Four experiments were conducted to test the hypotheses. In experiment 1, dogs were trained to identify Staph. aureus on agar plates; in experiments 2 and 3, dogs identified Staph. aureus inoculated in milk; and, in experiment 4, dogs identified Staph. aureus in milk samples from cows with clinical mastitis.
Pathogens for training and testing were isolated from milk samples from dairy cows with signs of clinical mastitis; that is, change in milk quality and swelling of the udder. Mastitis samples were sent to a commercial veterinary laboratory (Laboklin GmbH & Co. KG, Bad Kissingen, Germany; accreditation number D-Pl-13186-01-00). Milk samples were cultivated on Columbia Agar with 5% sheep blood; Columbia CNA agar, Endo agar, and Saboraud agar with gentamicin and chloramphenicol (all from Becton Dickinson, Heidelberg, Germany). Columbia 5% blood is not very selective; Pseudomonas
, aerobic gram-negative as well as gram-positive pathogens, and yeast can be cultivated; Columbia CNA is selective for gram positive; Endo agar is selective for Enterobacteriaceae
and coliforms; and Saboraud agar is selective for fungi (Candida
) or dermatophytes. Sampling amount per plate was 10 µL. Additionally, 1 mL of milk samples was placed into thioglycolate broth (in-house production) for enrichment. Plates for bacterial culture were incubated at 36 ± 1°C for 18 to 24 h. Enrichment broth was incubated at 36 ± 1°C for 18 to 24 h and was then plated on Columbia agar with 5% sheep blood and Endo agar for a second incubation at 36 ± 1°C for 18 to 24 h. Agar for yeast isolation was incubated at 36 ± 1°C. A first reading for yeast growth was done after 48 h and a final reading after 1 wk. After bacteriological growth occurred, pathogens were identified first by colony morphology, Gram strain, and biochemical reactions (catalase, hyaluronidase, oxidase, and esculin reaction). Then, identification was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF; Shimadzu, Duisburg, Germany;
Barreiro et al., 2010
- Barreiro J.R.
- Ferreira C.R.
- Sanvido G.B.
- Kostrzewa M.
- Maier T.
- Wegemann B.
- Böttcher V.
- Eberlin M.N.
- Dos Santos M.V.
Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
). Until use, pathogens were stored at −20°C in cryotubes (Pro-Lab Diagnostics, Richmond Hill, ON, Canada). Bacteria were isolated from 85 milk samples, and we received 35 isolates of Staph. aureus
and 10 isolates each of E. coli
, Strep. uberis
, Strep. dysgalactiae
, P. aeruginosa
, and C. albicans
For training and testing, pathogens were cultivated again on Columbia agar with 5% sheep blood for 24 h at 36 ± 1°C. For early trainings steps, some blank agar plates were placed in the same incubator for the same incubation period. Twelve hours before dog training, a sterile cotton swab (5 × 5 cm, Fuhrmann Verbandstoffe GmbH, Much, Germany) was placed in the top of the agar plate to absorb the bacterial odor or, for the negative control, the odor of the blank agar plate at 36 ± 1°C. The swab had no direct contact with the blood agar. The swabs were then placed in a plastic container (1 L, Jockey Plastik, Wippeführt, Germany) with a perforated lid. To avoid cross contamination, a container was used for one type of pathogen only. For training, the containers including the swab were used for a maximum of 2 wk. Between training sessions, they were kept refrigerated at 6°C. For dog testing, new samples were produced by culturing pathogens on blood agar and exposing cotton swabs to the culture odor for 12 h. The swabs were placed into new containers.
For experiment 2, bacterial colonies from same species as in experiment 1 were captured with an inoculation loop from the plate (1,000 cfu/mL) and dissolved in 2 mL of fresh bulk tank milk with SCC <120,000/mL. Of this milk, 1 mL was pipetted onto a cotton swab and placed in the bottom of the plastic container. In experiments 2 and 3, bacterial concentrations were 102 and 108 cfu/mL, respectively.
Bacterial cell pellets were produced for experiment 3 by incubating 20 strains of Staph. aureus, Strep. uberis, and Enterococcus spp. each (all isolated from milk samples of cows with clinical mastitis) for 24 h at 36 ± 1°C in 10 mL of brain-heart broth. From this broth, 100 µL was transferred into 10 mL of sterile brain-heart broth and again incubated at 24 h at 36 ± 1°C under aerobic conditions. After incubation, 2 mL of broth of each strain (approximately 2 × 108 cfu/mL) was transferred into a reaction tube (Eppendorf, Wezzlingen-Berzdorf, Germany) and centrifuged (600 × g) for 15 min. The cell pellet was solved in 100 µL of H2O-free glycerin and the cell glycerin suspension stored in cryotubes at −20°C. For dog training and testing, the cell glycerin suspension was solved in 2 mL of fresh bulk tank milk (SCC <120,000/mL; total plate count <10,000 cfu) and 1 mL was pipetted on a cotton swab. We used bulk milk instead of sterile milk in order to train the dogs to ignore low concentrations of ubiquitous bacteria. The cotton swabs were placed into plastic containers as described above.
For experiment 4, milk samples from cows with signs of clinical mastitis (see above) were used. Milk sample were sent to the Freie Universität Berlin from various dairy farms in Brandenburg, Germany. Aliquots of 10 mL were shipped to 3 mastitis diagnostic laboratories and 1 aliquot was refrigerated at −20°C. Samples were only included in the experiment if at least 2 laboratories made the same bacteriological diagnosis. Samples with Staph. aureus (n = 11), E. coli (n = 5), Strep. uberis (n = 8), and Trueperella pyogenes (n = 1) were included. For dog testing, 1 mL of the milk sample was pipetted on a cotton swab and placed into a new plastic container. Up to 10 test samples were generated from 1 aliquot.
In experiments 1 and 2, dogs had to indicate 1 container holding a swab with Staph. aureus odor out of 10 containers holding swabs with odor of 1 of 4 other bacterial species and 1 yeast. The composition of the 9 containers was random. Randomization both for samples within a trial and the order of trials were generated with the random number function of Excel (Microsoft Corp., Redmond, WA).
For experiments 1 and 2, the prevalences of different pathogens within the 10 containers were Staph. aureus (10%); E. coli (5%), Strep. uberis (27%), Strep. dysgalactiae (28%), P. aeruginosa (20%), and C. albicans (10%). Therefore, the chance of identifying the correct container by chance was 1:10. Each dog performed 10 trials with 10 containers each, resulting in 100 samples searched per dog. The protocol was modified for experiment 3. The number of containers was reduced to 7 per trial. Dogs performed 15 trials for a total of 105 samples searched. In experiment 3, the containers to discriminate were holding swabs with odor of Strep. uberis (23%), Enterococcus (24%), or bulk milk without bacteria (39%). Two trials served as negative controls without a container with Staph. aureus, and 2 trials were done with 2 containers with Staph. aureus. The order of trials was randomized, and the prevalence of Staph. aureus was 15%.
In the fourth experiment, 10 trials were performed with 7 containers each (i.e., total of 70 samples). The container to differentiate included Strep. uberis (31%), Strep. dysgalactiae (3%), E. coli (10%), T. pyogenes (2%), Enterococcus (16%), and bulk milk (25%). The prevalence of Staph. aureus in this test was 13%.
Containers were positioned in a circle with a radius of approximately 80 cm, with each container approximately 70 cm apart. For every trial, unused containers were included. Dogs, handlers, and any other persons in the experimental room were blinded to the position of the sample to avoid hidden clues. The test was videotaped and transferred to another room where the experimenter was seated. Dog handlers were instructed to make sure that the dogs examined every container, even if the dog had already indicated one as being positive. After the dog had searched each container, the dog handler announced the number of the positive container to the experimenter via video transmission. The experimenter gave feedback to the dog handler so that the dog was eventually rewarded. Indications were documented as correct positive, correct negative, false positive, or false negative.
Test characteristics including number of dogs, samples, trials, and odorant used in each experiment are summarized in Table 3
Table 3Test characteristics of dogs for detecting Staphylococcus aureus in experiment 1 (blood agar), experiment 2 (inoculated milk, 103 cfu/mL), experiment 3 (inoculated milk, 108 cfu/mL), and experiment 4 (mastitis milk)