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s13567 017 0418 5 PDF
s13567 017 0418 5 PDF
Abstract
Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health
and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic
losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal
infection diseases and control management in farms and field conditions. Current techniques employed to diagnose
pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as
enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to dis‑
tinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR)
and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease
in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments,
being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care
biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we pro‑
vide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different
sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at
development level some are validated according to standards of the World Organization for Animal Health and are
commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sens‑
ing systems for implementation in field condition are included in this review.
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publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Vidic et al. Vet Res (2017) 48:11 Page 2 of 22
itself. The extensive antibiotic use in animal production Eltzov and Marks [20, 21] have proposed a point-of-
has incidence on the spread of multidrug-resistant bac‑ care detection system based on stacked paper mem‑
teria and on antibiotic-resistant infections in humans. In branes that quantify E. coli within less than 5 min.
most developed countries, serious consideration is being When liquid samples containing bacteria are added
undertaken to regulate and severely restrict the use of onto the bottom membrane layer, the liquid starts to
antibiotics in animal production. The rapid and accurate migrate from the lower to the upper layers. As each
diagnostic of virulent E. coli strains is vital for assessing layer becomes wet, E. coli cells from contaminated
the antibiotic resistance information, for administration samples push through to the next membrane layers.
of appropriate treatment and thus for avoiding useless During migration, the bacterial cells bind with the spe‑
antibiotics utilization. cific antibody, itself conjugated with horseradish per‑
The main issue in E. coli diagnostic is to distin‑ oxidase (HRP) enzyme to allow signal measurement.
guish between the closely related strains in order to In target-free samples, the HRP-labeled anti-E. coli
distinguish between pathogenic and non-pathogenic antibodies from migrating are stopped by previously
species. Commonly used methods for E. coli detec‑ immobilized target bacteria on the capture layer. The
tion and identification include culture, fermentation, upper-most layer contains the HRP enzymatic sub‑
enzyme linked immunosorbent and PCR assay. These strate producing a measurable signal only with sam‑
methods show disadvantages in terms of long identi‑ ples containing target E. coli. This portable and rapid
fication time (typically few days), high labor and rea‑ immunoassay was shown to have around 1000-folds
gent cost [9, 10]. Novel biosensors based on specific higher sensitivity than ELISA since only 100 cells/mL
biochemical recognition strategies have been reported were successfully quantified.
for rapid and specific E. coli detection as those based
on PCR [11], quartz crystal microbalance system [12], 4 Detection of avian influenza viruses
surface plasmon resonance [13], chemiluminescence Aquatic birds constitute the main reservoir for avian
[14] and electrochemistry [15–17]. The biosensors for influenza viruses (AIVs) [22]. These viruses represent a
E. coli diagnostics are elaborated to assure two major global threat to animal health and international poultry
steps: the capture of target bacteria from the biological industry. Particularly high concern represents pandemic
or environmental samples and subsequent identifica‑ emergences which may cause enormous economic losses.
tion of captured bacterial sub-type. A huge variety of AIVs are divided into low and highly pathogenic strains
high affinity antibodies against E. coli that bind to sur‑ regarding their pathogenicity for chicken. The highly
face/flagella proteins is available, as well as appropri‑ pathogenic AIV (HPAIV) spreads rapidly in domestic
ate labels that can be employed for the amplification of poultry and result in high mortality rate. The low path‑
detectable signal (as enzymes, biofunctionalized nano‑ ogenic AIV (LPAIV) may cause mild respiratory or gas‑
particles or fluorophores). These allow development of trointestinal symptoms, but usually without any signs of
various sandwich-type immunosensors and immuno‑ illness. However, LPAIV strains may acquire high patho‑
assays for rapid detection of E. coli in infected samples. genicity during multiple infections in a chicken popula‑
Immunosensor developed by Jaffrezic-Renault et al. tion [23].
[18] is based on the following strategy: addressable Based on their antigenic specificity, Influenza A
magnetic nanoparticles coupled with anti-LPS anti‑ viruses infecting birds, are divided into 16 hemagglu‑
bodies were used for the generic capture of Gram- tinins (HA, H1-16) and 9 neuraminidases (NA, N1-9)
negative bacteria onto the graphite ink electrode. The subtypes. To date, naturally occurred HPAIV that pro‑
use of immunomagnetic beads allow detection of a duce high mortality in chickens, turkeys and other
biomarker contained in complex sample matrices. birds of economic importance have been restricted
Conductometric measurements allowed real-time, only to H5, H7 and H9 subtypes. Of these viruses, some
sensitive detection of E. coli or Serratia marcescens H5N1, H7N2 and H7N7 viruses have shown to cause up
cultures from 1 to 103 CFU/mL. The conductometric to 100% mortality with 48 h in infected chickens. Many
immunosensor permitted also the direct detection of outbreaks of HPAIV occurred in domestic poultry
10 to 103 CFU/mL of Pseudomonas aeruginosa and production systems as it was the case with the highly
Acinetobacter baumannii strains that were undetect‑ pathogenic avian influenza H5N8 virus since 2014
able using standard immunoblot methods. Gram-pos‑ [24]. H5N1 viruses have affected the poultry indus‑
itive bacteria such as Staphylococcus epidermidis were try in numerous countries for the past 15 years and
not detected indicating the specificity of detection have resulted in the deaths of millions of birds. Con‑
[19]. sequently, a global influenza virological surveillance
Vidic et al. Vet Res (2017) 48:11 Page 4 of 22
in poultry populations and migratory birds is recom‑ Influenza hemagglutinin (HA) surface protein binds to
mended by both by World Organization for Animal sialic acid glycan residues (α-2,6 and α-2,3 sialic acids)
Health (OIE) and World Health Organization (WHO) on the surface of human and bird cells. HAs expressed
[25, 26]. Availability of a swift and sensitive AIV diag‑ by AIV bind specifically to the α-2,3 sialic acid which is
nosis tool that allows virus strain identification will be preferentially expressed in the intestine of water-flow,
highly useful for disease surveillance as well as for opti‑ while HA proteins from human-adapted viruses prefer to
mizing biosecurity measures on farms. bind to the α-2,6 sialic acid glycan, mainly expressed on
Avian influenza virus belong to the Influenza A virus the epithelial cells of the human upper respiratory tract.
family Orthomyxoviridae. The genome of AIV is consti‑ Based on this difference in sialic acid linkages, biosensors
tuted by eight segments of negative-stranded RNA. Ten were developed for detecting and differentiating between
main proteins are encoded by viral genome: the poly‑ avian and human influenza viruses. Glycan-immobilized
merase basic protein 1 (PB1) and 2 (PB2), hemagglutinin field effect transistor biosensor was shown to detect and
(HA), nucleoprotein (NP), neuraminidase (NA), the poly‑ discriminate between human (H1) and avian (H5) influ‑
merase acidic protein (PA), matrix proteins 1 and 2, and enza viruses at attomolar-level sensitivity [37]. Surface
non-structural protein 1 and 2 [27]. Some other influenza plasmon resonance [38, 39], optical waveguides [40] and
A virus proteins were found as PB1-F2, PB1-N40, PA-X, quartz crystal microbalance [41] were also employed in
NEP, M42, PA-N155, PA-N182 [28–31]. Potentially all glycan-based AIV detection. HA binding to sialic acid
these viral proteins, as well as their corresponding cod‑ attached to gold nanoparticles allows detection of influ‑
ing RNA sequences, are AIV biomarkers. Nevertheless, enza virus in solution without any pretreatment or ampli‑
frequent mutations in the AIV genome lead to changes in fication step [42]. This reaction may produce a signal in
antigenic properties which restrained the choice of bio‑ colorimetric test that is linearly proportional to the virus
markers. HA, M2 and NA are the dominant targets for titer (Figure 2). A recent study reported that glycan can
the host antibody response, anti-viral drug development be printed onto glass slides to generate microarray [43].
and represent also the targets in diagnostics assays. The microarray was shown to capture different strains of
A broad of traditional serological diagnostic methods influenza virus with a clinical relevant limit of detection
like haemagglutination (HA) test, Hemagglutination- (ten plaque forming units) allowing virus diagnostics.
inhibition (HI) test, neutralization (NT) and ELISA test Most of portable antibody-based methods for influenza
are available as well as those based on virus propagation virus diagnosis, including those being commercialized,
and isolation from cell culture or embryonated chicken are lateral flow tests [44]. Lateral flow tests may detect a
eggs. These methods are effective and sensitive but they specific influenza biomarker in a complex media thanks
require relatively important amount of virus particles and to their chromatography component consisting of series
special sample collection and handling tend to be costly, of pads that transport samples spontaneously during the
labor intensive and time-consuming. Moreover, HPAIV, test (Figure 3).
as H5N1, are quite virulent for eggs, killing them quickly. Samples to be detected for influenza virus by lateral
This makes standard egg culture amplification procedure flow test are solubilized in a detergent containing solu‑
quite difficult. Alternative molecular methods based on tion and deposited on the sample pad. This step initi‑
PCR and RT-PCR are more sensitive but need extracted ates lateral flow of sample components. In the first step
genetic material and require the use of equipment which of detection a specific influenza virus biomarker is rec‑
is available only in diagnostic or scientific laboratories. ognized by antibodies carried by gold-nanoparticles
In consequence, on-site detection of avian influenza pre-adsorbed on the conjugate pad. Usually antibodies
viruses is rare until now for both early diagnostics and raised against preserved epitopes in viral nucleoprotein
monitoring [32–34]. Similarly, a variety of different sero‑ are used for this step. Influenza viruses complexed with
logical tests that detect the response of the infected host immune-gold nanoparticles reach the test lines. Two
are established but they are not adapted to strains which test lines with pre-immobilized antibodies that specifi‑
have a pandemic potential. Moreover they are not robust cally recognize either influenza A or B virus bind to dif‑
and are difficult to be employed in point-of-care condi‑ ferent epitopes of the virus and, in that way accumulate
tion [35]. There are several bedside tests which allow a immune-gold nanoparticles carrying the viruses. The
relatively quick (up to 30 min) detection of viral antigens accumulation of gold nanoparticles results in an appear‑
[36]. Unfortunately, these tests provide low sensitivity ance of a visible red line. Usually, antibodies recognizing
and often produce false negative results, especially dur‑ specific epitopes in HA proteins are used for the test line.
ing later stages of the disease development. Low sensitiv‑ Finally, non-bound immune-gold nanoparticles arrive at
ity is the main reason why these tests are rarely used in the control line which harbors the secondary antibody,
routine diagnostics of influenza virus. showing the second visible red line. In the absence of
Vidic et al. Vet Res (2017) 48:11 Page 5 of 22
Figure 2 Colorimetric sensor for detection of influenza A virus. Sialic-mediated colorimetric detection of Influenza virus. Gold nanoparticles
are stabilized with sialic to specifically bind HA protein on Influenza virus surface. Sialic-acid gold nanoparticles alone show the absorbance at
510 nm, while virus-bound nanoparticles absorbed at 600–610 nm. This allows label-free colorimetric readout for virus detection. Cartoon adapted
from [42].
Figure 3 Lateral flow strips for detection of influenza A and B viruses. Schematic presentations of a lateral flow tests realized on a nitrocellu‑
lose strips with immobilized antibodies against influenza virus A and B. A sample containing influenza virus flow by capillarity from the sample pad
to bind test and control lines. In contrast, a sample without target virus flows from the sample application pad and binds only to the control line.
Vidic et al. Vet Res (2017) 48:11 Page 6 of 22
viral particles in the sample, the immune-gold nanopar‑ subtyping. Some of them combine lateral flow test after
ticles flow alone and bind only to the control line. Thus, specific labeled primer-set amplification to increase the
two colored lines stand for positive result while a single sensitivity of lateral flow system. It will be interesting to
colored line corresponds to negative result (Figure 3). In have efficient integrations of available and stable ampli‑
most cases antibodies can distinguish between influenza fication and detection assays, for instance, the combi‑
A and B viruses but still not able to differentiate within nation of PCR/isothermal amplification and biosensor
subtypes and, thus, between LPAIV and HPAIV stains. technology. For instance, a portable and rapid assay
In contrast, aptamers generated against specific AIVs or for the detection of the emerging avian influenza A
specific RNA/DNA primers immobilized on the test lines (H7N9) virus has been developed in a form of diagnos‑
allow AIV sub-typing. tics suitcase. The test is based on reverse transcription
The extreme simplicity to use, efficiency, reliability recombinase polymerase amplification (RT-RPA) assay,
and label-free detection characterized lateral flow tests. isothermal amplification and a fluorescence detection
However, their sensitivity is sufficient for detecting pro‑ machine. The workflow consisted of viral nucleic acid
teins but has to be improved to allow detection of viral extraction, isothermal target nucleic acid fragment
particles in a complex medium as fecal swab sample. It amplification and fluorescence detection [48]. Portable
was shown that silver nanoparticles added to the test nucleic acid thermocyclers including PCR and isother‑
line can amplify the colorimetric signal up to 1000-fold mal amplification has become applicable for rapid on-
[45]. A limit of detection 0.09 ng/mL was estimated for site viral nucleic acid detection despite of the need of
AIV detection by lateral flow test amplified with quan‑ genetic materials isolation procedure. The combination
tum dots [46]. For comparison, this test showed 100- of nucleic acid extraction, amplification and detection
fold higher sensitivity than ELISA performed with the methods can be varied for different viral strain detec‑
same antibodies. Recently, a quantum-dot based lat‑ tion (Figure 4). RPA assays for AIV detection can be
eral-flow immunoassay system was proposed for quan‑ combined with lateral flow test. This combination pro‑
titative detection of influenza A virus subtypes H5 and vides a qualitative but not quantitative result (Figure 5).
H9 [47]. The modification of specific antibodies with Recently, an electrochemical immunosensor based on a
quantum-dot amplified signal and permitted a quanti‑ specific anti-M1 antibody was shown to detect all sero‑
tative read-out of the virus detection under an ultravio‑ types of influenza A virus [49] with sensitivity similar to
let lamp. classical molecular methods (80–100 × 103 PFU/mL).
Several commercial PCR, RT-qPCR, RT-RPA kits A lower effective limit of 1 × 103 PFU/mL was achieved
and portable machines, in a mobile diagnostic suit‑ by coupling the anti-M1 monoclonal antibody to gold
case, are available on the market for AIV detection and nanoparticles in a quartz crystal microbalance assay
Figure 4 Detection of influenza virus RNA. Available combinations of portable nucleic acid extraction, amplification and detection methods for
virus detection.
Vidic et al. Vet Res (2017) 48:11 Page 7 of 22
Figure 5 Lateral flow kits for detection of influenza viruses. RT-RPA with lateral flow Influenza virus detection kits for influenza A, influenza B,
Influenza H5 and H7 subtypes. Pictures provided from Hawk Scientific Co., Ltd and GenProNex Biomedical INC. (Flu A: influenza A, Flu B: influenza B).
Figure 6 Aptamer-based detection of influenza viruses. Schematic representations of aptamer development and virus detection. Selex
procedure is applied for selection of specific aptamers. These sensing elements are immobilized on the sensor surface to bind efficiently to the viral
proteins in infected samples. The recognition signal is proceeded to provide diagnostic.
microarray utilizes a panel of primers for multiplex PCR and virus subtyping [63]. Current developments are
amplification of the HA, NA and M1 genes of influenza focused on miniaturization and automatization of micro‑
viruses. Li et al. [59] coupled a DNA microarray and mul‑ array biosensors for building portable diagnostics plat‑
tiplex reverse transcriptase PCR microarrays to provide forms for avian influenza viruses.
subtyping of influenza virus strains. Similarly, Kessler
et al. [60] developed a three-dimensional DNA flow- 5 Detection of Mycoplasma and other bovine
through biochip for typing and subtyping of influenza mastitis pathogens
viruses. Wang et al. [61] designed a microarray-based Mycoplasmas are the smallest bacterial cells that lack a
detection and genotyping of a wide range of respira‑ cell wall around their cell membrane, which makes them
tory viruses. In one another study, Townsend et al. [62] insensible to many common antibiotics. Antibiotics
described a microarray, for the relatively rapid identifica‑ help preventing some clinical signs but cannot eliminate
tion of influenza A virus subtypes H1N1, H3N2 viruses infection. Various mycoplasmae strains infect animals,
circulating in the human population as well as the highly but usually play a secondary role in infections by exac‑
pathogenic avian A/H5N1 virus that was isolated from erbating pre-existing disease. Nevertheless, Mycoplasma
poultry in Southeast Asia and that spread to Europe. This bovis (M. bovis) can play a primary role of pneumonia,
test demanded extraction and amplification of the viral mastitis or arthritis in cattle. M. bovis is considered as
RNA, and thus cannot easily be used on-site. However, one of the most pathogenic and the most frequent Myco-
the device provided the absolutely correct types and sub‑ plasma species. It is estimated that M. bovis infection
types for an average of 72% of the isolates. The authors causes €144 million in the European cattle industry [64].
emphasized that the failures were due to the nucleic Methods used for diagnosis of Mycoplasma infection
acid amplification step rather than limitations in the include bacterial culture, fluorescent antibody-based test,
microarray. serological tests and PCR. Serological tests cannot be
Although microarrays are typically used a fluores‑ applied for early diagnosis or for detection of acute infec‑
cent read-out there is a great potential of electrochemi‑ tion since serum antibody biomarkers rise at 10–14 days
cal microarrays for detection of influenza viruses. For after acute infection. The somatic cell count as a refer‑
instance, the microarray silicon chip for the detection of ence method for monitoring milk quality allows mastitis
influenza A virus based on more than 12 500 electrodes, diagnosis. This simple but labor-intensive assay implies
each carrying specific oligomers allowed the genotyping methods as microscopic analysis or cytometry for raw
and identification of HA and NA influenza A proteins milk. Both methods are slow and require well-trained
Vidic et al. Vet Res (2017) 48:11 Page 9 of 22
staff to provide result accuracy and reproducibility. Early PCR inhibitors. Various procedures are established for
diagnosis is however of the extreme importance due to DNA extraction and purification from the raw milk. Rea‑
the high costs of treatments. Some portable somatic cell gents for these additional steps are included in commer‑
counters have been validated and are used as cow-side cial kits which additionally increase reaction time and
test for mastitis control at a dairy farm. Although they are the price per analysis. Nevertheless some PCR-based kits
very simple to apply, they have low sensitivity and speci‑ allow identification of total bacterial strains causing mas‑
ficity and usually cannot identify the pathogen strain [65, titis and detection of their antibiotic resistant gens [70].
66]. Especially, the early diagnosis of bovine mastitis is Advances in the development of the nucleic acid micro‑
important regarding its huge impact on farm economics array have permitted automatization of the protocols and
due to treatment cost and reduction in milk production. development of multiplex biochips for detection of vari‑
Biosensors have been developed to detect specific ous dairy pathogens [71]. For instance, a biochip based
Mycoplasma biomarkers in a rapid and easy-to-use diag‑ on DNA amplification of genes characteristic for mastitis
nosis manner. Majority of in-field biosensors are devel‑ causing pathogens was shown to efficiently detect six other
oped to detect NAGase and haptoglobin biomarkers pathogens in addition to M. bovis in bovine milk with a
in milk samples. Expression of both bacterial proteins limit of detection of 103 CFU/mL [72]. More recently a
is characteristic for the acute phase inflammation. For test that combined a rapid PCR with a nucleic acid micro‑
instance, oxido/reduction process of 1-naphthol, which is array immunoassay was proposed [73]. This colorimetric
a substrate of NAGase can be easily detected and quanti‑ test allowed simultaneous detection and identification of
fied using carbon electrodes. Pamberton et al. [67] have six strains from four different mastitis causing pathogens
reported an electrochemical sensor based on a screen- within less than 3 h. In the microarray a set of specific anti‑
printed carbon electrode which detected NAGase pro‑ bodies that recognize tags attached to specific PCR frag‑
tein in milk samples with a limit of detection of 10 mU/ ments were immobilized by printing onto nitrocellulose
mL. Surface plasmon resonance was employed to moni‑ membrane. After PCR with tagged primers, the double-
tor binding of haemoglobin to haptoglobin immobilized tagged amplicons were captured between the antibodies
on the surface of the chip [68]. The formation of this pro‑ printed onto the nitrocellulose and carbon nanoparticles
tein complex resulted in changes in mass attached to the carrying alkaline–phosphatase. In the presence of the alka‑
chip surface which indicates mastitis. Though, when this line–phosphatase substrate, the black spots appear on the
biomarker is targeted to test milk samples false positive membrane that can be easily seen by the naked eye (Fig‑
results are obtained in some milk samples that contained ure 7). The selectivity of the test was obtained by attach‑
blood traces. ing different tags to primers specific for each pathogen of
A single-stranded DNA aptamer showing high affinity interest. This experimental approach may be integrated
and specificity against P48 protein of M. bovis has been into a self-contained, simple and disposable cassette for
used in a competitive enzyme-linked aptamer assay for point-of-care multi-pathogen molecular diagnostics.
the detection of M. bovis in sera [69]. P48 protein is an An electrochemical sensor based on electrochemical
optimal biomarker for M. bovis since it is an invariable impedance spectroscopy was developed for detection of
protein that is localized on the membrane surface of M. pathogenic Staphylococcus aureus ATCC25923 bacteria
bovis. A competitive enzyme-linked aptamer assay using by Braiek et al. [74]. A linear relationship between the
the biotin-labeled aptamer of P48 protein was applied in increment in the electron transfer resistance and the log‑
an indirect diagnostic test. The sensitivity and selectivity arithmic value of S. aureus concentration was observed
of the test were similar to commercial ELISA kits. between 10 and 106 CFU/mL. The limit of detection was
Mastitis as an inflammatory infection may be caused low as 10 CFU/mL, and the reproducibility was calcu‑
not only by Mycoplasma, but by many other bacteria. The lated to 8%. In addition, a good selectivity versus E. coli
most frequent clinical infections in dairy cattle are caused and S. epidermidis was demonstrated.
by about ten different bacterial pathogens as Staphylococ-
cus aureus, Streptococcus agalactiae, S. bovis, S. canis, E. 6 Detection of Clostridium perfringens
coli. Consequently for controlling the disease spread and Clostridium perfringens (C. perfringens) is a gram-posi‑
for targeting antimicrobial therapy a rapid strain identi‑ tive, anaerobic, fermentative, spore-forming soil bacte‑
fication and quantification of the microbial load in milk rium that may produce one or several exotoxins. At least
samples is needed. Nowadays, PCR or RT- PCR-based 17 different C. perfringens toxins have been identified
tests are employed for laboratory testing. As mentioned [75, 76], some of them being very potent causing ani‑
above, these methods are rapid and sensitive but their mal and food-borne illnesses, and are even considered
application for milk samples is sometimes impeded as to be bioweapons. C. perfringens are classified into five
milk contains calcium ions and proteinases that act as isotypes (A, B, C, D and E) regarding five major toxins
Vidic et al. Vet Res (2017) 48:11 Page 10 of 22
Figure 7 Microarray for detection of bacterial nucleic acids. Schematic representation of microarray for mastitis bacteria detection realized
on porous nitrocellulose membrane slides. Illustration adapted from Mujawar et al. [73].
they produce [alpha, iota, alpha, beta (1 and 2), epsilon increase dramatically. In consequence, the improving
toxins, and enterotoxin E] [77]. In addition, novel toxins diagnostics and surveillance of C. perfringens are crucial.
have been identified, as NetB, which was isolated from Diagnosis of enteric diseases produced by C. perfrin-
chickens with necrotic enteritis [78]. The toxin produc‑ gens is usually difficult since these bacteria can nor‑
tions in animal gut are associated with specific entero‑ mally colonize bird gut. Moreover, it was shown that
toxemias characterized by a variety of symptoms and alpha-toxin of C. perfringens is not an essential viru‑
traumatic infections. C. perfringens strains may infect lence factor in the pathogenesis of necrotic enteritis in
birds, dogs, horses, pigs, lambs, cattle, sheep, goats and chickens. Thus, the main challenge is to determine the
other domestic animals. Although C. perfringens is an biological activity of C. perfringens in terms of rapid
important enteric pathogen for almost all domestic ani‑ strain identification and differentiation of pathogenic
mals, the most advance studies on its pathogenicity have from non-pathogenic strains. A solution can be found
been made for broiler chicken. For instance, alpha- and in cell-based detection systems which are emerging bio‑
beta-toxin of C. perfringens are responsible for clostrid‑ sensor technologies that detect the biological activity of
ial enteric disease in poultry. This necrotic enteritis pathogens or toxins. Those biosensors have mammalian
cause significant economic losses to the global poultry cells as sensing elements and allow monitoring pertur‑
industry due to high mortality rates. Furthermore, some bations in cell physiological activities following expo‑
strains of C. perfringens that produce enterotoxins at sure [80]. Cell-based biosensors are capable of detecting
sporulation are responsible for foodborne disease in the presence of pathogens or active toxin in clinical,
humans. environmental and food samples [2] rendering accurate
The isolation of C. perfringens alone is not sufficient estimation of the risk associated with the agent identi‑
for diagnosis but has to be confirmed with histological fication. Different device designs are proposed from a
evaluation of lesions. The lesions in the lamina propria 96-well plate to modified electrodes carrying mamma‑
associate with a strong inflammation is characteristic for lian cells [80].
early stages of infection, while in later stages, necrosis of Each isotype of C. perfringens carries a defined sub‑
mucosa and association of gram-positive rods with the set of virulence genes coding for toxin-producing
lesions is characteristics [79]. The infections are usually sequences. Sergeev et al. [81] immobilized specific oli‑
treated by antibiotics. In farms that stopped using antibi‑ goprobes onto a multipathogen oligonucleotide micro‑
otic growth promoters, outbreaks of clostridial infections array to detect six toxin-producing sequences in C.
Vidic et al. Vet Res (2017) 48:11 Page 11 of 22
perfringens. Sequences encoding the different toxins In contrast to bluetongue virus which mainly affects
hybridized strongly and specifically to the correspond‑ sheep, strains of epizootic hemorrhagic disease virus,
ing oligoprobes. The microarray-based test was applied another distinct species within the genus Orbivirus, may
on fluorescently-labeled amplicons obtain by initial exhibit high mortality and morbidity in both sheep and
PCR amplification step [81–83]. After hybridization, cows. As both diseases have similar clinical symptoms,
the microarray was scanned to measure the presence or both can be transmitted by the same insect and both
absence of signal above background as an indicator of have significant negative impacts on trade, the diagnosis
bacterial presence in the tested sample. This experimen‑ of Orbiviruses is important. Current methods for blue‑
tal approach was used for simultaneous analysis for sev‑ tongue and epizootic hemorrhagic disease virus include
eral bacterial stains, their toxin genes or drug resistance numerous nucleic acid amplification assays, genotyp‑
determinants on a single-chip platform [84]. ing viral isolates, DNA microarray and next-generation
Finally, toxins produced by C. perfringens may be sequencing [91]. Promising advanced methods, such as
detected using specific antibodies integrated onto an fluorescent microsphere assays that can be adapted to
immunosensor. Such a device was demonstrated for sen‑ single-tube multiplexing or multiple-well multiplexing,
sitive and label-free detection of epsilon toxin produced allow simultaneous detection of various viral RNA by
by C. perfringens [85]. The sensor was obtained using an in-solution hybridization [92]. These tests are currently
epsilon-toxin specific monoclonal antibody immobilized under validation in some laboratories. However, other
onto single walled carbon nanotubes. By controlling the alternative amplification methods, as loop-mediated iso‑
morphology of the carbon nanotube assembly the sensor thermal amplification which can allow pen-site testing,
was adapted for detection of analytically relevant concen‑ or lateral flow test have still not be applied for Orbivirus
trations of toxin (nM range) and with sensitivities com‑ detection. Nevertheless, a rapid lateral flow test for the
parable to those of ELISA. detection of bluetongue virus-specific antibodies has
been commercialized and recently validated [93].
7 Detection of bluetongue and epizootic Danielli et al. [94] reported a rapid detection of Iba‑
hemorrhagic disease viruses raki virus causing epizootic hemorrhagic disease at
Bluetongue a major non-contagious infectious disease of picomolar concentrations by magnetic modulation and
domestic and wild ruminants (mainly sheep, cattle, deer) synchronous detection. This assay based on fluores‑
is caused by the bluetongue virus which is an orbivirus cent-labeled oligonucleotide detection in a homogene‑
of the Reoviridae family. Bluetongue virus is transmit‑ ous solution can be integrated into the portable device
ted by the bite of a female of certain species of midges and use for in field rapid virus diagnosis. The cDNA
of the insect family Ceratopogonidae but can also infect of the nonstructural NS3 protein expressed by the
embryos via the placenta or be transmitted via seminal Ibraki virus served as a biomarker. The complementary
fluid and colostrum [86]. World Organisation for Ani‑ nucleic acid probe was labeled with three different tags:
mal Health (Office International des Epizooties, OIE) a fluorescent dye and biotin were attached as a double-
listed bluetongue virus because of its economic impact. tag at the 5′ end, while a dark quencher was attached at
The worldwide losses, estimated to 3 billion US$ a year the 3′ end (Figure 8). Following a PCR cycle, the probe
[87], mainly affect ovine and bovine rearing industries. was attached to the streptavidin-coated magnetic bead
The estimated cost of bluetongue outbreaks in Scotland via the biotin tag. It was shown that each magnetic bead
is £100 million per year (£30 million in direct losses and may attach thousands of labeled probed, which allowed
£70 million in indirect losses [88]), while the US losses in to concentrate probes into the detecting area. Sample
trade and associated testing of cattle for bluetongue virus concentration and separation can be easily performed
status has been estimated at $130 million a year [89]. It by external magnetic field. In this assay, during PCR,
is worth noting that although the mortality and morbid‑ the fluorescent energy transfer (FRET)-based probe
ity of bluetongue in cattle are rare; cattle can be infected hybridized with its complementary sequence (Figure 8).
by the virus without showing any clinical sign and in that Then, FRET-based probe is cleaved by Taq-polymerase
way acting as reservoirs and contributing to the virus which allows the fluorescent light to be produced. The
transmission. In consequence, economic losses come not intensity of the fluorescent light can be calibrated to the
only from direct damages caused by death, or reduced pathogen concentration, and used as measure of viral
meat and milk production, but also from indirect effects titer in infected samples. In comparison, authors shown
due to the restriction in animal, semen or serum export‑ that their system distinguish target from control probe
ing. The clinical manifestations of bluetongue disease after a single cycle of PCR while classical RT-PCR gave
range from an unapparent or mild disease, to pyrexia, significant signal only after 12 cycles, while laser scan‑
tachypnea, lethargy and even fatal disease [90]. ning microscopy required 18 amplification steps to give
Vidic et al. Vet Res (2017) 48:11 Page 12 of 22
suggested that, although non-quantitative, LAMP-based a capsid composed of four structural peptides VP1-4.
diagnostics of coccidiosis parasite show many advan‑ There are seven immunologically and genetically distinct
tages: (i) the colorimetric read-out provided an instant serotypes. There is no cross protection between different
result as no requirement for electrophoresis step; (ii) serotypes which implies that an outbreak diagnostic of
test sensitivity allowed parasite detection upon ongoing both virus and of their serotype have to be done. Classi‑
infection, and at early stages when any visible lesions can cal methods for the food-and-mouth diagnostics involve
be resolved; (iii) LAMP was less sensitive to PCR inhibi‑ virus isolation, ELISA and RT-PCR tests [109]. Recently
tors present in tested samples as some metal-ions or pro‑ an improved duplex one-step RT-PCR assays was vali‑
teases compared to PCR and, finally, (iv) the completed dated [110]. The main advantage of this test is in the
test was cheaper than ~£1 per sample. Thus, simplicity, co-amplification of foot-and-mouth virus RNA and host
low cost and no requirement for sophisticated laboratory β-actin mRNA. Host mRNA served as an intern control
equipment make the LAMP-colorimetric paper test a to ensure accuracy and to avoid false negative results that
good candidate for in field applications. occur when quality of RNA in samples is poor. Although
the assay was validated as specific and sensitive, it is only
9 Detection of foot‑and‑mouth disease viruses adapted to laboratory diagnostics.
Foot-and-mouth disease virus causes an extremely infec‑ Fowler et al. [111] reported a lateral flow device for
tious and contagious disease of cloven-hoofed animals recovery of food-to-mouth viral RNA. In their assay
as cattle, pigs, sheep and many wild species. Although the virus detection and subtype identification were per‑
showing a low mortality, foot-and-mouth disease has the formed in laboratory facilities while the lateral flow
global impact estimated between US$6.5 and 21 billion a device was only proposed to improve field sample pres‑
year, due to high morbidity of the disease and the huge ervation during transport. To test the proof of concept,
numbers of animals affected [108]. It was estimated that the authors applied epithelial suspension or cell culture
one infected cow can infect more than 70 cattle, which infected with a food-to-mouth virus representing various
makes foot-and-mouth virus the most infectious human serotypes on the antigen lateral flow strip. Specific anti‑
and animal pathogen known. The clinical symptoms of gen–antibody reaction resulted in development of the
food-and-mouth disease are formation of painful fluid- test line. This lateral flow device was then employed as a
filled vesicles (blisters) or some erosion on the mouth tis‑ dry, thermos-stable and non-hazardous transport system
sues as tongue and lips, or other parts of the body where of infected sample for subsequent nucleic acid amplifica‑
the skin is thin, like between the two toes of the feet. The tion, sequencing or virus recovery in laboratory facilities.
pain causes many supplementary symptoms as depres‑ Reid et al. [112] developed a rapid and simple chroma‑
sion, excessive salivation, lameness, anorexia and reluc‑ tographic strip foot-and-mouth diagnostic test for field
tance to move or stand. application. This device was shown to detect specifically
The developed countries have eradicated the foot- foot-and-mouth disease virus antigen in laboratory and
and-mouth disease but widespread and long distance field condition. The chromatographic strip test contained
movements of animals sometimes re-incur virus. The a specific monoclonal antibody with broad reactivity for
outbreaks in disease free regions cause economic losses foot-and-mouth disease viruses. The device was vali‑
of about US$1.5 billion a year. Therefore a rapid imple‑ dated with laboratory-based tests on a range of samples
mentation of the measures and a coordination between as nasal swabs, epithelial suspensions and probangs from
regions and countries are needed to control disease clinical samples or from animals infected experimentally
spread [109]. However, even nowadays the virus iden‑ as well as in supernatant fluids resulting from their pas‑
tification cannot be done in all endemic regions. Many sage in cell culture.
developing countries during the suspected outbreaks Another example of a preliminary validated point-
have to send infected samples to international laborato‑ of-care test for foot-and-month disease diagnosis is a
ries because of the lack of resource and laboratory equip‑ device that coupled reverse transcription loop-mediated
ment. The transportation represents a major biosecurity isothermal amplification (RT-LAMP) with a lateral flow
risk, increases time of analysis and risk of sample degra‑ strips [113]. The test provided diagnosis within less than
dation and has additional cost. In consequence, there is 1 h with no requirement for instrumentation because the
a need for a portable device for in field foot-and-mouth test line is visible by a naked eye. Moreover, the test can
diagnosis to assure better disease control, surveillance be performed in a standard water bath or heating block
and management in outbreaks. because RT-LAMP technique does not require a ther‑
The food-and-mouth virus belongs to the Aphthovirus mocycler as it amplifies specific nucleotide sequences
genus of the Picornaviridae family. It consists of a single- at a constant temperature. The validation of the device
stained positively-charged RNA genome surrounded by has shown a successful detection of foot-and-month
Vidic et al. Vet Res (2017) 48:11 Page 14 of 22
disease virus RNA from epithelial suspensions without strain differentiation between C. jejuni and C. coli is diffi‑
the need for prior RNA extraction. All these advantages cult using conventional cellular or biochemical methods,
together with a possibility that a RT-LAMP method can because of the similar characteristics of the two species
be adapted for a high throughput system make this assay [118]. Second, transport can stress bacteria making them
attractive for field use. viable but-not culturable on selective agar plates, thus
making plate count methods inefficient.
10 Detection of Campylobacter Molecular biology methods, that use nucleic acids as
Campylobacter is a Gram-negative spiral-shaped bac‑ target for the tests allow detection of the viable but-not
terium, mobile with flagella, which belongs to the culturable microorganisms, reduce the time required to
Campylobacteriaceae family. It is microaerophilic and obtain results and improve specificity. Various kinds of
thermophilic microorganism that can grow well at the specific and sensitive PCR-based tests have been devel‑
temperature range between 37 and 42 °C. Campylobac- oped that are usually run in laboratories. Thus, they
ter can cause diseases in humans and animals including require the transportation of the samples from the site of
wild animals, pets and livestock species. C. jejuni, C. coli analysis to the equipped facilities. Regarding the sensitiv‑
and C. fetus strains have been found worldwide. Particu‑ ity of Campylobacter to a variety of environmental condi‑
larly, C. jejuni and C. coli have been found in sheep, cat‑ tions, the transport should be fast to reduce the loss of
tle, chickens, turkeys, dogs, cats and pigs while C. fetus viability.
has been found mostly in sheep, goats and cattle. C. A portable device could help farmers to obtain a rapid
jejuni, mostly found in poultry, may colonize the intes‑ detection of the pathogen, reducing the spread of the
tine of turkeys, chickens and waterfowl. Clinical signs of microorganism in the livestock and consequently of the
infection are enteritis with diarrhea, vomiting and fever, food that could cause human illness. Barletta et al. [119]
but also abortions and infertility, bovine genital campy‑ proposed a test based on melting-point curve analysis
lobacteriosis, ileitis in hamsters, colitis in ferrets, enteri‑ which identifies post-PCR products of C. jejuni. They
tis in porcine. Lesions of infected young animals include standardized a classical PCR test with primers already
edema of the mucosa of the ileum and cecum, mononu‑ reported against 16S rRNA of the Campylobacter spp.
clear infiltration of the submucosa, villous atrophy with The test can be run using a multiple PCR for perform‑
intraluminal accumulation of mucus. In addition, animals ing detection of Salmonella, Shigella and Campylo-
can be infected asymptomatically with any of Campylo- bacter spp. Although selective, the method requires
bacter strains. For instance, C. jejuni is not considered to agarose gel migration of samples, making it slow and
be pathogenic in birds. Still, some cases of C. jejuni infec‑ not adapted for screening high number of samples.
tion in chickens have been reported to cause enteritis and In contrast, fluorescence-based RT-PCR methods are
death. more robust and faster because no post-PCR protocols
Poultry is considered as the main source of human are required. Unfortunately, both protocols require the
campylobacteriosis [114, 115], the disease caused by extraction of DNA and, thus, depend on the efficiency
the infection with Campylobacter. The WHO recom‑ of the enzymatic reaction. As results may depend on
mended a control and surveillance of Campylobacter in the reagents used for enzymatic reaction, PCR-meth‑
poultry to reduce the risk for humans since this bacte‑ ods avoiding the amplification step would be more
rium has a significant impact on public health. In fact, accurate.
contaminated, undercooked poultry is responsible for In last 10 years various kind of sensors have been
50–80% of cases of campylobacteriosis investigated. Nev‑ developed to improve the rapidity, specificity and sim‑
ertheless, contaminated beef and pork products are also plicity of Campylobacter detection. For this, proteins,
responsible for some infections of people. Costs due to C. nucleic acids, aptamers, antibodies and whole cells have
jejuni infections are estimated between US$1.5 billion to been used as bioreceptors for the development of spe‑
US$8.0 billion a year and about €2.4 billion a year, in the cific point-of-care devices. Among them DNA-biosen‑
United States and Europe, respectively [116, 117]. sors seem to be the most attractive diagnostic tool for
The recommended procedure for detection of Campy- their rapidity, specificity and cost effectiveness. Indeed,
lobacter requires 4 days to provide response on its pres‑ DNA-biosensors allow rapid, real-time monitoring of
ence or absence, while up to 7 more days are required hybridization with the target nucleic acids. Specificity
for Campylobacter strain identification. Microaerophilic of the system relies on oligonucleotide probes cova‑
conditions, a specific temperature, and selective enrich‑ lently immobilized on the sensing surface. Techniques,
ment media are required to grow Campylobacter in lab‑ such as optical [120], acoustic [121], electrochemical
oratory. However, classical plate based methods are not [122], microwire [123] and localized surface plasmon
quite suitable for diagnostics Campylobacter. First, the resonance [124], have been proposed for traducing the
Vidic et al. Vet Res (2017) 48:11 Page 15 of 22
hybridization with the specific target nucleic acid to the Possibility to have multi-pathogen in field diagnosis will
pathogen detection. For instance, an organic light emit‑ significantly reduce time of detection allowing the acti‑
ting diode (OLED) biosensor can be employed for the vation of control measures in a short time. The improv‑
detection of Campylobacter in poultry meat samples, ing of the hygiene of poultry will reduce the number of
using a DNA probe attached to a glass slide (Figure 9). infections caused by Campylobacter in farms and thus
The labelling of the secondary DNA probe with an the costs of human hospitalization.
Alexa Fluor fluorophore allowed reaching a sensitivity
of 0.37 ng/μL DNA and 1.5 × 101 CFU/g of Campylo- 11 Detection of Salmonella
bacter [125]. The method was demonstrated to be high Salmonella is a Gram-negative, rod-shaped, faculta‑
sensitive even when no preliminary nucleic acid enrich‑ tive anaerobic, non-spore-forming, motile enterobacte‑
ment was performed. In addition, it is robustness, as ria, that can grow over the temperature range from 7
zero false positive or false negative response have been to 48 °C, and at optimum pH from 6.5 to 7.5. Salmo-
obtained. nella is a cause of foodborne illness worldwide and is
Zhang et al. [126] described the utilization of magneto responsible of livestock infections that can be transmit‑
strictive particle coated with three layers of silica to bind ted from animals to humans. Poultry are considered
anti-C. jejuni antibodies. Such functionalized magnetic as one of the most important Salmonella reservoirs.
particles were shown to detect C. jejuni in water released Various serotypes of Salmonella overlap between farm
from chicken factories. Although bacterial cells are typi‑ animals and humans [130]. Animals are mainly infected
cally diluted in water, the easy manipulation of magnetic through feed, drinking water or environmental sources.
particles allowed bacterial cells concentration giving a Poultry farmers which suffers flock losses due to Salmo-
sensitive detection with the detection limit of 102 CFU/ nella infection, suffers also for eggs and other consum‑
mL. In another study, Wei et al. [127] proposed a bio‑ able poultry products losses. Various different strains
sensor based on surface plasmon resonance for identi‑ of Salmonella have been identified although only two
fication of C. jejuni. Using specific antibodies against C. species have been recognized: S. enterica and S. bon-
jejuni a sensitivity of 103 CFU/mL was reached. gori. Salmonella enterica can be subdivided in subspe‑
Gnanaprakasa et al. [128] developed an optical bio‑ cies, Salmonella enterica subsp. enterica (I), Salmonella
sensor for the detection of C. jejuni based on diffraction enterica subsp. salamae (II), Salmonella enterica subsp.
optic technology and surface plasmon resonance. The arizonae (IIIa), Salmonella enterica subsp. diarizonae
hippuricase gene (hipO) of C. jejuni was used as a tar‑ (IIIb), Salmonella enterica subsp. houtenae (IV), Sal-
get sequence. After being thiolated the complementary monella enterica subsp. houtenae (VI). Salmonella bon-
probe was immobilized on the gold surface of the sens‑ gori was originally designated as S. enterica subspecies
orchip. On-chip hybridization was traduced to an optical V. The most important subspecies involved in poultry
signal which allowed C. jejuni quantification in infected infections are Salmonella Typhimurium, Salmonella
samples with a detection limit of 5.0 nM. Enteritidis, Salmonella Virchow, Salmonella Hadar.
Wald et al. [129] used a lateral flow method to detect Among all strains, S. Enteritidis is the serovar mostly
C. jejuni or C. coli reaching a sensitivity of about 7.3 implicated in salmonellosis worldwide. Affected birds
log of CFU/g. Though not highly sensitive this device are anorectic, weak, have diarrhea and may die shortly
is portable and can be adapted to multiplex detection. after hatching. Those who survive rest small in size and
frequently carry localized infection of the ovary. In
consequence survived hens may transmit the infection
to the eggs and produce infected chicks.
The ISO 6579:2002 (AnnexD) method is currently
the EU standard method for diagnosis of Salmonella in
poultry samples. Other standard culture-based meth‑
ods are available. Some of them have been validated by
other regulatory agencies. Most of these conventional
Figure 9 Visualization and detection of Campylobacter. A culture-based methods comprise the following steps: (i)
Transmission electron microscopy of Campylobacter cell. Bar, 2 µm. liquid pre-enrichment on non-selective medium followed
B OLED biosensor probing of a negative control sample containing by that on (ii) liquid selective media containing inhibit‑
a non-Campylobacter DNA at 12.5 ng/µL, using a probe at 50 ng/µL.
The spot was obtained upon deposition of 1 µL of the sample on the
ing compounds towards non-Salmonella bacteria, (iii)
sensor surface. C OLED biosensor detection of Campylobacter DNA culture on solid selective agar, and (iv) isolation of sus‑
sequence at 12.5 ng/µL, using 50 ng/µL of the probe. The spot was pected colonies from the solid media, their incubation in
obtained with 1 µL of the sample as previously explained [125]. a specific solid media followed by additional biochemical
Vidic et al. Vet Res (2017) 48:11 Page 16 of 22
and serological tests for strain identification. All culture- able to detect only one cell of the pathogen in an infected
based methods need complex laboratory equipment and sample. The cantilever bends lightly, due to the capture
at least 1 week to give results. Moreover, some serotypes of a bacterium by the specific peptide, and a laser acti‑
cannot be detected on the selective media used for iso‑ vates the alarm. The authors used phage-derived peptides
lation, thus, false negatives can be provided by the test. to obtain an array for parallel detecting of eight serovars.
For instance, Salmonella Virchow cannot be cultivated Phage display uses genetic modification of viruses that
on agar. infect bacteria, bacteriophages, to insert a sequence of
In consequence, immunological and serological tests the peptide that specifically recognizes one biomarker
have been developed for Salmonella diagnosis. The iden‑ into a phage coat protein gene. In that way, the phage
tification step that uses biochemical assays detecting a starts to express the peptide on its surface. The display‑
specific biomarker has reduced the volume of the rea‑ ing phages can be attached on the biosensor surface and
gents allowing the simultaneous inoculum of many wells assure specific Salmonella detection.
on the same strip leading to a reaction that can give the A lateral flow system for Salmonella AD, produced by
answer within 24 h. Many of such kits are commercially the DuPont, uses two-step enrichment (16–22 h plus
available (as API 20E, bioMerieux; Enterotube II, BD 6–8 h) before the sample insertion on the strip. Strips
diagnostics, etc.). Nevertheless, it is difficult to incorpo‑ contained colloidal gold conjugated anti-Salmonella
rate immunoassays into an array to detect multiple tar‑ antibodies coated onto the sample pad. The test provides
gets due to the high cross-reactivity between antibodies. results in 10 min. Strips have been validated for Salmo-
On the other side, ELISA, although high sensitive and nella detection on raw meat, chicken sera, eggs, pro‑
fast, still can give false negative results, cross-reactivity cessed meat, fruits, vegetables, etc. This method was also
problems, and need a pre-enrichment step [131]. confirmed on meat samples spiked with 1–4 CFU/25 g
The utilization of molecular biology and PCR-based before the enrichment.
methods, such RT-PCR and Digital-PCR, can reduce the Bulut [134] reported the development of a lateral flow
time for Salmonella detection to several hours. These immunochromatographic test platform for Salmonella
methods may be employed even when serotyping fails using the invA gene as a target. This gene encodes the
due to the change or loss of the surface antigens. Vari‑ protein responsible for the invasivity of the pathogen. The
ous kinds of rapid tests for Salmonella have been devel‑ platform requires no labeling of the target as it detects
oped and approved, including the VIDAS Salmonella amplicons obtained by using specific primers followed by
(SLM) method, the immuno-concentration Salmonella sandwich hybridization. Nevertheless, the method is not
the most economically important and outstanding wel‑ Cai et al. [137] proposed a label-free electrochemi‑
fare issues for industrialized beef cattle producers and cal biosensor based on molecular beacon for detection
animal health organizations. The virus seems to spread of target mRNA from RSV. Molecular beacons are oli‑
by an aerosol route and can be easily transmitted by gonucleotide hybridization probes in a form of a hair‑
contact with respiratory secretions from infected cattle. pin shaped molecules. Within the hairpin structure a
Frequently, BRSV infection is associated with secondary streptavidin binding aptamer sequence was blocked for
bacterial infections that are treated by the massive use of this assay. Upon hybridization of the beacon with specific
antibiotics. In consequence, BRSV represents an addi‑ nucleic acid sequence, the hairpin opens to allow bind‑
tional public health-concern through the risk of antibi‑ ing to streptavidin-HRP protein complex. In that way,
otic-resistance developing. the hybridization event can be quantified by the enzy‑
BRSV is a single-strained, negative-sense RNA envel‑ matic reaction using a HRP substrate, tetramethylben‑
oped virus classified as a Pneumovirus of Paramyxoviri- zidine (TMB). The sensor was shown to detect 13 amol
dae family. The genome of BRSV encodes for 11 proteins: of RSV mRNA in a 4 µL total volume of a complex bio‑
nucleocapsid (N), phosphoprotein, large polymerase, logical media. The high specificity and selectivity of the
transcriptional antitermination factor M2-1 and RNA device was demonstrated as sensor detected specific RSV
regulatory protein M2-2 that all associate the genomic mRNA but not one base mismatched sequence.
RNA, then attachment (G), fusion (F) and small hydro‑ In another study, the HRP-TMB reaction was used for
phobic protein that are transmembrane surface glyco‑ electrochemical detection of RSV specific antigens [138].
proteins, while matrix or membrane protein (M) is a For this, the polystyrene array slide that allows antibody
nonglycosylated and associates with the inner face of the immobilization was attached with disposable screen-
envelope. In addition, two nonstructural proteins, named printed electrodes. The RSV fusion protein was captured
NS1 and NS2, accumulate within the infected cells. in a sandwich by a specific monoclonal antibody attached
After the incubation period estimated to take between to the polystyrene surface and a second antibody-coupled
2 and 5 days, BRSV infection is usually limited to the with HRP. The enzymatic reaction HRP-TMB was tra‑
upper respiratory tract giving clinical signs that may duced into an amperometric signal by the electrode. The
range from minimal to severe with dyspnea and death. whole test needed only 25 min to produce result show‑
Affected calves can have tachypnea, ocular serous secre‑ ing similar selectivity and sensibility to commercial RT-
tions, dry muzzle, reduced activity, anorexia, and high PCR or immunofluorescent tests when probed on clinical
fevers. The clinical signs may last until 7–10 days post- samples.
infection. Interestingly, the antibody response is fre‑ Perez et al. [139] described the detection of RSV by
quently developed few days before clinical signs appear. nanoparticle amplification of immuno-PCR assay. Their
Thus, testing serum samples pooled from a number of test detects RSV by viral particles capturing into a sand‑
animals in a respiratory outbreak may help diagnosis. wich formed between two different antibodies specific
Still, although clinical signs may suggest BRSV infection, against the F protein. The first antibody was attached to
diagnosis of BRSV requires a laboratory confirmation. magnetic beads to allow extraction, while the second was
PCR is commonly used as a molecular method for co-immobilized onto gold nanoparticles with specific
BRSV diagnosis. However, the virus isolation is difficult, DNA sequences. After extraction the hybridized DNA
except in some cases upon the acute phases of infection. can be released by heating and detected via RT-PCR. It
Detection of negative-sense RNA genome or mRNA of was shown that this nanoparticle amplified immune-
the virus is even more delicate than detection of viral PCR assay not only provided better virus sampling but
proteins. First, RNAs are sensitive to RNAse digestion also reduce the effect of PCR-induced variation in sam‑
and degradation and have shorter life time than proteins. ple replicates by increasing the tag DNA concentration
Second, RSV mRNA is expressed cyclically, while protein and by lowering the background signaling. Compared to
expression is stable and increases in a regular manner ELISA and RT-PCR detection, the nanoparticle amplified
over time. In consequence, ELISA test that quantify the immune-PCR showed a several 1000-fold improvement
RSV fusion protein subunits F0 and F1, expressed by all in the limit of detection as it detected 4.1 PFU/mL.
RSV strains, provides an efficient diagnostic tool. Simi‑
larly, the detection of specific antibodies in serum sam‑ 13 Conclusions
ples has proved useful to establish a diagnosis of BRSV We show some advanced biotechnological approaches
antigen in the acute sample. Interestingly, the antibody that allow early detection of pathogens that affect
titer in animals with developed clinical signs of the dis‑ domestic livestock and poultry. The pathogens pre‑
ease is usually higher than in the sample taken several sented in this review entail significant economic losses
weeks later. due to weaken food production systems, increased
Vidic et al. Vet Res (2017) 48:11 Page 18 of 22
veterinary costs and may pose a direct threat to global have to undergo inter-laboratories testing to be har‑
food security. In addition, infection diseases of animal monized and validated. To be used outside research
population carry public health risks of outbreaks as well laboratories, a biosensor usually needs adaptation for
as sporadic and endemic zoonoses. Conventional diag‑ implementation in field conditions in order to reduce
nostic techniques are frequently time consuming, labor the risk of obtaining false positive or negative results.
intensive and require to be performed on sophisticated Among presented detection strategies, the nucleic acid-
equipment by trained professionals with certain experi‑ based biosensors appear being highly suitable for swift
ence. New technologies are employed to overcome these and sensitive testing. Their limitation lays in sample
difficulties and to provide accurate, simple and afford‑ preparation steps that include nucleic acids extraction
able biosensor devices that can give rapid response to and amplification. The analytical properties of anti‑
test. Biosensors have been designed to detect the tar‑ body- and aptamer-based biosensors depend strongly
get (protein or nucleic acid sequence) related to the on selectivity of these biomolecules and also on the sta‑
pathogen by using sensitive and selective recognition bility obtained after their immobilization on the sensor
properties of antibodies, aptamers, glucans and DNA surface. Furthermore, over the long term, we believe
probes. These sensing elements are associated with a that biosensors technology combining nanotechnolo‑
transducing element (electrochemical, optical or colori‑ gies, advances nucleic acid amplification methods, and
metric) that emits a direct signal when the target is rec‑ next-generation sequencing analysis will be a powerful
ognized. Although most of presented analytical devices systemic tool for pathogens detection and surveillance
are used only in research laboratories, it is expected system to control animal disease outbreaks and preven‑
that more biosensors will emerge in the future given tion. Development of reference materials, harmoniza‑
the rapid spread of livestock infectious diseases. There tion of sampling methods, mobile analysis and data
is a strong need to develop portable, miniaturized and networking will significantly support developing of high
multitargeting devices that can be used directly in the sensitive and selective biosensors for real-time in field
field by veterinaries or by competent national authori‑ monitoring (Figure 10). Surveillance big data obtained
ties responsible for organizing disease controls. To be from those diagnostic technologies will help disease
recommended by the World Animal Health Organiza‑ prevention and pathogens control to livestock and poul‑
tion, biosensors for animal infectious disease diagnosis try industrials and will improve animal welfare.
Figure 10 Elements of integrated portable diagnostic laboratory. Advanced analytical technologies will offer to veterinary practitioners a
suitcase containing consumables, reagents and devices to perform in field diagnostics with laboratory-grade performance.
Vidic et al. Vet Res (2017) 48:11 Page 19 of 22
Competing interests 13. Yazgan I, Noah NM, Toure O, Zhang S, Sadik OA (2014) Biosensor for
The authors declare that they have no competing interests. selective detection of E. coli in spinach using the strong affinity of deri‑
vatized mannose with fimbrial lectin. Biosens Bioelectron 61:266–273
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JV designed the structure of the review and took the lead in drafting of the Sensitive chemiluminescence immunoassay for E. coli O157: H7 detec‑
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helped with the revision of the draft. All authors read and approved the final Anal Chem 86:1115–1122
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Acknowledgements rolling circle amplifications coupled peroxidase-mimicking DNAzyme
This work was funded through the grant of the Agence Nationale de la amplification. Biosens Bioelectron 75:315–319
Recherche SENSAIV (ANR-15-MRSE-0028-01) to JV, BioAsia Grant 35976PH to 16. Kim M, Jung T, Kim Y, Lee C, Woo K, Seol JH, Yang S (2015) A microflu‑
JV, and MOST Grant, Southern Taiwan Medical Device Industry Cluster (CY-08- idic device for label-free detection of Escherichia coli in drinking water
04-22-105) Taiwan to CMC. using positive dielectrophoretic focusing, capturing, and impedance
measurement. Biosens Bioelectron 74:1011–1015
Author details 17. Idil N, Hedström M, Denizli A, Mattiasson B (2017) Whole cell based
1
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of Medical Biotechnology and Laboratory Science, College of Medicine, Chang and bio-bar code assays based on biofunctionalized magnetic micro‑
Gung University, Tao‑Yuan 33302, Taiwan. 4 Institute of Analytical Sciences, beads. Sensors 7:589–614
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