Sensitivity and specificity of different methods for the isolation of Salmonella from pigs.

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.     

A comparison of two enrichment and two plating media for the isolation of Salmonella sp. from broilers.

Twenty duplicate cloacal swabs and the intestines of 98 broilers were cultured using Rappaport-Vassiliadis and tetrathionate as enrichment broths. These were plated to brilliant green and modified dulcitol brilliant green agars at one, two and seven days. Salmonellae were recovered with greater frequency from tetrathionate plated to modified dulcitol brilliant green than the other combinations.     

Salmonella gona infection in sheep

A field outbreak of salmonellosis due to Salmonella agona in sheep and some subsequent experimental work is described. While the field outbreak in pregnant ewes and neonatal lambs caused severe losses the experimental disease in non-pregnant sheep was mild and transient. S agona was not isolated from the carcases of the experimental sheep killed after 28 days post infection but it persisted for 69 days in the faeces of one ewe which was kept alive for three months. Media comparisons indicated that selenite enrichment broths incubated at 43 degrees C and plated on to brilliant green agar gave the most satisfactory cultural results.     

Detection and characterization ofSalmonella typhimurium from a dairy herd in North Dakota

Nasal secretions, faecal samples and buffy coats were obtained from 102 cattle from a North Dakota dairy herd with a history of calf scours. Treated buffy coats, faecal samples and nasal secretions were inoculated into tetrathionate broth (TB), incubated at 37°C overnight, and plated onto brilliant green agar medium with novobiocin (BGAN). The TB was left at room temperature for 5 days and then used to inoculate fresh TB. The fresh TB was incubated at 37°C over night and plated onto BGAN medium. All the plates were incubated at 37°C over night and observed forSalmonella-like growth. Suspect colonies were further tested andSalmonella isolates were serotyped by the National Veterinary Services laboratory. Twenty-two of the 36 calves sampled harbouredS. typhimurium in their faeces, but no samples from cows were positive. NoSalmonella were isolated from the buffy coats, but 4 calves were shown to haveSalmonella in their nasal secretions. Extended enrichment of the faecal cultures in TB resulted in a significant increase inSalmonella isolations, although 2 samples were positive following the initial enrichment period and not after secondary enrichment. The typicalSalmonella isolate detected from this herd contained a transmissible R-plasmid encoding resistance to tetracycline, kanamycin, sulphisoxazole and ampicillin. This study confirmed that delayed secondary enrichment in TB is superior to primary enrichment for detection ofSalmonella from cattle.Abbreviations Ap ampicillin - BGAN brilliant green agar with novobiocin - Cm chloramphenicol - DSE delayed secondary enrichment - Gn gentamicin - Kn kanamycin - NA nalidixic acid - NADC National Animal Disease Center in Ames, Iowa - NVSL National Veterinary Services Laboratory in Ames, Iowa - PB polymyxin B - PE primary enrichment - Sm streptomycin - Su sulphisoxazole - TB tetrathionate broth - Tc tetracycline - TSI triple sugar iron agar     

Estimation of the diagnostic accuracy of the invA-gene-based PCR technique and a bacteriological culture for the detection of Salmonella spp. in caecal content from slaughtered pigs using Bayesian analysis

The goal of this study was to estimate the accuracy of the invA-gene-based polymerase chain reaction (PCR) and a culture technique based on pre-enrichment with buffered peptone water, three selective enrichment media (selenite, tetrathionate and Rappaport-Vassiliadis broths) and four selective, solid media (Xylose-Lysine-Tergitol-4, Salmonella/Shigella, Hekton-Enteric and MacConkey), for the detection of Salmonella organisms from caecal samples from slaughter pigs. For this purpose a latent-class (Bayesian) approach was used. Two hundred and three slaughtered pigs were used after grouping them into two groups of 96 and 107 animals. Sensitivity (Se) was estimated to be 56% (95% probability interval 40, 76) for culture and 91% (81, 97) for PCR. The specificity (Sp) of the PCR was 88% (80, 95) while the Sp of the culture had been considered 100% in the statistical analysis as all culture-positive samples were confirmed by serotyping. PCR Se was not affected by the Salmonella serotypes present in the samples analysed. Accordingly, a minimum of 25.5% of the pigs was estimated to harbour Salmonella organisms in their faeces. It was concluded that bacteriology on caecal samples alone was a poor diagnostic method, and that the PCR method could be considered a cost-effective alternative to culture in Salmonella monitoring programmes. However, given the moderate Sp of this molecular technique, PCR-positive samples should be further confirmed through bacteriology.     

Evaluation of plating media for recovering Salmonella from thermally treated egg albumen

Salmonella spp. present in pasteurized egg albumen are often difficult to recover by direct plating because of thermal injury and the presence of innate iron binding and other antimicrobials in egg white. The literature has reported a multiplicity of selective and nonselective media used to recover heat-injured Salmonella, to measure the proportion of injured cells, or both. This study compared the proficiency of selective and nonselective plating media for supporting colony development or for assessing bacterial injury of heat-stressed Salmonella from egg albumen. A 6-strain composite of Salmonella was added to albumen (pH 9.0), heated at 53.3°C for 3.1 min, and plated on 26 nonselective and 22 selective media. Recovery of heat-injured salmonellae varied little (≤0.52 log cfu/mL) among the nonselective media tryptic soy agar, plate count agar, dextrose tryptone agar, or brain heart infusion agar, regardless of the manufacturer. Selective media that were optimal for recovery of salmonellae from albumen included 3 brilliant green agars, Levine eosin methylene blue agar, and bismuth sulfite agar, which recovered more cells (P ≤ 0.05) than selenite-cystine, tetrathionate, xylose-lysine-tergitol-4, xylose lysine deoxycholate, or Rappaport-Vassiliadis agars. The results of this study may assist in choosing selective or nonselective media to maximize the recovery of Salmonella from thermally treated albumen.     

Selective effect of propolis in the isolation of Listeria monocytogenes (author's transl)]

Propolis is a substance produced by honeybees. It is inhibitory to some bacteria species, mainly Gram-positive bacteria, but less inhibitory to Listeria monocytogenes (L.m) than to the other Gram-positive bacteria tested. In order to obtain selective growth of L.m. from contaminated samples, the effect of propolis in plating media and broths on various strains of bacteria was examined. Table I shows the effect of increasing concentrations of propolis in tryptose-agar (TA). L.m. tolerated higher concentrations of propolis than Streptococcus viridans and Staphylococcus aureus. L.m. grew well in tryptosebroth (TB) that contained 0.15 mg propolis pr. ml medium, while Streptococcus viridans and Streptococcus agalactiae were completely inhibited as seen in Table II. Table III shows that when serum was added to the agar, the inhibitory effect was reduced. It can also be seen that Gram-negative bacteria grew quite well on media that contained 0.19 mg propolis pr. ml. To reduce the growth of Gram-negative bacteria, nalidixic acid was added to the medium. Table IV illustrates growth of various species of bacteria in tryptosephosphatebroth (TFB) with or without propolis and nalidixic acid. Most of the strains tested were inhibited, but Pseudomonas aeruginosa and to some extent faecal streptococci were able to grow in the medium that contained the selective substances. As a conclusion it seems that propolis may be a valuable additive to a medium for the selective isolation of L.m.     

Salmonella enterica level in French pig farms effluents: Experimental and field data

The study aimed to (1) validate the mini-MSRV-MPN method to quantify Salmonella enterica in pig slurry, (2) estimate the effect and interaction on temperature, time and initial Salmonella load on the survival of the 2 strains of Salmonella typhimurium (PF 1690 and DT104 100/706/037) during slurry storage and (3) identify Salmonella contaminated finishing pig batches and assess the level of contamination of their slurry. The mini-MSRV-MPN method was compared to direct isolations on brilliant green agar supplemented with rifampicin to quantify Salmonella in pig slurry. Doelhert uniform shell design was used to study the effect of different parameters on the survival of the 2 strains of Salmonella in pig slurry. Environmental samples of faecal material and a sample of the slurry of 50 batches of finishing pigs were analysed by bacteriological classical method to identify Salmonella. Quantification was performed on pools of faeces and in slurry using the mini-MSRV-MPN technique. This method proved to be suited to quantify Salmonella in pig slurry. Temperature, time of slurry storage and their interaction influenced Salmonella decrease. 12 batches of pigs tested Salmonella positive. Quantification was possible in 5 batches of faecal samples (2.4–350 MPN g^− 1 of Salmonella). Quantification was achieved in 2 out of 6 positive samples of slurry (1.6 and 110 MPN mL^− 1 of Salmonella).     

Isolation of Salmonella organisms from the mesenteric lymph nodes of horses at necropsy.

OBJECTIVES: To determine the prevalence of Salmonella infections in horses at necropsy. DESIGN: Cross-sectional prevalence survey. ANIMALS: 102 horses. PROCEDURE: Mesenteric lymph nodes were collected from horses that were necropsied. Horses had died or were euthanatized because of severe disease or at the request of the owner. Twenty-eight of the horses were racehorses euthantized following acute catastrophic injuries on the racetrack. Mesenteric lymph nodes were submitted for Salmonella culture via direct plating of tissue specimens on MacConkey agar and by use of 4 enrichment culture techniques that used tetrathionate and selenite enrichment broth and brilliant green and Salmonella-Shigella selective plating media. RESULTS: Salmonella typhimurium was isolated from the mesenteric lymph nodes of 2 foals (2/102, 1.96% of the horses). Salmonella organisms were not isolated from the mesenteric lymph nodes of adult horses. CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of Salmonella infections in horses of our study (1.96%) suggests that the results of cross-sectional surveys, using bacteriologic culture to determine prevalence of Salmonella infection, should be interpreted with caution. Prevalence of Salmonella infections determined in a single facility may not reflect the prevalence of Salmonella-infected horses in the general population; furthermore, obtaining a Salmonella isolate from a horse does not establish that the horse is a chronic Salmonella carrier.     

Comparison of bacterial culture and real-time PCR for the detection of Salmonella in grow-finish pigs in Western Canada using a Bayesian approach

The study objective was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) and a culture protocol used to detect Salmonella in the faeces of grow-finish pigs using a Bayesian approach. The RT-PCR was invA-gene-based assay, while the culture protocol included pre-enrichment in buffered peptone water, selective enrichment in tetrathionate and Rappaport-Vassiliadis broths, and isolation on semi-solid (modified semi-solid RV) or solid (XLT4, Rambach) agar plates. Bayesian analysis was performed using a two-test, two-population model with dependence between culture and RT-PCR and compared to a second model with conditional independence between these two tests. Two hundred and ninety three individual faecal and 294 pooled pen samples from grow-finish pig collected from 10 farms were tested and results were divided into two groups according to herd size (five herds <250 sows, five herds with >400 sows). In the dependence model, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively. Culture Se was 92% (75, 99), while culture Sp was considered 100% as all culture-positive samples were confirmed by serotyping. In the conditional independence model, RT-PCR Se and Sp, and culture Se, were 96% (93, 98), 99% (98, 100) and 97% (94, 100), respectively. The dependence model resulted in posterior estimates of Se that were lower and with broader probability intervals than the independence model, indicating that when RT-PCR and culture are evaluated relative to each other, the correlation between these tests is an important source of bias and should be adjusted for during analysis. The RT-PCR evaluated in this study performed almost comparably to culture; given the cost savings associated with using this test and more timely results, the RT-PCR may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low.     

Antibacterial activity of formic and propionic acids in the diet of hens on salmonellas in the crop

1. The inclusion of formic and propionic acids in the form of Bio‐Add to the food of hens made no difference to die pH of the intestinal tract, but resulted in higher concentrations of these acids in the contents of die crop and gizzard.

2. Organic acids in the crop contents were bactericidal for Salmonella serotype Enteritidis PT4 in vitro, and also caused sub‐lethal damage because fewer cells were recovered on selective salmonella media (brilliant green phenol red agar) than on non‐selective media (nutrient agar).

3. Inclusion of Bio‐Add in the food at 12g/kg may reduce the number of lactic acid‐producing bacteria in the crop, and hence the amount of naturally produced organic acids.     

Choice of the optimal method for the isolation of Salmonella from meat- and bone powder designed for industrial feed mixtures

The purpose of this paper was (1) comparison of four multi-step methods used for Salmonella isolation from meat- and bone powder; (2) elaboration of a new sensitive method of Salmonella isolation from this product; (3) evaluation of a new solid selective medium (BxLH) described by the authors for Salmonella isolation in comparison to brilliant green agar (BGE) according to Edel and Kampelmacher. The study was carried out on 173 meat- and bone powder samples naturally contaminated with Salmonella oranienburg. The samples were examined for the Salmonella presence by means of four compared methods (Methods 1 to 4). The new method of isolation proposed by the authors (Method 3) proved to be the most effective among all compared for Salmonella recovery. It seems that the superiority of Method 3 in comparison to the other applied was a result of, (1) homogenization of the investigated samples in distilled water before preincubation followed by maintenance at room temperature for 2–4 h; (2) the use of a new selective BxLH agar; (3) the use of multiple plating after selective enrichment. The BxLH medium was shown to be more suitable for Salmonella isolation than BGE agar because of more efficient inhibition of other bacterial growth with simultaneously abundant growth of the Salmonella organisms. The additional advantage offered by BxLH agar was the fact that lactose-positive salmonellas grow as typical representatives of this genus. This enables their identification, in contrast to the situation when lactose containing media are used, where the colonies of such salmonellas are similar to the colonies of, for example, Escherichia coli.     

Comparison of GN Hajna and tetrathionate as initial enrichment for salmonellae recovery from swine lymph nodes and cecal contents collected at slaughter.

An epidemiologic survey was conducted to determine the prevalence of salmonellae in swine from 5 farms of an integrated swine operation. The purpose of this study was to evaluate the recovery efficiencies for salmonellae from swine lymph nodes and cecal contents when GN Hajna and tetrathionate were compared as initial enrichments. Salmonellae were isolated from 61% of 645 pigs at slaughter; 324 positive cultures were from lymph nodes, and 224 were from cecal contents. Frequently, pigs had salmonellae isolated from both the lymph nodes and cecal contents. Total isolations, regardless of source, were similar for GN Hajna (247) and tetrathionate (301). There was no difference (P > 0.05) in the number of isolations from lymph nodes when GN Hajna enrichment was compared with tetrathionate enrichment (174 vs. 150). However, there was a significant (P < 0.05) advantage of utilizing tetrathionate when compared with GN Hajna for isolations from cecal contents (151 vs. 73).     

Sensitivity of methods for the isolation of Escherichia coli O157 from naturally infected bovine faeces

At present, no standard protocol has been described to detect the presence of Escherichia coli O157 in cattle faeces. Therefore, the sensitivity of 26 different isolation methods was determined in order to recommend a method of choice. Faeces samples from 17 different beef cattle at a farm previously found positive for E. coli O157 were subdivided into a total of 40 samples. It was not known whether the 17 cattle shed E. coli O157 at the time of sampling. At another farm where cattle have been found negative for E. coli O157 on different occasions, five faeces samples were collected. Two methods yielded the highest sensitivity (74%): 6h enrichment in modified tryptone soya broth supplemented with novobiocin (mTSBn) followed by an immunomagnetic separation (IMS) with (i) Dynal beads or (ii) Captivate beads and selective plating on Rainbow agar (RA) plates. Enrichment for 6h was significantly better than 24h enrichment. Only after 24h, buffered peptone water (BPw) was significantly better than mTSBn. A sensitivity of 82% was obtained only when the two most sensitive tests were done simultaneously. Because none of the tests gave 100% sensitivity, it can be concluded that isolation rates of E. coli O157 from bovine faeces using only one of the tested procedures results in an underestimation of the incidence of E. coli O157 in cattle. Performing more than one test on the samples must be considered.     

Application of different methods for the diagnosis of paratuberculosis in a dairy cattle herd in Argentina

Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.     

Evaluation of selective media for bifidobacteria in poultry and rabbit caecal samples.

Five media were evaluated to determine their selectivity for Bifidobacterium sp. in hen and rabbit caecal samples. The colonies arising on the plates inoculated with the caecal samples were Gram stained and screened for the presence of fructose-6-phosphate phosphoketolase activity. Rogosa agar modified by the addition of cysteine-hydrochloride (0.05% w/v), Beeren's agar (with 5 ml/l of propionic acid as a selective agent), BS 2 agar (containing per one litre sodium propionate 15 g, lithium chloride 3 g, paromomycin sulphate 50 mg, neomycin sulphate 200 mg), and Wilkins-Chalgren agar (MW) modified by the addition of acetic acid (1 ml/l) and mupirocin (100 mg/l) were selective for Bifidobacterium sp. from rabbit caecal samples. In contrast, only MW medium was suitable for the isolation and enumeration of bifidobacteria in hen caecal samples. In conclusion, the results suggest that MW agar showed the greatest selectivity. A further advantage of this medium is its case of preparation. Therefore this agar could contribute to the study of the effects of the ingestion both probiotics and prebiotics. Finally, it could be noted that the bifidobacteria selective media should be chosen in respect of the animal species origin of the sample tested.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Comparison of an antigen-capture enzyme-linked immunosorbent assay with bacterial culture for detection of Salmonella in poultry-hatchery environmental samples

An antigen-capture, monoclonal-antibody-based enzyme-linked immunoassay (ELISA) that detects a broad range of Salmonella serovars in various serogroups was developed and compared with standard culture procedures for detection of Salmonella in 1055 field samples collected from poultry-hatchery environments. The diagnostic sensitivity of the ELISA relative to culture was 99.9% and the diagnostic specificity 99.6%. The extensive culture procedure included nonselective enrichment (NSE) as well as primary selective enrichment (PSE) and delayed secondary enrichment (DSE) with Hajna tetrathionate (TT) and Rappaport–Vassiliadis (RV) selective-enrichment broths. Significantly more Salmonella-positive samples were detected by ELISA and culture at the DSE stage than at the NSE and PSE stages (P < 0.05). Significantly more RV than TT broths were positive for Salmonella by culture and ELISA by the DSE stage (P < 0.05). This ELISA procedure could be a reliable screening test for the detection of Salmonella in hatchery samples.     

Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.     

Evaluation of Selective Media for Bifidobacteria in Poultry and Rabbit Caecal Samples

Five media were evaluated to determine their selectivity for Bifidobacterium sp. in hen and rabbit caecal samples. The colonies arising on the plates inoculated with the caecal samples were Gram stained and screened for the presence of fructose-6-phosphate phosphoketolase activity. Rogosa agar modified by the addition of cysteine-hydrochloride (0.05 % w/v), Beeren’s agar (with 5 ml/l of propionic acid as a selective agent), BS 2 agar (containing per one litre sodium propionate 15 g, lithium chloride 3 g, paromomycin sulphate 50 mg, neomycin sulphate 200 mg), and Wilkins-Chalgren agar (MW) modified by the addition of acetic acid (1 ml/l) and mupirocin (100 mg/l) were selective for Bifidobacterium sp. from rabbit caecal samples. In contrast, only MW medium was suitable for the isolation and enumeration of bifidobacteria in hen caecal samples. In conclusion, the results suggest that MW agar showed the greatest selectivity. A further advantage of this medium is its ease of preparation. Therefore this agar could contribute to the study of the effects of the ingestion both probiotics and prebiotics. Finally, it could be noted that the bifidobacteria selective media should be chosen in respect of the animal species origin of the sample tested.