Histophilus Somni-Induced Thrombotic Meningoencephalitis in Cattle from Northern Paraná, Brazil1

Pesq. Vet. Bras. 35(4):329-336, abril 2015 DOI: 10.1590/S0100-736X2015000400003

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil1

Selwyn A. Headley2*, Ana Paula F.R.L. Bracarense2, Victor H.S. Oliveira3, Gustavo R. Queiroz4, Werner Okano5 3, Karina K.M.C. Flaiban6, Júlio A.N. Lisbôa4 3 , Alice F. Alfieri ABSTRACT.- and Amauri A. Alfieri Histophilus somni-induced thrombotic meningoencephalitis Headley S.A., Bracarense in cattle A.P.F.R.L., from northern Oliveira Paraná, V.H.S., Queiroz Brazil. G.R., Pesquisa Okano Veterinária W., Alfieri BrasileiraA.F., Flaiban 35(4):329-336. K.K.M.C., Lisbôa Laboratório J.A.N. & Alfieride Patologia A.A. 2015. Animal, Departamento de Medicina Vete- rinária Preventiva, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR-445 Km [email protected] Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predo- 380,minantly Cx. Postal from 10.011,North America, Londrina, that PR 860571-970,is caused by Brazil.Histophilus E-mail: somni with sporadic descrip- tions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations

suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis characteristicwith vasculitis of and brain thrombosis dysfunction. of small Hematological vessels that and contained cerebrospinal discrete fluid neutrophilic analyses were and/or not

nerve ganglion of all animals. All tissues from the central nervous system used during this lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal- orescence assay. PCR and RT-PCR assays investigated the participation of infectious agents study were previously characterized as negative for rabies virus by the directH. somni immunoflu, Listeria monocytogenes - associated with bovine neurological disease by targeting specific genes of of H. somni from, bovine brain herpesvirussections of all -1 animalsand -5, bovine with histopathological viral diarrhea virus, diagnosis and ovine of TME; herpes all virus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA geneH. somni other PCR/RT-PCR assays were negative. These findings confirmedH. somni the from participation Brazil. of in the neuropathologicalHistophilus somni, thrombotic disease observed meningoencephalitis, in these animals, bovine extend disease, the neuropathology, geographical centraldistribution nervous of system,this disease, molecular and diagnostics,support previous histophilosis. findings of INDEX TERMS: RESUMO.- [Meningoencefalite trombótica-induzida por encephalitis- TME) é uma doença neurológica fatal de bovi- Histophilus somni em bovinos da região norte do Para- nos ocasionada por Histophilus somni. A infecção tem sido ná.] Meningoencefalite trombótica (Thrombotic meningo- descrita predominantemente na América do Norte e de for- ma esporádica em outros países. O objetivo deste estudo é relatar a ocorrência de TME em bovinos da região norte do 1 estado do Paraná, Brasil. A maioria dos animais apresenta- 2 Received on October 3, 2014. Laboratório de Patologia Animal, Departamento de Medicina Veteri- ram sinais clínicos neurológicos característicos de disfun- náriaAccepted Preventiva, for publicationUniversidade on Estadual February de 10, Londrina 2015. (UEL), Londrina, PR, [email protected] - 3 Laboratório de Biologia Molecular, Departamento de Medicina Veteri- rebrospinal não foram sugestivas de infecção bacteriana do 860571-970, Brasil. *Corresponding author: cérebro.ção cerebral A histopatologia aguda. Análises revelou hematológicas meningoencefalite e do fluido com ce 4 Clínica de Grande Animais, Departamento de Clínicas Veterinárias, - nária Preventiva, UEL, Londrina, PR 860571-970. 5 Laboratório de Patologia Animal, Escola de Medicina Veterinária, Uni- UEL, Londrina, PR, 860571-970. vasculite e trombose de pequenos vasos com discreto infil 6 Laboratório de Patologia Clínica, Departamento de Medicina Veteriná- todostrado neutrofílicoos animais. e/ouAs amostras linfocítico de mescladasistema nervoso com fibrina central no versidade Norte do Paraná, Arapongas, PR, 86702-670, Brasil. incluídastronco e córtex nesse cerebralestudo foram e no gânglio previamente do nervo caracterizadas trigêmio de ria Preventiva, UEL, Londrina, PR 860571-970. 329 Selwyn A. Headley et al.

330 como negativas para raiva por meio de técnica de imuno- Gross neurological lesions are characteristic and visible in associados à doença neurológica em bovinos foi avaliada hemorrhagic areas randomly distributed throughout the fluorescência direta. A participação de agentes infecciosos- most untreated cases, and consist of 1 – 30 mm diameter- cação parcial de genes de H. somni, Listeria monocytogenes, - por técnicas moleculares como PCR e RT-PCR para amplifi culitisbrain (Van and thrombosis,Dreumel et al. which 1970, predominantly Maxie & Youssef affects 2007, small Za herpesvírus ovino 2. As seções do cérebro de todos os ani- arteries,chary 2012). resulting Histologically, in tissue necrosis TME is with characterized a marked neutro by vas- maisherpesvírus com diagnóstico bovino 1 histopatológicoe 5, vírus da diarreia de TME viral foram bovina positi e- H. somni. - philic response (Inzana & Corbeil 2004, Maxie & Youssef presençavas em PCR de para DNA a dedetecção H. somni do nosgene fragmentos 16S rRNA de de cérebro 2007, George 2009); thrombi frequently contains intrale- avaliados.O sequenciamento As reações dos de produtos PCR/RT-PCR amplificados para todos confirmou os outros a sional bacterial colonies (Maxie & Youssef 2007, Zachary micro-organismos avaliados resultaram negativas. Os re- in2012). cases This of H. paper somni presents-induced the thrombotic clinical, hematological,meningoencepha ce- H. litisrebrospinal from northern fluid, and Paraná. neuropathological findings observed somni nos episódios de doença neurológica observada nos sultados desse estudo confirmaram a participação do MATERIALS AND METHODS - Animals, clinical history, and necropsy. All affected cattle traramanimais a avaliados, presença ampliade H. somni a distribuição em outras geográfica formas de da mani TME- festaçãoe ratifica clínicaestudos das prévios infecções realizados por essa no bactéria. Brasil que demons the signalment and principal clinical manifestations of the affec- originated from several breeds, and between 1.5-6 years of age; Histo- an ongoing study that is investigating the cause of neurological philus somni, doença de bovinos, neuropatologia, sistema nervoso diseaseted cattle in are cattle given from in Tablethe state 1. Further, of Paraná. these Most animals properties were part ( =4) of TERMOS DE INDEXAÇÃO: Meningoencefalite trombótica, n were small subsistence farms, while herd no 5 raised cattle for fattening. Cattle at all herds are routinely vaccinated against Foot central, diagnóstico molecular, histofilose. INTRODUCTION and Mouth Disease and clostridiosis, maintained predominantly on green pastures, and supplemented with commercially prepa- Histophilosis is a multisystemic disease entity of rumi- red mineral salts; water was given ad libitum. nants caused by the Gram-negative bacterium Histophilus somni (formerly known as Haemophilus somnus) which three cases occurring during August, September, and December produces several clinical syndromes including thrombo- The first two cases occurred in late July 2013,n=4) were with inthe perma other- tic meningoencephalitis (TME), bronchopneumonia, pleu- nent recumbency, a few were blind (n=2), and with convulsions ritis, polysynovitis, septicemia, myocarditis, otitis media, (2013.n=2); All only animals one animal were depressed, was febrile but (n mosto ( manifestations until spontaneous death or euthanasia was acute in most cases (n=4), with variation of 2 1). to The8 days, course but ofanimal clinical no infertility, abortion, and mastitis (Inzana & Corbeil 2004,- 2 was reportedly ill during 22 days. This animal was hospitalized Pérez et al. 2010). Histophilosis has been a major problem due to a history of progressive emaciation and muscular weakness, and was maintained on supportive therapy. Additionally, of cattle in North America (Van Dreumel et al. 1970, Ma- most animals (n=4) were euthanized in extremis, with owners recDonald economic et al. loss 1973, to Georgethe affected 2009) livestock and Australia industry (Lancaster (Saun- consent, due to the severity of clinical manifestations; animal et al. 1984, Hick et al. 2012) for decades, resulting in seve no of histophilosis from countries such as the UK (Wessels & (CSF) of all animals were collected before death for laboratory ders et al. 1980). Additionally, there are sporadic reports analyses. 4 died spontaneously. Blood samples and cerebrospinal fluid - Routine necropsies were performed soon after death; selec- losisWessels in cattle2005), from Argentina different (Descarga geographical et al. 2002), regions and of Japan Bra- ted tissue fragments (brain, liver, lungs, kidneys, spleen, intesti- (Tanaka et al. 2005). Recently, we have described histophi ne, myocardium, and lymph nodes) from each animal necropsied - routinely processed for histopathological evaluation. Neurologi- zil due to the characterization of systemic disease in five were fixed by immersion in 10% buffered formalin solution and- animals (Headley et al. 2013b), and reproductive manifes threattations to have the beefbeen cattle confirmed feedlot in industry bovine fetuses of Brazil (Headley and South et cerebellum,cal tissue fragments thalamus, of and all animalsthe trigeminal were obtainednerve ganglion from five and pre the al. 2015). Further, we believe that histophilosisH. somni ismight conside be a- -defined anatomical locations of the brain: brainstem, cerebrum, red as an emerging disease pathogen of beef cattle feedlots the organs mentioned above were collected freshly during ne- America (Headley et al. 2014a), while carotid rete miriabile (TNG-CRM) complex. Duplicate sections of TME is a fulminating neurological disease of cattle that diagnostics. Additionally, to avoid cross contamination, necropsy occursin Argentina after septicemia (Descarga inducedet al. 2002). by H. somni equipmentcropsy, and used maintained for sample at -80°Ccollection until were processed cleaned for and molecular then im- which produces fatality rates in affected herds and affects - predominantly feedlot cattle relative to dairy(George or pastured 2009), en each collected sample. mersedThis in study a mixture adhered of antiseptic to the guidelines and disinfectant for the usage solution of animals/ betwe - samples in studies as required by CONCEA (approved by CEUA/ monstrate lateral recumbency, profound depression, a sle- animals (Fecteau & George 2004). Affected cattle can de Characterization of rabies virus. - UEL protocol # 32340.2012.04). epy appearance (MacDonald et al. 1973; Fecteau & George The direct immunofluor- 2004), head tilt, nystagmus, strabismus, blindness, coma, escence assay was performed for the identification of Lyssavirus and convulsions (MacDonald et al. 1973, George 2009). from brain samples of all animals submitted to the Official Dia Pesq. Vet. Bras. 35(4):329-336, abril 2015 Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

331 Table 1. Signalment, principal clinical manifestations, and outcome of cattle with thrombotic meningoencephalitis from the northern Paraná Animal nº Signalment, Principal clinical Course Clinical herd size, and manifestations (days) outcome geographical location

1 Nellore, male, 1.5 years lateralBlindness, recumbency, depression, tetraparesis, fever, flaccid reduced anal sphincter, tongue tonus flaccid tail, loss of 6 Euthanasia 50 cattle, Curiúva county facial sensibility, loss of flexor reflex (all limbs), nystagmus, permanent

2 Girolando, male, 1.5 years sleepyDepression, appearance motor incoordination, muscular weakness, permanent 22 Euthanasia 30 cattle, Londrina lateral recumbency, progressive emaciation, respiratory difficulty,

3 Gir, male, 6 years Depression, flaccid tail, loss of flexor reflex (all limbs), permanent 6 Euthanasia 4 Crossbreed,20 cattle, Londrina female, 4 years Convulsions,sternal recumbency depression left and right cervical movement, muscular 8 Spontaneous

3 cows, Londrina weakness, permanent lateral recumbency, no menace reflex death

5 Nellore, female, 2.5 years Absence of cutaneous and palpebral reflexes, bilateral blindness, 2 Euthanasia 250 cattle, Alto Paraná bruxism, convulsions, depression, flaccid tongue, opisthotonos gnostic Laboratory (Centro de Diagnóstico Marcos Enrietti; Cur- nucleotide sequences of H. somni obtained during this investiga- itiba, Paraná, Brazil). tion were deposited at GenBank. PCR/RT-PCR characterization of infectious agents asso- Phylogenetic trees and sequence alignments were created by ciated with neurological disease in cattle. Nucleic acids were brainstem, TNG, and thalamus) of all animals as described (Boom using MEGA 6.01 software (Tamura et al. 2011), constructed by the extracted from frozen brain fragments (cerebrum, cerebellum,- geneNeighbor-Joining of Pasteurellaceae method, family; based distances on 1,000 values bootstrapped were calculated data sets by usingof the the nucleotide Kimura 2 sequences parameter of model. selected strains of the 16S rRNA thenet al. used1990), in in PCR/RT-PCR association assayswith Proteinase designed Kto (Ambion, identify infectiousGrand Is agentsland, NY, associated USA), and with a modified neurological method disease (Alfieri of cattle et al. from2006), Brazil. and RESULTS Hematological and biochemical analyses of blood and These protocols targeted specific amplicons of the glycoprotein cerebrospinal fluid C gene of bovine herpesvirus type 1 (BoHV-1) andListeria -5 (Claus mono et- Overall CSF analysis from all animals did not indicate cytogenesal. 2005), and the 5’-UTR region of the bovine viralHistophilus diarrhea somnivirus (Vilček et al. 1994), the listeriolysin gene of the complete blood cell count (CBC) of these animals. He- herpesvirus(Wesley type 2 et (OvHV-2) al. 2002), was the also 16s targeted rRNA gene to detectof DNA of matologicaldefinite bacterial analyses infections, revealed but (Table there 2) wereneutrophilia variations in all to (Angen et al. 1998). The tegument protein gene of ovine sheep-associated malignant catarrhal fever, SA-MCF (Baxter et al.- animals; hyperfibrinogenemia (animals nº 3 and 4), and 1993). andleucocytosis 5), with (nº increased 2) were enzymatic observed. Significantactivity of biochemicalcreatine ki- Positive controls consisted of extracted nucleic acid from pro abnormalities included elevated urea concentration (nº 1 totype strains of BoHV-1 (Los Angeles) and -5 (AA-Par) amplifiedL. mo- in Madin-Darby bovine kidney cells (Claus et al. 2005); and field nocytogenes H. somni nase (nº 1, 3-5), aspartate aminotransferase (nº 1-4), and strains of previous cases of OvHv-2 (Headley et al. 2013c), - observed only in animal nº 5 and consisted of hyperprotei- ase-free water(Headley (Invitrogen et al. Corp.,2013a; Carlsbad, Headley etCA, al. USA) 2014b), was used as norraquiaalkaline phosphatase and mononuclear (nº 1, 2pleocytosis and 5). CSF (Table alterations 3). were negative(Headley controls et al. 2013b), in all andPCR/RT-PCR OvHV-2 (Headley assays. All et RT-PCR/PCRal. 2013c). Nucle pro- - Pathological findings The most constant gross neurological alterations obser- ductsSequencing were separated and phylogeneticby electrophoresis analyses. in 2% agarose gels, stai ved in these animals were moderate congestion of menin- ned with ethidium bromide, and examined under ultraviolet light. geal vessels (n=3), shallow sulci with distended gyri (n=2), The amplified PCR o UK)products and submittedwere then forpurified direct (illustra sequencing GFX PCRusing DNA the and forward Gel Band and - Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire,- servedand foci in of animal hemorrhage no 5 (Table at the 4). cerebral cortex of animal n lity analysis of chromatogram readings by using the PHRED sof- 4 (Fig.Histologically, 1A-B); significant thrombi pathological occurred predominantly findings were notwithin ob reverse primers. The obtained sequences were examined for qua small vessels of the meninges and in all regions of the brain Consensustware (http://asparagin.cenargen.embrapa.br/phph); sequences were then generated by the CAP3 sequences program were only accepted if base quality was equal to or greater than 20. TME within the brain sections of these animals is given in pl), after which the partial nucleotide sequences obtained were (Fig. 1C-E) of all animals investigated; the distribution of (http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3. (n=5), cerebellar white matter (n=2), grey and white matter ofTable the thalamus4. Briefly, (TMEn=3) andwas brainstemobserved at(n =5),the andcerebral at the cortex TNG- compared by the BLAST program (http://www.ncbi.nlm.nih.gov/ BLAST) with similar sequences deposited in GenBank to confirm sequence identity and validate the isolates identified. The partial Pesq. Vet. Bras. 35(4):329-336, abril 2015 332 Selwyn A. Headley et al.

Table 2. Hematological and biochemical findings of cattle with thrombotic meningoencephalitis Laboratory Parameters Reference Animals nº

Hemogram values 1 2 3 4 5

Packed Cell Volume (%) 27 – 48 42.8 36.8 44.1 42.1 38.0 Red Blood cells (x10⁶/µL) 6 – 11.6 8.51 8.20 7.88 7.6 7.97 Hemoglobin (g/dL) 8.50 – 16.5 11.2 11.1 11.1 10.7 12.6 Total plasma protein (g/dL) 6.6 – 7.8 7.8 7.0 8.0 9.8 7.4 Fibrinogen (mg/dL) 300 - 700 400 400 1,000 800 600 Leucocytes (x10³/ µL) 6,200 – 12,200 10,100 14,200 8,800 7,600 7,000 Lymphocytes (x10³/ µL) 3,300 – 8,000 4,141 5,112 1,496 1,672 2,030 Eosinophils (x10³/ µL) 0 – 2,400 0 0 0 0 0 Monocytes (x10³/ µL) 200 - 700 0 284 0 0 0 Bastonetes (x10³/ µL) 0 - 200 0 0 88 304 0 Segmented neutrophils (x10³/µL) 1,300 – 3,400 5,959 8,804 7,216 5,624 4,970 MCV (fL) 30.5 – 55.5 50.3 44.9 56.0 55.5 47.6 Serum MCHC biochemistry (%) 28 – 38 26.1 30.1 35.1 25.4 34.0 MCH (pg) 9.7 – 19 13.1 13.5 14.0 14.0 15.8

Albumin (g/dL) 2.7 – 4.3 2.3 1.5 2.6 2.3 2.6 Creatinine (mg/dL) 1 – 2.7 2.7 0.60 1.5 1.2 2.3 Urea (mg/dL) 5.8 – 35.9 111 15 35 39 70 Creatine kinase, CK (UI/L) 35 – 280 952 136 4,117 4,164 5,676 Aspartate aminotransferase, AST (UI/L) 32 – 71 207 88 962 399 ND Gamma-glutamyl transferase GGT (UI/L) 3.7 – 31.3 34 45 23 41 30 Alkaline phosphatase, ALP (UI/L) 33 – 100 256 160 ND ND 198 Total bilirubin (mg/dL) 0.32 – 0.74 0.47 0.55 0.54 0.22 0.47 Conjugated bilirubin (mg/dL) 0.01 – 0.34 0.35 0.35 0.12 0.05 0.06 ND = not determined. Unconjugated bilirubin (mg/dL) 0.16 – 0.67 0.12 0.20 0.42 0.17 0.41

Table 3. Cerebrospinal fluid analyses of cattle with thrombotic meningoencephalitis o 2, Parameters Reference Animals whognosis had of H.TME somni (Table 4). Bacterial DNA was amplified from nervemultiple ganglion. sections In of summary, the brain H.from somni all animals, except n DNA identifiedn=5), only thalamus from the trigeminal(n=4), the Color valuesUnco- Unco-1 Unco-2 Unco-3 Unco-4 Unco-5 lored lored lored lored lored lored n=3), and DNA cerebellum was amplified (n=2) Aspect Clear Clear Clear Clear Clear fromof affected the TNG-CRM animals. All complex other PCR/RT-PCR ( assays provided brainstemnegative results and cerebral and all cortex animals ( were negative for rabies

Protein (mg/dL) 40 39.3 21.6 19.9 17.7 133.7 cellDensity count (mm3) >1,010 1,004 1,008 1,006 1,006 1,004 Total nucleated 10 2 0 0 2 14 ofvirus the by partial the immunofluorescence nucleotide sequence assay. of the H. somni Direct sequencing revealed the desired 408o base pairs Segmented 0 0 0 0 28 o 3), and the brainstem (no 5). 16 InitialrRNA neutrophils (%) BLASTgene from analyses the TNG-CRM revealed complexthat all (animalsnucleotide n sequences 1, 2, and Band 0 0 0 0 0 o o neutrophils (%) (GenBank4), cerebral Accession cortex (n n Mononuclear 0 0 0 0 72 no o o o cells (%) n=5). Thrombi consisted of discrete intra- KM374589, animal n 1; KM374590, vascular accumulations of neutrophils and/or lymphocytes with 2; KM374591,similar sequences n 3; KM374592, deposited in n GenBank. 4; and KM374593, Further, the n -CRM complex ( generated5) derived phylogeneticfrom this study tree demonstrated revealed that 99-100% all isolates identity of H. of the affected vascular endothelium; intralesional bacteria somni formed a large cluster, which was distinct from other wereadmixed not withfrequently fibrin andobserved moderate and thereto marked was no proliferation associated members of the Pasteurellaceae family (Fig. 2). degrees of cortical edema, gliosis, and hemorrhage were DISCUSSION perivascular inflammatory exudate. Additionally, varying non-neurological pathological alterations included throm- brain sections of these animals are consistent with those observed in the brain sections evaluated. Other significanto describedThe histopathological in H. somni-induced findings TME observed of ruminants within (Fecteau several 2 and 5), liver and intestine (no 5). bi within small vessels of the lung (Fig.1F), myocardium (n similar to the brain lesions previously described in cases of PCR, sequencing, and phylogenetic analysis & George 2004; Maxie & Youssef 2007; George 2009), and The H. somni - from the brain of all animals with histopathological dia- systemic (Headley et al. 2013b) and reproductiveH. somni from (Headley these PCR successfully amplified bacterial DNA et al. 2015) histophilosis from Brazil. Additionally, the amp lification and consequent sequencing of Pesq. Vet. Bras. 35(4):329-336, abril 2015 Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil 333

Histophilus somni induced thrombotic meningoencephalitis in cattle from northern Paraná. (A) Congestion of meningeal vessels and hemorrhage with distended gyri and shallow sulci. (B) Edema of the sectioned surface of Fig.1.the Gross cerebrum. and histopathological There are thrombi features (*) at ofthe meninges (C-D), carotid rete miriabile (E), and the lungs (F). (B) Scale in centimeters. (C- F C and F D-E

) HematoxylinTable 4. Principal and eosin gross stain. findings ( with) Bar the = 20µm.distribution ( ) 100µm. of thrombotic meningoencephalitis and Histophilus somni DNA in brain sections of cattle Histophilus somni DNA Brain- Cere- Cere- TNG- Thala- Brain- Cere- Cere- TNG- Thala- Animal nº Gross findings stemThrombotic bellum meningoencephalitis brum CRM mus stem bellum brum CRM mus

myocardial and endocardial petechial, 1 hemorrhage,Congestion of ulcerativeabomastits meningeal vessels; + - + + - + + + + +

congestion of meningeal vessels, hydropericardium, 2 hyperemiaCerebrum; shallowof intestinal sulci mucosa with distended gyri; + + + + + - - - + -

emphysema, and atelectasis, ulcerative abomastits 3 Congestion of meningeal vessels, pulmonary + - + + + + - + + + gyri, cranioventral consolidation of lungs, 4 Ascites, cerebrum: shallow sulci with distended + - + + - - + - + +

enlarged liver, hydrothorax, renal infarction, nottongue observed and oesophagus: ulcerations 5 Significant pathological alterations were + + + + + + - + + +

TNG-CRM = Trigeminal nerve ganglion-carotid rete miriabile complex, + present, - absent. Pesq. Vet. Bras. 35(4):329-336, abril 2015 334 Selwyn A. Headley et al.

The clinical manifestations, the acute incubation period culminating in spontaneous death or euthanasia, and the reduced number of animals affected at each herd during -

this study are frequent clinical and epidemiological find bacterialings associated isolation with was TME not (MacDonald attempted in et any al. instance1973, Fecteau since diagnoses& George 2004,of TME George were only 2009). achieved During after this investigation,histopathological evaluation. It is worthy to mention that the hemato- - dicative of classical bacterial infections of the brain (Scott logical and more specifically, the CSF findings were not in - terations2004, Stokol indicative et al. 2009). of bacterial Although disease the CBC with in cases an increase of TME inis nonspecific,the number ofthe nucleated CSF findings cells, normally elevated demonstrateprotein concen al-

Alterationstrations (Van to DreumelCSF levels et were al. 1970, only Fecteau observed & Georgein animal 2004, no5 Scottbut were 2004), indicative xanthochromia, of hyperproteinorraquia and hemorrhage (Scottand lympho 2004).-

cytic pleocytosis, without xanthochromia and hemorrhage. markedAlternatively, variation a retrospective in the three casesstudy ofthat TME classified described, the with CSF pleocytosisrelative to being neurological neutrophilic, disease lymphocytic, in 101 cattle, and histiocytic observed - tions observed in most animals are due to prolonged recumbency.(Stokol et al.However, 2009). theInterestingly, results of the the CSF biochemical presented alterain this study are important for the ante-mortem diagnosis and the understanding of TME, since there are few documentations of the alterations associated with CSF in spontaneous cases of neurological histophilosis and the classical represent- ation of CSF alterations in TME is not fully known (Scott

The distribution of TME was predominant at the brain- 2004). animals, while these lesions were not consistent at the cerebellumstem, cerebral and cortex, thalamus. and However, the TNG-CRM H. somni complex DNA of was all Fig.2. The phylogenetic relationship of selected strains of Histo- philus somni Pasteurel- followed by the thalamus ( - laceae family. The isolates from this study are highlighted (ar- n rows); the GenBank based onaccession the 16S numbers rRNA gene and country of the of origin stemrecovered (n=3), more and cerebellum frequently ( fromn=2). theThis TGN-CRM might suggest complex, that of the sequences used are given. E. coli was used as the out- =4), cerebral cortex, brain- -group. tion of TME in a particular anatomical region of the brain there is no definite correlationH. somni between DNA from the these demonstra areas. areas, suggest that this bacterium was associated with the Nevertheless, histopathological lesions of TME seem to H. somni and the amplification of DNA and sequencing were previously used to identify this - neuropathological lesions; the amplification of have predilection for the cerebral cortex (MacDonald et- Moreover, the negative PCRs and RT-PCRs results with the tributedal. 1973, throughoutMaxie & Youssef any part 2007, of theZachary brain 2012) (MacDonald and thal et absencepathogen of (Angen other ethistopathological al. 1998, Headley disease et al. 2013b,patterns 2015). sug- amus (Maxie & Youssef 2007), but can be randomly dis- gest that other common pathogens associated with neur- bution of TME observed during this investigation, and sug- ological disease of cattle were not involved in the develop- gestal. 1973). that fragmentsThese findings of multiple are in agreementanatomical with regions the distriof the ment of the clinicopathological manifestations observed brain must be collected to obtain an accurate histopathological diagnosis of TME, particularly in non-conventional negative PCR assay for OvHV-2, since this is a common areas of this disease. Additionally, since histopathological causeduring of this neurological investigation. disease Of extreme of cattle importance from Brazil was (Head the- H. somni DNA was study, lesions at the carotid rete miriabile were thrombotic neuroanatomicalconfirmation and region,the amplification of the brain, of particularly the ca- ley et al. 2012, 2013c). Additionally, in the cases from this- rotidconstant rete from miriabile, the TGN-CRM should always complex be ofinvestigated all animals, when this TME is suspected. and not the characteristic fibrinoid necrosis frequently dia gnosed in SA-MCF (Headley et al. 2012, 2013c). Pesq. Vet. Bras. 35(4):329-336, abril 2015 Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil 335

H. somni DNA from these cases with REFERENCES Fre- The identification of - quency of group A rotavirus in diarrhoeic calves in Brazilian cattle typical histopathological findings of TME represent the Alfieri A.A., Parazzi M.E., Takiuchi E., Medici K.C. & Alfieri A.F. 2006. thefirst geographical successfully distributioncharacterization of this of diseasethis important and pathogen. neuro herds, 1998-2002. Trop. Anim. Health Prod. 38:521-526. logical disease of cattle from Brazil, and definitely extends Haemophilus somnus Vet. This is because histophilosis was previously described Angen O., Ahrens P. & Tegtmeier C. 1998. Development of a PCR test for by our group in cattle from southern Brazil (Headley et identification of in pure and mixed cultures. Microbiol. 63:39-48. steers that demonstrated pulmonary distress at a feedlot Baxter S.I., Pow I., Bridgen A. & Reid H.W. 1993. 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CONCLUSIONS Headleyof encephalitic S.A., Bodnar listeriosis L., Fritzen in J.T.,small Bronkhorst ruminants D.E., from Alfieri northern A.F., Okano Parana, W. Histophilus somni - & Alfieri A.A. 2013a. Histopathological and molecular characterization ments of cattle with neuropathological manifestations and Brazil. Braz. J. Microbiol. 44:889-896. Histophilus somni-induced infections in cattle histopathological lesions DNA thatwas areidentified consistent from withbrain thosefrag Headley S.A., Oliveira V.H., Figueira G.F., Bronkhorst D.E., Alfieri A.F., Okano described in TME of cattle from North America. W. & Alfieri A.A. 2013b. Further, sequencing of the H. somni from southern Brazil. Trop. Anim. Health Prod. 45:1579-1588. - Headley2-induced S.A., malignant Queiroz G.R., catarrhal Fritzen fever J.T.T., in Lisboa a heifer. J.A.N., Semina, Alfieri Ciênc. A.F., Agrarias Oliveira ticipation of this pathogen in the lesions 16S herein rRNA described. gene from R.A.M., Bracarense A.P.F.R.L. & Alfieri A.A. 2013c. Ovine herpesvirus type affectedThese brain results fragments contribute of all to animals the documentation confirmed the of parthis 34:3895-3900. important emerging infectious disease of cattle from Bra- Histophilus somni is a potential threat to beef cattle feedlots from Headley S.A., Alfieri A.F., Oliveira V.H.S., Beuttemmüller E.A. & Alfieri A.A. 2014a. Brazil. Vet. Rec. 175:249. zil and South America, complements previous findings of Headleyencephalitic S.A., Fritzen bovine J.T.T., listeriosis Queiroz from G.R., southern Oliveira Brazil. R.A.M., Trop. Alfieri Anim. A.F., HealthSantis spontaneoushistophilosis cases by our of groupTME. (Headley et al. 2013b, 2014a, G.W.D., Lisbôa J.A.N. & Alfieri A.A. 2014b. Molecular characterization of 2015), and produces novel CSF findings associated with Acknowledgements.- Selwyn A. Headley, Ana Paula F.R.L. Bracarense, Prod. 46:19-25. Histophilus somni with Headleyspontaneous S.A., Voltarelli abortions D., Oliveirain dairy V.H.S., cattle Bronkhorst herds from D.E., Brazil. Alfieri Trop. A.F., Anim.Negri L.C., Okano W. & Alfieri A.A. 2015. Association of Brazil)Alice F. fellowships.Alfieri, Júlio This A.N. study Lisbôa, was and partially Amauri funded A. Alfieri by a CNPqare recipients grant (Pro of- the National Council for Scientific and Technological Development (CNPq, Health Prod. 47:403-413. posal #448797/2014-3) and a joint CNPq/Ministry of Agriculture (MAPA) Hick P.M., Read A.J., Lugton I., Busfield F., Dawood K.E., Gabor L., Hornitzky M. & Kirkland P.D. 2012. Coronavirus infection in intensively managed Thegrant authors (Protocols contributed #578645/2008-4 for publication and #478254/2012-1). of the article as follows: Haemophilus, Prescottcattle with J.F., respiratory Songer G. &disease. Thoen Aust.C.O. (Eds), Vet. J. 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