

Wild and farmed game meat consumption is considered as an emerging risk factor

for Toxoplasma gondii infection in humans (Kijlstra and Jongert 2008). The most

recent estimates provided by the European Food Safety Authority (EFSA 2007)

reported that approximately 50 % of wild game is seropositive for T. gondii. In

wild boar (Sus scrofa, Linnaeus, 1758), epidemiological surveys conducted in

Europe revealed seropositivity rates up to 38.4 % (Gauss et al. 2005). Parasitized

D. Ranucci (&) Á F. Veronesi Á I. Di Matteo Á R. Branciari Á D. Miraglia Á D. Piergili Fioretti

Dipartimento Scienze Biopatologiche ed Igiene delle Produzioni Animali ed Alimentari,

Università degli Studi di Perugia, Perugia, Italy

e-mail: david.ranucci@unipg.it

C. Marini

Istituto Zooprofilattico Sperimentale Umbria e Marche, Perugia, Italy

C. Boiti et al. (eds.), Trends in Veterinary Sciences,

DOI: 10.1007/978-3-642-36488-4_15, Ó Springer-Verlag Berlin Heidelberg 2013



D. Ranucci et al.

wild boar meat can be a source of infection for hunters during evisceration procedures, and for consumers (Choi et al. 1997; Cook et al. 2000). This makes the

implementation of monitoring plans beginning at the hunt, as provided by European legislation (Directive EC/99/2003), of utmost importance. Serologic testing is

not always practical in the hunted boar because of difficulties in sample collection.

Recently, an alternative approach based on antibody screening performed on meat

juice has been suggested in swine (Dubey et al. 2005; Berger-Schoch et al. 2011).

Encouraging results for the determination of antibodies to T. gondii in meat juice

from domestic pigs were obtained by applying an indirect immunofluorescent

antibody test (IFAT) method (Ranucci et al. 2010). The aim of this study was to

evaluate the applicability of an IFAT technique conducted on meat juice obtained

from the diaphragm muscle of hunted wild boar and compare it to the traditional

serum antibody research.

15.2 Materials and Methods

Serum and meat juice samples obtained from 160 wild boar hunted in 2009 and

2010 were analyzed. The blood of each animal was collected via intracardiac

puncture immediately after killing. Samples were centrifuged at 4,000 rpm for

15 min, and the separated serum was stored at -20 °C until analysis. A fragment

(2.5 9 2.5 cm) of the diaphragm muscle was taken for meat juice extraction.

Muscle samples were placed in containers (Sardstedt, Numbrecht, Germany),

maintained at -20 °C for 12 h, and then thawed at 4 °C for 8–12 h. Meat juice

was collected in test tubes and quantified. Blood and meat juice samples were

subjected to screening by IFAT for the determination of anti-T. gondii Immunoglobulin G (IgG) using commercial slides (Mega Cor Diagnostik, Horbranz,

Osterreich) obtained from T. gondii tachyzoites cultured on Vero cells and fluorescein isothiocyanate conjugated anti-swine IgG (anti-swine IgG FITC conjugate;

Sigma Immunochemicals, St. Louis, MO) diluted 1/32. Sera were tested at a

screening dilution of 1/40 (cut off) (Bartová et al. 2006), while for meat juice, in

the absence of accurate bibliographic references, a cut-off value of 1/5 was

adopted on the basis of previous evaluations. All positive samples were subjected

to 2-fold serial dilutions to determine the final titer (endpoint). The concordance

between serum and meat juice overall results was calculated by McNemar’s

Chi-square test, using 2 9 2 contingency tables where serum was considered as

the comparative test and meat juice as the alternative test. The overall agreement between test and the one relative to two distinct serum antibody level titers

(1/40, C 1/40) was evaluated by calculating the K value, interpreted according to

the following guidelines: \0.2 = slight agreement, 0.2–0.4 = fair, 0.4–0.6 =

moderate, 0.6–0.8 = substantial, [0.8 = almost perfect (Thrusfield 2007). All

statistical analyses were carried out using EpiInfo 6.01 free software (CDC,

Atlanta, GA) and the statistical significance level was set at p \ 0.05.

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13 Mycobacterium avium subsp. paratuberculosis as an Emergent Pathogen in Raw Ovine Milk Produced in Central Italy

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