Trop Anim Health Prod DOI 10.1007/s11250-014-0740-0


Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil Selwyn Arlington Headley & Daniele Voltarelli & Victor Henrique Silva de Oliveira & Dalton Evert Bronkhorst & Alice Fernandes Alfieri & Luiz Carlos Negri Filho & Werner Okano & Amauri Alcindo Alfieri

Received: 4 June 2014 / Accepted: 20 November 2014 # Springer Science+Business Media Dordrecht 2014

Abstract This study investigated the participation of infectious agents in spontaneous abortions and reproductive problems at eight dairy cattle herds from three geographical regions of Brazil. Fourteen aborted fetuses and the organ sections of one cow with history of repeated abortions were received for pathological evaluations and molecular diagnostics. PCR/RT-PCR assays targeted specific genes of abortifacient agents of cattle: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), Listeria monocytogenes, Neospora caninum, Leptospira spp., Brucella abortus, and Histophilus somni. Six fetuses were adequate for pathological investigations; one of these did not demonstrate remarkable pathological alterations. Significant histopathological findings included vasculitis, hemorrhage, and fibrinous thrombosis of the cerebrum (n=4); necrotizing myocarditis (n=3); and hemorrhagic enteritis (n=3). The placenta and uterus of the cow as well as the kidney, pancreas, and liver of her aborted fetus contained H. somni DNA and demonstrated histopathological evidence of histophilosis. All fetuses contained H. somni DNA in multiple organs. Coinfections of H. somni with B. abortus (n=2), N. caninum (n=2), BVDV (n=1), and BoHV-1 (n=1) were identified; two fetuses demonstrated S. A. Headley (*) Laboratory of Animal Pathology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445 Km 380, Campus Universitário, P.O. Box 10.011, 86057-970 Londrina, Paraná, Brazil e-mail: [email protected] D. Voltarelli : V. H. S. de Oliveira : A. F. Alfieri : A. A. Alfieri Laboratory of Molecular Biology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445 Km 380, Campus Universitário, P.O. Box 10.011, 86057-970 Londrina, Paraná, Brazil D. E. Bronkhorst : L. C. N. Filho : W. Okano Laboratory of Veterinary Pathology, School of Veterinary Medicine, Universidade Norte do Paraná, Arapongas, Paraná, Brazil

three pathogens. These findings suggest that H. somni was associated with the spontaneous abortions and reproductive problems of these herds. However, the exact cause of fetal death might not be attributed only to H. somni in all aborted fetuses, since some of these were infected with other abortifacient agents. Keywords Histophilosis . Bovine reproductive diseases . Abortifacient agents . Molecular diagnostics

Introduction Histophilus somni (previously known as Haemophilus somnus) is a Gram-negative bacterium associated with several clinical syndromes (collectively termed histophilosis) in ruminants including suppurative bronchopneumonia, thrombotic meningoencephalitis, pleuritis, polysynovitis, septicemia, myocarditis, infertility, abortion, and mastitis (Inzana and Corbeil 2004; Pérez et al. 2010). Histophilosis has been a major problem of cattle in North America (George 2009) and Australia (Lancaster et al. 1984; Hick et al. 2012), resulting in severe economic loss to the affected livestock industry (Saunders et al. 1980). Moreover, there are sporadic reports of H. somni in countries such as Hungary (Janosi et al. 2009), the UK (Wessels and Wessels 2005), and Argentina (Descarga et al. 2002). Descriptions of H. somni in cattle from Brazil have being restricted to the systemic disease in four steers and one aborted fetus (Headley et al. 2013b) and in feedlot cattle with respiratory distress (Headley et al. 2014a). Consequently, the occurrence of histophilosis in non-conventional geographical regions suggests that the disease is widespread and efforts must be

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implemented to reduce economic losses to the affected livestock. Although H. somni is a commensal of the upper respiratory and genital tracts of ruminants (Inzana and Corbeil 2004), this bacterium is considered as a sporadic abortifacient agent of cattle (van Dreumel and Kierstead 1975; Anderson 2007), with endometritis, cervicitis, and vaginitis terminating in infertility being common manifestations of reproductive disease (Schlafer and Miller 2007; van der Burgt et al. 2007; Pérez et al. 2010). Moreover, descriptions of H. somni-associated abortions and reproductive disease in cattle include older reports of experimental studies (Miller and Barnum 1983; Corbeil et al. 1986; Widders et al. 1986) and natural infections (van Dreumel and Kierstead 1975), with an outbreak of infertility and endometritis (van der Burgt et al. 2007). This study investigated the infectious causes of repeated episodes of spontaneous abortions, stillbirths, and infertility in dairy cattle herds from three geographical regions of Brazil.

Table 1 Herd no.

Materials and methods Geographical locations, herds affected, and reproductive history Cattle of eight dairy herds derived from three geographical regions (Southern, n = 6; Northeastern, n = 1; and Midwest, n = 1) of Brazil with clinical history of reproductive problems were investigated. Fourteen aborted fetuses (South, n = 11; Northeast, n = 2; and Midwest, n = 1) from these regions were used during this study. The distribution of the number of abortions and associated reproductive dysfunctions per herd affected is given in Table 1. Most farms reared cattle for commercial milk production; detailed clinical data was not obtained from all establishments. Two herds (A and C) were classified as free of bovine tuberculosis and brucellosis (MAPABrasil 2006); most practiced routine immunizations against bovine herpesvirus 1 (BoHV-1), bovine viral

The biological data, geographical locations, and principal reproductive problems of cattle within the herds investigated Aborted fetuses


Geographical locations

No. of cows

Principal reproductive problems

Reproductive immunization program

Abortions (n=15) Infertility Low conception rate Repeated estrus (n=50) Stillbirths (n=8) Abortions (n=5) Low conception rate Mummified fetuses Ovarian cysts Repeated estrus Abortions (n=8) Hydrops Low conception rate Miscarriages Stillbirths Abortions (n=2)

BVD IBR Leptospirosis

ID no.

Age (days)


1 2

270 265


Buriti dos Lopes, PI Northeastern Brazil






Arapongas, PR Southern Brazil



4 5 6 7 8 9

210 240 240 210 240 45


Tamarana, PR Southern Brazil



Jaguari, MS Midwestern Brazil



10 11

120 180


Cidade Gaúcha, PR Southern Brazil


Abortions (n=34)





Londrina, PR Southern Brazil


Abortions (n=7)





Ivaiporã, PR Southern Brazil


Abortions (n=11)





Tamarana, PR Southern Brazil


Abortions (n=3)


PR Paraná, MS Mato Grosso do Sul, PI Piauí, BVD bovine viral diarrhea, IBR infectious bovine rhinotracheitis


BVD IBR Leptospirosis

BVD IBR Leptospirosis BVD IBR Leptospirosis BVD IBR Leptospirosis BVD IBR Leptospirosis BVD IBR Leptospirosis

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diarrhea virus (BVDV), and leptospirosis. All cattle herds in Brazil are routinely immunized against brucellosis (MAPA-Brasil 2006). Moreover, herd A is a closed establishment, i.e., additional cattle were not introduced into this herd during the last 4 years. The common reproductive manifestation of all herds was the varying degrees of repeated abortions, despite routine immunization against BoHV-1, BVDV, and leptospirosis; some of the most frequent abortifacient agents of cattle in Brazil (Alfieri et al. 1998). The number of reported abortions varied from 2.1 % (2/95) in herd D to 27.2 % (34/125) at herd E. Additionally, abortions at these farms were not restricted to any definite gestational period: being early at herds D and H, mid-gestational (herds E, F, and G), and during the last phase (herds A, B, and C). Cattle at all farms are maintained predominantly on green pastures and supplemented with commercial mineral salt, hay, and corn silage during the dry seasons; water is administered at ad libitum from artesian wells. The consulting veterinarian at herd A related that there has been a drastic reduction in milk production (from an average of 3,500 to 2,500 l/day), reduced conception rate (from 65 to 25 %), irregular estrous cycles with repeated estrous (n=50), unresolved late-term abortions (n=15), and stillbirths (n=8). Additionally, most recently weaned calves have demonstrated reduced daily weight gain. In early September 2012, a cow from this farm with clinical history of repeated abortions gave birth to an aborted fetus (fetus no. 1). Two days after calving, this cow demonstrated hind limb paralysis, lateral decumbency, terminating in sudden death 1 day after the initial manifestations of disease. An onsite necropsy done by the consulting veterinarian revealed hemorrhages of the vaginal mucosa, kidneys, and liver; edema of the uterine mucosa that contained an extremely fetid secretion, hemopericardium, and epicardial hemorrhages were also observed. Tissue sections from this cow and her fetus as well as from another aborted fetus (fetus no. 2) were submitted for molecular diagnostic investigations. Additionally, serum samples (n= 42) of cows with repeated episodes of abortion, stillbirths, and irregular estrous cycles were received for antibody detection against BVDV and BoHV-1 by virus neutralization (VN), Leptospira spp. by microscopic agglutination test (MAT), and Brucella abortus by the tamponated acidified antigen (TAA) test. Herd C is an elite cattle holding that had been faced with unresolved reproductive problems including mummified fetuses, abortions, stillbirths, and the birth of weak calves during the last 2 years. During this period, five aborted fetuses were submitted for routine necropsy. Additionally, paired serum samples from 12 randomly selected cows that were used as embryo receptors were collected for VN against BVDV and BoHV-1, as well as MAT for Leptospira spp. and TAA for B. abortus. Moreover, these cows had history of repeated

abortions (n=7) or were pregnant (n=5); VN for BVDV and BoHV-1 was also done from the serum of two neonates that were alive at the time of collection. Pathological evaluations Six fetuses (nos. 3–8) from herds B (n=1) and C (n=5) were received in adequate conditions for routine necropsy and histopathological evaluations; six were received frozen and/or severely autolyzed. Additionally, tissue sections from the cow, her aborted fetus, and that of another fetus (herd A) were received for histopathological analysis. Selected tissue samples of the necropsied fetuses and of the samples received (cow, fetus nos. 1 and 2) were fixed by immersion in 10 % buffered formalin solution and routinely processed for histopathological evaluation. Duplicate sections of selected organs were maintained at −80 °C until processed for molecular diagnostics. Molecular investigation of infectious agents of bovine reproductive disease Nucleic acids (DNA and/or RNA) were extracted from the submitted tissue fragments of the cow and all aborted fetuses as described (Boom et al. 1990), in association with Proteinase K (Ambion, Grand Island, NY, USA), and a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate method (Alfieri et al. 2006). Due to the size of fetus nos. 9 and 14 and the advanced stage of postmortem autolysis, all major organs were pooled for molecular analyses. The extracted DNA/RNA was used in PCR/RT-PCR assays designed to amplify specific amplicons of infectious reproductive agents of cattle. These protocols targeted the glycoprotein C gene of BoHV-1 (Claus et al. 2005), the 5′UTR region of BVDV (Vilček et al. 1994), the listeriolysin gene of Listeria monocytogenes (Wesley et al. 2002), the 16s rRNA gene of H. somni (Angen et al. 1998), the Nc5 region of Neospora caninum (Marques et al. 2011), the 16S rRNA gene of Leptospira spp. (Merien et al. 1992), and the 31-kDa Brucella cell surface salt extractable protein (BCSP) of Brucella abortus (Baily et al. 1992). PCR for N. caninum was only done on clinical samples of the cerebrum and myocardium (Marques et al. 2011). Moreover, DNA was extracted from whole blood samples (PureLink Genomic DNA Kit, Life technologies, Grand Island, NY) of 42 cows from herd A and 12 cows from herd C and then used in PCR/RT-PCR assays as previously described. Positive controls consisted of extracted nucleic acid from Madin Darby bovine kidney cell culture adapted NADL and Los Angeles strains of BVDV and BoHV-1, respectively; and field strains of previous cases of L. monocytogenes (Headley et al. 2014b), H. somni

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(Headley et al. 2013b), and N. caninum (Marques et al. 2011); and housekeeping antigens of Leptospira spp. and B. abortus. Nuclease-free water (Invitrogen Corp., Carlsbad, CA, USA) was used as negative controls in all PCR assays. All 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 (illustra GFX PCR DNA and Gel Band Purification Kit, GE Healthcare, Little Chalfont, Buckinghamshire, UK) and submitted for direct sequencing using the forward and reverse primers. The obtained sequences were examined for quality analysis of chromatogram readings by using the PHRED software (http://asparagin.cenargen.; sequences were only accepted if base quality was equal to or greater than 20. Consensus sequences were then generated by the CAP3 program ( pl), after which the partial nucleotide sequences obtained were compared by the BLAST program (http://www.ncbi. with similar sequences deposited in GenBank to confirm sequence identity and validate the isolates identified. The partial nucleotide sequences of H. somni obtained during this investigation were deposited at GenBank. Serological determinations VN against BoHV-1 and BVDV was done from sera obtained from 42 cows of herd A and 12 cows and two neonates from herd C. VN for BoHV-1 (Los Angeles strain) was performed by using serial twofold dilutions of sera made in MEM (Médici et al. 2000); sera were tested for virus neutralizing antibodies through 1:1,024 dilutions. VN for BVDV-1 (NADL strain) was done (Dias et al. 2010), using twofold dilutions starting at 1:5 through 1:160. Sera were considered positive when titers that were ≥2 for BoHV-1 and ≥10 for BVDV were observed. Additionally, the TAA test was used to detect the presence of antibodies against B. abortus (Kit Brucellosis TECPAR, Instituto de Tecnologia do Paraná, Curitiba, Brazil), while the MAT assay (OIE 2013) was used to determine the presence of antibodies against 21 serovars of leptospira from all serum samples from herds A and C.

Results Gross and histopathological findings The principal gross and histopathological findings of the fetuses are summarized in Table 2. Only six fetuses

were in adequate conditions for necropsy and consequent histological evaluations; one of these (fetus no. 4) did not demonstrate significant histopathological alterations. Five fetuses received in advanced stages of postmortem autolysis were not processed for routine histopathology. Significant necropsy alterations included marked hyperemia of intestinal mucosa of four fetuses (Fig. 1a) and congestion of the meningeal vessels of three. Additionally, there were renal cortical infarctions (Fig. 1b), an extremely pale and enlarged liver, and fibrinous serositis (Fig. 1c) in two fetuses. Petechial hemorrhages of the mesentery (Fig. 1d) were observed in one fetus. Significant histopathological findings were observed in fetus nos. 3, 4, 6, 7, and 8 as well as the organs received from the cow, her aborted fetus (fetus no. 1), and fetus no. 2. Four fetuses (nos. 3, 4, 6, and 8) necropsied demonstrated vasculitis and fibrinous thrombosis of the meningeal vessels and the white and gray matter of the cerebrum (Fig. 2a–b); moreover, severe vascular congestion and hemorrhage were observed in the brainstem and cerebellum of fetus nos. 3 and 8 (Fig. 2c–d). Necrotizing myocarditis with vasculitis was observed in three fetuses (nos. 3, 4, 6, and 8) and within the myocardium of the cow, her fetus (fetus no. 1), and fetus no. 2 from herd A. Renal cortical infarctions and hemorrhage were observed in fetus nos. 6 and 7. Hemorrhagic enteritis without thrombosis was observed in three fetuses. Additionally, thrombosis of the hepatic artery at the portal trait of the liver and splenic arteries was also observed.

Molecular characterization of infectious agents of bovine reproductive disease The results of the molecular investigation are summarized in Table 3. H. somni DNA was amplified by the PCR assays from different organ samples of the cow, her aborted fetus (fetus no. 1), and all other fetuses. H. somni DNA was more predominant within the cerebrum (n=9), lungs (n=7), myocardium (n=6), and kidneys (n=6) of all aborted fetuses. Moreover, the intestinal segments of the cow and of fetus no. 2 (non-diarrheic) and no. 3 (hemorrhagic enteritis) contained H. somni DNA. Interestingly, H. somni DNA was amplified from the uterus and placenta of the cow as well as from the kidney, liver, and pancreas of her aborted fetus (fetus no. 1). The myocardium of the cow as well as organs from fetus nos. 3, 12, and 13 contained the BCSP fragment of B. abortus. BVDV was detected within the organs of fetus nos. 5 and 8; BoHV-1 was identified within the hemorrhagic kidney of fetus no. 8. The blood samples of all cows from herds A and C were negative by the PCR/RT-PCR assays

Trop Anim Health Prod Table 2 Principal gross and histopathological findings observed in aborted bovine fetuses


Fetus no. (age days)

Gross alterations



3 (270)

Congestion of meningeal vessels Hyperemia of intestinal mucosa

Cerebrum: vasculitis, hemorrhage, and thrombosis

Myocardial hemorrhage

Hemorrhagic enteritis Necrotizing myocarditis with vasculitis and thrombosis


4 (210)

Ascites Hydrothorax Renal hemorrhage Enlarged lymph nodes

5 (240)

Myocardial hemorrhage Enlarged lymph nodes Fibrinous serositis

6 (240)

7 (210)

8 (240)

Pleural adhesions Ascites

Spleen: vasculitis and thrombosis Cerebrum: vasculitis, hemorrhage and thrombosis Necrotizing myocarditis with vasculitis Significant histopathological alterations were not observed

Congestion of meningeal vessels

Cerebrum, thrombosis of meningeal vessels

Fibrinous serositis

Hemorrhage enteritis

Hyperemia of intestinal mucosa

Necrotizing myocarditis and vasculitis

Pulmonary hemorrhage with cranioventral consolidation

Renal cortical infarctions and hemorrhage

Severely distended gallbladder Cortical renal infarctions Enlarged lymph nodes

Lymph nodes, edema and lymphoid depletion

Hyperemia of intestinal mucosa

Myocarditis and vasculitis

pale Liver

Renal cortical infarctions and hemorrhage

Mesentery hemorrhage Bloody intestines Congestion of meningeal vessels

Cerebrum: vasculitis, hemorrhage and thrombosis

Enlarged lymph nodes

Cortical renal hemorrhages

Liver pale and hemorrhagic

Hemorrhagic enteritis

Myocardial hemorrhages

Liver, central lobular thrombosis

Renal hemorrhage

Myocarditis and vasculitis Pulmonary hemorrhage

that targeted specific genes of BoHV-1, BVDV, and H. somni. Concomitant abortifacient agents were observed in 53.8 % (7/13) of the fetuses evaluated. However, fetus nos. 8 and 13 were more severely infected; these demonstrated the presence of three pathogens (fetus no. 8, H. somni, BVDV, and BoHV1; fetus no. 13, H. somni, B. abortus, and N. caninum) simultaneously. Dual infections in fetuses were due to simultaneous H. somni with B. abortus (n=2), N. caninum (n=2), and BVDV (n=1).

pool), no. 10 (cerebrum), no. 11 (myocardium), no. 12 (spleen), no. 13 (cerebrum), and the cow (intestine and placenta). The sequences derived from this study have been deposited in GenBank (Accession nos. KF770461, KF770462, KF770463, KF770464, KF770465, KF770466, KF770467, KF770468, KF770469, KF770470, KF770471, KF770472, KF770473, KF770474, and KF770475). BLAST analyses revealed that the isolates from this study demonstrated 99–100 % identity with similar sequences of H. somni deposited in GenBank.

Sequencing of the 16S rRNA gene of H. somni

Serological determinations

The partial sequences of the 16S rRNA gene of H. somni were obtained from fetus no. 1 (pancreas), no. 2 (liver), no. 3 (liver), nos. 4 to 6 (cerebrum), no. 7 (lung), no. 8 (kidney), no. 9 (fetal

All serum samples of the 42 cows from herd A demonstrated titers that were ≤64 for BoHV-1 and ≤80 for BVDV. However, serum samples of 80 % (4/5) of the

Trop Anim Health Prod Fig. 1 Gross demonstrations of lesions observed in spontaneously aborted bovine fetuses. Observe the diffuse hyperemia of the intestinal mucosa (a), hemorrhage of meningeal vessels (b), fibrinous exudate forming a mat over the serous surfaces of organs within the abdominal cavity (c), and petechial mesenteric hemorrhages (d)

pregnant cows from herd C demonstrated titers that were ≥256 with one having titer of 64 for BoHV-1; most cows (85.71 %; 6/7) with history of abortions had titers ≥128 for BoHV-1. SN titers ≥80 for BVDV were demonstrated in 20 % (2/5) of the pregnant cows and in a cow with a history of abortion. Both neonates from herd C were positive for BVDV and BoHV-1 by VN; one of these was born from a BVDV- and BoHV1-positive cow. Additionally, all serum samples from herds A and C were negative for B. abortus by the TAA assay, and Leptospira spp. by MAT. Fig. 2 Histopathological demonstration of alterations observed in aborted bovine fetuses. There is the formation of hyaline thrombosis within cerebral vessels at the meninges (a) and the cerebrum (b), with congestion and hemorrhage of the brainstem (c) and cerebrum (d). Hematoxylin and eosin stain; bar, a, b, and d = 20 μm; c, 200 μm

Discussion The thrombotic meningoencephalitis (TME) observed in most fetuses and the histopathological findings identified within several organs of the cow and the five aborted fetuses necropsied have been described in histophilosis (Schlafer and Miller 2007; George 2009; Pérez et al. 2010) and are similar to those observed in a recent report of H. somni-associated diseases in cattle from southern Brazil (Headley et al. 2013b). Although B. abortus produces vasculitis in affected aborted fetal tissues (Neta

+ + + + + + − NC NC NC

+ + +b + − + + + NC NC

− + + NC − − − NC NC NC

− − − − + + − 0 + +






+ − − + − NC − NC NC NC

Fetus 4

Herd C

The pool of tissues of the 45 and 60 days old fetus

Organs that were PCR/RT-PCR positive for bovine viral diarrhea virus

Organs that were PCR/RT-PCR positive for bovine herpesvirus 1

Organs that were PCR/RT-PCR positive for B. abortus

Organs that were PCR/RT-PCR positive for N. caninum

− negative, + positive, NC not collected, 0 not present/located

Cerebrum Kidney Liver Lung Myocardium Small intestine Spleen Thymus Uterus Placenta Poolse

Fetus 3

Fetus 1


Fetus 2

Herd B

Herd A

The distribution of H. somni DNA in organs of cattle from Brazil

Organs evaluated

Table 3

+ + + −d +d NC − NC NC NC

Fetus 5 + − − + − NC NC − NC NC

Fetus 6 − − + + + − + NC NC NC

Fetus 7 − +c − − −d − − NC NC NC

Fetus 8


Fetus 9

Herd D

+ + + + +a NC − NC NC NC

Fetus 10

Herd E

+a NC NC + +a NC NC NC NC NC

Fetus 11

+b − + −b −b NC + NC NC NC

Fetus 12

Herd F

+a, b −b −b −b − NC − −b NC NC

Fetus 13

Herd G


Fetus 14

Herd H

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et al. 2010), only fetus no. 3 that was examined by histopathology contained B. abortus and H. somni DNA. Nevertheless, confirmation of the possible participation of H. somni as an abortifacient agent was obtained by the successful amplification and sequencing of the partial fragment of 16S rRNA gene of this organism from multiple organs of all fetuses evaluated. H. somni was previously isolated from aborted fetuses (van Dreumel and Kierstead 1975; Corbeil et al. 1986; Headley et al. 2013b), cows with vaginitis and cervicitis (Last et al. 2001), and in experimentally reproduced histophilosis (Miller and Barnum 1983). Moreover, the direct association of this pathogen as an abortifacient agent was demonstrated by the identification of typical lesions within the myocardium of the cow and the amplification and sequencing of H. somni DNA within her uterus and placenta. Additionally, H. somni DNA was identified within the kidney, pancreas, and liver of her aborted fetus (fetus no. 1) with histopathological evidence of histophilosis. Bacterial dissemination in H. somni-associated abortions probably occurs due to bacteremia between the pregnant uterus and the fetus (Kirkbride 1992; Inzana and Corbeil 2004; Anderson 2007) after vaginal or respiratory histophilosis (Schlafer and Miller 2007); this would explain the concomitant presence of H. somni within multiple organs of the dam and her aborted fetus, as well as several organs of the other fetuses. Further, dissemination in histophilosis is related to virulence factors of the pathogen associated with the compromised defense systems of the host due to stress-induced conditions and/or intense management systems (Inzana and Corbeil 2004). We believe that in these cases, the fetuses were probably contaminated by the infected dams who transferred the bacterium hematogenously, via placenta, resulting in disseminated fetal disease. Nevertheless, since more than 50 % of the fetuses investigated were concomitantly infected by other abortifacient agents, fetal death in all cases cannot be attributed exclusively to H. somni. The cases of H. somni-associated abortions described herein, coupled with a previous description by our group (Headley et al. 2013b), might represent the largest collection of recently documented cases of spontaneous abortive bovine histophilosis within a country. Previous epidemiological/ retrospective studies investigating the etiology of bovine abortions in North America have identified H. somni as a causative agent in relatively few cases (Kirkbride 1993; Jamaluddin et al. 1996; Khodakaram-Tafti and Ikede 2005). Alternatively, an Argentina study (Campero et al. 2003) and a recent retrospective investigation of bovine abortions in southern Brazil (Antoniassi et al. 2013) did not include H. somni as an abortifacient agent. These results would suggest that H. somni is an infrequent, opportunist, and sporadic agent of abortions in cattle (Kirkbride 1993; Anderson 2007), while its

participation as a reproductive pathogen is controversial (Kwiecien and Little 1991). However, most of these studies did not use molecular biology techniques to identify infectious agents of bovine abortions; diagnosis was based on histopathological findings associated with bacterial culture/isolation and immunohistochemistry (Kirkbride 1992; Campero et al. 2003; Khodakaram-Tafti and Ikede 2005; Antoniassi et al. 2013). Although the study done in southern Brazil (Antoniassi et al. 2013) did not include H. somni as a possible etiologic agent of bovine abortion, the cause of abortions in more than half (53.7 %; 263/490) of the fetuses evaluated and the associated etiology of the histopathological lesions observed in 40 fetuses were not determined. Therefore, since PCR assays are more sensitive and specific than these diagnostic methods, it is likely that cases of H. somni-associated abortions might have not been detected during these investigations resulting in the few or the absence of cases described. Unfortunately, bacterial isolation and/or culture were not attempted during this investigation because the fetuses received were not adequately collected for these purposes. This investigation has demonstrated H. somni DNA in bovine abortions from three geographical regions (Northeastern, Southern, and Midwestern) of Brazil. Further, we have recently diagnosed histophilosis from different regions of the state of Paraná, Southern Brazil (Headley et al. 2013b), while additional cases of H. somni-associated respiratory distress (Headley et al. 2014a) and TME (manuscript in preparation) have been confirmed. These findings suggest that histophilosis is widespread within cattle herds of the state of Paraná and possibly in Northeastern and Midwestern Brazil. This might imply that histophilosis was not adequately diagnosed/investigated in cases of bovine mortality or probably neglected as an important contributor to livestock disease and mortality in Brazil. Most of the pathological lesions observed during this study have been described in cases of histophilosis worldwide (Kwiecien and Little 1991; Inzana and Corbeil 2004; Pérez et al. 2010), as well as from Brazil (Headley et al. 2013b). However, to the best of the authors’ knowledge, the fibrinous serositis observed in fetus nos. 5 and 6 was not previously associated with H. somni; multiple tissues of both fetuses contained H. somni DNA. Although fibrinous serositis has been described in B. abortus-induced abortions in cattle (Neta et al. 2010), B. abortus DNA was not amplified from tissues of these fetuses (Table 3). Nevertheless, fibrinous pleuritis is a frequent sequel of bronchopneumonia in cases of pulmonary histophilosis (Divers 2008; Woolums et al. 2009), as was observed in fetus no. 5, and could be expected in severely infected animals, particularly in those without a competent immunological system. The relatively low titers of serum antibodies against BVDV and BoHV-1 demonstrated at herd A were interpreted as vaccine responses to these infectious

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agents. However, the severely elevated titer of VN antibodies against BVDV and BoHV-1 at herd C suggests that the cows and the calves were infected by these pathogens, but the cows were not bacteriemic since H. somni DNA was not identified by PCR from the blood samples evaluated, nor was B. abortus or Leptospira spp. detected. The concomitant identification of H. somni with B. abortus, N. caninum, BoHV-1, and BVDV within several organs of the aborted fetuses during this study is not surprising; these infectious agents are endemic and frequently associated with abortions in cattle herds throughout Brazil (Alfieri et al. 1998; Takiuchi et al. 2005). Coinfections in aborted bovine fetuses from southern Brazil were also recently described (Antoniassi et al. 2013). Alternatively, since L. monocytogenes is not a common pathogen of livestock in Brazil (Headley et al. 2013a, b, 2014b), the non-detection of listerial DNA during this study was expected. Surprising, Leptospira spp., a common agent of bovine abortion, was not identified during this investigation. The identification of N. caninum DNA in tissues of three aborted fetuses during this study must be interpreted with caution, since this protozoa is maintained within cattle herds predominantly via vertical transmission (Moore 2005; Dubey et al. 2006; Dubey and Schares 2006). Additionally, N. caninum is endemic within the state of Paraná (where positive cases were identified during this study), with a prevalence of 14–34 % in dairy cattle (Marques et al. 2011), and also in most states of Brazil (Moore 2005). Further, transmission of N. caninum between the infected dam and fetus is efficient (Dubey et al. 2006), and it is difficult to confirm N. caninum as an abortive agent, since an infection can also occur in the aborted fetus or its mother even when this protozoa was not the cause of abortion (Dubey and Schares 2006). Consequently, the identification of N. caninum DNA in aborted fetuses from this region does necessarily indicate that this protozoa participated in the cause of these abortions, particularly since three fetuses were concomitantly infected by other pathogens. Collectively, the abortions investigated during this study occurred within all phases of the bovine gestational cycle; this random pattern is a frequent manifestation of H. somni-induced abortions in cattle (Firehammer 1959; Chladek 1975; Corbeil et al. 1986; van der Burgt et al. 2007). Reviews of the phases of occurrence and the principal pathological alterations of common abortive infectious agents of cattle (Anderson 2007; Cabell 2007) suggest that most abortifacient agents have a definite pattern of occurrence during the gestational phases. Additionally, it was not possible to investigate sporadic infectious agents of bovine abortions, such as Escherichia coli, Salmonella spp., and fungi, so the possibility of other abortifacient agents associated with the abortions

herein described cannot be ignored. Nevertheless, these results demonstrate the potential economic impacts that H. somniassociated abortions might have on cattle herds in Brazil and suggest that this pathogen should be included in the list of differential diagnoses particularly in cattle herds with abortions but without any definite abortion-related pattern. Acknowledgments The authors thank Drs. Eduardo E. A. Bezerra, Shiguedy Katto, and Luiz F. C. Cunha Filho for submitting some of these cases for evaluation. Additionally, we are extremely grateful to Dr. Julio C. Freitas and Dr. Lucienne G. P. Giordano, Department of Veterinary Preventive MedicineUniversidade Estadual de Londrina, for performing the leptospirosis and brucellosis serology, respectively. Selwyn A. Headley, Alice F. Alfieri, and Amauri A. Alfieri are recipients of the National Council for Scientific and Technological Development (CNPq; Brazil) fellowships. Ethical standards This study adhered to the guidelines for the usage of animals/samples in studies as required by the Universidade Estadual de Londrina. Additionally, all clients were informed relative to the utilization of their animals/samples during this investigation, consent was obtained, and the investigation adhered to a high standard of veterinary care. Conflict of interest The authors declare that they have no competing interests.

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Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil.

This study investigated the participation of infectious agents in spontaneous abortions and reproductive problems at eight dairy cattle herds from thr...
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