Contents lists available at ScienceDirect Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres Short communication Short communication: Isolation frequency of bacteria causing lymphadenitis and abscesses in small ruminants in central Spain Ricardo de la Fuentea,⁎, Moisés de las Herasa, Carlos Torrijosa, Paloma Diez de Tejadaa, Marta Pérez-Sanchob, Francisco J. Carrióna, José A. Ordena, Gustavo Dominguez-Bernala a Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain b Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, 28040 Madrid, Spain A R T I C L E I N F O Keywords: Caseous lymphadenitis Abscess disease Treuperella pyogenes Actinomyces hyovaginalis A B S T R A C T Infectious lymphadenitis in small ruminants caused by Corynebacterium pseudotuberculosis and Staphylococcus aureus subsp. anaerobius are widely distributed throughout the world, and result in significant economic losses. Trueperella pyogenes has also been associated with lymphadenitis in sheep and goats. In order to determinate the isolation frequency of the different agents associated with lymphadenitis and abscesses, 171 pus samples (135 from sheep and 36 from goats) from 46 flocks were investigated. Isolated bacteria were identified by MALDI-TOF method. S. aureus subsp. anaerobius was the most frequently detected agent. It was identified in 76 animals (59 sheep and 17 goats) from 24 of the surveyed flocks. Of these infected animal, 25 (32,9%) were over one year old, confirming that abscess disease may occur in a significant percentage of adult animals. C. pseudotuberculosis was identified in 45 of the sampled animals (36 sheep and 9 goats) from 24 flocks. Only 5 of animals suffering caseous lymphadenitis were under one year old. T. pyogenes was isolated from 17 animals (13 sheep and 4 goats) in 11 flocks. Seven of these samples were taken from subcutaneous abscesses located in not lymph nodes regions. A notable finding of this work is the isolation of Actinomyces hyovaginalis from 5 of the samples analyzed all of them taken from subcutaneous abscesses located in superficial lymph nodes regions. Thus, T. pyogenes and A. hyovaginalis should be included in the differential diagnosis of lymphadenitis in small ruminants. In 19 of the 46 surveyed flocks at least two of the four agents were detected, which underlines the need to analyze samples from several animals to reach an accurate diagnosis in flocks affected by lymphadenitis. 1. Introduction Two specific lymphadenitis are currently recognized in small ru- minants, caseous lymphadenitis and abscess disease. These diseases are widely distributed throughout the world and result in significant eco- nomic losses. Caseous lymphadenitis is a chronic, contagious disease of worldwide distribution caused by Corynebacterium pseudotuberculosis. The condition affects mainly sheep and goats and is characterized by abscess formation in superficial lymph nodes (external form) or within internal organs and lymph nodes (internal form) (Williamson, 2001). The disease is most frequently seen in adult animals. C. pseudotu- berculosis infection may also occur in other animal species, and it is relatively common in horses, llamas and alpacas and buffalos (Fontaine and Baird, 2008). Human cases, although rare, have been reported especially in Australia and New Zealand, and it is considered an occu- pational zoonosis (Dorella et al., 2006). Abscess disease produced by Staphylococcus aureus subsp. anaerobius affects mainly young animals up to 6 months of age and is characterized by the presence of abscesses in superficial lymph nodes (De la Fuente and Suarez, 1985; De la Fuente et al., 1985, 2011). Subcutaneous ab- scesses not involving lymph node are occasionally reported. The geo- graphic distribution of abscess disease is much more limited than caseous lymphadenitis. In Europe, the condition has been mainly di- agnosed in France, where the disease was first described in 1911, and Spain. The condition has also been detected in Italy, Poland and Croatia, and a unique outbreak of the disease has been described in both Denmark and Hungary in animals imported from France. In Africa, abscess disease has been reported in Tunisia, Kenya, Nigeria, Egypt, Sudan and Somalia. The condition has also been diagnosed in occi- dental Asia (Saudi Arabia and Iran) (De la Fuente et al., 2011). Trueperella pyogenes causes a variety of clinical manifestations in domestic animals, being bovine mastitis the most common (Quinn et al., 2011; Radostits et al., 2007). T. pyogenes infection has also re- cently been associated with lymphadenitis and abscesses in sheep and http://dx.doi.org/10.1016/j.smallrumres.2017.06.022 Received 21 April 2017; Received in revised form 23 June 2017; Accepted 26 June 2017 ⁎ Corresponding author. E-mail addresses: rifuente@vet.ucm.es (R. de la Fuente), moises_delasheras@hotmail.com (M. de las Heras), carlosto@ucm.es (C. Torrijos), maperezs@visavet.ucm.es (M. Pérez-Sancho), fjcarrion@vet.ucm.es (F.J. Carrión), jaorden@vet.ucm.es (J.A. Orden), gdbernal@vet.ucm.es (G. Dominguez-Bernal). Small Ruminant Research 154 (2017) 5–8 Available online 27 June 2017 0921-4488/ © 2017 Elsevier B.V. All rights reserved. MARK http://www.sciencedirect.com/science/journal/09214488 http://www.elsevier.com/locate/smallrumres http://dx.doi.org/10.1016/j.smallrumres.2017.06.022 http://dx.doi.org/10.1016/j.smallrumres.2017.06.022 mailto:rifuente@vet.ucm.es mailto:moises_delasheras@hotmail.com mailto:carlosto@ucm.es mailto:maperezs@visavet.ucm.es mailto:fjcarrion@vet.ucm.es mailto:jaorden@vet.ucm.es mailto:gdbernal@vet.ucm.es http://dx.doi.org/10.1016/j.smallrumres.2017.06.022 http://crossmark.crossref.org/dialog/?doi=10.1016/j.smallrumres.2017.06.022&domain=pdf goats (Ribeiro et al., 2015). In recent years, MALDI-TOF technique has been demonstrated as a rapid and reliable technique used for identification of microorganisms in the routine clinical laboratory both in human and veterinary medi- cine (Angeletti, 2017; Randall et al., 2015; Wattal et al., 2017). This study reports the isolation frequency of agents causing lym- phadenitis and abscesses in small ruminants in a convenience sample of 171 animals (135 sheep and 36 goats) from 46 flocks located in 5 provinces in central Spain. 2. Materials and methods 2.1. Sampling procedure In April 2015, small ruminant practitioners working in 4 provinces of central Spain (Madrid, Ávila, Toledo and Palencia) were contacted, asking for their collaboration for sampling cases of lymphadenitis and abscesses in sheep and goats. Pus samples from subcutaneous abscesses located or not in lymph nodes regions were collected by veterinarians using a sterile syringe. Submitted samples were accompanied by a re- cord sheet containing information on the host species of the flock (sheep, goat or both), breed, size of the flock, sex and age of each sampled animal and site of collection. Significant differences in the frequencies of S. aureus subsp. anae- robius infection between sheep breeds were determined by the Fisher exact test. Statistical significance was set at P ≤ 0.05. 2.2. Bacteria isolation and identification Samples were inoculated in two plates of Columbia agar supple- mented with 5% sheep blood (bioMérieux, Marcy l’Etoile, France), which were subsequently incubated for 48 h at 37 °C, one aerobically, and the other microaerophilically (candle jar system). Firstly, identification of the isolates was performed by MALDI-TOF technique. Isolates were subjected to a protein-peptide extraction pro- tocol according to the manufactureŕs instructions (Bruker Daltonik GmbH, Germany). Bacterial extracted proteins were deposited on a polished steel target plate, let dry at room temperature and overlaid HCCA matrix solution (α-cyano-4-hydroxycinnamic acid in 50% acet- onitrile and 2.5% trifluoroacetic acid). After air-dried at room tem- perature, spectra acquisition was performed using an UltrafleXtreme equipment (Bruker Daltonik) in the linear and positive mode using the FlexControl software (version 3.4) in an automatic mode. Isolates were identified by comparing their MALDI profiles with those included in the BDAL Bruker database (5989 entries) using MALDI Biotyper software (Bruker Daltonik) according to the manufactureŕs instructions. Identification results was interpreted as follows: log-scores below 1.699 was considered not reliable while score values between 1.700-1.999 were considered probable identification at genus level. Finally, score values greater than 2.000 was interpreted as identification at species level being highly probable identification when scores were higher than 2.300. Those isolates which showed score values lower than 1.700 were identified by partial sequencing of the 16S rRNA gene using the uni- versal primers 20F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1500 (5′- GGTTACCTTGTTACGACTT-3). (Weisburg et al., 1991). These analyses were performed using a C1000 ThermalCycler (Bio-Rad, Hercules, CA, USA) and the PCR products were checked in a 2% agarose gel (Biotools, Queensland, Australia). Molecular identification was performed by comparison of individual 16S rRNA gene sequences with those included in the Nucleotide BLAST NCBI (National Center for Biotechnology In- formation, www.ncbi.nlm.nih.gov). 3. Results and discussion From May 2015 to June 2016, 171 pus samples (135 from sheep and 36 from goats) from 46 flocks (28 sheep flocks, 9 goat flocks and 9 mixed sheep and goat flocks) were submitted. Flocks size ranged from 142 to 1700 (median 460). Sampled animals were from one month to 10 years of age (median 2 years). Fifty-five of the animals were less than one year old. The number of submitted samples per flock ranged from one to 9 (median 3). Only 17 of the samples were from sub- cutaneous abscesses located in not lymph nodes regions. Forty-eight of the sheep samples were taken from Assaf breed, 46 from Laucane breed, 9 from Spanish Castellana breed, 6 from Spanish Colmenareña breed and the rest 21 from crosses. The isolation frequencies of the different agents identified in this study are shown in Table 1. S. aureus subsp. anaerobius was the most commonly detected agent. Thus, abscess disease was diagnosed in 76 animals (59 sheep and 17 goats) from 24 of the surveyed flocks (18 sheep flocks, 4 goat flocks and 2 mixed), which shows the widespread of this condition in the analyzed herds. Twenty-five of these infected animals (32.9%) were over one year old. It has traditionally been considered that abscess disease affects mainly young animals up to 5–6 months of age (De la Fuente and Suárez, 1985; De la Fuente et al., 2011). However, in an epidemiological study performed in an enzootic area (Segovia, Spain) Tejedor et al. (1991) found that between 0.5 and 2.5% of 1–2 year old sheep were affected by the disease, and suggested that these animals can act as reservoir. The results of this study confirm that abscess disease may occur in a significant percentage of adult an- imals. Only 7 of the S. aureus subsp. anaerobius positive samples (4 from sheep and 3 from goats) were taken from abscesses located in not lymph nodes regions such as flanks, chest, back or croup. The most frequently affected lymph nodes were mandibular, parotid, lateral retropharyngeal and superficial cervical. The infection frequency of sheep from Assaf breed with S. aureus subsp. anaerobius (33/48, 68,7%) was significantly higher than those from Laucane breed (19/46, 41,3%) and crosses (7/ 26, 26,9%). None of the sheep from Spanish breeds were infected with this bacterium. This results support field observations of the collabor- ating practitioners that Assaf sheep breed is more susceptible to abscess disease than other breeds. Caseous lymphadenitis was also widespread in the surveyed flocks. C. pseudotuberculosis was identified in 45 of the sampled animals (36 sheep and 9 goats) from 24 flocks (15 sheep flocks, 6 goat flocks and 3 mixed). Only 5 of animals suffering caseous lymphadenitis were under one year old, corroborating that the disease affects mainly adult Table 1 Isolation frequency of the identified bacteria in the 171 clinical samples studied. Identified bacteria Number of isolates % Refered to the samples studied Staphylococcus aureus subsp. anaerobius 76a 44.4 Corynebacterium pseudotuberculosis 45b 26.3 Treuperella pyogenes 17c 9.9 Actinomyces hyovaginalis 5d 2.9 Staphylococcus lentus 5e 2.9 Streptococcus ovis 4f 2.3 Staphylococcus simulans 3g 1.7 Staphylococcus warneri, Staphylococcus capraeh, Staphylococcus equorum, Corynebacterium camporealensis, Pseudomonas aeruginosa, Pseudomonas hydrophila, Actinobacillus pleuropneumoniae, Vagococcus lutrae, Acinetobacter spp. 1 0.6 Negative or non-significant growth 15 8.8 a One in mixed infection with T. pyogenes. b Two in mixed infection with S. lentus and one with S. simulans. c One in mixed infection with A. hyovaginalis. d One in mixed infection with S. ovis. e One in mixed infection with C. camporealensis. f One in mixed infection with Acinetobacter spp. g One in mixed infection with V. lutrae. h In mixed infection with P. hydrophila. R. de la Fuente et al. Small Ruminant Research 154 (2017) 5–8 6 http://www.ncbi.nlm.nih.gov animals (Al-Gaaraby et al., 2009). Caseous lymphadenitis remains an important subject of veterinary concern because, once introduced into a flock, it is very difficult to control. T. pyogenes was isolated from 17 animals (13 sheep and 4 goats) in 11 flocks (8 sheep flocks, 2 goat flocks and one mixed). Seven of these samples were taken from subcutaneous abscesses located in not lymph nodes regions. Only 3 of the animals infected with T. pyogenes were under one year old. Our results agree with those recently described in a retrospective study by Ribeiro et al. (2015) in which they found a high frequency of lymphadenitis and abscesses caused by T. pyogenes in sheep. In addition, they also detected this bacterium in the same pro- cesses in goats. T. pyogenes has been associated with a wide variety of clinical manifestations in domestic animals (Quinn et al., 2011; Radostits et al., 2007; Ribeiro et al., 2015). A notable finding of this work is the isolation of Actinomyces hyo- vaginalis from 5 of the samples analyzed (4 from sheep and one from goat) all of them taken from abscesses located in lymph nodes regions. The identification of 3 of these isolates was performed by sequencing the 16S rRNA due to the low score obtained with the MALDI-TOF method. In 2 cases A. hyovaginalis was isolated in mixed culture, one with T. pyogenes and the other with Streptococcus ovis. A. hyovaginalis has been associated predominantly with pigs, and isolated from samples of purulent vaginal discharges or aborted fetuses (Collins et al., 1993; Hogg et al., 2012) and disseminated necrotic pulmonary lesions (Aalbeck et al., 2003). However, recently Foster et al. (2012) have described for the first time the isolation of A. hyovaginalis from samples of 9 sheep and a mouflon. Six of this 9 A. hyovaginalis sheep isolates came from abscess samples, and at least 3 of the samples were referred to the diagnostic laboratory as suspicious cases of caseous lymphade- nitis. On the other hand, Schumacher et al. (2009) isolated A. hyova- ginalis from a case of lymphadenitis in a goat that was negative to C. pseudotuberculosis. The postmortem examination of this case described lesions very similar to typical caseous lymphadenitis with abscesses located in the tracheobronchial lymph nodes. The pathological significance of all the other bacteria identified in this study, apart from the four already discussed, including S. ovis and coagulase negative staphylococci (CNS), is not evident. Two of the 4 S. ovis found in this study were in mixed culture along with other bac- terium (Table 1). S. ovis has previously been isolated from 7 clinical specimens from sheep, but 5 of the 7 strains were isolated in mixed cultures with at least two other bacteria (Collins et al., 2001). In ru- minants CNS are mainly associated with intramammary infections, and some of the species are considered minor pathogens (Vanderhaeghen et al., 2015). Six of the 11 CNS isolates identified in this work were in mixed culture along with other bacterium (Table 1). They are most probably opportunistic invading from skin. Mixed cultures of two bacteria were found in 10 of the samples analyzed, being the most significant a case of S. aureus subsp. anaerobius along with T. pyogenes and another case of T. pyogenes together with A. hyovaginalis (Table 1). In 19 of the 46 surveyed flocks two (16 flocks) or three (3 flocks) of the four main agents causing specific lymphadenitis were detected (Table 2). In 9 flocks both abscess disease and caseous lymphadenitis were diagnosed. This implies that, in the geographical area studied, the analysis of a single or very few samples for the etiological diagnosis of lymphadenitis may lead to a partial view of the problem. 4. Conclusion The differential etiologic diagnosis of infectious lymphadenitis in small ruminants should include not only C. pseudotuberculosis and S. aureus subsp. anaerobius but also T. pyogenes and A. hyovaginalis. Moreover, due to the fact that more than one of these agents are fre- quently found in the same flock, it is necessary to analyze samples from several animals (4–6, depending on the prevalence, and covering, where appropriate, the different ages) to reach an accurate diagnosis in flocks affected by lymphadenitis. Conflicts of interest None. Acknowledgement This study was supported by a grant from the Universidad Complutense de Madrid-Banco Santander (GR3/14). References Aalbeck, A., Christensen, H., Bisgaard, M., 2003. Actinomyces hyovaginalis associated with disseminated necrotic lung lesions in slaughter pigs. J. Comp. Pathol. 129, 70–77. Al-Gaaraby, M.H., Osman, S.A., Oreiby, A.F., 2009. 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