Abstract
Background: Formerly, Arcanobacterium pyogenes was recently renamed Trueperella pyogenes. This opportunistic bacterium is related to miscellaneous pyogenic infections in animals. Most studies involving T. pyogenes are case reports, whereas few surveys have focused the major aspects of T. pyogenes infections involving a case series study design.
Objective: The aim of this study was to retrospectively evaluate selected epidemiological and clinical aspects, as well as the in vitro antimicrobial susceptibility pattern of 144 cases of T. pyogenes infections among domestic animals from 2002 to 2012.
Animals and methods: T. pyogenes was isolated from different clinical specimens from cattle, goats, sheep, pigs, horses, dogs, and buffaloes. Correlations were assessed by the Chi-square or Fisher's exact tests.
Results: Mastitis (45.1%), abscesses (18.0%), pneumonia (11.1%), and lymphadenitis (9.0%) were the most common clinical manifestations. In addition, the organism was also isolated from other miscellaneous clinical specimens from cases of septicemia, encephalitis, pyometra, prostatitis, orchitis, seminal vesiculitis, pericarditis, and omphalitis. No statistical association was observed between T. pyogenes infections and age, gender, or season across the study. The most effective drugs against the pathogen were florfenicol (99.1%), cefoperazone (96.0%), cephalexin (95.0%), and ceftiofur (94.8%). High resistance rates were observed against trimethoprim-sulfamethoxazole (49.3%), followed by norfloxacin (10.9%) and tetracycline (9.2%).
Conclusions: This study highlights the diversity of clinical manifestations and the opportunistic behavior of T. pyogenes infections in domestic animals, with predominance of mastitis, abscesses, pneumonia, and lymphadenitis. It also reinforces the importance of knowing the susceptibility profile before initiating therapy, to improve antimicrobial therapy approaches.
1. Introduction
Formerly, Arcanobacterium pyogenes was recently renamed Trueperella pyogenes (T. pyogenes) based on phylogenetic studies (16S rRNA), phospholipid composition, and the presence of menaquinone (vitamin K2) (Yassin et al. Citation2011).
T. pyogenes is a well-recognized Gram-positive, non-motile, non-spore-forming, short, rod-shaped ‘coryneform’ bacterium. This opportunistic organism belongs to the Actinomycetales order, which is taxonomically related to important organisms that are pathogenic to humans and animals from the genera Mycobacterium, Corynebacterium, Actinomyces, Rhodococcus, Dermatophilus, and Nocardia (Quinn et al. Citation2011).
This micro-organism is ubiquitous, usually found on the skin, oropharynx, upper respiratory, urogenital, and gastrointestinal tracts of animals (Songer & Post Citation2005, Quinn et al. Citation2011). In addition, it may contaminate farm utensils or to be transmitted by biting flies that cause bovine mastitis. T. pyogenes infections have been usually described involving pyogenic infections in livestock (Radostits et al. Citation2007). In contrast, T. pyogenes infections are unusual in companion animals (Greene Citation2012) and humans (Kavitha et al. Citation2010), since the bacterium is not an inhabitant of the normal microflora of this species. Among humans, T. pyogenes infections are predominantly restricted to cases of occupational exposure (Gahrn-Hansen & Frederiksen Citation1992).
The virulence of T. pyogenes is attributed to several mechanisms. To date, pyolisin, a potent cytolisin related to tissue damage, is considered the primary virulence factor of the organism. In addition, neuraminidases (nanH and nanP genes), fimbriae (fimA), and collagen-binding protein (cbpA) are also involved in the pathogenicity, and associated to mucosal adherence and colonization (Jost & Billington Citation2005). Development of pyogranulomatous reactions in tissues and organs is another pathogenic mechanism of T. pyogenes (Radostitis et al. Citation2007). Due to opportunistic behavior of the micro-organism, several routes of transmission are possible (Jost & Billington Citation2005).
A great variety of clinical manifestations has been attributed to T. pyogenes infections in domestic animals, including mastitis, pneumonia, metritis, arthritis, lymphadenitis, otitis, peritonitis, pyodermitis, umbilical thickening, pyodermitis, endocarditis, organ abscesses, osteomyelitis, and urinary and genital tract infections (Songer & Post Citation2005; Radostits et al. Citation2007; Quinn et al. Citation2011; Greene Citation2012). Bovine mastitis in heifers and dry cows appears to be the most common clinical picture of T. pyogenes infections in livestock (Radostits et al. Citation2007; Ruegg Citation2010). Nevertheless, the knowledge on T. pyogenes infections among domestic animals is fragmented and restricted to the report of cases or outbreaks (Motta et al. Citation2011). Thus, few studies have focused on the major clinical and epidemiological aspects of T. pyogenes infections involving a great number of animals. We describe here the main epidemiological and clinical aspects, as well as the antimicrobial sensitivity pattern of 144 T. pyogenes cases affecting various domestic species over an 11-year period.
2. Materials and methods
Microbiological culture data, and selected clinical and epidemiological findings from 144 T. pyogenes cases among different domestic animals admitted to the Veterinary Hospital of the School of Veterinary Medicine and Animal Science, UNESP/Botucatu, SP, Brazil, for an 11-year period (2002–2012), were analyzed retrospectively. The major clinical manifestations were investigated in the study.
Selected epidemiological data were represented by age, season, gender, and breed. T. pyogenes was isolated in both males and females, in different breeds, and in cross-bred animals. From 65 cases of mastitis, only seven were identified in summer months. The remaining cases were observed in the spring (n = 24), autumn (n = 23), and winter (n = 11).
Among animals affected by pneumonia, only four cases were observed in winter, whereas the remaining cases were identified in summer (n = 3), autumn (n = 2), and spring (n = 2). The most common age of bovine mastitis occurrence ranged from 6- to 7.9-years-old (n = 26), followed by 4- to 5.9-years-old cows (n = 9), and 2- to 3.9-years-old (n = 7) cows. In contrast, other clinical manifestations studied were more common among 0–1.9- (n = 24), and 2–3.9-years-old animals (n = 20), followed by 4–5.9-years-old animals (n = 8), and 6–7.9-years-old animals (n = 9).
Cultured material was obtained mainly from milk, abscesses, broncho-alveolar lavage fluid, lymph nodes, blood, cerebrospinal fluid, uterus secretion, semen, and various tissues at necropsy. All the samples were plated on defibrinated sheep blood agar (5%), and simultaneously incubated aerobically and at 5% CO2 atmosphere, at 37 °C for 96 hours. The same samples were also plated on MacConkey media and kept under the same aerobic conditions described above. When required, the same samples were also plated on sheep blood agar and incubated under anaerobic conditions, and kept at 37 °C for up to 7 days. Colonies morphologically compatible with T. pyogenes were submitted to conventional phenotypic tests (Quinn et al. Citation2011), including CAMP test with S. aureus ATCC 13565, aesculin and gelatin hydrolysis, nitrate reduction, urease, and acid production from glucose, maltose, mannitol, sucrose, and xylose.
Pneumonia was diagnosed by isolation of pathogen from broncho-alveolar lavage fluid and samples from lung abscesses (Quinn et al. Citation2011). Clinical and subclinical mastitis were diagnosed by conventional strip cup test and California Mastitis Test (2+ and 3+ score), respectively (Radostits et al. Citation2007). Clinical and subclinical cases were grouped and considered as mastitis manifestation.
From a total of 144 cases, 113 isolates were submitted to the antimicrobial disk diffusion test according to the NCCLS guidelines for aerobic actinomycetes (NCCLS Citation2006), with some modifications. Plates containing Mueller-Hinton agar were supplemented with 0.5% sheep blood and 0.5% Tween 80 (Martinez-Martinez et al. Citation1995). Briefly, strains were subcultured twice on sheep blood agar (5%) under aerobic conditions at 37 °C to ensure purity. After 48 hours, the isolates were inoculated in brain-heart-infusion broth and incubated at 37 °C for 48 hours. Sterile glass beads were added to decrease clump formation, which are commonly observed during actinomycetes growth. In order to inoculate the recommended amount of colony forming units, the isolates were vortexed until the appropriate optical density (OD) was reached in the McFarland scale, a critical step in actinomycete culture (Ambaye et al. Citation1997; Condas et al. Citation2013). The final inocula were adjusted to OD equivalent to a 0.5 McFarland standard. Due to the lack of standard breakpoints for T. pyogenes interpretative antimicrobial susceptibility categories, those established by the NCCLS for other Gram-positive bacterium and other fastidious Gram-positive organisms, such as Streptococcus pneumoniae, were used to define inhibition zones, according to similar studies involving coryneform bacterium (Zastempowska & Lassa Citation2012). The isolates were classified as susceptible or resistant after 48 hours of incubation. Fourteen commercially available antimicrobials used in livestock and companion animal veterinary practice were used in the sensitivity tests as follows: amoxicillin (10 μg), ampicillin (10 μg), cephalexin (30 μg), cefoperazone (75 μg), ceftiofur (30 μg), ciprofloxacin (5 μg), enrofloxacin (5 μg), florfenicol (30 μg), penicillin G (10 units), gentamicin (10 μg), norfloxacin (10 μg), rifampicin (5 μg), trimethoprim-sulfamethoxazole (25 μg), and tetracycline (30 μg).
The Chi-square or Fisher's exact tests were used to (1) compare the proportion of mammary infections by T. pyogenes and age of animals, (2) mammary infections versus season, and (3) pneumonia versus season. Statistical analyses were conducted using SPSS version 14 for Windows (SPSS Inc., Chicago, IL, USA) and the level of statistical significance was set at 0.05.
3. Results
After 48 hours, small colonies of 1 mm in diameter, beta-hemolytic on sheep blood agar under aerobic and 5.0% CO2 conditions were observed in all samples, which were suggestive of T. pyogenes. Panoptic and Gram staining revealed small pleomorphic micro-organisms (‘coryneform’ aspect). The isolates were positive in CAMP test with S. aureus ATCC 13565, and combined to results of different phenotypic tests and substrate utilization, the organisms were classified as T. pyogenes. Among 144 samples, 132 (91.7%) were cultured only in aerobic conditions, whereas 12 (8.3%) were cultured for both aerobic and anaerobic atmospheres. From these 12 samples cultured anaerobically, 7 showed positive cultures for the genera Peptostreptococcus, Fusobacterium, and Clostridium.
Data on clinical manifestations versus domestic species infected by T. pyogenes are shown in . One hundred forty-four T. pyogenes cases affecting domestic animals were evaluated in this study. From these, mastitis (45.1%), abscesses (18.0%), pneumonia (11.1%), and lymphadenitis (9.0%) were the most common clinical manifestations. However, the organism was also isolated among other miscellaneous diseases, such as septicemia, encephalitis, pyometra, prostatitis, orchitis, seminal vesiculitis, pericarditis, interdigital phlegmon, and omphalitis.
Table 1. Occurrence of clinical manifestations caused by Trueperella pyogenes in domestic species. Brazil, 2002–2012.
Cattle (62.5%) and sheep (19.4%) were the species most frequently affected by T. pyogenes infections, followed by a few cases among goats (9.0%), pigs (3.5%), horses (2.8%), buffaloes (1.4%), and dogs (1.4%).
More than 80.0% of cows revealed clinical mastitis. No statistically significant association was observed between mastitis caused by T. pyogenes versus season (P = 0.1573) and occurrence of mastitis versus age of cows (P = 0.2424). Also, no statistically significant association was observed between T. pyogenes pneumonia and season (P = 0.1991).
The results on the most relevant antimicrobial susceptibility pattern of the isolates are described in . The most effective drugs (>90% efficacy) against the isolates were florfenicol (99.1%), cefoperazone (96.0%), cephalexin (95.0%), ceftiofur (94.8%), amoxicillin (94.6%), penicillin G (91.2%), and gentamicin (91.8%). High resistance rates among the isolates were recorded against trimethoprim-sulfamethoxazole (49.3%), followed by norfloxacin (10.9%) and tetracycline (9.2%).
Table 2. In vitro antimicrobial susceptibility profile of Trueperella pyogenes isolated from domestic animals. Brazil, 2002–2012.
4. Discussion
Over the last decades, T. pyogenes has been implicated as a cause of different clinical manifestations in domestic animals (Radostits et al. Citation2007; Quinn et al. Citation2011; Greene Citation2012). In fact, besides the major occurrence of mastitis, pneumonia, lymphadenitis, and abscesses, miscellaneous other less frequent infections among livestock were identified in this study, including septicemia, encephalitis, pyometra, prostatitis, orchitis, seminal vesiculitis, pericarditis, omphalitis, and interdigital phlegmon. This result confirms the opportunistic behavior of T. pyogenes infections, since the pathogen is found in mucous membranes of animals and in the environment, and may potentially infect any tissue or organ from domestic species (Jost & Billington Citation2005; Quinn et al. Citation2011).
T. pyogenes is a common pathogen causing diverse suppurative clinical manifestations among cattle and pigs, although it is specially reported as a causative agent of bovine mastitis. The organism is typically related to mammary infections in heifers and dry cows, and occasionally as a secondary invader in lactating cows after teat injuries (Ruegg Citation2010). Two types of severe bovine mastitis are caused by T. pyogenes, called pyogenes mastitis and summer mastitis. Pyogenes mastitis is a sporadic suppurative form of infection, predominant in housed cattle. In contrast, summer mastitis is reported particularly as outbreaks in pastured cattle with access to ponds and wet areas during summer months in Europe (Radostits et al. Citation2007). In Brazil, an outbreak of bovine mastitis caused by T. pyogenes was described in a dairy farm with history of a sudden increase of clinical cases after a long rainy period and a peak in the fly population, which was characterized as summer mastitis (Motta et al. Citation2011). In this study, 65 (45.1%) cases of mastitis were described, confirming mammary infections as a major manifestation of T. pyogenes infections in this species, predominantly bovine clinical cases. However, no statistically significant difference was observed across the study between the occurrence of bovine mastitis and season. This circumstantial evidence indicates that mammary infections caused by T. pyogenes occur all over the year in this country, probably because of the typical tropical climate that favors annual proliferation of vector flies and animal-to-animal spread of the pathogen. Despite the fact that the major prevalence of cows with mastitis ranged from 4 to 8 years old in this study, no statistical association was observed between mammary infections and age. This result probably reflects the common age of the lactating cows sampled, increasing exposure of animals to biting flies, udder injuries, and/or environmental contamination by T. pyogenes. In addition, since T. pyogenes is usually associated with bovine mastitis during the dry period (Ruegg Citation2010; Radostits et al. Citation2007), lack of dry cow therapy in some cows probably contributed to increase of mastitis at this common age (4 to 8 years old) during lactation in this study.
Chronic suppurative pneumonia is another common clinical manifestation of T. pyogenes infections among livestock (Buddle & O'Hara Citation2005). In this study, no statistical association was observed between pneumonia and season. This result was unexpected, since occurrence of livestock pneumonia is considered most common among cold months. Thus, besides the recognized risk factors – cold immunosuppression and housing of domestic animals in stalls in cold periods – other predisposing conditions may be involved in the establishment of pneumonia caused by T. pyogenes, such as long transportation of cattle to feedlots, collection and mixing animals in farms, deprivation of feed and water for long periods, and marked changes in weather (Radostits et al. Citation2007). In Europe, in vivo and postmortem prevalence of respiratory disorders in veal calves, as well as major predisposing risk factors, was investigated in a cross-sectional study involving 174 farms from 3 main meat-producing countries. Among different potential risk factors considered, the characteristics of the batch were predominant, besides other factors related to housing, management, and feeding equipment were also relevant to respiratory disorders in calves (Brscic et al. Citation2012)
Another relevant finding in our retrospective study was the high frequency of sheep lymphadenitis and abscesses caused by T. pyogenes. These results are consistent with some reports around the world that also have identified T. pyogenes as an usual agent of lymphadenitis (Radostits et al. Citation2007; Quinn et al. Citation2011) and abscesses (Doré et al. Citation2007) in livestock, including in Brazil (Lara et al. Citation2011). Despite the fact that Corynebacterium pseudotuberculosis is considered the primary cause of ovine and goat caseous lymphadenitis worldwide (Radostitis et al. Citation2007; Dorneles et al. Citation2014), our results indicate that T. pyogenes should be included in the differential diagnosis of the disease, because the formation of similar colonies on culture media and the microscopical ‘coryneform’ aspect of this organism makes T. pyogenes another possibility in the etiology of lymphadenitis.
Bovine metritis and infertility, especially in the postpartum period, are also common infections caused by T. pyogenes (Songer & Post Citation2005). Recently, the relationships between Escherichia coli, Fusobacterium necrophorum, and T. pyogenes, as well as specific virulent factors of micro-organisms present in the uterus of cows at different stages of lactation, were investigated (Bicalho et al. Citation2012). In this study, six animals showed reproductive tract infections, including cows, ewes, mares, and goats, reinforcing the involvement of T. pyogenes as a causative agent of reproductive disorders in livestock.
Unlike in livestock, T. pyogenes infections are infrequently described in dogs and cats, probably because this pathogen is not considered to be a normal inhabitant of the mucous membranes of companion animals (Billington et al. Citation2002; Greene Citation2012). In this study, only one case of pneumonia and another of encephalitis were observed in two dogs, reinforcing that T. pyogenes is an uncommon clinical pathogen among companion animals.
Apparently, cephalosporins, penicillin and other beta-lactams, and tetracyclines are the antimicrobial therapies of choice in T. pyogenes infections in livestock (Radostits et al. Citation2007; Quinn et al. Citation2011). Recently, in vitro minimal inhibitory concentration of 55 T. pyogenes strains of bovine mastitis from Poland was investigated using the broth microdilution test. The most sensitive antimicrobials against the isolates were beta-lactams, including penicillin, ampicillin, cephalothin, ceftiofur, and a penicillin–novobiocin combination (Zastempowska & Lassa Citation2012). In this study, the most effective drugs against isolates were florfenicol (99.1%), cefoperazone (96.0%), cephalexin (95.0%), ceftiofur (94.8%), amoxicillin (94.6%), gentamicin (91.8%), and penicillin G (91.2%). Despite high in vitro sensitivity of our isolates to some antimicrobials (>90%), poor prognosis has been observed after in vivo antimicrobial therapy involving T. pyogenes infections in livestock, particularly in bovine mastitis cases (Ruegg Citation2010). The poor prognosis of therapy is mainly related to the ineffectiveness of conventional antimicrobials in reaching the micro-organisms inside the pyogranulomatous foci of the lesions (Songer & Post Citation2005; Radostits et al. Citation2007; Quinn et al. Citation2011). In addition, the improper accuracy and lateness in diagnosis also contribute with poor prognosis of T. pyogenes therapy in domestic animals, particularly in suppurative pneumonia (Radostitis et al. Citation2007).
Recently, a similar study identified resistance of T. pyogenes strains from bovine mastitis mainly to tetracycline (85.5%) and erythromycin (9.1%) (Zastempowska & Lassa Citation2012). Likewise, high resistance rates of our isolates were recorded against trimethoprim-sulfamethoxazole (49.3%), followed by norfloxacin (10.9%) and tetracycline (9.2%). The inappropriate use of antimicrobials increases the selection rate of multidrug resistant bacteria (Giguère et al. Citation2010). Based on these findings, the selection of first-choice antimicrobial treatment should be based on local in vitro resistance patterns. In fact, the responsible use of antimicrobials for animals is an emergent one health concern, to conserve these drugs for human therapy approaches.
Routine diagnosis of T. pyogenes infections among domestic animals is based on microbiological culture and phenotypic identification of the bacteria from lesion material (Songer & Post Citation2005; Quinn et al. Citation2011), as done in this study. Co-infections of T. pyogenes and strict anaerobic micro-organisms, such as Peptostreptococcus indolicus, F.necrophorum, and Prevotella melaninogenica have been identified in livestock elsewhere, particularly from animals suffering of mastitis, liver abscesses, and suppurative foot infections (Songer & Post Citation2005; Radostits et al. Citation2007). The detection of anaerobic bacterium in low numbers in this study may be associated with more frequent requests for aerobic microbiological culture. Nevertheless, the importance of T. pyogenes co-infections with anaerobic organisms cannot be neglected, since experimental mammary infections with this bacterium combined with P. indolicus may cause much more severe disease (Radostits et al. Citation2007).
Recently, the use of molecular techniques have shown to be a valuable tool in the detection of T. pyogenes, in the identification of major virulence factors (Zastempowska & Lassa Citation2012; Bicalho et al. Citation2012), and in genomic sequencing of the bacterium (Machado & Bicalho Citation2014). Surveillance studies focusing on the major virulence factors of this opportunistic animal pathogen are critical to provide information regarding the molecular epidemiology and pathogenicity of T. pyogenes isolates causing distinct clinical manifestations, as well as hypothetical differences among the virulence of pathogen strains from different geographical areas or countries.
There are no specific control measures to prevent T. pyogenes infections in domestic animals. Vaccination with culture filtrates or inactivated T. pyogenes has been ineffective in protecting domestic animals. Pyolisin is a potential antigen to subunit vaccines (Jost & Billington Citation2005), although no commercial products are widely available for prevention routine (Songer & Post Citation2005). Despite of the opportunistic behavior and the presence of the pathogen in environment and normal microflora of mucous membranes in livestock, control of fly vectors populations, reduced contact of udder with water or humid environment, and isolation and/or culling of animals with severe draining abscesses may limit animal-to-animal spread of infection or T. pyogenes outbreaks.
5. Conclusion
This study highlights the diversity of clinical manifestations caused by T. pyogenes infections in livestock, with predominance of bovine mastitis and pneumonia, sheep abscesses and lymphadenitis, and reinforces the importance of isolation and the performance of in vitro susceptibility testing to improve antimicrobial therapy approaches.
Disclosure statement
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the manuscript.
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References
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