Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene

Journal Pre-proof Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene M. Mabrok, P. Chokmangmeepisarn, B.R. LaFrentz, P. Kayansamruaj, H.T. Dong, C. Rodkhum PII:

S0044-8486(19)31885-X

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734597

Reference:

AQUA 734597

To appear in:

Aquaculture

Received Date: 24 July 2019 Revised Date:

11 October 2019

Accepted Date: 11 October 2019

Please cite this article as: Mabrok, M., Chokmangmeepisarn, P., LaFrentz, B.R., Kayansamruaj, P., Dong, H.T., Rodkhum, C., Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734597. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1

Development of a species-specific polymerase chain reaction for highly sensitive

2

detection of Flavobacterium columnare targeting chondroitin AC lyase gene

3 4

Mabrok M1,

2, 6

, Chokmangmeepisarn P1, 6, LaFrentz BR3, Kayansamruaj P4, Dong

5

HT5, Rodkhum C1, 6*

6 7

1

8

University, Bangkok, Thailand.

9

2

Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn

Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Suez

10

Canal University, Egypt.

11

3

12

Agricultural Research Service, Auburn, AL, USA.

13

4

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Thailand.

15

5

16

University, Bangkok, Thailand.

17

6

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Chulalongkorn University, Bangkok, Thailand.

Aquatic Animal Health Research Unit, United States Department of Agriculture‐

Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok,

Department of Science, Faculty of Science and Technology, Suan Sunandha Rajabhat

Fish Infectious Diseases Research Unit (FID RU), Faculty of Veterinary Science,

19 20

*Corresponding author:

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Channarong Rodkhum :E‐mails: ([email protected]; [email protected] )

22 23

Data Availability

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The authors declare that they do not have any shared data available

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Conflicts of Interest

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The authors declare that there are no conflicts of interest.

28 29

Short head: Flavobacterium columnare species-specific PCR.

30 31 1

32

Abstract

33

Columnaris disease, caused by Flavobacterium columnare, is an acute infection of gills and

34

fins, that exists worldwide and causes remarkable losses in freshwater fish. The 16S rRNA

35

gene is not a good candidate for primer design, since this gene has high identity among

36

species in the same genus. In the present study, we developed a species-specific PCR for

37

detection of Flavobacterium columnare based on the chondroitin AC lyase (cslA) gene.

38

The cslA gene sequence from 13 F. columnare strains were aligned to design a specific

39

primer set, Fcol-F and Fcol-R, based on conserved region of the gene. The new primer set

40

produced a specific amplicon of 287 bp from 83 isolates of F. columnare but not from

41

related or other bacterial pathogens. The PCR was sensitive and detected up to 3 pg of

42

genomic DNA and as few as 7 colony-forming units of bacteria. The sensitivity was

43

decreased tenfold when F. columnare gDNA was spiked into tissue samples. The designed

44

primers precisely amplified F. columnare from skin and gills of infected fish, but not in

45

those of clinically healthy fish, reflecting their possible use for diagnostic purposes.

46 47

Keywords: Flavobacterium columnare, chondroitin AC lyase DNA, PCR, Columnaris

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disease.

49 50 51 52 53 54 55 56 57 58 59 60 61

2

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1. Introduction

63

Flavobacterium columnare, the causative agent of columnaris disease, is considered one of

64

the most threatening pathogens limiting the culture of many important fish species

65

(Declercq et al., 2013). The ubiquitous nature of bacteria in the freshwater environments,

66

besides their subsistence existence on skin and gills, makes F. columnare among the most

67

eminent pathogens in cultured, ornamental, and wild fish populations (Faisal et al., 2017;

68

Scott and Bollinger, 2014; Shotts and Starliper, 1999). F. columnare affects a wide range of

69

freshwater fish, including tilapia (Oreochromis sp.), channel catfish (Ictalurus punctatus),

70

common carp (Cyprinus carpio), eels (Anguilliformes), goldfish (Carassius auratus),

71

rainbow trout (Oncorhynchus mykiss), salmonids, and brook trout (Salvelinus fontinalis)

72

(Avendaño-Herrera et al., 2011; Dong et al., 2015; Evenhuis et al., 2014; Suomalainen et

73

al,, 2005; Wagner et al., 2002) and results in large economic losses worldwide (Arias et al.,

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2012).

75

To date, there are no credible means to control columnaris infections in warm water fish.

76

Research on disease prevention and control have been conducted including the use of

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commercial vaccines, antibiotics and chemotherapeutics such as salt, potassium

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permanganate, and copper sulfate (Birnbaum, 1998; Plumb, 1999; Shoemaker et al., 2011).

79

Approved antibiotics are commonly used for treatment; however, there is concern for the

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emergence of drug-resistant bacteria (Serrano, 2005). Thus, implementing good preventive

81

measures is currently the only fundamental tool for reducing the incidence of disease,

82

which mainly depends on the early and precise diagnosis of the causative agents.

83

A definitive diagnosis of the disease can be achieved through clinical examination,

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traditional plating techniques, biochemical tests, and molecular characterization of the

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causative agent. Clinical signs are not always reliable diagnostic tools, since they can be

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produced by many pathogens (Mabrok et al., 2016; McVicar, 1979). Additionally, isolation

87

of F. columnare using plating technique can be problematic (Groff and Lapatra, 2000). The

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fastidious nature and slow growth of F. columnare allows for other bacteria to grow,

89

resulting in a rigged detection of the colonies among the mixed population (Hawke and

90

Thune, 1992; Tiirola et al., 2002). Furthermore, the predominance of other bacteria

91

occasionally obscures and prohibits the formation of separate bacterial colonies (Dalsgaard,

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1993). Typically, F. columnare requires a minimum of 24 h of incubation to be isolated 3

93

from external lesions and infected tissues, and an additional 24 h for identification that

94

involves the interpretation of several phenotypic characters including the colony

95

morphology and detailed biochemical testing (Shotts and Starliper, 1999). Therefore, all

96

traditional methods required additional time, resulting in high fish mortalities with massive

97

economic losses.

98

Hence, the presumptive diagnosis of columnaris should rely on a molecular basis.

99

Polymerase chain reaction (PCR) has been used extensively to detect and identify many

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fish pathogens, including: Aeromonas hydrophilia, Edwardsiella tarda, Vibro anguillarum,

101

V. vulnificus, Pseudomonas anguilliseptica (Arias et al., 1995; Cascón et al., 1996; Fadel et

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al., 2018; Hiney and Smith, 1998; Martinez-Picado et al., 1996). Diversity in intergenic

103

spacer regions (ISR) among various rRNA gene sequences have been selected to clarify

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various taxa of bacteria and to distinguish between closely related species (Avaniss-

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Aghajani et al., 1994; Barry et al., 1991; Zavaleta et al., 1996). Several PCR protocols for

106

detection of F. columnare have been developed using species-specific sets of primers

107

targeting 16S rRNA or ISR gene fragments (Bader and Shotts, 1998; Bader et al., 2003;

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Darwish et al., 2004; Toyama et al., 1994; Wakabayashi, 1999; Welker et al., 2005).

109

However, some of the designed primers lack specificity or sensitivity because of intra-

110

specific genomic variation and are difficult to use because of hairpins and primer-dimer

111

formation. The 16S rRNA gene was not a good target for a specific primer design, since

112

this gene had high identity among species in the same genus. The most commonly used

113

PCR for F. columnare (Welker et al., 2005) can result in multiple sized PCR products and

114

multiple bands, which can be confusing for interpreting results. Additionally, our

115

preliminary results showed cross reactivity of these primers with another bacterial species

116

(F. indicium).

117

Therefore, relying on a different conserved gene to detect the identity of bacteria is the

118

most reliable way for proper and effective diagnosis. Chondroitin AC lyase is a tissue-

119

degrading enzyme that is produced extracellularly by F. columnare and was found to be

120

significantly related to the bacteria virulence (Stringer‐Roth et al., 2002). The gene has

121

been cloned from F. columnare, and was reported to share less than 50% similarity to the

122

gene found in Bacteroides (Xie et al., 2005). The current study aimed to design a specific

4

123

and sensitive PCR for detection of F. columnare based on the chondroitin AC lyase (cslA)

124

gene sequences taking into consideration all previous limitations.

125 126

2. Materials and Methods

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2.1. Bacterial isolates and growth condition

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A total of 117 bacterial isolates were used in the current study. Of these, 42 isolates of F.

129

columnare, classified into 4 genetic groups (LaFrentz et al., 2018) , as well as 21 isolates of

130

closely related Flavobacterium spp. were used for assessing PCR specificity (Table 1). The

131

remaining bacteria included 41 Thai F. columnare isolates, 7 related Flavobacterium

132

species, and 6 other bacterial fish pathogens (Table 2). These bacterial isolates were

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retrieved from infected freshwater fish in Thailand from 2012 to 2018, designated with

134

organism code and Gene Bank accession numbers (if available; Table 2). The type strain of

135

F. columnare, American Type Culture Collection (ATCC) 23463T, belonging to genetic

136

group 1, was also included as a reference strain in this study. Flavobacterium columnare

137

isolates and other closely related Flavobacterium species were cultured on Anacker and

138

Ordal’s agar (AOA) or broth (AOB; Anacker and Ordal, 1959) or modified Shieh agar

139

(LaFrentz and Klesius, 2009). All other bacteria species were cultured in Tryptic Soy Agar

140

(TSA) and Tryptic Soy Broth (TSB). Briefly, a loop of bacterial stock was plated on AOA

141

or TSA and incubated at 28 ˚C for 48 h. Single colonies were then subcultured in AOB or

142

TSB and grown for 48 h at 150 rpm under the same culture conditions. Bacterial pellets

143

were then harvested at 13,000 rpm for 5 min and were used for DNA extraction.

144 145

2.2. Preparation of bacteria genomic DNA

146

The genomic DNA of the bacterial isolates was extracted using a genomic DNA extraction

147

kit (Promega, USA) following the manufacturer’s protocol for genomic DNA extraction

148

from bacterial cells. For Gram-positive bacteria, an additional pretreatment step with

149

lysozyme (10 mg mL-1) was used to enhance cell lysis and increase the yield. Genomic

150

DNA samples were quantified using a Nanodrop (Nanodrop 1000, Thermo Scientific, UK),

151

adjusted to 100 ng µl-1, and stored at -20 ˚C until being used as template for PCR.

152 153 5

154

2.3. Primers design procedures

155

Species-specific PCR primers were designed from the chondroitin AC lyase (cslA) gene of

156

F. columnare. The cslA gene sequences from 13 isolates, representative of the four genetic

157

groups of F. columnare, were retrieved from the GenBank database. The sequences were

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subjected to a multiple sequence alignment (CLUSTER W method) (Thompson et al.,

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1994) and trimmed using the Molecular Evolutionary Genetics Analysis MEGA v6.0

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software (Tamura et al., 2013). Conserved regions within the cslA gene were selected as

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potential primer sites (Fig. 1) and primers were designed using the Oligo 7 primer analysis

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software. The best primer pairs that match PCR primer design guidelines from the point of

163

string-based alignment scores, melting temperature, primer length, and GC content, and

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gave a properly sized amplicon with a constant oligonucleotide-target duple were selected

165

(McGraw et al., 1990). The primers, Fcol-F and Fcol-R (Table 3), which amplify a 287 bp

166

amplicon were synthesized commercially (Ward medic, Bangkok, Thailand).

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2.4. PCR procedures

169

The designed primer set was initially optimized to amplify the cslA gene sequence of F.

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columnare, using 25 µL reaction volume containing 12.5 µL Green Master Mix (Dream

171

TagTM, Thermo Scientific), 0.2 µM of each primer, and 3 µL DNA template. PCR

172

amplification was conducted in a T100TM gradient thermocycler (BIO-RAD) and

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Mastercycler personal (Eppendorf) apparatus. A thermal gradient from 58 to 62°C was used

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to determine the optimal annealing temperature for the specific binding of the primers to

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the template DNA. The preliminary study demonstrated that 60 °C was optimal and

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resulted in no faint or non-specific bands (data not shown). Subsequently, samples were

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subjected to an initial denaturation at 94 °C for 5 min; followed by 30 cycles of denaturing

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at 94 °C for 45 s; annealing at 60 °C for 45 s; and extension at 72 °C for 30 s. The DNA

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template from the type strain of F. columnare (ATCC 23463T) was included as a positive

180

control, while a DNA free sample (molecular biology water) was used as a negative

181

control. Amplified products were detected by horizontal 1.5% (w/v) agarose gel

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electrophoresis for 25 min at 100 V in Tris-borate-EDTA (TBE) 1× electrophoresis buffer,

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visualized using 5 µl of RedSafe TM (INTRON BIOTECH) and photographed under UV

6

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light and computer digitized (INGENIUS 3, SYNGENE). A gene ruler 100 bp plus DNA

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ladder (Thermo Scientific) was used as a molecular mass marker.

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2.5. Evaluation of the PCR specificity

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The specificity of the primers (Fcol-F/Fcol-R) was screened against a panel of

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microorganisms listed in Tables 1 and 2 using the optimized conditions, and was compared

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with the primer set (FCISRFL/FCISRR1) previously published by Welker, Shoemaker,

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Arias, Klesius (2005) (Table 3). The production of the expected amplicon size (287 bp)

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from a genomic DNA template was considered a positive result with the Fcol-R/Fcol-R

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primers. To ensure that the amplified product belonged to a fragment of the cslA gene,

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three amplicons were randomly selected following PCR amplification. The products were

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purified using the NucleoSpin Extract II Kit (Macherey-Nagel) and subjected to direct

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sequencing (1st BASE Pte Ltd) using the same set of primers. Sequencing was performed

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using ABI Big Dye Terminator v3.1 chemistry on an ABI PRISM 3130XL DNA Analyzer

198

(Applied Biosystems). The edited sequences from each amplicon were assembled using

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BioNumerics and were blasted against the expected amplified fragment of F. columnare

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cslA gene.

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2.6. Evaluation of the PCR sensitivity

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To determine the sensitivity of the PCR, varying concentrations of gDNA, tenfold serial

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dilutions of DNA template, ranging from 300 ng to 0.003 pg (10-8 dilution) were used in 25

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µL PCR amplifications following the same conditions outlined above. Furthermore, serial

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dilutions ranging from 100 to 10-10 of a pure culture of F. columnare grown at 28°C for 48 h

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and adjusted at 0.5 McFarland standard (expected to have 108 CFU mL-1) were performed

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using a sterile 1% phosphate buffered saline (PBS). Five µL of each dilution was amplified

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in a similar PCR reaction volume under the same conditions. Simultaneously, 100 µL of

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each dilution were plated to determine the actual number of colony-forming units

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(CFU)/dilution (CFU mL-1). The number of CFUs determined from plate counts were

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ranged from 0 to 4 × 106.

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To investigate the sensitivity of the method in fish samples, gill, skin, and kidney (20 mg)

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collected from apparent healthy Nile tilapia (Oreochromis niloticus) were spiked with serial 7

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tenfold dilutions of extracted F. columnare genomic DNA ranging from 300 ng to 0.003 pg

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(10-8 dilution). DNA from tissue was extracted using silica-membrane technology

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(NucleoSpin® Tissue Kit, MACHEREY-NAGEL, USA). Tissues (approximately 20 mg)

218

were homogenized with a sterile pestle in a 2 mL DNA free microcentrifuge tube

219

containing 180 µL of a pre-lysate buffer. The homogenate was then treated with 25 µL of

220

proteinase K enzyme and incubated at 56 °C for 1 h. Manufacturer’s instructions were

221

followed for the remainder of the DNA isolation process. Finally, the genomic DNA was

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recovered from the spin-column using 30 µL of elution buffer provided by the company,

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adjusted to 100 ng µL-1 using a Nanodrop (Nanodrop 1000, Thermo Scientific, UK), and

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stored at -20 ˚C until being used as a template for PCR.

225 226

2.7. Clinical validation of new primers set

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To ensure the validity of the primers for field diagnosis, tissue samples of skin (n=10) and

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gills (n=10) were aseptically excised from 10 moribund and 10 apparently healthy hybrid

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red tilapia, Oreochromis sp. The animal procedures were approved by the Chulalongkorn

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University Animal Care and Use Committee under protocol no. 13310085. Twenty fish

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weighing 17.76±1.8 were randomly collected from two tilapia farms (10 fish/ farm),

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located at different geographical areas in Thailand. The first farm (Ratchaburi province)

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had a history of natural outbreaks of columnaris during the period of 2017-2019 with

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characteristic gill necrosis and saddleback lesions in the affected fish and mass mortalities,

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while the second farm (Nakornprathom province) had no history of infection where all fish

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were apparently healthy. DNA from 20 mg of the collected tissues (skin and gills) was

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extracted using silica-membrane technology (NucleoSpin® Tissue Kit, MACHEREY-

238

NAGEL, USA) following the manufacturer’s instruction for tissues (outlined above). A

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standard plating technique was also used to isolate F. columnare from those tissues. PCR

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was conducted as outlined above and each reaction included approximately 100 ng DNA.

241 242 243

3. Results 3.1. Primer specificity

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The designed primers yielded the expected amplified fragment, 287 bp, from all the

245

genomic DNA samples of F. columnare but not from those of other closely related and 8

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non-relevant bacteria species, demonstrating the specificity of these primers for the

247

detection of F. columnare (Fig. 2). Three of the amplicons were randomly selected and

248

subjected to sequencing. Evaluation of the sequences revealed that all PCR products were

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identical to the expected sequence of the amplified fragment of the F. columnare cslA gene.

250

In contrast, evaluation of the FCISRFL and FCISRR1 primer pair previously designed by

251

Welker, Shoemaker, Arias, Klesius (2005) showed specificity for F. columnare but also

252

cross-reactivity with F. indicum (Fig. 3), generating an amplicon of 400 bp.

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3.2. Primer sensitivity

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To estimate the sensitivity of the cslA gene-based primers in PCR detection of F.

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columnare, DNA template in eluted buffer was exposed to tenfold serial dilutions. The

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results showed that the designed primer set is highly sensitive, with a detection limit of

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approximately 3 pg per reaction (10-5 dilution) (Fig. 4). Moreover, the designed primers

259

could detect as little as 4 CFU mL-1 of F. columnare when pure cultures were used (Fig. 5).

260

Additionally, artificially inoculated fish samples, spiked with serial tenfold dilutions of F.

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columnare genomic DNA were positive following amplification with PCR (Fig. 6). As

262

predicted, the sensitivity was tenfold decreased to 30 pg (10-4 dilution) when a complex

263

DNA sample was used as template. Neither control nor tissue sample treated with lower

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concentrations of F. columnare genomic DNA was amplified.

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3.3. Clinical validation of new primers set

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The newly designed primers were able to amplify and detect F. columnare in naturally

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infected fish organs. As indicated in Fig. 7, the F. columnare cslA gene was amplified in

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skin and gills of infected fish (lanes 1–10), but not in those of clinically healthy fish (lanes

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11-20). The pathogen was isolated from infected tissues (skin and gills) using a direct

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plating technique, where characteristic yellowish and rhizoid-shaped colonies were

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obtained.

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4. Discussion

275

Flavobacterium columnare is considered as one of the most threatening pathogens limiting

276

the culture of freshwater fish species of commercial value and induced prodigious fiscal 9

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losses in distinct geographical areas of the world (Loch and Faisal, 2015). Proper fish

278

health management begins with disease prevention rather than treatment, which can be

279

assisted with the early detection of the disease using sets of diagnostic tools (Assefa and

280

Abunna, 2018). Indeed, the distinction between F. columnare and other genetically and

281

phylogenetically related species based on morphology and traditional culture techniques

282

has shown some obstacles (de Alexandre Sebastião et al., 2019; Loch and Faisal, 2015).

283

Hence, research has been conducted to identify alternative diagnostic tools based on the

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molecular typing of this pathogen. The 16S rRNA gene and 16S-23S ISR are the most

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widely recognized target genes for the molecular analysis of many pathogenic and

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opportunistic bacteria (Yıldırım et al., 2011). Heterogeneity in 16S rRNA gene sequences

287

has been exploited to clarify various taxa of bacteria and to develop species-specific PCR-

288

based diagnostic tools (Avaniss-Aghajani et al., 1994). However, the application of these

289

tools for further detection of some bacteria including F. columnare are still being developed

290

with respect to specificity, reasonability and ease of use.

291

Several 16S rRNA based PCR primer sets have been designed for the specific detection of

292

F. columnare (Bader et al., 2003; Darwish et al., 2004; Toyama et al., 1996; Wakabayashi,

293

1999; Welker et al., 2005). However, some of the designed primers lack specificity and/or

294

sensitivity because of intra-specific genomic variation and are difficult to use because of

295

hairpins and primer-dimer formation. Bader and Shotts (1998) designed species-specific

296

sets of primers targeting 16S rRNA gene based on the sequences from strain ATCC 14902.

297

The authors reported that the designed primer sets work efficiently against those isolates

298

but required a protracted period of denaturation to prevent primer-dimer formations and to

299

allow amplicon formation. Furthermore, the initially designed primer sets did not

300

consistently produce specific PCR products under generally accepted PCR conditions and

301

showed no specificity against F. columnare evaluated strains in double-blind studies

302

conducted by Bader et al. (2003). Toyama et al. (1996) also successfully established a PCR

303

detection method to amplify a fragment of F. columnare 16S rRNA gene, but the designed

304

primer set was found to amplify other related Flavobacterium species.

305

Fundamentally, poor primer design associated with the limited numbers of evaluated strains

306

probably reduced the technical precision and resulted in false detection of amplified targets.

307

For instance, the primer sets that were designed by Bader and Shotts (1998) not only 10

308

amplified an 800 bp fragment length of the 16S rRNA gene of F. columnare but also

309

amplified the ATCC 43622 strain that has been reclassified as F. johnsoniae by Welker et

310

al. (2005). Similarly, in the present study, the evaluation of the FCISRFL and FCISRR1

311

primer pair previously designed by Welker et al. (2005) cross reacted with F. indicum.

312

Indeed, the intraspecies diversity in the 16S rRNA gene sequence of F. columnare may

313

restrain the detection of bacteria using a single primer set (Toyama et al., 1996;

314

Wakabayashi, 1999). Thus, several attempts have been developed to cope with this problem

315

of genetic variability. Toyama et al. (1996) successfully developed a PCR technique in

316

which two sets of primers were used to ensure detection and to differentiate F. columnare

317

from other closely related species, thereby increasing the complexity of the method.

318

Moreover, Triyanto et al. (1999) developed a nested PCR technique to identify F.

319

columnare strains of different genomovars where the initial PCR product amplified by

320

universal 16S rRNA primers was used as a template for the specific primers reaction. Thus,

321

the technique required two-step procedures, which increased the risk of cross-

322

contamination and increased the cost and time of detection.

323

Based on the previous studies, it appears that the 16S rRNA gene was not the best target for

324

designing specific primers since this gene showed high identity among other

325

Flavobacterium species. Therefore, the present study aimed to design a new set of primers

326

targeting the chondroitin AC lyase (cslA) gene. The specificity of Fcol-F and Fcol-R primer

327

pair was screened against 83 isolates of F. columnare retrieved from infected fish, 28 other

328

representatives from the genus Flavobacterium and 6 non-Flavobacterium bacteria

329

pathogenic to freshwater fish. The newly designed primer set gave an expected amplified

330

fragment of approximately 287 bp from all the genomic DNA samples of F. columnare but

331

not from those of other closely related and non-relevant bacteria species, reflecting the

332

specificity of these primers for F. columnare. Our primers were also able to identify

333

isolates from the four genetic groups of F. columnare represented by the 42 strains tested,

334

contrary to Toyama et al. (1996). This intra-species specificity was plausible because the

335

genomic differences in the cslA gene were considered during the design of the primers.

336

Regarding the results of PCR optimization, our primers set worked well under the accepted

337

temperature ranges of PCR (57-62°C) with the highest specificity at 60 °C, where no faint

338

or non-specific amplification bands were obtained. The ability of the newly designed 11

339

primers to form primer-dimers or primer-primer dimers was considered during the initial

340

design. Consequently, our new primer set unlike Bader and Shotts (1998) set, did not

341

require a pre-PCR melting period or long cycles and gave highly repeatable results within a

342

short time frame.

343

Concerning the sensitivity of the primers, the cslA primers were highly sensitive with a

344

detection limit of 3 pg of DNA template per reaction (10-5 dilution). Furthermore, the

345

primers could detect as few as 4 CFU, which is comparable to the results of Bader et al.

346

(2003). However, a two-step PCR procedure was required using Bader’s method to obtain a

347

higher level of sensitivity. Our PCR sensitivity was relatively higher when compared to

348

those designed by Welker et al. (2005). The amplified fragment in our method was smaller

349

(287 bp compared to 400-500 bp) and may account for its greater sensitivity. Sensitivity

350

can be decreased when amplifying large fragments of genomic DNA (Bader et al., 2003).

351

Additionally, our new primer set was able to amplify the target in tissue samples spiked

352

with F. columnare genomic DNA; however, the sensitivity was decreased tenfold to 30 pg

353

(10-4 dilution). The sensitivity might be decreased because of the inhibitory effect of the

354

convoluted tissue samples, which still required further investigations. The beneficial use of

355

PCR for detection of fish pathogens in both fresh and preserved tissues has been fully

356

illustrated (Argenton et al., 1996; Martinez-Picado et al., 1996). In this study, we clearly

357

demonstrated the ability of the new PCR assay to detect F. columnare in fish tissues even at

358

low concentrations. Our results, therefore, encourage the use of the newly developed PCR

359

assay for initial detection of freshwater columnaris in conjunction with plate cultivation,

360

which can be difficult because F. columnare is fastidious and easily overgrown by other

361

fast growing-bacteria (Tiirola et al., 2002).

362

Adherence to the gills and colonization of the skin are critical aspects of the pathogenesis

363

of columnaris disease (Declercq et al., 2013). The skin and gills were shown to be the

364

major release sites of this pathogen and were often used for molecular detection of F.

365

columnare (Tripathi et al., 2005). In the current study, the PCR was able to detect F.

366

columnare in skin and gills of infected fish, but not in those of clinically healthy stocks.

367

The results reported here reflect the clinical validation of our new primer set and provide

368

insights into the potential use of the assay for the field diagnosis of columnaris disease.

12

369

In conclusion, our study has demonstrated that the newly designed cslA gene-based primer

370

set is a promising tool for the diagnosis of F. columnare. It provides a sensitive and

371

reproducible means of detection with no further limitations commonly reported in previous

372

publications. The successful amplification of bacteria from artificially spiked tissues and

373

naturally infected tissues reflected the potential use of the new primer set for diagnostic

374

purposes; however, the precise value of this set to detect columnaris disease among

375

symptomatic and asymptomatic carriers remains questionable and requires further

376

investigation.

377 378

Acknowledgements

379

This research project received financial support from Chulalongkorn University

380

Ratchadaphiseksomphot Endowment Fund for Postdoctoral Fellowship, Chulalongkorn

381

University

382

GR_61_013_31_003) and grant for Fish Infectious Diseases Research Unit (FID RU),

383

Faculty of Veterinary Science, Chulalongkorn University. We thank all staff and students at

384

Fish Infectious Diseases Research Unit (FID RU), Faculty of Veterinary Science,

385

Chulalongkorn University, for providing technical assistance and laboratory equipment.

Ratchadaphiseksomphot

Endowment

Fund

(Grant

number:

CU‐

386 387

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561

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19

562

Table 1. Flavobacterium columnare and other Flavobacterium spp. used for determining the specificity of the PCR. Bacterial

No

Strain code

*Genetic

species

group 1-9

IA-S-4; CSF-298-10; S15-63; ALG-03-063; 14-051; ARS-15-4; ARS-DRB-1-10; ISRAEL; RCO2503

10

F4-HK

11, 12

PT-14-00-151; FBCC-CC-12K;

13- 20

94-081; C#2; EE923; A2502; AL-02-36; TI2429; ALG-00-530; MS-02-475

21

CC1351

22

TI2063

23- 32

AU-LMB-08-5; TN-3-2012; ARS-15-12; 90-106; GA-02-14; ALM-05-111; ALM-05-140; ALM-05-69;

F. columnare

1

2

3

ARS-1; Grizzle 33-42 43-63

TI1354B; TI1371; BZ-1-02; BZ-5-02; TI1690; TI2056; TI472; Costa Rica 04-02-TN; TI1677; TI982 T

T

4

T

F. amniphilum LMG 29727 ; F. anhuiense DSM 22555 ; F. aquatile ATCC 11947 ; F. aquidurense CCUG 59847T; F. araucananum CCUG 61031T; F. brevivitae LMG 29004T; F. frigidarium ATCC

Flavobacterium

700810T; F. frigidimaris DSM 15937T; F. hydatis ATCC 29551T; F. inkyongense JCM 31385T; F.

related species

johnsoniae ATCC 17; F. lacunae LMG28710T; F. oncorhynchi 631-08T; F. pectinovorum ATCC 19366T; F. psychrophilum ATCC 49511; F. resistens BD-B365t; F. spartansii T16T; F. terrae LMG 28395T; F. tiangeerense JCM 15087T; F. verecundum LMG 29005T; F. vireti AB2014312T

563

NA, not applicable

564

*Genetic grouping of the tested F. columnare strains were assigned according to LaFrentz et al. (2019)

565 566

20

NA

567

Table 2. Description of various bacterial isolates used to evaluate the specificity of Fcol primers designed in the present

568

study, including Gene Bank accession number, geographic origin, source, and the year of isolation. Bacteria species

Flavobacterium columnare

No. 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

Strain Code CUVET1201 CUVET1202 CUVET1212 CUVET1213 CUVET1214 CUVET1215 CUVET1221 CUVET1336 CUVET1339 CUVET1346 CUVET1347 CUVET1348 CUVET1349 CUVET1350 CUVET1351 CUVET1352 CUVET1353 CUVET1354 CUVET1361 CUVET1363 CUVET1365 CUVET1369 CUVET1370 CC1801 CC1803 CC1804 CC1805 CC1808 Sp1802 Sp1805 Sp1806 Sp1808 AT1702 AT1703

Gene Bank accession Number KF274033 KF274034 ND KF274038 KF274039 KF274040 ND ND ND KF774290 ND ND ND KF774291 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

21

Geographic origin Ratchaburi Ratchaburi Phetchaburi Phetchaburi Phetchaburi Phetchaburi Bangkok Kanchanaburi Kanchanaburi Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Kanchanaburi Kanchanaburi Kanchanaburi Kanchanaburi Kanchanaburi Chachoengsao Chachoengsao Chachoengsao Chachoengsao Chachoengsao Samutprakan Samutprakan Samutprakan Samutprakan Ayutthaya Kanchanaburi

Host RT RT RT RT RT RT KC RT RT RT RT RT RT RT RT RT RT RT RT RT RT RT RT SB SB SB SB SB SB SB SB SB RT RT

Source Organ Gill Gill Tail Kidney Gill Kidney Ulcer Skin Gill Gill Gill Gill Gill Skin Skin Gill Skin Skin Gill Gill Gill Gill Kidney Skin Skin Skin Skin Skin Gill Gill Gill Gill Gill Gill

Year Isolated 2012 2012 2012 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2018 2018 2018 2018 2018 2018 2018 2018 2018 2017 2017

Flavobacterium indicum Chryseobacterium massiliae Chryseobacterium taichungense Chryseobacterium indologenes Flectobacillus roseus Aeromonas hydrophila Aeromonas vernoii Pseudomonas aeruginosa Streptococcus agalactiae Edwardsiella tarda Edwardsiella ictaluri

98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117

AT1704 CN1807 CN1808 UD1806 UD1809 CC1802 CC1807 CUVET1225 CUVET1205 CUVET1220 CUVET1217 CUVET1218 CUVET1219 CUVET1207

T1-1

ND ND ND ND ND ND ND KJ190180 KJ190166 KJ190177 KJ190174 KJ190175 KJ190176 KJ190168 ND ND ND ND ND ND

Uttaradit Chinart Chinart Uttaradit Uttaradit Chachoengsao Chachoengsao Nhongkai Nhongkai Ratchaburi

RT RT RT RT RT SB SB KC NT RT RT RT RT NT NT NT SCF

Gill Gill Gill Gill Gill Skin Skin Skin ulcer Skin Kidney Skin Kidney Kidney Skin ulcer Kidney Kidney Kidney

2017 2018 2018 2018 2018 2018 2018 2016 2016 2016 2016 2016 2016 2016 2015 2015 2014

569

RT, red tilapia, Oreochromis sp.; KC, koi carp, Cyprinus carpio L.; NT, Nile tilapia, O. niloticus (L.); SB, Seabass,

570

Dicentrarchus labrax; SCF, striped catfish, Pangasianodon hypophthalmus; ND, not determined.

571 572 573 574 575 576 577 578 579 22

580

Table 3. List of primers used in the current study. Primer acronym

Sequence (5’– 3’)

length

Amplicon size (bp)

Target gene

Reference

Fcol-F

AATGACTTCAACTAGAACAGTAGGTGCTGA

30

287

cslA

This study

Fcol-R

CTTGTTTATCATAGATCATAGCTGATGCTCC

31

FCISRFL

TGCGGCTGGATCACCTCCTTTCTAGAGAC

29

450-550

16S rRNA

(Welker et al., 2005)

FCISRR1

TAATYRCTAAAGATGTTCTTTCTACTTGTTTG

32

581 582 583 584 585 586 587 588 589

23

590

Figure legends

591

Fig. 1. Alignment of the cslA gene of 13 F. columnare isolates targeted for PCR. The

592

sequence data were aligned and trimmed by the Molecular Evolutionary Genetics Analysis

593

MEGA v6.0 software (Tamura et al., 2013) using multiple alignment analysis (CLUSTER

594

W method) (Thompson et al., 1994). Conserved sequences used for designing the Fcol-F

595

and Fcol-R primers are indicated.

596 597

Fig 2. Specificity of the newly designed primer set for the detection of F. columnare. Lane

598

numbers matching with the number of bacteria listed in Table 1, 2. Lanes marked M refer

599

to 100 bp molecular mass ladder (Thermo Scientific). CP is the positive control (F.

600

columnare ATCC 23463T), and CN is the negative control (DNA free template). Ten µL of

601

each PCR amplification was loaded into a well of an agarose gel (1.5%) containing Safe

602

Red. The amplified product with the expected amplicon size (287 bp) is considered as a

603

positive result. Molecular weight markers (bp) are indicated to the right and the left of the

604

gel.

605 606

Fig 3. PCR amplification of 14 bacterial species with FCISRFL and FCISRR1 primer pair

607

described by Welker, Shoemaker, Arias, Klesius (2005). Lanes marked M refer to 100 bp

608

molecular mass ladder (Thermo Scientific). CN is the negative control (DNA free

609

template). Lane 1: the specific DNA product of about 500 bp amplified from F. columnare

610

CUVET 1232; Lane 2: the specific DNA product of about 400-500 bp amplified from

611

Flavobacterium indicum; Lane 3- 14: the specific DNA product amplified from Aeromonas

612

hydrophila, Aeromonas vernoii, Pseudomonas aeruginosa, Streptococcus agalactiae,

613

Edwardsiella tarda, Edwardsiella ictaluri, Chryseobacterium massiliae CUVET1205,

614

Chryseobacterium massiliae CUVET1220, Chryseobacterium taichungense CUVET1217,

615

Chryseobacterium

616

CUVET1219, and Flectobacillus roseus CUVET1207, respectively. Ten µL of each PCR

617

amplification was loaded into a well of an agarose gel (1.5%) stained with Safe Red.

618

Molecular mass markers (bp) are indicated to the right and the left of the gel.

taichungense

CUVET1218,

619

24

Chryseobacterium

indologenes

620

Fig 4. Sensitivity of the newly designed primer set for the detection of F. columnare cslA

621

gene. DNA template was diluted in a serial tenfold dilution. Lanes marked M refer to 100

622

bp molecular mass ladder (Thermo Scientific). CN is the negative control (DNA free

623

template). Lanes 1: 300 ng DNA per reaction (no dilution); Lane 2: 30 ng DNA per

624

reaction (10-1 dilution); Lane 3: 3 ng DNA per reaction (10-2 dilution); Lane 4: 300 pg DNA

625

per reaction (10-3 dilution); Lane 5: 30 pg DNA per reaction (10-4 dilution); Lane 6: 3 pg

626

DNA per reaction (10-5 dilution); Lane 7: 0.3 ng DNA per reaction (10-6 dilution); Lane 8:

627

0.03 ng DNA per reaction (10-7 dilution); Lane 9: 0.003 ng DNA per reaction (10-8

628

dilution). Ten µL of each PCR amplification was loaded into a well of an agarose gel

629

(1.5%) stained with Safe Red. Molecular mass markers (bp) are indicated to the right and

630

the left of the gel.

631 632

Fig 5. Sensitivity of the newly designed primer set for the detection of F. columnare cslA

633

gene. Lanes marked M refer to 100 bp molecular mass ladder (Thermo Scientific). CP is

634

the positive control (F. columnare ATCC 23463T), and CN is the negative control (DNA

635

free template). Lane 1: PCR containing 4 × 106 CFU of F. columnare; Lane 2: 4 × 105

636

CFU; Lane 3: 4 × 104 CFU; Lane 4: 4 × 103 CFU; Lane 5: 4 × 102 CFU; Lane 6: 4 × 101

637

CFU; Lane 7: 4 × 100 CFU; Lane 8: 4 × 10-1 CFU; Lane 9: 4 × 10-2 CFU; Lane 10: 4 × 10-3

638

CFU; Lane 11: 4 × 10-4 CFU. Ten µL of each PCR amplification was loaded into a well of

639

an agarose gel (1.5%) stained with RedSafe. Molecular mass markers (bp) are indicated to

640

the right and the left of the gel.

641 642

Fig 6. Sensitivity of the newly designed primer set for the detection of F. columnare cslA

643

gene. Artificially inoculated tissue templates were previously adjusted to 100 ng and spiked

644

with serial tenfold dilutions of F. columnare genomic DNA. Lanes marked M refer to 100

645

bp molecular mass ladder (Thermo Scientific). CP is the positive control (F. columnare

646

ATCC 23463T), and CN is the negative control (DNA free template). Lanes 1: 300 ng DNA

647

per reaction (no dilution); Lane 2: 30 ng DNA per reaction (10-1 dilution); Lane 3: 3 ng

648

DNA per reaction (10-2 dilution); Lane 4: 300 pg DNA per reaction (10-3 dilution); Lane 5:

649

30 pg DNA per reaction (10-4 dilution); Lane 6: 3 pg DNA per reaction (10-5 dilution); Lane

650

7: 0.3 ng DNA per reaction (10-6 dilution); Lane 8: 0.03 ng DNA per reaction (10-7 25

651

dilution); Lane 9: 0.003 ng DNA per reaction (10-8 dilution). Ten µL of each PCR

652

amplification was loaded into a well of an agarose gel (1.5%) stained with Safe Red.

653

Molecular mass markers (bp) are indicated to the right and the left of the gel.

654

Fig 7. Clinical validation of F. columnare cslA gene based primers among the tissues of

655

naturally infected and clinically healthy hybrid red tilapia, Oreochromis sp, previously

656

collected from two different localities in Thailand. CP is the positive control (F. columnare

657

ATCC 23463T), and CN is the negative control (DNA free template). Lanes 1–10, tissues

658

from F. columnare infected fish; Lanes 11–20, tissues from uninfected fish. Lanes 1, 3, 5,

659

7, 9, 11, 13, 15, 17, and 19, skin; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, gills. Ten µL of

660

each PCR amplification was loaded into a well of an agarose gel (1.5%) stained with

661

RedSafe. Molecular mass markers (bp) are indicated to the left of the gel.

26

662 663

Mabrok et al. Fig. 1.

664 665

27

M CN CP 1 2

3

4

5

6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 M

M CN 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 M

287 bp

287 bp

300 bp 200 bp

300 bp 200 bp

M NN CP 43 44 45 46 47 48 49 50 51 52 53 45 55 56 57 58 59 60 61 6 2 63 M

M CN CP 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 M

287 bp

287 bp

300 bp 200 bp

300 bp 200 bp M CN CP 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 M

M

287 bp

CP

105 106 107 108 109 110 111 112 113 114 115 116 117 M

287 bp 300 bp 200 bp

300 bp 200 bp

666 667

CN

Mabrok et al. Fig. 2.

668 669

28

M CN

1

2

3

4

5

500 bp 400 bp

7

8

9

10

11

12

13

14 M

500 bp 400 bp

670 671

6

Mabrok et al. Fig. 3.

672 673 674 675 676 677 678 679

29

M

CN

1

2

3

4

5

6

7

8

9

M

287 bp 300 bp 200 bp

300 bp 200 bp

680 681

Mabrok et al. Fig. 4.

682 683 684 685 686 687 688 689 690

30

M CN

1

2

3

4

5

6

7

8

9 CP

M

287 bp 300 200

300 200

691 692

Mabrok et al. Fig. 5.

693 694 695 696 697 698 699 700 701

31

M CN

1

2

3

4

5

6

7

8

9 CP

M

287 bp 300 bp 200 bp

300 bp 200 bp

702 703

Mabrok et al. Fig. 6.

704 705 706 707 708 709 710 711 712 713

32

M CN CP 1 2

3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20

287 bp 300 bp 200 bp

714 715

Mabrok et al. Fig. 7.

716 717 718

33

1- The 16S rRNA gene is not a good candidate for primer design, since this gene has

high identity among species in the same genus. 2- The present study developed for the first time a species-specific PCR for detection

of Flavobacterium columnare based on the chondroitin AC lyase (cslA) conserved gene. 3- The new primers produced a specific amplicon of 287 bp from F. columnare

strains but not from related or other bacterial pathogens and covered all limitations found in the previous publications. 4- The designed primers correctly amplified field isolates in a double-blind study, reflecting their possible use for diagnostic purposes.

The Editor Journal of AQUACULTURE

Thailand, 2019-07-24

Subject: Submission of manuscript to Journal of Aquaculture

Dear Editor, Please find enclosed a manuscript entitled "Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene, authored by Mahmoud Mabrok,, Putita Chokmangmeepisarn, Benjamin LaFrentz, Pattanapon Kayansamruaj, Ha Thanh Dong and Channarong Rodkhum, which I would like to submit to your consideration for eventual publication in Journal of Aquaculture. Since Flavobacteriosis or columnaris diseases is devastating desease in tilapia culture worldwide. Sensitive and specific diagnostic tool is urgently needed. The current work gave insight into the potential use of a novel species-specific polymerase chain reaction for sensitive and specific detection of columnaris disease, which thought to be more significant and will be of interest to the readers of your journal. On behalf of myself and the co-authors, I state that the present work is original and has not been published, nor is being considered for publication in another journal. We know of no conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome. As Corresponding Author, I confirm that the manuscript has been read and approved for submission by all the named authors. Moreover, the current manuscript was revised for language and grammars and has been edited by an English-speaking native, as recommended. We hope you find our manuscript suitable for publication and look forward to hearing from you in due course. Yours sincerely,

Channarong Rodkhum

Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. E-mail: [email protected] [email protected]