# Trueperella pyogenes andBrucella abortus Coinfection in a Dog and a Cat on a Dairy Farm in Egypt with Recurrent Cases of Mastitis and Abortion

Authors: Gamal Wareth; Mohamed El-Diasty; Falk Melzer; Jayaseelan Murugaiyan; Amir Abdulmawjood; Lisa D. Sprague; Heinrich Neubauer
Journal: Veterinary Medicine International
 (2018)

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## Abstract

Trueperella pyogenes was isolated from a dog and a cat with a mixed infection withBrucella abortus. Both lived on a dairy cattle farm with a history of regular cases of abortion and mastitis. Identification of the bacteria was done by means of MALDI-TOF MS, loop-mediated isothermal amplification (LAMP) based oncpn60, partial 16S rRNA sequencing, and growth on Loeffler Serum Medium. Isolation ofTrueperella pyogenes on the dairy farm highlights its neglected role in reproduction failure and draws attention to its effects in the dairy industry in Egypt. Diagnosis and control of abortion in Egypt should includeTrueperella pyogenesas one of possible causes of abortion.


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## Body

## 1. Introduction

Trueperella (T.) pyogenes is Gram positive, haemolytic, nonmotile, non-spore-forming, facultative anaerobic coccobacillus. The species was originally known asCorynebacterium pyogenes andActinomyces pyogenes and then asArcanobacterium pyogenes. Based on the 16S rRNA signature nucleotide comparisons and menaquinone and phospholipid compositions, the genus was divided intoArcanobacterium andTrueperella gen. nov., in honour of the German microbiologist Hans G. Trüper. Currently, the genusTrueperellaencompasses five different species, which are,T. pyogenes, T. abortisuis, T. bonsai, T. bernardiae, andT. bialowiezensis[1].Trueperella pyogenes is a commensal and an opportunistic pathogen which can cause a variety of suppurative infections in livestock and wildlife and can elicit sporadic cases of abortion due to endometritis and placentitis at any stage of gestation [2]. The bacteria can affect a wide range of animal species, including cattle, camel, horse, swine, antelope, bison, chicken, pigeon, turkey, deer, elephant, gazelle, macaw, and reindeer [3], as well as companion animals such as dogs and cats [4].T. pyogenes has recently been isolated from lung abscesses of slaughtered one-humped camels in Cairo presenting with clinical and subclinical pulmonary infection [5]. Different suppurativeT. pyogenes infections in livestock and companion animals are associated with a variety of virulence factors, particularly exotoxin pyolysin and adhesins (fimbriae, neuraminidases, and collagen-binding protein) [6].The present study describes the isolation ofT. pyogenes in the uterine discharge of a bitch after abortion and from a cat presenting with pyometra. Both animals lived on a dairy farm with recurrent cases of abortion and mastitis due toBrucella (B.) abortusbiovar (bv) 1 infections [7]. The aim of the current study was to identify and highlight a neglected pathogen in Egypt which can circulate in pet animals in contact with livestock and cause reproductive failure within the dairy industry.


## 2. Material and Methods

### 2.1. Bacteriology

Uterine discharges were collected from the bitch and the cat under sterile conditions after positiveBrucella serology using Rose Bengal Test and Complement Fixation Test and sent to the OIE reference laboratory of brucellosis at the Friedrich-Loeffler-Institut forBrucella identification and biotyping. During cultivation on blood agar forBrucella biotyping, two types of bacterial colonies were found. Identification and biotyping of the putativeBrucella isolates were done by assessing colony morphology, biochemical reactions, CO2 requirement, production of H2S, growth in the presence of the dyes thionine and fuchsine, reaction with monospecific antisera (A, M, and R), and phage lysis (Wb, Tb, and F25) [8]. PutativeTrueperella strains were additionally grown on Loeffler Serum Medium (Himeda, Mumbai, India) and tested for catalase activity.


### 2.2. MALDI Based Species Identification

The microbial species identification was carried out using matrix-assisted laser desorption/ionization (MALDI-TOF MS) as described elsewhere [9]. In brief, bacteria from single colonies were inactivated by the addition of 300 μL of HPLC grade water and 900 μL of absolute ethanol. For protein extraction, the suspensions were centrifuged at 11290g for 2 min, the supernatants discarded, and the resulting cell pellets air-dried to remove traces of ethanol. Each pellet was reconstituted in 50 μL of 70% formic acid and 50 μL of acetonitrile. The samples were then sonicated (100% amplitude and 1.0 duty cycle) for 1 min on ice using a sonicator (UP100H; Hielscher Ultrasound Technology, Teltow, Germany). Next, samples were centrifuged at 11290g for 5 min at room temperature and the clear supernatant was collected. One μL of each supernatant was spotted onto the MALDI target (MSP 96 target polished steel (MicroScout Target) plate; Bruker Daltonik, Bremen, Germany), air-dried, and overlaid with 1.0 μL of saturated α-cyano-4-hydroxycinnamic acid matrix solution (in 50% acetonitrile and 0.25% trifluoroacetic acid). The MALDI measurements were carried out using a Microflex LT (Bruker Daltonics, Bremen, Germany) instrument and MBT Compass Explorer 4.1 software (Bruker Daltonics, Bremen, Germany). The MALDI Biotyper manufacturer’s recommendation on log score value of 0–3 for species identification was followed. Score values between 2.3 and 3.0 indicate “highly probable species identification”; values between 2.0 and 2.29 indicate a “secure genus identification and probable species identification”; values between 1.7 and 1.99 indicate “probable genus identification”; and values between 0 and 1.69 indicate “no reliable identification.”


### 2.3. DNA Extraction, AMOS-PCR, and Partial 16S rRNA Sequencing

Genomic DNA was extracted from heat inactivated individual bacterial colonies using the High Pure PCR template preparation kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s instructions. AMOS-PCR forB. abortus, B. melitensis, B. ovis, andB. suis was done as previously described [7, 10]. Partial 16S rRNA genes of the bacterial isolates were amplified by PCR with the 16SUNI-L (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 16SUNI-R (5′-GTG TGA CGG GCG GTG TGT AC-3′) primer pair (Jena Bioscience GmbH, Jena, Germany) to generate amplicons of approx. 1,400-bp [11]. PCR products were analyzed by agarose gel electrophoresis, bands were cut out, and DNA was purified using a Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Cycle sequencing of the partial 16S rRNA genes was done in both directions by using forward and reverse amplification primers with a BigDye Terminator Version 1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) according to the recommendations of the manufacturer. Sequencing products were analyzed with an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Identification of isolates was done by a BLAST search (https://www.ncbi.nlm.nih.gov/blast/) using 16S rRNA gene sequences.


### 2.4. Loop-Mediated Isothermal Amplification (LAMP)

A newly designed loop-mediated isothermal amplification (LAMP) assay based oncpn60 (encoding for chaperonin 60) was carried out in a total volume of 25 μL containing 0.5 μL each of pho-F3 and Pyo-B3 primer (25 pmol/μL) equivalent to 0.5 μM final concentration, 1 μL each of Pyo-LoopF and Pyo-LoopB primer (25 pmol/μL) equivalent to 1.0 μM final concentration, 2 μL each of Pyo-FIP and Pyo-BIP primer (25 pmol/μL) equivalent to 2.0 μM final concentration, and 15 μL Isothermal Master Mix Iso-001 (Optigene, Horsham, UK). Subsequently, 3 μL DNA was added as a template. The LAMP assay was run at 70°C for 30 min with a melting curve analysis step (annealing curve 98°C to 80°C ramping at 0.05°C per s) in a portable real-time fluorometer (Genie II®, Optigene, West Sussex, UK) according to the manufacturer’s instructions as previously described [12].


## 2.1. Bacteriology

Uterine discharges were collected from the bitch and the cat under sterile conditions after positiveBrucella serology using Rose Bengal Test and Complement Fixation Test and sent to the OIE reference laboratory of brucellosis at the Friedrich-Loeffler-Institut forBrucella identification and biotyping. During cultivation on blood agar forBrucella biotyping, two types of bacterial colonies were found. Identification and biotyping of the putativeBrucella isolates were done by assessing colony morphology, biochemical reactions, CO2 requirement, production of H2S, growth in the presence of the dyes thionine and fuchsine, reaction with monospecific antisera (A, M, and R), and phage lysis (Wb, Tb, and F25) [8]. PutativeTrueperella strains were additionally grown on Loeffler Serum Medium (Himeda, Mumbai, India) and tested for catalase activity.


## 2.2. MALDI Based Species Identification

The microbial species identification was carried out using matrix-assisted laser desorption/ionization (MALDI-TOF MS) as described elsewhere [9]. In brief, bacteria from single colonies were inactivated by the addition of 300 μL of HPLC grade water and 900 μL of absolute ethanol. For protein extraction, the suspensions were centrifuged at 11290g for 2 min, the supernatants discarded, and the resulting cell pellets air-dried to remove traces of ethanol. Each pellet was reconstituted in 50 μL of 70% formic acid and 50 μL of acetonitrile. The samples were then sonicated (100% amplitude and 1.0 duty cycle) for 1 min on ice using a sonicator (UP100H; Hielscher Ultrasound Technology, Teltow, Germany). Next, samples were centrifuged at 11290g for 5 min at room temperature and the clear supernatant was collected. One μL of each supernatant was spotted onto the MALDI target (MSP 96 target polished steel (MicroScout Target) plate; Bruker Daltonik, Bremen, Germany), air-dried, and overlaid with 1.0 μL of saturated α-cyano-4-hydroxycinnamic acid matrix solution (in 50% acetonitrile and 0.25% trifluoroacetic acid). The MALDI measurements were carried out using a Microflex LT (Bruker Daltonics, Bremen, Germany) instrument and MBT Compass Explorer 4.1 software (Bruker Daltonics, Bremen, Germany). The MALDI Biotyper manufacturer’s recommendation on log score value of 0–3 for species identification was followed. Score values between 2.3 and 3.0 indicate “highly probable species identification”; values between 2.0 and 2.29 indicate a “secure genus identification and probable species identification”; values between 1.7 and 1.99 indicate “probable genus identification”; and values between 0 and 1.69 indicate “no reliable identification.”


## 2.3. DNA Extraction, AMOS-PCR, and Partial 16S rRNA Sequencing

Genomic DNA was extracted from heat inactivated individual bacterial colonies using the High Pure PCR template preparation kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s instructions. AMOS-PCR forB. abortus, B. melitensis, B. ovis, andB. suis was done as previously described [7, 10]. Partial 16S rRNA genes of the bacterial isolates were amplified by PCR with the 16SUNI-L (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 16SUNI-R (5′-GTG TGA CGG GCG GTG TGT AC-3′) primer pair (Jena Bioscience GmbH, Jena, Germany) to generate amplicons of approx. 1,400-bp [11]. PCR products were analyzed by agarose gel electrophoresis, bands were cut out, and DNA was purified using a Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Cycle sequencing of the partial 16S rRNA genes was done in both directions by using forward and reverse amplification primers with a BigDye Terminator Version 1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) according to the recommendations of the manufacturer. Sequencing products were analyzed with an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Identification of isolates was done by a BLAST search (https://www.ncbi.nlm.nih.gov/blast/) using 16S rRNA gene sequences.


## 2.4. Loop-Mediated Isothermal Amplification (LAMP)

A newly designed loop-mediated isothermal amplification (LAMP) assay based oncpn60 (encoding for chaperonin 60) was carried out in a total volume of 25 μL containing 0.5 μL each of pho-F3 and Pyo-B3 primer (25 pmol/μL) equivalent to 0.5 μM final concentration, 1 μL each of Pyo-LoopF and Pyo-LoopB primer (25 pmol/μL) equivalent to 1.0 μM final concentration, 2 μL each of Pyo-FIP and Pyo-BIP primer (25 pmol/μL) equivalent to 2.0 μM final concentration, and 15 μL Isothermal Master Mix Iso-001 (Optigene, Horsham, UK). Subsequently, 3 μL DNA was added as a template. The LAMP assay was run at 70°C for 30 min with a melting curve analysis step (annealing curve 98°C to 80°C ramping at 0.05°C per s) in a portable real-time fluorometer (Genie II®, Optigene, West Sussex, UK) according to the manufacturer’s instructions as previously described [12].


## 3. Results and Discussion

In addition toB. abortus bv1, a Gram positive, catalase negative, aerobic, nonmotile, ß-haemolytic bacterium was recovered from a bitch who had recently aborted and from a cat suffering from an open pyometra. Both animals lived on a dairy farm with regular cases of abortion and mastitis [7]. These recovered bacteria produced H2S and were able to grow under aerobic conditions with and without CO2. They did not react with the tested monospecific antisera and phages specific forBrucella and produced no amplicon in the AMOS-PCR. MALDI-TOF MS identified these bacteria asT. pyogenes with a log score of 2.18 (dog isolate) and a log score of 2.02 (cat isolate), respectively. These log score values confirm the identification at the species level. MALDI-TOF MS forTrueperella differentiation with a log score around 2 shows comparable discriminating power with molecular methods; it is a rapid and accurate tool forT. pyogenes diagnosis [13]. Partial 16S rRNA sequencing confirmed the isolates to beT. pyogenes and cultivation on Loeffler serum revealed the typical pitting of the serum slope. The species was also identified using thecpn60 LAMP assay. A loop amplification signal of the LAMP products was observed for bothT. pyogenes isolates and for the reference strainT. pyogenesDSM 20594 and none in the negative control containing water and LAMP-Mastermix (Figure 1). The mean of the annealing temperature of the amplicons was 89,6°C sd ± 0.05 s (Figure 2). Thecpn60 LAMP assay allowed a reliable, rapid, and low cost identification ofT. pyogenes (Table 1).Table 1
Results of LAMP including detection time and annealing temperature of each isolate and positive and negative control.
Number
Isolate number 1
Isolate number 2
Positive control
Negative control
Sample ID
15RB7429H
15RB7430H
T. pyogenes DSM 20594
HPLC water and Master mix
Result
+ve
+ve
+ve
−ve
Detection time
10:00
7:30
17:30
00
Annealing
89.7
89.6
89.6
00Figure 1
Loop amplification signal of the LAMP products of the twoT. pyogenes isolates, the reference strainT. pyogenesDSM 20594, and the negative control.Figure 2
Annealing reaction curves of the respective amplicons.The chaperonin 60 encoding gene has been previously used for the identification of various Gram positive bacteria and a chaperonin sequence database containing a large collections of sequences including genecpn60 ofT. pyogenes is available. This was comparable to the previously described LAMP-mediated identification ofA. pluranimalium usingpla gene [14].Trueperella pyogenes is a ubiquitous occurring organism and is frequently found as a commensal in the oropharynx, upper respiratory tract, and gastrointestinal tract of livestock [2]. However, underlying chronic illness, innate immunity, poor animal husbandry, and production methods appear to influence the virulence of the agent [15–18]. It can readily be transmitted by biting flies and contaminated farm and dairy equipment [6].In Egypt, the dairy industry suffers from large financial losses due to reduced fertility and milk yield as a consequence of uterine infections and mastitis caused not only by brucellosis [5] but also by other agents such asE. coli,Fusobacterium necrophorum, andT. pyogenes [19, 20]. AlthoughT. pyogenes is a well-known agent causing reproductive disorders in male and female livestock species [4], its role is neglected as primary cause of abortion in cattle, as diagnosis of abortion is focusing only on predominately classical agents, such asBrucella species. Circulating of the bacterium in dog and cat kept in dairy farm with history of abortion and mastitis is alarming and representing a potential reservoir of infection for livestock. Thus screening for this agent should be considered in cases of seronegativity toBrucella. On the other hand, eradication of pet animals has to be implemented and taken into account in control measures to reduce dissemination of pathogens.T. pyogenesisis not a sufficiently known pathogen in animals and humans because of inadequate identification of this bacterium that should be better known to clinical microbiologists. Diagnosis of abortion in cattle and control has to includeT. pyogenesisespecially in farm suffering from mastitis and sporadic abortion.



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*Source: 2056436-2018-03-20.xml*