Pyelonephritis caused by Mannheimia varigena in a Holstein calf

Abstract

A 7-day-old calf died following development of mild respiratory symptoms. Postmortem examination revealed the kidneys were inflamed, and Gram-negative bacteria was detected in the kidneys, supporting the diagnosis of suppurative pyelonephritis. Mannheimia varigena antigen was found in the lesions and the cytoplasm of macrophages and neutrophils in the renal cortex. The Gram-negative bacilli from the kidney were identified as M. varigena by sequencing the 16S rDNA. Although M. varigena is known to cause bovine respiratory disease syndrome, shipping fever, and meningitis, it was unknown that it could also cause suppurative pyelonephritis. Our study provides the first evidence of suppurative pyelonephritis caused by M. varigena in cattle and information that would improve our understanding, diagnosis, and treatment for M. varigena infections.

Mannheimia is a Gram-negative bacterium genus that encompasses M. haemolytica, M. varigena, M. glucosida, M. granulomatis, and M. ruminalis [3]. The most commonly studied Mannheimia is M. haemolytica, which is associated with diseases such as shipping fever, bovine respiratory disease syndrome [7], and peritonitis [9] in cattle. However, M. varigena infection is also associated with a number of conditions such as lung disease [2], shipping fever [10] and could also cause lesions such as suppurative meningitis [5], mastitis, endocarditis, and enteritis [3]. M. varigena is normally found within the oral cavity and the gastrointestinal tract [3], but could also be isolated from the upper respiratory tract of healthy calves [4] and the uterus of dairy cows [18]. Although M. varigena infection had been reported, there are few studies on histopathology and drug resistance studies using clinically isolated M. varigena.

Antibiotic resistance is an emerging global problem in livestock animals, and multi drug resistance had been reported in M. haemolytica [14] and drug resistance gene was not only M. haemolytica [14] but also M. varigena [12, 13], however, the information of drug resistance using clinically isolated M. varigena is limited.

Here, we report a fatal case of pyelonephritis caused by M. varigena infection, providing histopathological and bacterial characteristic of M. varigena in a neonatal calf.

A 7-day-old female Holstein calf at a farm housing 45 crossbred and 45 Holstein cattle was presented with rough breathing and fever (38.7°C) in January 2016. Despite ampicillin treatment, the calf died the next day. The animal was brought to Aichi Prefectural Chuo Livestock Hygiene Service Center where a postmortem necropsy was performed. Mycoplasma bovis was isolated from a nasal and ear swab of two calves that had died within a week of each other and no symptoms were detected in the adult cows and the other calves.

Necropsy examination revealed that both kidneys were inflamed, and small, dark-red lesions were scattered throughout the renal cortex (Fig. 1a). Hemorrhage was found in the renal pelvis on the cut surface of the kidney. Furthermore, the lungs showed dark red lobules. However, no visible lesions were found in the other organs. Tissue samples of the liver, spleen, left kidney, heart, lung, thymus, and brain were fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin wax, sectioned at approximately 3 µm thickness, and stained with hematoxylin and eosin (H&E) or Gram stain for histological examination. To label the M. varigena antigen, 3-µm thick sections of formalin fixed samples of liver, spleen, kidney, and lung were treated with 3% hydrogen peroxide in methanol to suppress endogenous peroxidase activity. Antigen retrieval was performed by treating the sections with 0.1% actinase E solution in phosphate buffered saline at 37°C for 20 min. The tissues were then incubated with rabbit anti-M. varigena 971 strain serum (National Institute of Animal Health, Tsukuba, Japan) as primary antibody for 60 min at room temperature followed by a secondary antibody (Histofine Simple Stain MAX-PO (Multi; Nichirei Bioscience Inc., Tokyo, Japan) for 30 min at room temperature. The sections were then incubated with aminoethyl carbazole (AEC) substrate solution (Histofine Simple Stain AEC solution; Nichirei Bioscience Inc.) at room temperature for 5 min. and then counterstained with hematoxylin. Sections of tissues containing M. varigena and pieces of liver (into which M. haemolytica serotypes A1, A2, A5-A9, A12-A14, A16, Bibersteinia trehalosi serotypes T3, T4, T10, T15 and M. glucosida had been injected) were used as positive controls in order to verify the immunohistochemical specificity of the reaction. Pasteurella haemolytica biotype A had 13 serotypes and P. haemolytica biotype T had 4 serotypes [16]. Subsequently, in 1999, based on the results from DNA-DNA hybridization and 16S RNA studies, all serotypes of P. haemolytica biotype A were grouped under a newly created genus Mannheimia [16]. All serotypes under biotype A became M. haemolytica except A11, which became M. glucosida. All four serotypes of P. haemolytica biotype T were named as P. trehalosi retaining the genus Pasteurella [16]. Further taxonomical analysis in 2007 resulted in the creation of a new genus, Bibersteinia, under which were included all the four P. trehalosi serotypes, later named as B. trehalosi [16].

Fig. 1.

a. Kidney, swollen and small, dark-red lesions scattered throughout the cortex are visible. b. Suppurative lesion with congestion and hemorrhage in the renal pelvis. H&E staining. Bar=500 µm. c. Infiltration of many oat-like cells (arrows) and neutrophils in the suppurative lesion of renal pelvis. H&E staining. Bar=20 µm. d. Mannheimia varigena antigen was detected in the suppurative lesion of renal pelvis. Bar=100 µm. e. M. varigena antigen was detected in the cytoplasm of macrophages and neutrophils in the interstitium of renal cortex. Bar=20 µm.

Suppurative lesions were widely detected in the renal pelvic mucous membrane and renal papilla (Fig. 1b) and showed numerous neutrophils and oat-like cells (Fig. 1c). Gram-negative bacilli were detected in the lesions and the lumen of the renal pelvis.

Moderate to severe congestion and hemorrhage were observed surrounding the suppurative lesion in the inner medulla (Fig. 1b), and thrombi were also scattered in the lesions. Cellular debris and neutrophils were found in some of the collecting and renal tubules; the neutrophils had also infiltrated the interstitium surrounding the tubules. In other organs, thymocytes were decreased in the thymus cortex, and neutrophils were found around the white pulp of the spleen. Slight edema and congestion were observed in the lung. No significant lesions were detected in other organs. Gram-negative bacteria with M. varigena antigens were detected in the suppurative lesion and lumen of renal pelvis (Fig. 1d) and cytoplasm of macrophages and neutrophils in the interstitium (Fig. 1e). No antigen was detected in the liver, spleen and lung. In addition, a positive reaction was detected only in the positive control sections of tissues containing M. varigena, but not in the other positive controls. The rabbit anti-M. varigena 971 strain serum specifically reacted with M. varigena by immunohistochemical analysis.

To isolate the bacteria, tissue homogenates prepared from the liver, spleen, left kidney, heart, lung, and brain were spread onto normal sheep blood agar, chocolate agar, and deoxycholate-hydrogen sulfide-lactose agar plates. The agar plates were incubated at 37°C in 5% CO₂ with and without oxygen. Gram-negative bacilli were isolated from only the left kidney sample. No bacteria were isolated from any of the other samples tested. The isolates from the kidney were applied to biochemical testing using the API 20 NE system (BioMérieux, Inc., Marcy l’Etoile, France), according to the manufacturer’s instructions, which confirmed the presence of M. haemolytica (1420004).

To detect Mycoplasma, lung homogenate supernatant was incubated in NK liquid medium (Kanto Chemical Co., Inc., Tokyo, Japan) at 37°C in aerobic conditions for 3 days. After incubation, medium was centrifuged and sediment was used for DNA extraction. Genomic DNA was extracted from both isolates and sediment by using Instagene Matrix (Bio-Rad Laboratories, Hercules, CA, U.S.A.) according to the manufacturer’s instructions.

To identify if M. haemolytica, M. glucosida, M. ruminalis, M. granulomatis, and M. varigena were present in the isolate, specific PCR assay for 16S ribosomal DNA was performed and sequenced [3, 11]. The partial sequence analysis of the 16S rDNA from the isolate (1,468 bp region) showed 99.0% similarity with M. varigena type strain, supporting the presence of M. varigena in the isolate. The 16S rDNA sequences were deposited to DNA Data Bank of Japan (Accession Number: LC466640). M. bovis [6] and M. dispar [15] were not detected from the DNA sample of sediment using PCR.

To determine the susceptibility of the M. varigena to antibiotics, minimum inhibitory concentration (MIC) was calculated using broth dilution method according to clinical laboratory standards institute (CLSI) method [8] against ampicillin (ABPC), ceftiofur (CTF), tetracycline (TC), florfenicol (FF), enrofloxacin (ERFX), and danofloxacin (DNFX). The breakpoints of these antibiotics were referred from the breaking point of M. haemolytica based on CLSI [8] and Alexander et al. [1]. The isolate analyzed in the current study was susceptible to all antimicrobial drugs (Table 1).

Table 1. Antimicrobial susceptibility tests.

Class	Antimicrobial agents	MIC	Breaking point	Reference
Penicillin	Ampicillin (ABPC)	≤1	16	[1]
Cephem	Ceftiofur (CTF)	≤0.12	8	[8]
Tetracycline	Tetracycline (TC)	0.5	8	[8]
Amphenicol	Florfenicol (FF)	≤1	8	[8]
New Quinolone	Enrofloxacin (ERFX)	≤0.03	2	[8]
	Danofloxacin (DNFX)	≤0.03	0.25	[8]

MIC: minimum inhibitory concentration.

Our results confirmed M. varigena infection in the dead Holstein calf, which could be the cause of the suppurative lesions in the renal pelvis. M. bovis was not detected in the lung homogenates, and therefore, it is unlikely to be associated with the pulmonary lesions. Although M. varigena could cause various lesions such as suppurative meningitis [5], mastitis, endocarditis, enteritis [3], and pneumonia [2], it is unknown if M. varigena is associated with pyelonephritis, and therefore, this is the first report of pyelonephritis caused by M. varigena.

In the current study, suppurative lesions and bacterial isolation were limited only to the kidney. Gram staining and histological examination revealed that M. varigena was present only in the suppurative lesions of kidney. Although the bladder or ureter were not examined, M. varigena would be infected via urethra because of limited detection of M. varigena.

In our case, the isolates were misidentified as M. haemolytica by commercial biochemical test. However, the 16S rDNA profiles differ between the different Mannheimia species [11] and we determined the isolated bacteria to be M. varigena using this method. It was reported that M. varigena is the most frequently isolated bacteria from bovine respiratory disease after M. haemolytica [17], and reliable detection methods for M. varigena such as PCR would be useful for accurate and rapid diagnosis.

Drug resistance is one of the biggest concerns in raising livestock. Although tetracycline [13] and multi-drug resistance genes [12] had been detected previously in M. varigena [4], the isolate in this study did not show drug resistance to any of the tested drugs. Although the calf in this study was treated with ampicillin immediately following presentations of breathing difficulties, the symptoms were not alleviated despite lack of resistance to ampicillin. This could be explained in part by the fact that kidney functions were attenuated beyond recovery before ampicillin treatment. Therefore, it is important that diagnosis is made rapidly in these situations.

In conclusion, we have reported a unique case of pyelonephritis caused by M. varigena in a calf. Although M. varigena could cause a number of diseases, pyelonephritis had not been associated with M. varigena infection. M. varigena could be misidentified by using commercially available biochemical test and 16S rDNA sequence analysis is required to correctly identify M. varigena infection. This study provides evidence that M. varigena could cause pyelonephritis and correct identification of the infectious agent and drug resistance would be useful for future treatments.