INTRODUCTION

Mastitis is the disease that leads to the most important economic losses in the dairy industry [44, 48, 50]. Etiology is conditioned by determinants and predisposing factors [49, 55], corresponding to the last ones, situations which favor the infectious agents action as climatic elements, stabling conditions, pre and postmilking mammary gland hygiene, mechanized milking apparatus state, nutritional factors and immunocompetence, besides physiological variables linked to the animals like number of parity, lactation period and others [1, 3, 5, 32, 41, 44, 50].

Summer mastitis owns its name by appearance of clinical episodes in bovine (Bos taurus and Bos indicus) herds during July-October periods in North Hemisphere Countries. Nevertheless, its incidence is also increased during physiological circumstances [14, 44] and in other year seasons [28, 42]. It is an acute pathology and some authors describe it with etiology associated to Peptoniphilus indolicus and Trueperella (Arcanobacterium) pyogenes [28, 30, 51], most of the described cases in cows are related to the last one [3, 10, 14, 28, 29, 30, 36, 39], perhaps due to the technical constraints to the P. indolicus isolation by its condition as an strict anaerobic germ [14, 51].

T. pyogenes has a wide geographic distribution and principally inhabit mucous membranes of domestic animals [11, 13, 28, 45] except humans, whom cause an occupational disease [11, 27] and is very infrequent [45, 46]; this is an agent that can remain in fomites [31] and flies, which act as mechanical vectors [3, 28, 42, 44]. Moreover, the cited environmental conditions notably impair the sanitary settings on milking settlements [36, 50], which could lead to immunosuppressive states in animals, making them more vulnerable to opportunistic infections [5, 6, 26, 50, 52], as T. pyogenes [27, 46].

T. pyogenes is a bacterium with a large quantity of virulence factors [46], the most representative is a hemolytic exotoxin with cytolytic actions against neutrophils and macrophages [27, 31]. Additionally, it presents neuraminidases, fimbria and matrix-binding extracellular proteins that facilitate host tissue adhesion, it produces proteases and forms biofilms [27, 30], and these elements have been described in most of the strains isolated on clinical mastitis [57].

Phenotypic assays usually are done to the characterization of this bacterium [9, 11, 14, 45, 46, 57], whereas definitive identification tests are done on reference labs. Microscopically it is recognized as a diphteriform and pleomorphic coccobacilli, Gram positive and asporogenic [27]. The microbial growing is suspected, by inconspicuous hemolytic zones at 24 hours (h) of incubation at 37ºC on bovine blood agar, thereafter at 48 h and with an exhaustive macroscopic examination, it is possible to see white pin point colonies with 1-2 mm diameter, surrounded by a more prominent total (β) hemolysis [9, 46, 57], it is partially acid-alcohol resistant and has a fermentative metabolism [35]; unlike clinical samples, cultures do not have a characteristic odor; CAMP test is helpful to confirm the identification [13, 31] and differentiation from Arcanobacterium haemolyticum, wich inhibit Staphylococcus aureus β haemolysin [47].

Antimicrobial treatment for this disease is usually unsuccessful although susceptibility in vitro is seen. This happens due to the massive purulent discharge and inflammatory reaction, which somehow abate medicaments effectivity [36, 57] so, most of the affected cows are early slaughtered or submitted to chemical ablation of mammary quarter [14, 28, 35, 36], even in other domestic species [12, 42], exacerbating the scenario for livestock producers, whom before taking drastic decisions try to save animals with antibiotics and alternative treatments. This phenomenon has been described in other clinical forms of T. pyogenes infection, including other animal species [11].

Mastitis by T. pyogenes is infrequent [27] and its appearance is linked to meager hygiene conditions after milking because microorganisms, like other mammary pathogens, are vulnerable to iodized solutions during nipple sealing [36, 55]. Latin America outbreaks have been described in Brazil [36, 40], in other domestic animal species [12, 37, 42] and with alternative clinical forms on bovines [9].

In this opportunity, isolation of T. pyogenes is demonstrated as an agent associated to an outbreak of clinical mastitis in a bovine farm at Cacique Manaure County, Falcón State (Venezuela), affecting some Carora breed animals (B. taurus) during rainy season ending 2011, with recurrence in other cows lots from the same property beginning in 2012. This document constitutes the first report of cases in Venezuela.

MATERIALS AND METHODS

Study area

Cacique Manaure County has approximately 225 km² area [33], even though it is one of smallest in Falcón State, it has at least 33 farms so it has a high density of bovine production units [34]. Historically, higher precipitations in the region are from September until February (mean 50 mm³ and maximum 200-275 mm³) according to the closest pluviometric station records (Coro City) [23], while mean values of lowest temperature recorded at the closest climatological station (Tocuyo de la Costa) are registered in this period as well (25,0º-25,5ºC), and relative humidity slightly descends in comparison with summertime (74-78%) [24] then, coinciding with the rainy season. From August 2011 to January 2012, precipitations reached levels above historical average at the two closest pluviometric stations (in Coro and Valencia Cities), almost reaching the registered top [17, 18, 19, 20, 21, 22, 25], even in Coro City was counted a pluviometric surplus of 401% with 122 mm³ over the mean [22].

Clinical descriptions and samplings

The production unit is located at Yaracal, the capital from Cacique Manaure County, which has plenty of farms dedicated to bovine livestock, being one of the most important for Falcón State [34]. The farm focused in this opportunity is committed to the breeding of several animal species, including double proposal bovines. Three hundred and twenty Carora breed cows (B. taurus) in lactation were submitted to double mechanical milking per diem, with semi-intensive management. Sanitary actions were limited to maintenance and cleaning with chlorinated solution of milking apparatus. Animals committed to dairy production composed a heterogeneous group with various age strata and different lactation times, detecting in the post-partum lot the first clinical mastitis cases focused in this report (three animals) (September 2011), while the second outbreak circa January 2012 involved two cows, also at the peripartum stage; although there were previous episodes of clinical mastitis among the bovine herds, no one corresponded to the clinical manifestations detected in this opportunity.

Affected bovines on the farm were easily recognized by macroscopic alterations in the milk (fetid odor and serous-watery aspect), without clots, pus or bloody traces, with prominent alterations on mammary glands during palpation (spontaneously draining purulent abscess and crepitancy). Only five animals were analyzed through microbiological diagnosis, because were the unique with clinical manifestations.

Aseptic samples were taken from affected cows, cleaning up mammary gland with clear water and dried with disposable napkins. Initial three spirts by manual milking were discarded, and samples of milk were taken on plastic and sterile collectors, which were maintained on refrigeration until microbiological analyses [6]. In the second mastitis outbreak, besides milk specimens, a purulent sample from mammary gland was taken in an aseptic way through aspiration with a syringe from a cow, for microbiological studies as well, disinfecting surface with iodine solution, transportation and preservation conditions were similar to the aforementioned.

Laboratory assays

Once at the laboratorio de Microbiología Veterinaria from Universidad Nacional Experimental “Francisco de Miranda”, in order to avoid more than 24 h after sampling, specimens were inoculated on primary isolation culture media on plates (MacConkey, salad mannitol, Saboraud and 5% bovine defibrinated blood agar) (HiMedia Laboratories, Mumbay, India), and incubated at 37ºC in aerobic atmosphere, except blood plates which were submitted to microaerophilic conditions (CO2 5-10%). Cultures were daily inspected until 72 h of incubation, looking for microbial biomass or any other macroscopic evidence. Microscopic characterization was done with smears and Gram staining from culture samples, besides other phenotypic assessments (TABLE I). These procedures have been widely applied to identify this bacterium [9, 11, 13, 14, 36, 57].

To antimicrobial susceptibility tests, disk diffusion technique was applied according to Clinical and Laboratory Standards Institute (CLSI) protocols [7, 16], which have been previously used to analyze T. pyogenes isolates [11, 36]. There is no T. pyogenes specific protocol available so, Streptococcus pneumoniae reference methods were taken as models [8, 36, 45, 57], because both microorganisms have very similar nutritional demands, therefore, plating was done on Müller Hinton agar plus 5% bovine defibrinated blood, and incubated at 37ºC with 5-10% CO₂. Antimicrobials tested were selected according to previous reports [28, 36] and availability (TABLE II).

RESULTS AND DISCUSSION

Clinical manifestations detected on animals during the two outbreaks were similar to the previously described according to literature, with swollen and hyperthermic mammary glands, with occasional suppurant abscesses [10, 14, 31, 36, 40], coinciding with previous reports in sheep (Ovis orientalis aries) [42] and goats (Capra aegagrus hircus) [12]. Besides, macroscopic appearance of mastitic milk on T. pyogenes infections detected in this opportunity has been formerly documented, although it is not the unique finding reported in association with this bacterium [57], including fetid milk odor [12, 28, 36]; nevertheless, this sign should not be directly associated with T. pyogenes, instead it could be talking in favor of a mixed infection with P. indolicus [28, 30, 51], which is a butyric acid producer [38] and generates a very unpleasant odor, especially when it is linked to sulphure groups. This feature was seen only on clinical samples and it disappears in subculture conditions, as recognized previously [36], maybe because in the lab procedures were given conditions to the exclusive growing to T. pyogenes [14], which was principally described as a long chain fatty acids producer, that are commonly odorless [2, 56].

Macroscopic analysis of cultures at 24 h allowed to perceive tiny hemolytic zones, at 48 h whitish pin point isolated colonies were seen with a greater hemolysis, and without another colonial morphological types, discarding other species in the samples. Similar to previous papers, isolated colonies in this opportunity show traditional macroscopic characteristics [36, 57]. Microscopic visualization revealed pleomorphic Gram positive cocobacilli. No variations on phenotypic characters were registered among the five strains isolated on the two outbreaks (TABLE I).

Identification of T. pyogenes could be confused with A. haemolyticum because both have great phenotypical similarity [56], but much of the strains of the first have pyrrolidonylarylamidase, amylase and leucine aminopeptidase, besides fermentation of lactose and trehalose [35] so, these characters together with the other ones detected in this opportunity, conjugated with clinical data, confirm the etiologic diagnosis of these outbreaks associated to T. pyogenes, such as other researchers have done [28, 35, 36, 57]. Other key differentiating features are the origin of microorganism and the kind of hemolysis, because A. haemolyticum is a commensal on human microbiota and produces a greater β haemolysis area [43, 54], meanwhile T. pyogenes is principally isolated on domestic animals [11, 45, 46], generates a lesser β hemolysis area [46] and CAMP test with S. aureus β haemolysin is different [47].

Regarding other interesting epidemiological aspects, the findings in this opportunity are coincident with ones from Brazil [10, 12, 36] and from other latitudes [14]. Although T. pyogenes mastitis is associated to summer time on Northern Hemisphere Countries, there are reports that relate its appearance with other physiological circumstances (drying period and short postpartum lapse) [44] and environmental conditions [14, 36], even on highly rainy and temperature seasons, maybe due to intense proliferation of flies which can act as mechanical vectors [28, 36], coinciding again with the conditions registered in this opportunity (rainfall and cows on 4-6 weeks postpartum).

Summer mastitis frequency is associated to poor sanitary conditions on milking areas, by presence of mud and high humidity corollaries to persistent rain [36], and absence of good hygienic postmilking practices, which were collectively seen on this farm, so that animals had clear signs of behavioral alterations, being refractory to circulate on mud terrain, leading to reduction of milking episodes to one per day.

Correlation between mastitis by nonconventional pathogens and rainy seasons has been previously detected in rural areas similar to Yaracal [5, 6, 52], highlighting important considerations that should be taken about immunocompetence when stressing conditions are persistently present, converting animals more susceptible to opportunistic microbes [1, 5, 6, 48], a circumstance that could be exacerbated when germ virulence factors are confronted, even they can exert immunomodulant regulations like the reported to short chain fatty acids produced by P. indolicus, although this bacteria was not registered in these episodes at Yaracal, its etiologic association could be inferred considering the clinical evidence [4, 53].

This document is a retrospective analysis of recorded clinical cases, with detailed report of several findings linked to two consecutive clinical mastitis outbreaks, which have been diagnosed as summer mastitis by T. pyogenes. According the analysis of overall evidences, the conjunction of the reviewed elements could be highlighted as a potential “marker” of the disease; nevertheless, systematic studies on epidemiological risks factors specific to mastitis by T. pyogenes, must be undertaken to corroborate these ideas, especially about the possibility P. indolicus coinfection, which could dramatically change the considerations to design empirical antibiotic treatments. It is necessary to mention the report of Ribeiro et al. [45], who did not find statistical association between general T. pyogenes infections and age, gender and seasonality.

Regarding antibiotic susceptibility test, few differences on strain profiles were perceived among the two episodes, including the diameter on disk diffusion tests (TABLE II). The evaluation of antimicrobial susceptibility on T. pyogenes strains isolated on bovine mastitis is not frequent [57], in this opportunity the assays were done to guide veterinary physician about therapeutic possibilities, getting a general sensibility to different antimicrobials groups.

β lactamics (AM, P y AmC) have proven in vitro action against T. pyogenes [11, 28, 36, 44, 57] and are traditionally applied on clinical veterinary activities, with few reports of resistant isolates [11, 36, 46], as could be seen on second outbreak (TABLE II), the exceptions are Penicillin G and Oxacillin [11, 44, 46]; nevertheless, Ribeiro et al. reported different susceptibility. Neither cephem resistance has been observed [28, 36, 44] nor to fenicols (Florfenicol and C30) [28, 36], as could be seen in this report. About the rest of antimicrobials evaluated (TABLE II), partial concordances were detected to previous documents, i.e. sensible and intermedium strains were registered to tetracyclines like findings on Brazil [36], but in other latitudes a high frequency of tetracyclines resistance has been recorded [28], even linked to macrolides resistance (E) [57]; low quinolones effectivity were seen too (enrofloxacin) [36] and intermedium susceptibility to CC [28], being different to TABLE II data.

Experts recommend implementing parenteral (intravenous) treatment on acute cases like summer mastitis [15, 46], because intramammary or local administration might not be the adequate route, due to intense pus discharge and inflammatory reaction: Considering that in vitro assays can show a wide spectrum of susceptibility to different antimicrobial families [28, 44], will be appropriate to evaluate antibiotics with high bioavailability on mammary gland or milk as P, sulfonamides and macrolides like E or tylosin [15]; nevertheless, there are several reports of T. pyogenes strains resistant to the them [11, 44, 46] so, the use of these antibiotics should be considered according to results of respective antimicrobial susceptibility tests.

On the other hand, there are specialists who prefer chemical ablation of mammary quarters as a more traditional alternative in cases were pharmacological treatment fail [36]. Nevertheless, in most of the cases premature culling is the irremediable procedure [14, 36], as occurred on the episodes at Yaracal, where animals were treated with diverse antibiotics (oxitetracyclin, cephem and penicillin) and there were not satisfactory results, in a similar way, García et al. [11] reported a failed antibioterapic treatment in a pig with multiple abscesses.

CONCLUSIONS

This is the first diagnostic report of summer mastitis on Venezuelan bovine herds, being highly similar to previous ones about clinical manifestations, epidemiological risk factors and phenotypical characteristics for one of the etiologic agents (T. pyogenes). Although bacteriological protocols did not consider methodologies to detect anaerobic bacteria, clinical signs let to suspect the association with P. indolicus, given a highly suggestive scenario which could preliminary helps veterinarians in rural areas to have a presumptive diagnosis, who must corroborate their suspects with laboratory tests.

Antimicrobial susceptibility profiles of isolates show sensibility to numerous antibiotics, although they were not effective in vivo, in a similar way with previous clinical reports, maybe due to the absence of a complementary treatment to combat anaerobic germens.

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Author notes