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Mannheimia haemolytica A1-induced Fibrinosuppurative Meningoencephalitis in a Naturally-infected Holstein–Friesian Calf
J. Comp. Path. 2013, Vol. 149, 167e171
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Mannheimia haemolytica A1-induced Fibrinosuppurative Meningoencephalitis in a Naturally-infected HolsteineFriesian Calf S. Aschenbroich*, N. Nemeth*, R. Rech*, R. Briggs‡, S. Sanchez† and C. Brown* * Department of Pathology, † Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Athens, Georgia and ‡ USDA/ARS/NADC, Ames, Iowa, USA
Summary Mannheimia haemolytica is an opportunistic bacterium that is widely recognized among the bovine respiratory disease complex as the predominant pathogen causing broncho- and pleuropneumonia in cattle. Among the characterized M. haemolytica serotypes, A1 is the major cause of severe pulmonary lesions in cattle. This report describes post-mortem findings in a HolsteineFriesian calf with fibrinosuppurative meningoencephalitis and fibrinonecrotizing, haemorrhagic broncho- and pleuropneumonia, from which M. haemolytica and bovine viral diarrhoea virus (BVDV) were isolated. Microscopical evaluation showed expansion of the brainstem and cerebellar leptomeninges by neutrophils and fibrin, associated with gram-negative coccobacilli. Occasional blood vessels within the midbrain and cerebellum contained fibrin thrombi. Bacterial culture of cerebellum and lung yielded M. haemolytica with unusually high haemolytic activity. The isolates were confirmed as serotype A1 by rapid plate agglutination. Lung tissue was positive for BVDV by polymerase chain reaction. The broncho- and pleuropneumonia in this calf were consistent with typical mannheimiosis due to serotype A1; however, extrapulmonary infections due to M. haemolytica, as seen in this case, are rarely reported. To our knowledge, this is the first documentation of a natural BVDV and M. haemolytica co-infection associated with fibrinosuppurative meningoencephalitis in a calf. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: bovine viral diarrhoea virus; calf; Mannheimia haemolytica; meningoencephalitis
Mannheimia haemolytica is a commensal of the upper respiratory tract of healthy ruminants, but can become a significant cause of pneumonia in dairy and feedlot cattle. Disruption of the commensal relationship can lead to the establishment of M. haemolytica A1 as the dominant serotype (Al-Ghamdi et al., 2000; Zecchinon et al., 2005; Griffin et al., 2010). Immunomodulatory factors, such as weaning, shipping, commingling, changes in climatic conditions and bacterial and viral co-infections, may affect the bovine host and facilitate the development of serotype A1-induced fibrinosuppurative and necrotizing bronchopneumonia (Griffin, 2000; Zecchinon et al., 2005; Rice et al., 2007; Singh et al., 2011). Correspondence to: S. Aschenbroich (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.02.001
A 5-month-old neutered male HolsteineFriesian calf was submitted for necropsy examination to the Athens Veterinary Diagnostic Laboratory, Athens, Georgia, with a history of weight loss, lethargy, hind-limb ataxia and rare crackles in the left cranial lung quadrant. Following treatment with florfenicol (single dose 6 ml; NuflorÔ; Merck Animal Health Summit, New Jersey, USA) and daily oral electrolytes, the calf’s body condition improved and clinical signs appeared to resolve. However, improvement was short-lived, with the calf becoming laterally recumbent prior to its demise. The bovine viral diarrhoea virus (BVDV)-exposure status of the calf prior to vaccination with Bovishield Gold 4 (modified live virus strains of infectious bovine rhinotracheitis virus), BVDV types 1 and 2 and parainfluenza 3 virus Ó 2013 Elsevier Ltd. All rights reserved.
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(Pfizer Animal Health, New York, USA) was unknown. The calf had stunted growth, was dehydrated and in poor nutritional condition. The surfaces of the occipital lobe of the cerebrum, brainstem and cranial cerebellum were covered by multifocal, petechial to ecchymotic haemorrhages (Fig. 1). The leptomeninges of the brainstem and cerebellum were cloudy and had numerous, scattered fibrin strands. There was caudal herniation of the cerebellum (Fig. 1). The cranioventral aspect of the lungs was bilaterally mottled dark red, black and tan, and both cranial lobes were adhered to the parietal pleura by multiple fibrin strands. On cut section, the pulmonary parenchyma was disrupted by multifocal, variably sized, firm nodules. Multifocal petechiae were present throughout the myocardium, abomasal mucosa, pharyngeal tonsils and peripheral lymph nodes, while the pulmonary and renal parenchyma had more widespread ecchymotic haemorrhages. Both renal capsules contained multiple, pale white foci, and the cortices had multifocal acute infarcts. Pharyngeal tonsils and peripheral lymph nodes were markedly enlarged. There was serous atrophy of epicardial fat. The abomasal mucosa was multifocally ulcerated and numerous whipworms (Trichuris spp.) were embedded in the caecal mucosa. Other organs, including joints and spleen, had no gross evidence of disease. Tissues were collected for bacterial culture and histopathology. Fresh tissues were stored at 20 C for further diagnostic testing. For histopathology, tissues were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were stained with haematoxylin and eosin (HE).
Fig. 1. Multifocal, petechial to ecchymotic haemorrhages on the surface of the cerebrum, brainstem and cranial cerebellum. The leptomeninges of the brainstem and cerebellum are cloudy (arrowhead) and scattered, but frequent, fibrin strands are present at the cerebellopontine angle (arrow).
Microscopically, leptomeningitis was most severe in the brainstem and cerebellum, but also affected the cerebrum. The leptomeninges overlying the brainstem and cerebellum were multifocally expanded by abundant neutrophils, admixed with fibrin, haemorrhage and occasional clusters of coccobacilli (Fig. 2), which were characterized as gramnegative by Lillie Twort stain. Microhaemorrhages and multifocal, perivascular neutrophilic inflammation were present within the parenchyma of the brainstem, cerebrum and cerebellum (Fig. 2). Rare, dilated blood vessels within the cerebellar leptomeninges contained scant to moderate amounts of fibrin, while vasculature within the parenchyma of the midbrain contained multiple fibrin thrombi (Fig. 3). The choroid plexus was infiltrated by numerous neutrophils and plasma cells, with abundant haemorrhage and fibrin. The pulmonary parenchyma had multiple, extensive, foci of infarction. Pulmonary vessels contained fibrin thrombi and numerous alveoli were filled with fibrin. There was interlobular oedema and the pulmonary pleura was expanded by oedema and fibrin. The renal medulla had multiple infarcts and the cortical interstitium contained multiple, scattered aggregates of macrophages, with fewer lymphocytes and plasma cells. The liver exhibited mild to moderate periportal hepatitis with predominantly plasma cells and macrophages, accompanied by generalized Kupffer cell hyperplasia. The spleen had marked lymphoid hypoplasia and necrosis, with lymphoid depletion of germinal centres. No significant microscopical lesions were observed in skeletal muscle, pancreas, thyroid, heart, rumen, small intestine and large intestine.
Fig. 2. Severe cerebellar leptomeningitis characterized by an abundance of neutrophils with associated fibrin, haemorrhage and occasional clusters of coccobacilli. HE. Bar, 100 mm. Inset. HE. Bar, 50 mm.
Mannheimia haemolytica Meningoencephalitis in a Calf
Fig. 3. Dilated blood vessels within the midbrain parenchyma contain fibrin thrombi in a calf infected with Mannheimia haemolytica serotype A1. HE. Bar, 200 mm.
Fresh lung and cerebellum were incubated overnight at 37 C on tryptic soy agar (TSA) plates containing 5% defibrinated bovine blood at 7% CO2. These culture conditions were selected specifically for the recovery of multiple pulmonary bacterial pathogens, particularly Histophilus somni; the latter organism was not isolated from either lung or cerebellum. Instead, culture of lung and cerebellum yielded a light and heavy growth of M. haemolytica, respectively. M. haemolytica isolates from lung and cerebellum exhibited atypically large zones of b-haemolysis, as compared with reference A1 bovine pneumonic isolates NADC D153 and L101 (Tatum et al., 1998). Polymerase chain reaction (PCR) assays revealed bands representing the leukotoxin structural gene in M. haemolytica isolates from both lung and cerebellum (Tatum et al., 1998). Western blot analysis of parallel 3 h culture supernatants probed with neutralizing anti-leukotoxin monoclonal antibody 2C9-1E8 (Tabatabai et al., 2010) yielded bands, consistent with leukotoxin, that were approximately fourfold more intense by scanning densitometry than those produced by the previously mentioned reference isolate NADC D153 (Tatum et al., 1998). These results indicated that both isolates produced relatively high levels of leukotoxin. Rapid plate agglutination further classified lung and cerebellum isolates as serotype A1 (Frank and Wessman, 1978). Analysis of 16S rDNA sequence confirmed both isolates to be M. haemolytica A1 and revealed 100% homology across a 489 base pair segment designed to differentiate between species (MicroSEQ 500 16S rDNA bacterial identification kit, Applied Biosystems, Foster City, California, USA). Both isolates also displayed identical
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minimum inhibitory concentrations across a 14-antibiotic sensitivity panel on a 96-well microtitre plate, further supporting the likelihood that the cerebellum and lung isolates were genotypically and phenotypically identical (Sensititre; Trek Diagnostic Systems, Weslake, Ohio, USA). Due to the unusually high levels of ruminantspecific leukotoxin and haemolytic activity, and to the uncharacteristic lesions noted in the brain of the calf, further investigation for concomitant viral infection was undertaken. BVDV RNA was detected in a sample of lung by PCR (Letellier and Kerkhofs, 2003). Gross and microscopical lesions, including leptomeningeal haemorrhage and fibrin, fibrin thrombi within the central nervous system (CNS) vasculature and concurrent pleuropneumonia, in this calf supported Histophilus somni as the most likely aetiology (Fecteau and George, 2004; Confer, 2009). However, characteristic histological features such as marked vasculitis or inflammation and necrosis within the CNS parenchyma were not present, and H. somni was not isolated from either lung or cerebellum. Although it is possible that the heavy growth of M. haemolytica present in the cerebellum may have hindered the detection of small numbers of H. somni, such a light infection with H. somni would not likely have been sufficient to contribute to the severity of the lesions observed grossly and microscopically. Despite the gross and microscopical resemblance to H. somni, the bacteria cultured from both lung and cerebellum and the presumed aetiological agent was determined to be M. haemolytica. Although M. haemolytica is a primary pathogen in stressed, post-weaning cattle, it is also a normal inhabitant of the nasopharynx and tonsils of healthy calves (Confer, 2009; Lawrence et al., 2010; Singh et al., 2011). Among the twelve serotypes of M. haemolytica characterized to date, typical commensals that colonize the nasopharynx of healthy, non-stressed cattle include serotypes A2 and A4, with A1 being the least frequently isolated (Rice et al., 2007; Griffin et al., 2010; Singh et al., 2011). A shift from resident commensals to a rapidly proliferating population of predominantly M. haemolytica serotype A1 can occur secondarily to various combinations of environmental stressors. Serotype A1 is the most commonly isolated pathogen in cases of severe pneumonic mannheimiosis (Griffin et al., 2010; Lawrence et al., 2010; Singh et al., 2011). As in the present calf, pneumonia due to serotype A1 is typified by haemorrhagic and fibrinonecrotizing bronchopneumonia (Al-Ghamdi et al., 2000; Zecchinon et al., 2005; Confer, 2009). However, meningoencephalitis and fibrin thrombi in the vasculature of the brain parenchyma, as seen in this calf, are not typical features of mannheimiosis.
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While M. haemolytica is considered a primary cause of respiratory disease in cattle, it rarely leads to systemic illness. In certain systemic cases, a septicaemic process is suspected (Zeman et al., 1993; Lamm et al., 2009). It has been suggested that naturally-infected calves with advanced, pneumonic mannheimiosis and neonates and young calves with failure of passive transfer of colostrum can develop systemic infections due to M. haemolytica (Zeman et al., 1993). Extrapulmonary infections due to M. haemolytica have been reported sporadically and include mastitis, otitis, sepsis and abortion (Lamm et al., 2009). In naturally-occurring cases of concurrent otitis and pneumonia in calves, M. haemolytica was isolated from the tympanic bullae (Gagea et al., 2006). Concurrent necrosuppurative myositis and pneumonia due to M. haemolytica A1 in a naturally-infected heifer has also been reported. The authors of that report proposed that the myositis originated from septicaemia incited by severe pulmonary disease (Lamm et al., 2009). In addition, M. haemolytica serotype A1 was rarely isolated from brain, liver, heart and joints of some calves vaccinated with an avirulent, live M. haemolytica A1 vaccine strain, indicating a possible disseminated infection secondary to vaccination (Zeman et al., 1993). Significant lesions in these vaccinated calves included purulent meningitis and polyarthritis and the serotype isolated from tissues matched the vaccine strain (Zeman et al., 1993). While M. haemolytica has been noted to cause focal meningeal infections in naturally-infected and recently vaccinated ruminants (Maxie and Youssef, 2007), prior to this report, this bacterium had not been reported to cause disseminated infections that include meningitis, encephalitis and fibrin thrombi within the CNS. Despite the absence of grossly-evident septicaemia, the calf in the present case was likely to be septicaemic, given the large numbers of bacteria and histological evidence of vascular disturbance within the lung. Septicaemia may have facilitated the spread of the bacterium to the leptomeninges and neuroparenchyma and likely contributed to thrombi formation. The high levels of haemolysis and increased production of ruminant-specific leukotoxin in both cerebellum and lung isolates may indicate a greater degree of strain virulence (Griffin, 2000; Rice et al., 2007; Griffin et al., 2010) and may have contributed to the ability of M. haemolytica to disseminate from the lungs to the CNS in the present calf. The presence of BVDV nucleic acid within lung was suggestive of persistent viral infection. The timing and effects of a concurrent BVDV infection in this calf are unknown, but may have predisposed to a fatal, systemic infection by enhancing M. haemolytica-associ-
ated respiratory disease through immunosuppression (Zeman et al., 1993; Confer et al., 2005). In addition, the synergistic role of BVDV in bovine respiratory disease could have increased the pathogenicity of the established M. haemolytica A1 strain (Ghoneim et al., 2010) and facilitated bacterial dissemination. To our knowledge, this is a novel report of a naturallyinfected calf with concurrent BVDV and M. haemolytica serotype A1 infection resulting in fibrinosuppurative meningoencephalitis with fibrin thrombi and fibrinonecrotizing broncho- and pleuropneumonia.
Acknowledgments The authors thank A. Phillips of the Athens Veterinary Diagnostic Laboratory for laboratory assistance.
Conflict of Interest Statement None of the authors has any financial and personal relationships with other people or organizations that could have inappropriately influenced this work.
References Al-Ghamdi GM, Ames TR, Baker JC, Walker R, Chase CCL et al. (2000) Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States. Journal of Veterinary Diagnostic Investigation, 12, 576e578. Confer AW (2009) Update on bacterial pathogenesis in BRD. Animal Health Research Reviews, 10, 145e148. Confer AW, Fulton RW, Step DL, Johnson BJ, Ridpath JF (2005) Viral antigen distribution in the respiratory tract of cattle persistently infected with bovine viral diarrhea virus subtype 2a. Veterinary Pathology, 42, 192e199. Fecteau G, George LW (2004) Bacterial meningitis and encephalitis in ruminants. Veterinary Clinics of North America: Food Animal Practice, 20, 363e377. Frank GH, Wessman GE (1978) Rapid plate agglutination procedure for serotyping Pasteurella haemolytica. Journal of Clinical Microbiology, 7, 142e145. Gagea MI, Bateman KG, Dreumel TV, McEwen BJ, Carman S et al. (2006) Diseases and pathogens associated with mortality in Ontario beef feedlots. Journal of Veterinary Diagnostic Investigation, 18, 18e28. Ghoneim HA, Hassan NI, Elhalawany HA, Nabih AM (2010) Mixed infection of bovine viral diarrhea virus, Mycoplasma species and Mannheimia haemolytica in calves showed chronic pneumonia with reference to the histopathological findings of the affected lungs. Journal of American Science, 6, 538e555. Griffin D (2000) Bovine pasteurellosis and other bacterial infections of the respiratory tract. Veterinary Clinics of North America: Food Animal Practice, 26, 57e71. Griffin D, Chengappa MM, Kuszak J, McVey DS (2010) Bacterial pathogens of the bovine respiratory disease complex. Veterinary Clinics of North America: Food Animal Practice, 26, 381e394.
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Lamm CG, Step DL, Krehbiel CR, Love BC (2009) Myositis due to Mannheimia haemolytica infection in a beef heifer. Bovine Practitioner, 43, 23e26. Lawrence PK, Kittichotirat W, Bumgarner RE, McDermott JE, Herndon DR et al. (2010) Genome sequences of Mannheimia haemolytica serotype A2: ovine and bovine isolates. Journal of Bacteriology, 192, 1167e1168. Letellier C, Kerkhofs P (2003) Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus. Journal of Virological Methods, 114, 21e27. Maxie MG, Youssef S (2007) Nervous system. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 5th Edit., MG Maxie, Ed., Saunders Elsevier, St Louis, p. 339. Rice JA, Carrasco-Medina L, Hodgins DC, Shewen PE (2007) Mannheimia haemolytica and bovine respiratory disease. Animal Health Research Reviews, 8, 117e128. Singh K, Ritchey JW, Confer AW (2011) Mannheimia haemolytica: bacterialehost interactions in bovine pneumonia. Veterinary Pathology, 48, 338e348.
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Tabatabai LB, Zimmerli MK, Zehr ES, Briggs RE, Tatum FM (2010) Ornithobacterium rhinotracheale North American field isolates express a hemolysin-like protein. Avian Diseases, 54, 994e1001. Tatum FM, Briggs RE, Srinand S, Zehr ES, Hsuan SL et al. (1998) Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence. Microbial Pathogenesis, 24, 37e46. Zecchinon L, Fett T, Desmecht D (2005) How Mannheimia haemolytica defeats host defense through a kiss of death mechanism. Veterinary Research, 36, 133e156. Zeman D, Neiger R, Nietfield J, Miskimins D, Libal M et al. (1993) Systemic Pasteurella haemolytica infection as a rare sequel to avirulent live Pasteurella haemolytica vaccination in cattle. Journal of Veterinary Diagnostic Investigation, 5, 555e559.
June 19th, 2012 ½ Received, Accepted, February 12th, 2013
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE
Mannheimia haemolytica A1-induced Fibrinosuppurative Meningoencephalitis in a Naturally-infected HolsteineFriesian Calf S. Aschenbroich*, N. Nemeth*, R. Rech*, R. Briggs‡, S. Sanchez† and C. Brown* * Department of Pathology, † Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Athens, Georgia and ‡ USDA/ARS/NADC, Ames, Iowa, USA
Summary Mannheimia haemolytica is an opportunistic bacterium that is widely recognized among the bovine respiratory disease complex as the predominant pathogen causing broncho- and pleuropneumonia in cattle. Among the characterized M. haemolytica serotypes, A1 is the major cause of severe pulmonary lesions in cattle. This report describes post-mortem findings in a HolsteineFriesian calf with fibrinosuppurative meningoencephalitis and fibrinonecrotizing, haemorrhagic broncho- and pleuropneumonia, from which M. haemolytica and bovine viral diarrhoea virus (BVDV) were isolated. Microscopical evaluation showed expansion of the brainstem and cerebellar leptomeninges by neutrophils and fibrin, associated with gram-negative coccobacilli. Occasional blood vessels within the midbrain and cerebellum contained fibrin thrombi. Bacterial culture of cerebellum and lung yielded M. haemolytica with unusually high haemolytic activity. The isolates were confirmed as serotype A1 by rapid plate agglutination. Lung tissue was positive for BVDV by polymerase chain reaction. The broncho- and pleuropneumonia in this calf were consistent with typical mannheimiosis due to serotype A1; however, extrapulmonary infections due to M. haemolytica, as seen in this case, are rarely reported. To our knowledge, this is the first documentation of a natural BVDV and M. haemolytica co-infection associated with fibrinosuppurative meningoencephalitis in a calf. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: bovine viral diarrhoea virus; calf; Mannheimia haemolytica; meningoencephalitis
Mannheimia haemolytica is a commensal of the upper respiratory tract of healthy ruminants, but can become a significant cause of pneumonia in dairy and feedlot cattle. Disruption of the commensal relationship can lead to the establishment of M. haemolytica A1 as the dominant serotype (Al-Ghamdi et al., 2000; Zecchinon et al., 2005; Griffin et al., 2010). Immunomodulatory factors, such as weaning, shipping, commingling, changes in climatic conditions and bacterial and viral co-infections, may affect the bovine host and facilitate the development of serotype A1-induced fibrinosuppurative and necrotizing bronchopneumonia (Griffin, 2000; Zecchinon et al., 2005; Rice et al., 2007; Singh et al., 2011). Correspondence to: S. Aschenbroich (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.02.001
A 5-month-old neutered male HolsteineFriesian calf was submitted for necropsy examination to the Athens Veterinary Diagnostic Laboratory, Athens, Georgia, with a history of weight loss, lethargy, hind-limb ataxia and rare crackles in the left cranial lung quadrant. Following treatment with florfenicol (single dose 6 ml; NuflorÔ; Merck Animal Health Summit, New Jersey, USA) and daily oral electrolytes, the calf’s body condition improved and clinical signs appeared to resolve. However, improvement was short-lived, with the calf becoming laterally recumbent prior to its demise. The bovine viral diarrhoea virus (BVDV)-exposure status of the calf prior to vaccination with Bovishield Gold 4 (modified live virus strains of infectious bovine rhinotracheitis virus), BVDV types 1 and 2 and parainfluenza 3 virus Ó 2013 Elsevier Ltd. All rights reserved.
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(Pfizer Animal Health, New York, USA) was unknown. The calf had stunted growth, was dehydrated and in poor nutritional condition. The surfaces of the occipital lobe of the cerebrum, brainstem and cranial cerebellum were covered by multifocal, petechial to ecchymotic haemorrhages (Fig. 1). The leptomeninges of the brainstem and cerebellum were cloudy and had numerous, scattered fibrin strands. There was caudal herniation of the cerebellum (Fig. 1). The cranioventral aspect of the lungs was bilaterally mottled dark red, black and tan, and both cranial lobes were adhered to the parietal pleura by multiple fibrin strands. On cut section, the pulmonary parenchyma was disrupted by multifocal, variably sized, firm nodules. Multifocal petechiae were present throughout the myocardium, abomasal mucosa, pharyngeal tonsils and peripheral lymph nodes, while the pulmonary and renal parenchyma had more widespread ecchymotic haemorrhages. Both renal capsules contained multiple, pale white foci, and the cortices had multifocal acute infarcts. Pharyngeal tonsils and peripheral lymph nodes were markedly enlarged. There was serous atrophy of epicardial fat. The abomasal mucosa was multifocally ulcerated and numerous whipworms (Trichuris spp.) were embedded in the caecal mucosa. Other organs, including joints and spleen, had no gross evidence of disease. Tissues were collected for bacterial culture and histopathology. Fresh tissues were stored at 20 C for further diagnostic testing. For histopathology, tissues were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were stained with haematoxylin and eosin (HE).
Fig. 1. Multifocal, petechial to ecchymotic haemorrhages on the surface of the cerebrum, brainstem and cranial cerebellum. The leptomeninges of the brainstem and cerebellum are cloudy (arrowhead) and scattered, but frequent, fibrin strands are present at the cerebellopontine angle (arrow).
Microscopically, leptomeningitis was most severe in the brainstem and cerebellum, but also affected the cerebrum. The leptomeninges overlying the brainstem and cerebellum were multifocally expanded by abundant neutrophils, admixed with fibrin, haemorrhage and occasional clusters of coccobacilli (Fig. 2), which were characterized as gramnegative by Lillie Twort stain. Microhaemorrhages and multifocal, perivascular neutrophilic inflammation were present within the parenchyma of the brainstem, cerebrum and cerebellum (Fig. 2). Rare, dilated blood vessels within the cerebellar leptomeninges contained scant to moderate amounts of fibrin, while vasculature within the parenchyma of the midbrain contained multiple fibrin thrombi (Fig. 3). The choroid plexus was infiltrated by numerous neutrophils and plasma cells, with abundant haemorrhage and fibrin. The pulmonary parenchyma had multiple, extensive, foci of infarction. Pulmonary vessels contained fibrin thrombi and numerous alveoli were filled with fibrin. There was interlobular oedema and the pulmonary pleura was expanded by oedema and fibrin. The renal medulla had multiple infarcts and the cortical interstitium contained multiple, scattered aggregates of macrophages, with fewer lymphocytes and plasma cells. The liver exhibited mild to moderate periportal hepatitis with predominantly plasma cells and macrophages, accompanied by generalized Kupffer cell hyperplasia. The spleen had marked lymphoid hypoplasia and necrosis, with lymphoid depletion of germinal centres. No significant microscopical lesions were observed in skeletal muscle, pancreas, thyroid, heart, rumen, small intestine and large intestine.
Fig. 2. Severe cerebellar leptomeningitis characterized by an abundance of neutrophils with associated fibrin, haemorrhage and occasional clusters of coccobacilli. HE. Bar, 100 mm. Inset. HE. Bar, 50 mm.
Mannheimia haemolytica Meningoencephalitis in a Calf
Fig. 3. Dilated blood vessels within the midbrain parenchyma contain fibrin thrombi in a calf infected with Mannheimia haemolytica serotype A1. HE. Bar, 200 mm.
Fresh lung and cerebellum were incubated overnight at 37 C on tryptic soy agar (TSA) plates containing 5% defibrinated bovine blood at 7% CO2. These culture conditions were selected specifically for the recovery of multiple pulmonary bacterial pathogens, particularly Histophilus somni; the latter organism was not isolated from either lung or cerebellum. Instead, culture of lung and cerebellum yielded a light and heavy growth of M. haemolytica, respectively. M. haemolytica isolates from lung and cerebellum exhibited atypically large zones of b-haemolysis, as compared with reference A1 bovine pneumonic isolates NADC D153 and L101 (Tatum et al., 1998). Polymerase chain reaction (PCR) assays revealed bands representing the leukotoxin structural gene in M. haemolytica isolates from both lung and cerebellum (Tatum et al., 1998). Western blot analysis of parallel 3 h culture supernatants probed with neutralizing anti-leukotoxin monoclonal antibody 2C9-1E8 (Tabatabai et al., 2010) yielded bands, consistent with leukotoxin, that were approximately fourfold more intense by scanning densitometry than those produced by the previously mentioned reference isolate NADC D153 (Tatum et al., 1998). These results indicated that both isolates produced relatively high levels of leukotoxin. Rapid plate agglutination further classified lung and cerebellum isolates as serotype A1 (Frank and Wessman, 1978). Analysis of 16S rDNA sequence confirmed both isolates to be M. haemolytica A1 and revealed 100% homology across a 489 base pair segment designed to differentiate between species (MicroSEQ 500 16S rDNA bacterial identification kit, Applied Biosystems, Foster City, California, USA). Both isolates also displayed identical
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minimum inhibitory concentrations across a 14-antibiotic sensitivity panel on a 96-well microtitre plate, further supporting the likelihood that the cerebellum and lung isolates were genotypically and phenotypically identical (Sensititre; Trek Diagnostic Systems, Weslake, Ohio, USA). Due to the unusually high levels of ruminantspecific leukotoxin and haemolytic activity, and to the uncharacteristic lesions noted in the brain of the calf, further investigation for concomitant viral infection was undertaken. BVDV RNA was detected in a sample of lung by PCR (Letellier and Kerkhofs, 2003). Gross and microscopical lesions, including leptomeningeal haemorrhage and fibrin, fibrin thrombi within the central nervous system (CNS) vasculature and concurrent pleuropneumonia, in this calf supported Histophilus somni as the most likely aetiology (Fecteau and George, 2004; Confer, 2009). However, characteristic histological features such as marked vasculitis or inflammation and necrosis within the CNS parenchyma were not present, and H. somni was not isolated from either lung or cerebellum. Although it is possible that the heavy growth of M. haemolytica present in the cerebellum may have hindered the detection of small numbers of H. somni, such a light infection with H. somni would not likely have been sufficient to contribute to the severity of the lesions observed grossly and microscopically. Despite the gross and microscopical resemblance to H. somni, the bacteria cultured from both lung and cerebellum and the presumed aetiological agent was determined to be M. haemolytica. Although M. haemolytica is a primary pathogen in stressed, post-weaning cattle, it is also a normal inhabitant of the nasopharynx and tonsils of healthy calves (Confer, 2009; Lawrence et al., 2010; Singh et al., 2011). Among the twelve serotypes of M. haemolytica characterized to date, typical commensals that colonize the nasopharynx of healthy, non-stressed cattle include serotypes A2 and A4, with A1 being the least frequently isolated (Rice et al., 2007; Griffin et al., 2010; Singh et al., 2011). A shift from resident commensals to a rapidly proliferating population of predominantly M. haemolytica serotype A1 can occur secondarily to various combinations of environmental stressors. Serotype A1 is the most commonly isolated pathogen in cases of severe pneumonic mannheimiosis (Griffin et al., 2010; Lawrence et al., 2010; Singh et al., 2011). As in the present calf, pneumonia due to serotype A1 is typified by haemorrhagic and fibrinonecrotizing bronchopneumonia (Al-Ghamdi et al., 2000; Zecchinon et al., 2005; Confer, 2009). However, meningoencephalitis and fibrin thrombi in the vasculature of the brain parenchyma, as seen in this calf, are not typical features of mannheimiosis.
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While M. haemolytica is considered a primary cause of respiratory disease in cattle, it rarely leads to systemic illness. In certain systemic cases, a septicaemic process is suspected (Zeman et al., 1993; Lamm et al., 2009). It has been suggested that naturally-infected calves with advanced, pneumonic mannheimiosis and neonates and young calves with failure of passive transfer of colostrum can develop systemic infections due to M. haemolytica (Zeman et al., 1993). Extrapulmonary infections due to M. haemolytica have been reported sporadically and include mastitis, otitis, sepsis and abortion (Lamm et al., 2009). In naturally-occurring cases of concurrent otitis and pneumonia in calves, M. haemolytica was isolated from the tympanic bullae (Gagea et al., 2006). Concurrent necrosuppurative myositis and pneumonia due to M. haemolytica A1 in a naturally-infected heifer has also been reported. The authors of that report proposed that the myositis originated from septicaemia incited by severe pulmonary disease (Lamm et al., 2009). In addition, M. haemolytica serotype A1 was rarely isolated from brain, liver, heart and joints of some calves vaccinated with an avirulent, live M. haemolytica A1 vaccine strain, indicating a possible disseminated infection secondary to vaccination (Zeman et al., 1993). Significant lesions in these vaccinated calves included purulent meningitis and polyarthritis and the serotype isolated from tissues matched the vaccine strain (Zeman et al., 1993). While M. haemolytica has been noted to cause focal meningeal infections in naturally-infected and recently vaccinated ruminants (Maxie and Youssef, 2007), prior to this report, this bacterium had not been reported to cause disseminated infections that include meningitis, encephalitis and fibrin thrombi within the CNS. Despite the absence of grossly-evident septicaemia, the calf in the present case was likely to be septicaemic, given the large numbers of bacteria and histological evidence of vascular disturbance within the lung. Septicaemia may have facilitated the spread of the bacterium to the leptomeninges and neuroparenchyma and likely contributed to thrombi formation. The high levels of haemolysis and increased production of ruminant-specific leukotoxin in both cerebellum and lung isolates may indicate a greater degree of strain virulence (Griffin, 2000; Rice et al., 2007; Griffin et al., 2010) and may have contributed to the ability of M. haemolytica to disseminate from the lungs to the CNS in the present calf. The presence of BVDV nucleic acid within lung was suggestive of persistent viral infection. The timing and effects of a concurrent BVDV infection in this calf are unknown, but may have predisposed to a fatal, systemic infection by enhancing M. haemolytica-associ-
ated respiratory disease through immunosuppression (Zeman et al., 1993; Confer et al., 2005). In addition, the synergistic role of BVDV in bovine respiratory disease could have increased the pathogenicity of the established M. haemolytica A1 strain (Ghoneim et al., 2010) and facilitated bacterial dissemination. To our knowledge, this is a novel report of a naturallyinfected calf with concurrent BVDV and M. haemolytica serotype A1 infection resulting in fibrinosuppurative meningoencephalitis with fibrin thrombi and fibrinonecrotizing broncho- and pleuropneumonia.
Acknowledgments The authors thank A. Phillips of the Athens Veterinary Diagnostic Laboratory for laboratory assistance.
Conflict of Interest Statement None of the authors has any financial and personal relationships with other people or organizations that could have inappropriately influenced this work.
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June 19th, 2012 ½ Received, Accepted, February 12th, 2013