Histophilus somni-associated syndromes in sheep from Southern Brazil

Selwyn A. Headley Alfredo H.T. Pereira Luciana C. Balbo Giovana W. Di Santia Ana P.F.R.L. Bracarense Luiz F.C. Cunha Filho Jackson Schade Werner Okano Priscilla F.V. Pereira Fábio Morotti Lucienne G. Preto-Giordano Rogério A. Marcasso Alice F. Alfieri Júlio A.N. Lisbôa Amauri A. Alfieri About the authors

Abstract

Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n = 4), septicemia (n = 4), bronchopneumonia (n = 4), and myocarditis (n = 3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.

Keywords:
Histophilosis; Molecular testing; Bronchopneumonia; Myocarditis; Thrombotic meningoencephalitis

Introduction

Histophilus somni (formerly Haemophilus somnus) is a Gram-negative bacterium that is associated with a disease complex (collectively termed histophilosis) that can produce several clinical syndromes including thrombotic meningoencephalitis (TME), pleuritis, polysynovitis, arthritis, bronchopneumonia, septicemia, myocarditis, otitis media, infertility, abortion, and mastitis in affected ruminants.11 Harris FW, Janzen ED. The Haemophilus somnus disease complex (Hemophilosis): a review. Can Vet J. 1989;30(10):816-822.

2 Inzana TJ, Corbeil L. Haemophilus. In: Gyles CL, Prescott JF, Songer G, Thoen CO, eds. Pathogenesis of Bacterial Infections in Animals. Ames, IA, USA: Blackwell Publishing; 2004:243–258.

3 Corbeil LB. Histophilus somni host-parasite relationships. Anim Health Res Rev. 2007;8(2):151-160.
-44 Pérez DS, Pérez FA, Bretschneider G. Histophilus somni: pathogenecity in cattle an update. An Vet Murcia. 2010;26:5-21. These syndromes have been referred to as the H. somni disease complex, HSDC.55 O'Toole D, Sondgeroth KS. Histophilosis as a natural disease. Curr Top Microbiol Immunol. 2016;396:15-48.

Histophilosis or the HSDC, occurs predominantly in cattle with sporadic reports of disease occurring in several species of small ruminants. This bacterium has been isolated from healthy goats in Mexico66 Perez-Romero N, Aguilar-Romero F, Arellano-Reynoso B, Diaz-Aparicio E, Hernandez-Castro R. Isolation of Histophilus somni from the nasal exudates of a clinically healthy adult goat. Trop Anim Health Prod. 2011;43(5):901-903. and Hungary77 Janosi K, Hajtos I, Makrai L, et al. First isolation of Histophilus somni from goats. Vet Microbiol. 2009;133(4):383-386. and in sheep from Ethiopia.88 Tesfaye B, Sisay Tessema T, Tefera G. Diversity of bacterial species in the nasal cavity of sheep in the highlands of Ethiopia and first report of Histophilus somni in the country. Trop Anim Health Prod. 2013;45(5):1243-1249. Reports of H. somni associated diseases in sheep include TME,99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390.

10 Cassidy JP, McDowell SW, Reilly GA, et al. Thrombotic meningoencephalitis associated with Histophilus ovis infection in lambs in Europe. Vet Rec. 1997;140(8):193-195.
-1111 Romero A, Quinteros C, Marinho P, O'Tool D, Dutra F. Thrombotic meningoencephalitis (TME) by Histophilus somni in sheep in Uruguay. Veterinaria (Montevideo). 2013;49(191):38-47. pneumonia,99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390. reproductive disorders,1212 Lees VW, Meek AH, Rosendal S. Epidemiology of Haemophilus somnus in young rams. Can J Vet Res. 1990;54(3):331-336. and endometritis99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390.,1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138.; spontaneous1414 Palomares G, Aguilar F, Hernández L, et al. Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis. Small Rum Res. 2005;60(3):221-225. and experimentally induced epididymitis1515 Diaz-Aparicio E, Tenorio-Gutierrez VR, Arellano-Reynoso B, Enriquez-Verdugo I, Aguilar-Romero F. Pathogenicity of different strains of Histophilus somni in the experimental induction of ovine epididymitis. Can J Vet Res. 2009;73(2):157-160. have also been described. Moreover, H. somni was associated with pneumonia in bighorn sheep.1616 Ward AC, Weiser GC, Anderson BC, et al. Haemophilus somnus (Histophilus somni) in bighorn sheep. Can J Vet Res. 2006;70(1):34-42.

Descriptions of the HSDC in Brazil are incipient with abortions,1717 Headley SA, Voltarelli D, Oliveira VHS, et al. Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil. Trop Anim Health Prod. 2015;47(2):403-413. respiratory impairment,1818 Headley SA, Alfieri AF, Oliveira VHS, Beuttemmüller EA, Alfieri AA. Histophilus somni is a potential threat to beef cattle feedlots from Brazil. Vet Rec. 2014;175(10):249. systemic disease,1919 Headley SA, Oliveira VH, Figueira GF, et al. Histophilus somni-induced infections in cattle from southern Brazil. Trop Anim Health Prod. 2013;45(7):1579-1588. and TME2020 Headley SA, Bracarense APFRL, Oliveira VHS, et al. Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil. Pesqui Vet Bras. 2015;35(4):329-336. being described in cattle from different geographical regions; we believe that histophilosis might be a threat to the local beef cattle industry.1818 Headley SA, Alfieri AF, Oliveira VHS, Beuttemmüller EA, Alfieri AA. Histophilus somni is a potential threat to beef cattle feedlots from Brazil. Vet Rec. 2014;175(10):249. However, the participation of H. somni associated disease in sheep from Brazil is restricted to a single case report of a ewe with endometritis.1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138. Consequently, to identify additional cases of histophilosis in sheep from Brazil, this study investigated the possible association of H. somni in sheep mortality from different regions of Paraná State, Southern Brazil (Fig. 1).

Fig. 1
Gross demonstration of Histophilus somni associated lesions in sheep. There is multifocal necrotizing myocarditis (A) and fibrinous bronchopneumonia with abscess (B-C). Observe the severely congested meningeal vessels (D), petechial epicardial hemorrhages (E), pulmonary adhesions (F), and fibrinous bronchopneumonia with pulmonary abscess (G). Scale in centimeters.

Materials and methods

Farms affected, clinical history, autopsy, and bacteriological identification

Animals from this study were from different cities within the state of Paraná, Southern Brazil. Sheep from all farms were submitted to the Laboratory of Animal Pathology, UEL for routine autopsy and diagnostics. The geographical locations of the five farms affected, the biological data of affected sheep, and the clinical outcome are resumed at Table 1. Three farms (A, B, and C) are in northern Paraná, and the others in the northeastern (D) and southeastern (E) regions of this State. All farms contained breeds of sheep reared primarily for meat production, with the number of sheep varying from 20 (Farm D) to 1250 (Farm E). Sheep at Farms A, C, and E were routinely immunized against clostridial diseases; in addition, those at Farm C were also immunized against contagious ectima and foot-rot. Sheep at the other farms were not routinely immunized against infectious disease agents; all sheep were maintained predominantly on grassland pastures; water was obtained from streams or artesian wells within these farms.

Table 1
Geographical location, principal clinical manifestations, and outcome of sheep with histophilosis.

Apathy (n = 6) and respiratory discomfort (n = 4) were the predominant clinical manifestations reportedly observed; sheep # 3 was found dead without any clinical manifestation of disease. A cesarean section was done on sheep # 4 due to ruminal atony, abnormal gait and apathy, and a 3-month old fetus that died intra-uterus was surgically removed. However, the ewe died spontaneously two days later. In addition, one neonate lamb (#1) had omphalitis, enlarged knee and elbow joints and locomotory difficulties. The evolution of clinical manifestations was acute in all cases and all died spontaneously.

A routine autopsy was done soon after the death of all animals; selected tissue fragments (cerebrum, cerebellum, lung, myocardium, liver, kidneys, and intestine) were fixed by immersion in 10% buffered formalin solution and routinely processed for histopathological evaluation. Duplicate sections of the organs mentioned above from all animals, as well as sterile swabs from the affected joints (knee and elbow) and the umbilical cord of animal #1 were collected freshly during necropsy, and maintained at -80 °C until processed for molecular testing. Additionally, samples collected freshly at necropsy were plated in BHI agar with 5% of sheep blood and processed for microbiological culture as described.1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138.

Extraction of nucleic acids, PCR assays, and sequencing

Nucleic acids extracted from tissue fragments (Table 2) of all animals as described,2121 Boom R, Sol CJ, Salimans MM, et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28(3):495-503. were used in PCR assays designed to amplify specific amplicons of bacterial pathogens associated with systemic disease in ruminants. These assays targeting the 16s rRNA gene of H. somni,2222 Angen O, Ahrens P, Tegtmeier C. Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures. Vet Microbiol. 1998;63(1):39-48. species and type-specific isolates of Pasteurella multocida,2323 Townsend KM, Frost AJ, Lee CW, Papadimitriou JM, Dawkins HJS. Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates. J Clin Microbiol. 1998;36(4):1096-1100. and the lktC-artJ intergenic region of Mannheimia haemolytica.2424 Angen O, Thomsen J, Larsen LE, et al. Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response. Vet Microbiol. 2009;137(1-2):165-171. Pulmonary fragments of all animals were evaluated to identify the RNA of bovine respiratory syncytial virus, BRSV.2525 Almeida RS, Spilki FR, Roehe PM, Arns CW. Detection of Brazilian bovine respiratory syncytial virus strain by a reverse transcriptase-nested-polymerase chain reaction in experimentally infected calves. Vet Microbiol. 2005;105(2):131-135. Purulent exudate from the abscesses of sheep were collected freshly at necropsy and used in a PCR assay designed to identify the 16S rDNA gene of Trueperella (Arcanobacterium) pyogenes (T. pyogenes).2626 Ulbegi-Mohyla H, Hijazin M, Alber J, et al. Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties. J Vet Sci. 2010;11(3):265-267. Positive controls included nucleic acids of H. somni,1919 Headley SA, Oliveira VH, Figueira GF, et al. Histophilus somni-induced infections in cattle from southern Brazil. Trop Anim Health Prod. 2013;45(7):1579-1588.P. multocida, M. haemolytica, and BRSV from previous reports,2727 Oliveira VHS. Molecular Diagnosis of Viral and Bacterial Infections Associated with an Outbreak of Respiratory Disease in Dairy Calves. Master's of Science Dissertation. Master's ofScience, Universidade Estadual de Londrina; 2014. and from housekeeping sample (T. pyogenes). Nuclease-free water was used as negative controls in all PCR assays. PCR products were separated by electrophoresis in 2% agarose gels, stained with ethidium bromide, and examined under ultraviolet light. The amplified PCR products were then purified and submitted for direct sequencing using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare, Little Chalfont, UK) with the forward and reverse primers in the 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, USA).

Table 2
Principal pathological findings, histophilosis syndromes, and distribution of Histophilus somni DNA in sheep.

The obtained sequences were examined for quality analysis of chromatogram readings by using the PHRED software (http://asparagin.cenargen.embrapa.br/phph); sequences were only accepted if base quality was equal to or greater than 20. Consensus sequences were then generated by the CAP3 program (http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3.pl), after which the partial nucleotide sequences were initially compared by the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST) with similar sequences deposited in GenBank. Phylogenetic tree and sequence alignments based on the 16s rRNA gene of the Pasteurellaceae family were then created by using MEGA 62828 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725-2729.; Model selection indicated the Jukes-Cantor model as being the most appropriate for determination of the phylogenetic relationship with the Maximum Likelihood method. The initial tree for the heuristic search was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach. E. coli was used as the out-group to provide stability to the generated tree.

Results

Pathological findings and histophilosis syndromes

The principal pathological alterations are resumed in Table 2. The most frequent gross findings (Fig. 2A-G) observed were cranioventral pulmonary consolidation (n = 6); congestion of meningeal vessels of the brain, petechial hemorrhages of the heart, and pulmonary edema were observed in four sheep. Multifocal myocardial necrosis was observed in sheep # 2, while pulmonary abscesses were observed in sheep #5. Omphalitis (navel-ill) and marked swelling of both knee and elbow joints (joint-ill) occurred in the neonatal lamb (# 1). Endometritis was suspected due to the abnormal uterine exudate observed in the sheep (#4) that had a dead fetus removed surgically.

Fig. 2
Histopathological findings associated with Histophilus somni in sheep. There is necrotizing myocarditis (A), bronchopneumonia (B), bacterial emboli within the brain (C) and lung (D), and thrombosis at the cerebrum (E) and choroid plexus (F). Bar: A, C-E, 0.02 mm; B and F, 0.1 mm.

The principal histopathological findings (Fig. 2A-F) observed in affected sheep included TME (n = 4), necrotizing myocarditis (n = 4), purulent (n = 2) and fibrinous (n = 3) bronchopneumonia, and pulmonary abscesses (n = 1). The lamb (# 1) with omphalitis and joint ill had TME, purulent bronchopneumonia with widespread thrombosis that affected the kidneys, lungs, and lymph nodes. Further, TME and necrotizing myocarditis were observed in the ewe (# 4), while vascular congestion of the brain, lungs, and kidneys, suggestive of septicemia, occurred in her 3-month-old fetus that suffered intrauterine fetal death.

The clinical syndromes associated with H. somni observed during this study were: (a) bronchopneumonia, with lesions in sheep # 5, 6, 7, and 8; (b) myocardial disease, observed in sheep # 1, 3, and 4; (c), TME, affecting sheep # 2, 4, 5, and 8, (d) septicemia, sheep # 1, 3, 4, and 5; and (e) omphalitis and polyarthritis in sheep #1.

Furthermore, repeated attempts at bacterial culture and identification were frustrating, since the growth of colonies of H. somni did not occur from any of the samples collected.

Molecular identification of H. somni DNA in tissues

H. somni DNA was amplified from multiple affected tissues of all sheep (Table 2). Further, the disseminated distribution of H. somni DNA was more widespread in sheep # 1, 2, 3, 4, and 5. In addition, H. somni DNA was amplified from the umbilical vein and joints (knee and elbow) of the neonatal lamb (# 1) that had navel and joint ill. It must be highlighted that H. somni DNA was identified in multiple tissues (cerebrum, cerebellum, brainstem, liver, kidney, and spleen) of the sheep # 4 as well as in the kidney and spleen of her 3-month-old fetus. Further, T. pyogenes DNA was amplified from the purulent exudate of the pulmonary abscess of sheep # 5, where H. somni DNA was also identified by PCR indicating that both pathogens participated in the development of the lung abscess of this sheep; all other PCR assays yielded negative results.

Partial sequences of the 16s rRNA gene of H. somni were obtained from the umbilical vein (GenBank accession No. KP419695) of sheep #1, the cerebrum of sheep # 2 (KU726866), 6 (KU726864), and 5 (KU726867), the liver (KU726865) of sheep #4, and the kidney (KU726862) and spleen (KU726863) of the 3-month-old-fetus. Initial BLAST analyses revealed that these sequences demonstrated 98-99% identity with similar isolates of H. somni deposited in GenBank. Phylogenetic analyses revealed that the sequences derived from this study clustered with other isolates of H. somni (Fig. 3); the nucleotide sequences used for phylogenetic analyses during this study are given in Fig. 3. Furthermore, the isolates derived from the cerebrum and liver of sheep # 5 were identical to those obtained from spleen and kidney of her 3-month-old fetus that suffered intrauterine death.

Fig. 3
Phylogenetic tree based on the 16S rRNA gene sequences of Histophilus somni and selected Pasteurellaceae members generated by MEGA 6. The tree was constructed by the Neighbor-Joining method, based on 1000 bootstrapped data sets; distances values were calculated by using the Jukes-Cantor parameter model. The GenBank accession numbers and country of origin of the sequences are given; the sequences derived from this study are highlighted (black dot).

Discussion

The importance of this report lies is the identification of H. somni DNA in multiple tissues of sheep with histopathological lesions that characterize several typical clinical syndromes of HSDC previously described in cattle,22 Inzana TJ, Corbeil L. Haemophilus. In: Gyles CL, Prescott JF, Songer G, Thoen CO, eds. Pathogenesis of Bacterial Infections in Animals. Ames, IA, USA: Blackwell Publishing; 2004:243–258.

3 Corbeil LB. Histophilus somni host-parasite relationships. Anim Health Res Rev. 2007;8(2):151-160.

4 Pérez DS, Pérez FA, Bretschneider G. Histophilus somni: pathogenecity in cattle an update. An Vet Murcia. 2010;26:5-21.
-55 O'Toole D, Sondgeroth KS. Histophilosis as a natural disease. Curr Top Microbiol Immunol. 2016;396:15-48. and sheep.99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390. These results add to the occurrence of this pathogen as an important disease agent of ruminants in this country, considering the recent descriptions of H. somni-associated diseases,1919 Headley SA, Oliveira VH, Figueira GF, et al. Histophilus somni-induced infections in cattle from southern Brazil. Trop Anim Health Prod. 2013;45(7):1579-1588. abortions,1717 Headley SA, Voltarelli D, Oliveira VHS, et al. Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil. Trop Anim Health Prod. 2015;47(2):403-413. and TME in cattle,2020 Headley SA, Bracarense APFRL, Oliveira VHS, et al. Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil. Pesqui Vet Bras. 2015;35(4):329-336. as well as endometritis in one sheep.1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138. Consequently, these findings represent the first study to effectively confirm several clinical syndromes associated with H. somni in sheep from Brazil; a previous study only identified this pathogen in one ewe.1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138. Collectively, these findings contribute to the hypothesis that histophilosis is indeed a threat not only to beef cattle from Brazil,1818 Headley SA, Alfieri AF, Oliveira VHS, Beuttemmüller EA, Alfieri AA. Histophilus somni is a potential threat to beef cattle feedlots from Brazil. Vet Rec. 2014;175(10):249. but to ruminants on a wider scale, principally due to the development of respiratory, reproductive, and neurological disease in susceptible herds. Further, we postulate that cases of histophilosis in Brazil are probably underdiagnosed since most veterinary clinicians might have confused these syndromes with other disease processes of ruminants.

The results from this study have identified the DNA of H. somni within multiple tissues as well as from the knee joint and affected umbilical cord of the neonatal lamb (#1), thereby characterizing systemic histophilosis. Moreover, nucleotide sequencing of the amplicons confirmed these findings, associating this bacterium with the pathogenesis of the omphalitis (navel-ill), arthritis (joint-ill), bronchopneumonia, and myocarditis herein described. In addition, this sheep also presented TME and myocarditis, clinical syndromes frequently associated with H. somni in cattle,2929 Van Dreumel AA, Curtis RA, Ruhnke HL. Infectious thromboembolic meningoencephalitis in Ontario feedlot cattle. Can Vet J. 1970;11(7):125-130.

30 Wessels J, Wessels ME. Histophilus somni myocarditis in a beef rearing calf in the United Kingdom. Vet Rec. 2005;157(14):420-421.
-3131 O'Toole D, Allen T, Hunter R, Corbeil LB. Diagnostic exercise: myocarditis due to Histophilus somni in feedlot and backgrounded cattle. Vet Pathol. 2009;46(5):1015-1017. and sheep99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390.,1010 Cassidy JP, McDowell SW, Reilly GA, et al. Thrombotic meningoencephalitis associated with Histophilus ovis infection in lambs in Europe. Vet Rec. 1997;140(8):193-195.; these syndromes were also demonstrated experimentally in sheep.3232 Rahaley RS. Pathology of experimental Histophilus ovis infection in sheep II. Pregnant ewes. Vet Pathol. 1978;15(6):746-752.

Another interesting finding during this study was the amplification of H. somni DNA from multiple tissues of sheep # 5 and her fetus, confirming the vertical transmission of this pathogen; similar results were described in dairy cattle from different geographical regions of Brazil.1717 Headley SA, Voltarelli D, Oliveira VHS, et al. Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil. Trop Anim Health Prod. 2015;47(2):403-413. Although this was not an abortion per se, infection by H. somni resulted in fetal death, suggesting that this pathogen is also associated with reproductive disorders in sheep, since H. somni DNA was identified in rams with epididymitis,99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390.,1414 Palomares G, Aguilar F, Hernández L, et al. Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis. Small Rum Res. 2005;60(3):221-225. and in ewes with endometritis.99 Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J. 1991;68(12):387-390.,1313 Rizzo H, Gregory L, Carvalho AF, Pinheiro ES. Histophilus somni isolation on sheep with endometritis first case report on Brazil. Rev Bras Reprod Anim. 2012;36(2):136-138. Alternatively, H. somni is a known abortifacient agent of cattle with descriptions of natural infections in countries including Canada,3333 van Dreumel AA, Kierstead M. Abortion associated with Hemophilus somnus infection in a bovine fetus. Can Vet J. 1975;16(12):367-370. the UK,3434 van der Burgt G, Clark W, Knight R, Colles K. Cattle fertility problems and Histophilus somni. Vet Rec. 2007;160(17):600. USA,3535 Corbeil LB, Widders PR, Gogolewski R, et al. Haemophilus somnus: bovine reproductive and respiratory disease. Can Vet J. 1986;27(2):90-93. and Brazil.1717 Headley SA, Voltarelli D, Oliveira VHS, et al. Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil. Trop Anim Health Prod. 2015;47(2):403-413.

The phylogenetic analysis demonstrated that the isolates of H. somni derived from this study clustered with those of cattle and sheep from different geographical locations; this suggest the close phylogenetic relationship of these strains irrespective of their geographical origin, confirming that the same pathogen affects cattle and sheep, as was previously demonstrated.3636 Angen O, Ahrens P, Kuhnert P, Christensen H, Mutters R. Proposal of Histophilus somni gen. nov., sp. nov. for the three species incertae sedis ‘Haemophilus somnus'. ‘Haemophilus agni' and ‘Histophilus ovis'. Int J Syst Evol Microbiol. 2003;53(Pt 5):1449-1456. Unfortunately, bacterial isolation would have been ideal to define the biochemical characteristics of this pathogen but growth of H. somni was not successful. Culture of H. somni from infected tissues is considered a difficult task,44 Pérez DS, Pérez FA, Bretschneider G. Histophilus somni: pathogenecity in cattle an update. An Vet Murcia. 2010;26:5-21.,2222 Angen O, Ahrens P, Tegtmeier C. Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures. Vet Microbiol. 1998;63(1):39-48. which may explain the frustrated attempts at culture during this investigation. In this regard, PCR identification with subsequent sequencing, as herein described, is recommended to identify this bacterial pathogen from infected tissues or environmental samples.2222 Angen O, Ahrens P, Tegtmeier C. Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures. Vet Microbiol. 1998;63(1):39-48.

Conclusion

H. somni DNA was identified in multiple tissues of sheep from different geographical regions of the state of Paraná with clinical syndromes of histophilosis confirmed by histopathological evaluations. These findings suggest that this bacterial pathogen was associated with sheep morbidity and mortality from these regions.

Acknowledgements

S.A. Headley, A.P.F.R.L. Bracarense., A.F. Alfieri, J.A.N. Lisboa, and A.A. Alfieri are recipients of the National Council for Scientific and Technological Development (CNPq; Brazil) fellowships and grants. This study was partially funded by a CNPq grant (Proposal #448797/2014-3) and a joint CNPq/Ministry of Agriculture (MAPA) grant (Protocols #578645/2008-4 and #478254/2012-1).

References

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    Harris FW, Janzen ED. The Haemophilus somnus disease complex (Hemophilosis): a review. Can Vet J 1989;30(10):816-822.
  • 2
    Inzana TJ, Corbeil L. Haemophilus. In: Gyles CL, Prescott JF, Songer G, Thoen CO, eds. Pathogenesis of Bacterial Infections in Animals Ames, IA, USA: Blackwell Publishing; 2004:243–258.
  • 3
    Corbeil LB. Histophilus somni host-parasite relationships. Anim Health Res Rev 2007;8(2):151-160.
  • 4
    Pérez DS, Pérez FA, Bretschneider G. Histophilus somni: pathogenecity in cattle an update. An Vet Murcia 2010;26:5-21.
  • 5
    O'Toole D, Sondgeroth KS. Histophilosis as a natural disease. Curr Top Microbiol Immunol 2016;396:15-48.
  • 6
    Perez-Romero N, Aguilar-Romero F, Arellano-Reynoso B, Diaz-Aparicio E, Hernandez-Castro R. Isolation of Histophilus somni from the nasal exudates of a clinically healthy adult goat. Trop Anim Health Prod 2011;43(5):901-903.
  • 7
    Janosi K, Hajtos I, Makrai L, et al. First isolation of Histophilus somni from goats. Vet Microbiol 2009;133(4):383-386.
  • 8
    Tesfaye B, Sisay Tessema T, Tefera G. Diversity of bacterial species in the nasal cavity of sheep in the highlands of Ethiopia and first report of Histophilus somni in the country. Trop Anim Health Prod. 2013;45(5):1243-1249.
  • 9
    Philbey AW, Glastonbury JR, Rothwell JT, Links IJ, Searson JE. Meningoencephalitis and other conditions associated with Histophilus ovis infection in sheep. Aust Vet J 1991;68(12):387-390.
  • 10
    Cassidy JP, McDowell SW, Reilly GA, et al. Thrombotic meningoencephalitis associated with Histophilus ovis infection in lambs in Europe. Vet Rec 1997;140(8):193-195.
  • 11
    Romero A, Quinteros C, Marinho P, O'Tool D, Dutra F. Thrombotic meningoencephalitis (TME) by Histophilus somni in sheep in Uruguay. Veterinaria (Montevideo) 2013;49(191):38-47.
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Publication Dates

  • Publication in this collection
    Jul-Sep 2018

History

  • Received
    29 May 2017
  • Accepted
    24 Dec 2017
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E-mail: bjm@sbmicrobiologia.org.br