INTRODUCTION

Trueperella pyogenes is a Grampositive, non-motile, facultative anaerobe, rodshaped bacterium common habitant of mucus membranes in animals (^{Jost & Billington, 2005}; ^{Santos et al., 2010}; ^{Rzewuska et al., 2019}). T. pyogenes infections occur in wild and domestic animals, and is characterized by the infection of any tissue, mainly associated with abscess formation, causing a reduction in productive performance (^{Ribeiro et al., 2015}; ^{Rzewuska et al., 2019}).

In cattle, the liver is the main organ where T. pyogenes-abscesses can be located, generally as a sequela of ruminal acidosis (^{Amachawadi & Nagaraja, 2016}), also reported in kidneys, lungs, subcutaneous tissue and heart (^{Kleen et al., 2003}; ^{Ribeiro et al., 2015}; ^{Abdela, 2016}; ^{Rzewuska et al., 2019}). This bacterium is an important cause of mastitis and metritis (^{Azawi, 2008}; ^{Sheldon & Owens 2017}; ^{Zhang et al., 2017}), and can lead to sporadic abortions, usually in the middle and final thirds of pregnancy in cattle (^{Arainga et al., 2003}; ^{Anderson, 2007}; ^{Costa et al., 2019}; ^{Morrell et al., 2019}). Uterine abscesses are rare in animals, though more observed in cattle related to local trauma by improper use of instruments during uterine manipulation (^{Schlafler & Foster, 2016}). Herein we describe an outbreak of multiple visceral abscesses caused by T. pyogenes in a heifer with atypical presentation in the uterus associated to ruminal acidosis.

CASE DESCRIPTION

The cluster of cases occurred in a beef cattle breeding farm located in Buenos Aires province, Argentina. Cases were detected in a paddock of 250, ~18 months old Hereford heifers. Four (1.6%) heifers were detected with multiple firm nodular formations in the reproductive tract during trans rectal palpation. Heifers had been grazing pastures until weaning (~8 months old) and then penned with high-grain diet composed of corn silage (20-30%), grain corn (70-80%) and pellet as protein source for another 10 months. Heifers reaching 18 months of age were transrectally palpated for assessing their reproductive development and then sent to an open paddock. During this procedure, uterine nodular formations had been occasionally detected in previous years in about 3 to 5 heifers per year (~1-2%). Later one, the affected heifers were showing a progressive loss of corporal condition, and therefore culled since they were not suitable for reproduction.

One severely affected heifer was euthanized according to the guidelines of the Committee for Care and Use of Experimental Animals of the Instituto Nacional de Tecnología Agropecuaria (INTA) and post mortem examination was carried out. Grossly, an intramural nodular abscess of 10 cm length was present in the ventral wall of the uterine body forming a rounded external bulge (Figures 1 A,B), that at cut surface it had a thick fibrotic capsule and contained white viscous material defined as pus (Figure 1C). Similar abscesses were also found attached to the omasum, ruminal serosa, peritoneum and mesoduodenum, varying from 2 to 4 cm of diameter. In the diaphragmatic surface of the liver two 1 cm abscesses with irregular edges surrounding by a thin capsule were observed. The mesenteric (duodenum and jejunum) and hepatic lymph nodes were mostly enlarged and edematous.

Figure 1. Uterus of an 18-month-old Hereford heifer with multiple visceral abscesses caused by Trueperella pyogenes. A) Enlargement of uterine body (yellow frame). B) Rounded intramural formation of 10 cm in diameter at ventral wall of uterine body. C) Cut surface of the intramural abscess, characterized by purulent content surrounding by a thick fibrotic wall 

Tissue samples including the nodular formations, heart, lung, liver, small and large intestine, lymph nodes, spleen, kidney, rumen, abomasum, central nervous system and uterus were fixed by immersion in 10% neutral buffered formalin and processed by standard techniques to obtain 4-5-µm sections stained with hematoxylin and eosin (HE) for histologic examination. Microscopically, abscesses from uterus and serosa of omasum and rumen were characterized by a central area of necrosis composed of viable and degenerate neutrophils and macrophages admixed with cellular debris, and surrounded by organizing granulation tissue with macrophages, dense collagen and neovascularization (Figure 2 A,B).

Figure 2. Microscopic lesions in a 18-month-old Hereford heifer with multiple abscesses caused by Trueperella pyogenes. A) Abscess from uterus characterized by a central area of necrosis composed of viable and degenerate neutrophils and macrophages admixed with cellular debris (left), surrounded by organizing granulation tissue with macrophages, dense collagen and neovascularization (right) H&E. B) Higher magnification for detail of abundant neutrophils admixed with cellular debris. H&E. C) Ruminal mucosa with micro abscess composed of neutrophils in stratum spinous (dark arrow) and multifocal marked cytoplasmic vacuolation of epithelial cells of spinous layer (arrowhead). H&E. D) Abundant clusters of Gram-positive short rods from uterine abscess 

The liver showed two foci formed by necrotic center with degenerated inflammatory cells (mainly neutrophils) and necrotic debris, surrounded by fibroblasts and macrophages.Additionally, in the mesentery, diffuse inflammatory infiltrate with macrophages, lymphocytes and neutrophils was present. The rumen showed, multifocal marked cytoplasmic vacuolation of epithelial cells of spinous layer, mixed with moderate number of neutrophils forming micro abscesses (Figure 2C), and additionally the mucosa and submucosa had a diffuse moderate infiltrate of neutrophils. Hepatic and mesenteric lymph nodes had depletion of the germinative centers and showed subcapsular moderate infiltrate composed by macrophages and lymphocytes. Other organs did not present relevant lesions. Direct Gram stain from several tissues (uterine, serosal and liver abscesses), showed clusters of Gram-positive short rods (Figure 2D).

From bacteriological culture, sterile samples of lymph nodes and purulent content of abscesses from uterus and ruminal serosa were collected and inoculated onto Columbia blood agar. After 24 h at 37 °C under aerobic conditions, very small beta hemolytic colonies developed and gram-positive bacilli and coccobacilli were observed by Gram staining. Colonies were identified as T. pyogenes by phenotypic and biochemical characteristics (^{Rogovskyy et al., 2018}).

DISCUSSION

In this report, clinical, pathological and bacteriological findings confirm the occurrence of multiple abscesses by T. pyogenes. Abscesses were present in 1.6% of the animals. This occurrence is lower compared with a previous report in which 2 to 20% of cattle from a feedlot were affected with liver abscesses (^{Tadepalli et al., 2009}). T. pyogenes is a commensal bacterium of skin and mucous membranes. During episodes of ruminal acidosis, the bacteria could gain access via bloodstream and spread to the liver, causing abscesses (^{Nagaraja & Chengappa, 1998}; ^{Sheldon & Owens, 2017}; ^{Tadepalli et al., 2009}; ^{Rzewuska et al., 2019}).

In this case, the high concentrate diet after calving, could led to acidosis and rumenitis facilitating the entry of T. pyogenes and the dissemination of the bacteria via bloodstream to several tissues causing secondary abscesses (^{Nagaraja & Chengappa. 1998}; ^{Plaizier et al., 2009}; ^{Amachawadi & Nagaraja, 2016}). Disseminated abscessation by T. pyogenes has also been diagnosed in goats and sheep affecting various tissues, suggesting hematogenous spread after immunosuppression related to stress factors (^{Dughaym, 2004}; ^{Lin et al., 2010}; ^{Ribeiro et al., 2015}).

Hematogenous spread from the gut is a common route of transmission of several infectious microorganisms which can affect, among others, the uterus provoking metritis in cows (^{Jeon et al., 2017}). T. pyogenes is considered an important uterine pathogen in postpartum infections of cows causing metritis and endometritis (^{Sheldon & Owens, 2017}; ^{Zhang et al., 2017}; ^{Rzewuska et al., 2019}) which can lead to late term abortions (^{Arainga et al., 2003}; ^{Anderson, 2007}; ^{Costa et al., 2019}; ^{Morrell et al., 2019}). Reproductive infection by T. pyogenes is associated to superficial inflammation of the endometrium and occasionally extending to stromal layer (^{Bicalho et al., 2012}; ^{Ribeiro et al., 2015}; ^{Zhang et al., 2017}). In this context, T. pyogenes can act either as primary agent or synergistically with other bacterial pathogens (Bacteroides, Porphyromonas and Fusobacterium); however, the formation of uterine abscesses is uncommon (^{Schlafer & Foster, 2016}; ^{Sheldon & Owens, 2017}; ^{Rzewuska et al., 2019}).

In cattle, uterine abscesses are associated to improper uterine manipulation involving instruments such as insemination pipettes and uterine catheters, located in the dorsal wall of the uterine body (^{Schlafer & Foster, 2016}; ^{Sachuk et al., 2020}). In this report, the abscess was in the ventral wall of the uterine body and no uterine manipulation was carried out before, discarding a local trauma as well as postpartum contamination given their non-pregnant status.

CONCLUSIONS