Characterization of Histophilus somni sialic acid uptake mutant (ΔnanP-ΔnanU) using a mouse septicemia and mortality model.

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 108 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

Role of biofilms in antimicrobial resistance of the bacterial bovine respiratory disease complex.

An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis are able to form biofilms in vitro and there is evidence that at least H. somni retains this ability in vivo. However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.

Vaginitis with purulent vaginal discharge caused by artificial insemination using frozen Histophilus somni-contaminated semen.

In April 2020, two cows in Japan, developed reproductive disorders accompanied by vaginitis with purulent discharge within 3 days of artificial insemination (AI) with the same lot of frozen semen. Histophilus somni was isolated from the vaginal swabs of both cows as well as from the same lot of frozen semen used for the AI. This incident marks the first reported case of H. somni infection in cattle through AI. The major outer membrane protein gene sequences and pulsed-field gel electrophoresis profiles of the isolates were identical. Moreover, we investigated the antimicrobial activity of 12 frozen semen straws against an H. somni isolate using a disk diffusion test. These straws were sourced from five AI centers and included the same lot of semen used for the AI. Although the composition of semen diluents from individual AI centers is not publicly available, both the same lot of frozen semen used in the AI and other lots produced by the same manufacturer showed lower antimicrobial activity than semen from other manufacturers. These results strongly suggest that the two vaginitis were caused by AI using H. somni-contaminated frozen semen because of insufficient antimicrobial activity to inhibit bacterial growth. The minimum inhibitory concentrations of the six antimicrobials recommended for addition to frozen semen in isolates were below the recommended concentrations, suggesting that proper addition could have prevented this incident. This highlights the importance of conducting periodical checks on the antibacterial activity of frozen semen to prevent the transmission of pathogens via AI.

Complete circular assembly of Histophilus somni PDS1537697-81-1 genome and phenotypic characterization of its antibiotic susceptibility.

A Histophilus somni isolate from a clinically healthy, fall-placed calf was obtained upon arrival to a commercial feedlot. Fall-placed calves are commonly viewed to be at high risk for the development of bovine respiratory disease. The isolate was phenotyped for antimicrobial susceptibility and sequenced to obtain a complete, circular, genome assembly.

Antimicrobial Resistance Genes in Respiratory Bacteria from Weaned Dairy Heifers.

Bovine respiratory disease (BRD) is the leading cause of mortality and antimicrobial drug (AMD) use in weaned dairy heifers. Limited information is available regarding antimicrobial resistance (AMR) in respiratory bacteria in this population. This study determined AMR gene presence in 326 respiratory isolates (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) from weaned dairy heifers using whole genome sequencing. Concordance between AMR genotype and phenotype was determined. Twenty-six AMR genes for 8 broad classes of AMD were identified. The most prevalent, medically important AMD classes used in calf rearing, to which these genes predict AMR among study isolates were tetracycline (95%), aminoglycoside (94%), sulfonamide (94%), beta-lactam (77%), phenicol (50%), and macrolide (44%). The co-occurrence of AMR genes within an isolate was common; the largest cluster of gene co-occurrence encodes AMR to phenicol, macrolide, elfamycin, ß-lactam (cephalosporin, penam cephamycin), aminoglycoside, tetracycline, and sulfonamide class AMD. Concordance between genotype and phenotype varied (Matthew's Correlation Coefficient ranged from -0.57 to 1) by bacterial species, gene, and AMD tested, and was particularly poor for fluoroquinolones (no AMR genes detected) and ceftiofur (no phenotypic AMR classified while AMR genes present). These findings suggest a high genetic potential for AMR in weaned dairy heifers; preventing BRD and decreasing AMD reliance may be important in this population.

Histophilus somni as a Unique Causative Agent of Puerperal Metritis (PM) in a Third-Lactation Holstein Cow.

This manuscript aims to report the clinical and laboratory diagnosis of puerperal metritis (PM) in a dairy cow, caused by H. somni as a unique pathogen. The cow showed signs of systemic illness, including a sudden drop in milk production, a rectal temperature of 40.4 °C, tachypnea, dehydration, and completely fluid, brown, and fetid uterine discharge. Pure cultures of H. somni were identified and submitted to the Kirby-Bauer disc diffusion method for antibiotic sensitivity. The study showed that H. somni was resistant to tetracyclines and cephalosporins (Ceftiofur), antibiotics commonly used to treat uterine infections in dairy cows. To the authors' knowledge, this case describes for the first time PM caused by H. somni as a primary pathogen. Our results should lead to the inclusion of H. somni as a primary pathogen of metritis in laboratory diagnoses on a routine basis, which, in turn, may help to elucidate the incidence of H. somni as a causative agent of uterine infections in cows. If the incidence of H. somni is remarkably high or frequent, researchers could consider the use of commercial vaccines nowadays destined for the prevention of bovine respiratory disease and which could perhaps be effective in the prevention of reproductive pathology caused by H. somni.

Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

Antimicrobial Susceptibility in Respiratory Pathogens and Farm and Animal Variables in Weaned California Dairy Heifers: Logistic Regression and Bayesian Network Analyses.

Weaned dairy heifers are a relatively understudied production group. Bovine respiratory disease (BRD) is the most common cause of antimicrobial drug (AMD) use, morbidity, and mortality in this production group. The study of antimicrobial resistance (AMR) is complicated because many variables that may affect AMR are related. This study generates hypotheses regarding the farm- and animal-level variables (e.g., vaccination, lane cleaning, and AMD use practices) that may be associated with AMR in respiratory isolates from weaned dairy heifers. A cross-sectional study was performed using survey data and respiratory isolates (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) collected from 341 weaned dairy heifers on six farms in California. Logistic regression and Bayesian network analyses were used to evaluate the associations between farm- and animal-level variables with minimum inhibitory concentration (MIC) classification of respiratory isolates against 11 AMDs. Farm-level variables associated with MIC classification of respiratory isolates included the number of source farms of a calf-rearing facility, whether the farm practiced onsite milking, the use of lagoon water for flush lane cleaning, and respiratory and pinkeye vaccination practices. Animal-level variables associated with a MIC classification included whether the calf was BRD-score-positive and time since the last phenicol treatment.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil / Meningoencefalite trombótica-induzida por Histophilus somni em bovinos da região norte do Paraná

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somni with sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.(AU)

Meningoencefalite trombótica (Thrombotic meningoencephalitis- TME) é uma doença neurológica fatal de bovinos ocasionada por Histophilus somni. A infecção tem sido descrita predominantemente na América do Norte e de forma esporádica em outros países. O objetivo deste estudo é relatar a ocorrência de TME em bovinos da região norte do estado do Paraná, Brasil. A maioria dos animais apresentaram sinais clínicos neurológicos característicos de disfunção cerebral aguda. Análises hematológicas e do fluido cerebrospinal não foram sugestivas de infecção bacteriana do cérebro. A histopatologia revelou meningoencefalite com vasculite e trombose de pequenos vasos com discreto infiltrado neutrofílico e/ou linfocítico mesclada com fibrina no tronco e córtex cerebral e no gânglio do nervo trigêmio de todos os animais. As amostras de sistema nervoso central incluídas nesse estudo foram previamente caracterizadas como negativas para raiva por meio de técnica de imunofluorescência direta. A participação de agentes infecciosos associados à doença neurológica em bovinos foi avaliada por técnicas moleculares como PCR e RT-PCR para amplificação parcial de genes de H. somni, Listeria monocytogenes, herpesvírus bovino 1 e 5, vírus da diarreia viral bovina e herpesvírus ovino 2. As seções do cérebro de todos os animais com diagnóstico histopatológico de TME foram positivas em PCR para a detecção do gene 16S rRNA de H. somni. O sequenciamento dos produtos amplificados confirmou a presença de DNA de H. somni nos fragmentos de cérebro avaliados. As reações de PCR/RT-PCR para todos os outros micro-organismos avaliados resultaram negativas. Os resultados desse estudo confirmaram a participação do H. somni nos episódios de doença neurológica observada nos animais avaliados, amplia a distribuição geográfica da TME e ratifica estudos prévios realizados no Brasil que demonstraram a presença de H. somni em outras formas de manifestação clínica das infecções por essa bactéria.(AU)

Prevalencia de anticuerpos contra Histophilus somni y factores de riesgo en ganado para carne en Yucatán, México / Prevalence of antibodies against Histophilus somni and risk factors in beef cattle in Yucatan, Mexico

The purpose of this study was to estimate the seroprevalence of H. somni infection and to determine some risk factors associated with the seropositivity in beef cattle in the livestock region of Yucatan, Mexico. Furthermore, the intraherd correlation coefficient (r e) and the design effect (D) were estimated. The animals were selected using a two-stage random sampling. Blood samples were collected from 490 animals from 35 herds, and sera were tested for the detection of antibodies against H. somni, using the double agar gel immunodiffusion test. Information about each herd and animal sampled was recorded by a questionnaire personally applied in the farm. Data were analyzed by chi-square tests. Ten herds had zero seropositive animals, 19 had one and six had two seropositive animals. Thirty one out of 490 animals were seropositive. The animal seroprevalence adjusted to herd size was 5.5% (95% confidence interval = 3.5%, 7.5%). Animal seroprevalence in the six municipalities ranged from 3.6% to 9.5%, but no significant differences (P = 0.89) were found. The r e and D values for H. somni seroprevalence were 0 (SE = 0.01) and 1 (SE = 0.19), respectively. The chi-square test did not show association (P > 0.10) between the presence of antibodies against H. somni and the risk factors investigated.

El propósito de este estudio fue investigar la seroprevalencia de la infección por H. somni y determinar algunos factores de riesgo asociados con su seropositividad en ganado para carne en la región ganadera de Yucatán, México. Asimismo, se estimó el coeficiente de correlación dentro de hatos (re) y el efecto de diseño (D). Los animales se seleccionaron usando un muestreo aleatorio en dos etapas. Las muestras de sangre se recolectaron de 490 animales en 35 hatos, y los sueros se sometieron a análisis para detectar anticuerpos contra H. somni, mediante la prueba de inmunodifusión doble en agar. La información acerca del hato y de cada animal muestreado se obtuvo mediante una encuesta aplicada personalmente en el rancho. Los datos se analizaron mediante pruebas de Ji-cuadrada. Diez hatos tuvieron cero animales seropositivos, 19 tuvieron uno, y seis tuvieron dos animales seropositivos. Treinta y uno de los 490 animales fueron seropositivos. La prevalencia animal ajustada por el tamaño del hato fue 5.5% (intervalo de confianza al 95% = 3.5%, 7.5%). La seroprevalencia de los animales en los seis municipios varió de 3.6% a 9.5%, pero no se encontraron diferencias significativas (P = 0.89). Los valores de r e y D para la seroprevalencia de H somni fueron 0 (EE = 0.01) y 1 (EE = 0.19), respectivamente. Las pruebas de Ji-cuadrada no mostraron asociación (P > 0.10) entre la presencia de anticuerpos contra H. somni y los factores de riesgo investigados.