VN February 2024

VET Februarie / February 2024 The Monthly Magazine of the SOUTH AFRICAN VETERINARY ASSOCIATION Die Maandblad van die SUID-AFRIKAANSE VETERINÊRE VERENIGING Brief Update on Myxomatous Mitral Valve Disease (MMVD) CPD THEME Pasturella Multocida nuus•news

Dagboek • Diary Ongoing / Online 2023 April 2024 August 2024 SAVETCON: Webinars Info: Corné Engelbrecht, SAVETCON, 071 587 2950, / Acupuncture – Certified Mixed Species Course Info: Chi University: SAVA Johannesburg Branch CPD Events Monthly - please visit the website for more info. Venue: Johannesburg Country Club Info: Vetlink - OP Village Centenary Festival 05-06 April 2024 Venue: OP Village Residence, Onderstepoort, Old Southpan Road, Pretoria Info: Marnus Zaaiman (082 779 8435) / SAVA Oranje Vaal Branch Mini Congress 09-11 August Venue: Parys – venue to be confirmed. Info: NVCG Bush Break 12-13 August Venue: Skukuza, Kruger National Park Info:

February 2024 1 Contents I Inhoud President: Dr Paul van der Merwe Managing Director: Mr Gert Steyn +27 (0)12 346 1150 Editor VetNews: Ms Andriette van der Merwe Bookkeeper: Ms Susan Heine (0)12 346 1150 Bookkeeper's Assistant: Ms Sonja Ludik (0)12 346 1150 Secretary: Ms Elize Nicholas +27 (0)12 346 1150 Reception: Ms Hanlie Swart +27 (0)12 346 1150 Marketing & Communications: Ms Sonja van Rooyen +27 (0)12 346 1150 Membership Enquiries: Ms Debbie Breeze +27 (0)12 346 1150 Vaccination booklets: Ms Debbie Breeze +27 (0)12 346 1150 South African Veterinary Foundation: Ms Debbie Breeze +27 (0)12 346 1150 Community Veterinary Clinics: Ms Claudia Cloete +27 (0)63 110 7559 SAVETCON: Ms Corné Engelbrecht +27 (0)71 587 2950 VetNuus is ‘n vertroulike publikasie van die SAVV en mag nie sonder spesifieke geskrewe toestemming vooraf in die openbaar aangehaal word nie. Die tydskrif word aan lede verskaf met die verstandhouding dat nóg die redaksie, nóg die SAVV of sy ampsdraers enige regsaanspreeklikheid aanvaar ten opsigte van enige stelling, feit, advertensie of aanbeveling in hierdie tydskrif vervat. VetNews is a confidential publication for the members of the SAVA and may not be quoted in public or otherwise without prior specific written permission to do so. This magazine is sent to members with the understanding that neither the editorial board nor the SAVA or its office bearers accept any liability whatsoever with regard to any statement, fact, advertisement or recommendation made in this magazine. VetNews is published by the South African Veterinary Association STREET ADDRESS 47 Gemsbok Avenue, Monument Park, Pretoria, 0181, South Africa POSTAL ADDRESS P O Box 25033, Monument Park Pretoria, 0105, South Africa TELEPHONE +27 (0)12 346-1150 FAX General: +27 (0) 86 683 1839 Accounts: +27 (0) 86 509 2015 WEB CHANGE OF ADDRESS Please notify the SAVA by email: or letter: SAVA, P O Box 25033, Monument Park, Pretoria, 0105, South Africa CLASSIFIED ADVERTISEMENTS (Text to a maximum of 80 words) Sonja van Rooyen +27 (0)12 346 1150 DISPLAY ADVERTISEMENTS Sonja van Rooyen +27 (0)12 346 1150 DESIGN AND LAYOUT Sonja van Rooyen PRINTED BY Business Print: +27 (0)12 843 7638 VET nuus•news Diary / Dagboek II Dagboek • Diary Regulars / Gereeld 2 From the President 4 Editor’s notes / Redakteurs notas Articles / Artikels 8 Pasteurella multocida: from Zoonosis to Cellular Microbiology 22 My Veterinary Journey: Guided by Passion and Supported by Sponsorship from V-Tech Association / Vereniging 24 CVC News 26 SAVA News 34 In Memoriam 38 Legal Mews Events / Gebeure 7 World Veterinary Association Congress 2024 28 Paws and Celebrations: A Roaring Success at the 2023 Veterinary Graduation Ball 30 Onderstepoort residence 100 years old 49 Onderstepoort Centenary Vet's Health / Gesondheid 37 Life Coaching Technical / Tegnies 36 Royal Canin Column 40 Dental Column Relax / Ontspan 48 Life Plus 25 Marketplace / Markplein 42 Marketplace Jobs / Poste 44 Marketplace/Jobs / Poste 47 Classifieds / Snuffeladvertensies 8 24 22

Vetnuus | Februarie 2024 2 « BACK TO CONTENTS You all probably heard the joke of the two patients with similar hip discomfort and pain. The one patient was seen within an hour, x-rayed, diagnosed and booked for a hip replacement in three days, whereas the other patient could get an appointment with the doctor in three days, had to wait for two days for x-rays to be taken for a diagnosis, then referred to a specialist for further intervention and eventually booked for a hip replacement in four months. The first patient was a Labrador at a veterinary clinic, whereas the second patient was a human. At the dawn of a new year, we all set new goals, goals with the intention to better life for ourselves, our personnel and our clients. In my previous work environment, a controlled environment with a set population of dogs, horses and wildlife without the restrictions of the owner's budget, our goal was always that an animal that needed veterinary care should receive that care within an hour, a very steep expectation indeed. To bridge the gap of disappointment between expectations and reality, one of 2 things has to happen. Either the expectation has to lower or the reality has to step up. Expectation is a personal act or feeling. It is driven by the desire for satisfaction. It can be internally or externally driven. Internal will have the effect of personal drive and external will be driven by personnel or clientele or the image one wants to maintain. The question is, knowingly or unknowingly, what are the expectations created by us towards our staff and clientele? Although the above was meant as a joke, that is often the reality, a reality acting as a stressor on the deliverer. Interestingly enough, in the survey done by SAVA, client dissatisfaction was one of the major issues impacting the mental health of veterinarians. Are we not the creators of our own demise? Why is it that people are willing to accept a certain level of service in the medical field, yet they have a much higher expectation of veterinarians? Is it because we deliver on that expectation, but at what cost? High expectations often come connected to other issues, such as perfectionism, low self-esteem, negative core beliefs, fear of failure, fear of change and fear of non-delivery. All issues with the person self and issues that were even highlighted by SAVA’s survey. Balancing expectations and fear of failure has to again happen both internally and externally. By building resilience within ourselves and amongst our colleagues we can address the internal fear of failure by breaking it down into the parts that create it and getting to the core. But, we also have a responsibility to manage the expectations of the personnel, clients and maybe the organisation, institution, practice and work environment. Should you join or be part of an existing structure that you did not create according to your own wishes, you should realise that you are also buying into a culture, work ethic, standards and even somebody else’s expectations. Make sure that your values and expectations are in line with those of the place you are joining. If not, it may cause incompatibility and great disappointment. Is it time for a service charter to be developed delineating our responsibilities versus the responsibilities of our client to clearly define what a client can expect from a veterinary service provider? Michael J. Fox said: “My happiness grows in direct proportion to my acceptance and in inverse proportion to my expectations.” Expectations created by us as veterinary service providers should be realistic taking circumstances into account, and we as well as our clients must learn to accept the service that can be rendered. I trust all members will have a month filled with the fulfilment of realistic expectations. v Kind regards, Paul van der Merwe From the President Dear members, Expectation is a strong belief that something will be the case or will happen.

February 2024 3 for pet parenting peace-of-mind In today’s hyper-connected world pet parents often search online for information about their pets, but it can be di cult to validate information and while some of it may sound believable, some of it might even be dangerous. Hill’s Pet Nutrition believes in helping enrich and lengthen the special relationship between people and their pets and one of the best ways to do just that is by enhancing the understanding of our pets’ needs and wellbeing. This is why they created Pawpedia Pet Parent Academy: a trusted and free resource that helps pet parents upskill themselves to better care for their pets. Pawpedia has launched with 15 courses: 8 for dogs and 7 for cats. Visit to see what’s available. The lessons are easy to learn on the go and are set up as podcasts, videos, and printouts. Cat and dog parents can complete courses containing information about pets, their various lifestages, as well as their unique and special needs. Content is created by independent and trustworthy experts, including vets, veterinary behaviourists, , physiotherapists, bereavement counsellors, cat experts, and even legal counsel. Courses are made up of a few lessons each, and each lesson takes 5 - 15 minutes to ensure topics are covered eectively. Start at any point and follow the journey that suits you. It is completely FREE and available on desktop as well as mobile devices.

Vetnuus | Februarie 2024 4 « BACK TO CONTENTS Change and adapting to it To be confronted with change takes us out of our comfort zones. Humans are creatures of habit. It is a scientific fact that matter wants to be in a state of equilibrium or rest. It is the path of the least resistance. (Now that is deep for 06h16 in the morning coming from a not-morning person) (I hope I am close to the facts as the last science lesson I had was in 1987). Early morning research throws at me an unfamiliar word. Repose. I realise my repose was rudely interrupted by some or other 04h24 event. Maybe it was the early morning thunder that caught me a little off-guard. February is the month after the longest month of the year. The 55 weeks of January are finally over so a couple of New Year’s resolutions may already be only a vague bit of guild. How do we cope with change? Not being a philosophy major (or minor for that matter) I take to the cartoons for inspiration to solve some issues. “You can either run from it or learn from it” – Rafiki. My daughter and her husband spent last year in Cape Town for studies. The hope was that her husband would get a placement for his ophthalmology specialisation. Dr C applied all over the country and was invited to a couple of interviews. On the 28th of December, they were notified that he had to start working at Charlotte Maxeke Hospital. Fortunately, they were renting a furnished flat in Cape Town. Within 3 days they packed up everything, loaded the two cars (including a cat who does not travel well) and left for Johannesburg on the 31st. Dr C reported bright and early on the second to start his dreamed of Specialisation. My daughter had this response: “We do it now and panic in two weeks). Everything fell pretty sweet into place for them. They found a petfriendly flat, on the ground floor, with a little garden within a day. Their vehicles were serviced and ready to go when they got the news. They managed to courier a sum of 16 boxes for R1600 (Yes it is possible at Pudo) up to J’burg and the whole journey went smoothly. Some success can be attributed to preparation but the majority is favour from God. Change happens, but we are not simply matter, we are human with the distinct ability to adapt to change. Life is going to throw curveballs, that is a fact. But how you adapt is how you survive. I was confronted with a situation where I had to make a decision for my elderly mother. She point blank said she refused, I point blank said she had no choice. Sometimes you have to take hardball even with yourself. The world does not owe you anything and your inability to adapt is not going to change the way it turns. When the curveball comes, pull up the big girl panties and look it square in the face. Seeing the positive, make that change that you fear and treat it like an adventure. In this month’s magazine look out for the OP Village Centenary Celebration poster, The World Vet Association congress and a very interesting dental column. News received after the layout was complete is the passing of Dr Rudolph Bigalke, we pass our condolences to all friends and family. In the Memoriam column, there is a non-veterinarian: Ms Erica vd Westhuizen whom many of you will remember as the librarian who retired in 2009. She was the saving grace of many students, I am sure. May everybody have a great Valentine’s month, the salaries should just come in time to spoil a loved one, even if it is four-legged. May you find time to Repose and Revitalise. Andriette v From the Editor Editor’s notes / Redakteurs notas We do now and panic later

February 2024 5 EARLY EXPOSURE TO ANIMAL CARE DURING YEAR 1 TRAINING AT ANTIBIOTIC-FREE FARMS AND QUANTITATIVE GENETICS TRAINING (FARM ANIMALS) EU ACCREDITED VETERINARY DEGREE ALLOWING PRACTICE WORLDWIDE FINANCIAL AID SCHOLARSHIPS AVAILABLE GLOBAL FACULTY EXPERTISE IN SMALL AND LARGE ANIMAL MEDICINE ADVANCED FACILITIES INCLUDING ANATOMY AND CLINICAL SKILLS LABORATORIES CURRICULUM ALIGNED AS REQUIRED BY RCVS AVMA, EAEVE, AND WORLD ORGANIZATION FOR ANIMAL HEALTH. STUDY VETERINARY MEDICINE IN CYPRUS DOCTOR OF VETERINARY MEDICINE (DVM) 5-Year Undergraduate Degree Programme Targeted for High School Leavers “The South African Veterinary Association aims to serve its members and to further the status and image of the veterinarian. We are committed to upholding the highest professional and scientific standards by utilising the professional knowledge, skill and resources of our members, to foster close ties with the community and thus promote the health and welfare of animals and mankind”. MISSION STATEMENT Servicing and enhancing the veterinary community since 1920! Tel: 012 346 1150 E-mail:

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Vetnuus | Februarie 2024 8 « BACK TO CONTENTS Pasteurella multocida: from Zoonosis to Cellular Microbiology Brenda A. Wilson * and Mengfei Ho* Author information Copyright and License information PMC Disclaimer: ABSTRACT SUMMARY In a world where most emerging and reemerging infectious diseases are zoonotic in nature and our contacts with both domestic and wild animals abound, there is growing awareness of the potential for human acquisition of animal diseases. Like other Pasteurellaceae, Pasteurella species are highly prevalent among animal populations, where they are often found as part of the normal microbiota of the oral, nasopharyngeal, and upper respiratory tracts. Many Pasteurella species are opportunistic pathogens that can cause endemic disease and are associated increasingly with epizootic outbreaks. Zoonotic transmission to humans usually occurs through animal bites or contact with nasal secretions, with P. multocida being the most prevalent isolate observed in human infections. Here we review recent comparative genomics and molecular pathogenesis studies that have advanced our understanding of the multiple virulence mechanisms employed by Pasteurella species to establish acute and chronic infections. We also summarize efforts being explored to enhance our ability to rapidly and accurately identify and distinguish among clinical isolates and to control pasteurellosis by improved development of new vaccines and treatment regimens. INTRODUCTION We now live in an era where two-thirds of human infectious diseases and three-quarters of emerging or reemerging infectious diseases are zoonotic in origin, i.e., diseases caused by animal-associated pathogens that can be shared with humans (1–4). Coupled with the globalization of air travel and commerce and the megamobilization of the food and trade industries, the spread of zoonotic diseases poses a threat to global public health and biosecurity (2, 4–10). With this backdrop, there are rising concerns among health care officials, policy makers, and the general public about human acquisition of zoonotic diseases from close encounters with pets and other wild or domestic animals (5, 8, 10–16). Over 60% of U.S. households have at least one pet (17, 18). Although cats and dogs still rank highest in the U.S. pet population (17), the popularity of nontraditional or exotic pets is growing (9, 19–21). Combined with the expanding impact of changes in land usage and other anthropogenic activities affecting wildlife habitats (22) and the associated movement of and exposure to animals and animal products (11, 12, 23, 24), these trends are thought to contribute to the increased risk of transmission of known and novel zoonoses (9, 14, 22–30). Of the hundreds of bacterial species known to commonly reside in the oral, nasal, and respiratory cavities of animals (31–33), Pasteurella species are among the most prevalent commensal and opportunistic pathogens found worldwide in domestic and wild animals (34). Pasteurellosis (symptomatic infection with Pasteurella) is a high-impact disease in livestock, according to the World Animal Health Organization (OIE) ( In both animals and humans, Pasteurella species, most notably P. multocida, are often associated with chronic as well as acute infections that can lead to significant morbidity (manifested as pasteurellosis, pneumonia, atrophic rhinitis, dermonecrosis, cellulitis, abscesses, meningitis, and/or hemorrhagic septicemia [HS]) and mortality, particularly in animals (34–36). Most likely due to routine prompt prophylactic treatment of animal bite wounds with antibiotics, pasteurellosis is still a relatively uncommon cause of mortality in humans (37, 38), even though deaths due to pasteurellosis have increased in recent years in the United States (Fig. 1). Nevertheless, pasteurellosis is often associated with significant morbidity due to complications resulting from animal bite or scratch wounds or from respiratory exposure (39–46). Roughly 300,000 (1%) annual visits to the emergency rooms in the United States are due to animal bite or scratch wounds (45, 47). Pasteurella species are isolated from infections resulting from 50% of dog bites and 75% of cat bites (48–50), and indeed, it has been observed “… that seemingly trivial animal bites can result in severe complications and that P. multocida is an important cause of infection …” (49). Other contact with animals, such as kissing or licking of skin abrasions or Clin Microbiol Rev. 2013 Jul; 26(3): 631–655. doi: 10.1128/CMR.00024-13 PMCID: PMC3719492 PMID: 23824375 Leading Article (Pasteurella multocida)

February 2024 9 mucosal surfaces (eyes, nose, and mouth), can also result in infection with P. multocida (20, 38, 51–53). In nearly all reported cases of P. multocida infection, evidence of prior animal exposure or contact was indicated. In this review, we provide an overview of the prevalence and pathogenic potential of P. multocida, particularly how it relates to animal infections, human-animal interactions, transmission from animal reservoirs, and subsequent human disease. We also summarize what is currently known about the phylogenetic relationships of P. multocida with other members of the Pasteurellaceae and the pathogenomics, cellular microbiology, and molecular virulence mechanisms that enable P. multocida to cause both acute and chronic disease in animals and humans. Finally, we summarize current antibiotic treatment modalities and efforts toward increasing our options for prevention and control of transmission through animal vaccine development. PASTEURELLA AND THE PASTEURELLACEAE FAMILY Comparative Genomics of the Pasteurellaceae The genus Pasteurella is a member of the Pasteurellaceae family, which includes a large and diverse group of Gram-negative Gammaproteobacteria, whose members are not only human or animal commensals and/or opportunistic pathogens but also outright pathogens (34, 54, 55). Ancestral relationships among bacterial taxa within the Pasteurellaceae family can be inferred by comparing their 16S rRNA genes (Fig. 2). Comparative genomic and phylogenetic analyses of the Pasteurellaceae have revealed that many members of this highly diverse family were poorly classified (54, 56). Indeed, a number of the Pasteurellaceae have already been renamed: Histophilus somni (formerly Haemophilus somnus, H. agni, and H. ovis) (57), Mannheimia (formerly Pasteurella) haemolytica (58), Bibersteinia (formerly Pasteurella) trehalosi (59), Actinobacillus (formerly Haemophilus) pleuropneumoniae (60), Actinobacillus (formerly Pasteurella) ureae (61), Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans (62), Aggregatibacter aphrophilus (formerly Haemophilus aphrophilus and H. paraphrophilus) (62), Aggregatibacter (formerly Haemophilus) segnis (62), Avibacterium (formerly Haemophilus) paragallinarum (63), Avibacterium (formerly Pasteurella) gallinarum (63), Avibacterium (formerly Pasteurella) volantium (63), Avibacterium (formerly Pasteurella) avium (63), Basfia (formerly Mannheimia) succiniciproducens (64), and Gallibacterium (formerly Pasteurella) anatis (65). However, as can been seen from the 16S rRNA phylogenetic tree shown in Fig. 2, further reclassification or renaming may be warranted. Based on conserved signature sequence insertions and deletions (indels) that are specific for certain subgroups of Pasteurellaceae species, it has been proposed that the Pasteurellaceae family be divided into at least two clades (66). Two other independent studies produced similar but not identical 2-clade clustering of the Pasteurellaceae by using 12 intracellular proteins (67) or 50 conserved proteins (68). However, this attempt to classify the Pasteurellaceae into two clades reflects only the phylogenetic relationships of the genes examined resulting from more recent events such as horizontal gene transfer. Such clustering is not congruent with the phylogenetic tree derived from 16S rRNA gene comparison (as shown in Fig. 2), and it is also not reflective of known host specificities or disease manifestations. The first Pasteurellaceae member to be genome sequenced was Haemophilus influenzae strain Rd KW20 (69). Since then, the complete or nearly complete genomes of over 28 members of the Pasteurellaceae family have been sequenced, including at least six complete genomes from the species Pasteurella multocida. Phylogenetic analysis of 16S rRNA genes alone shows that these six P. multocida strains tightly cluster and are distant from H. influenzae (Fig. 3A). Genome-wide comparison based on the fractions of common genes (Fig. 3B) or based on similarity among the common genes (Fig. 3C) revealed subtle differences in relatedness among these six P. multocida strains. This likely reflects the dynamics of frequent gene transfer events among the pool of P. multocida strains. While phylogenetic analysis can readily distinguish P. multocida strains from other Pasteurellaceae, ancestral relationships among P. multocida strains are more difficult to define. Consequently, comprehensive genome-wide comparisons (i.e., pathogenomics) are necessary to account for the extent of diversity observed for pathogenic phenotypes among the P. multocida isolates (Table 1). Leading Article (Pasteurella multocida) >>> 10 Figure 1. Pasteurellosis deaths in the United States, 1993 to 2006. Data are based on CDC general mortality tables ( Email: ADVERTISE IN VETNEWS MAGAZINE

Vetnuus | Februarie 2024 10 « BACK TO CONTENTS Figure 2. Phylogenetic relationships of Pasteurella multocida and related Pasteurellaceae bacteria based on 16S rRNA genes. The maximum-likelihood phylogenetic tree was calculated by using MEGA5 (575), based on full-length 16S rRNA gene sequences. Nodes with bootstrap values of greater than 30% after 1,000 replicates are indicated. Leading Article (Pasteurella multocida)

February 2024 11 Table 1 Genome features and phenotypes of sequenced P. multocida strains Strain Source Typing GenBank accession no., size (Mbp) No. of: Genes Proteins P. multocida subsp. multocida Pm70 Oviduct of chicken with fowl cholera Capsular serotype F:3, nontoxinogenica AE004439.1, 2.26 2,089 2,012 P. multocida subsp. gallicida X73 Fowl cholera Capsular serotype A:1, nontoxinogenica CM001580.1, 2.27 2,128 2,069 P. multocida subsp. gallicida P1059 Turkey liver Capsular serotype A:3, nontoxinogenica CM001581.1, 2.31 2,168 2,111 P. multocida 36950 Bovine respiratory infection Capsular serotype A,b nontoxinogenic,a ICEPmu1c CM001581.1, 2.31 2,168 2,111 P. multocida 3480 Lung of swine with pneumonia Capsular serotype A,b nontoxinogenica CP001409.1, 2.38 2,296 2,223 P. multocida subsp. multocida HN06 Diseased swine Capsular serotype D, toxinogenica CP003313.1, 2.41 (pHN06, 5,360 bp) 2,361 2,265 aBased on presence or absence of the toxA gene, which encodes PMT. bBased on sequence homology to strains X73 and P1059 within the cap locus, which carries genes for capsule biosynthesis (Fig. 4). cICEPmu1, integrative conjugative element of P. multocida (79, 80). Pathogenomics of P. multocida A number of genes or gene clusters, identified through signature-tagged transposon mutagenesis (70, 71), in vivo expression technology (72), and whole-genome expression profiling (73–75), have been implicated as important for virulence of P. multocida (76). Some of these genes encoding putative virulence factors are universally present in all six P. multocida genomes, and these include genes encoding outer Figure 3. Phylogenetic comparison among selected Pasteurella multocida and Haemophilus influenzae species with completed genome sequences. (A) Phylogenetic relationships among the strains based on 16S rRNA genes. The maximum-likelihood tree was calculated by using MEGA5 (575), based on the 16S rRNA genes from each of the indicated strains of P. multocida (Pm) or H. influenzae (Hi) with complete genome sequences. Nodes with bootstrap values of greater than 30% after 1,000 replicates are indicated. (B) Genome-wide comparison based on the fractions of common genes among the strains. The neighbor-joining tree was calculated by using MEGA5 with distances derived from the fraction of genes that are common between each pair of genomes and have >90% coverage in BLASTN alignment. (C) Genome-wide comparison based on the similarity among the common genes among the strains. The neighbor-joining tree was calculated by using MEGA5 with distances derived from the average BLASTN identity for common genes with >90% coverage in alignment. >>> 12 Leading Article (Pasteurella multocida)

Vetnuus | Februarie 2024 12 « BACK TO CONTENTS membrane proteins (ompA, ompH, and ompW), iron acquisition genes (exbB-exbD-tonB, hgbA, and fur), thiamine metabolism genes (tbpA, thiP, and thiQ), and the adhesion/Flp pilus assembly gene cluster (tadZABCDEFG). Homologs of the tad gene locus are also present in many other Pasteurellaceae and Gram-negative bacteria, where they play key roles in biofilm formation, colonization, and pathogenesis (77). Potential virulence genes in P. multocida can also be inferred from a list of virulence genes found in the phylogenetically related H. influenzae (78).Unique genes correlated with virulence are present in almost each of the sequenced P. multocida genomes. For instance, P. multocida strain 36950, isolated from bovine lung, contains the large integrative conjugative element (ICE) ICEPmu1 of 82 kbp that carries 88 genes, including 12 antimicrobial resistance genes (79, 80). This ICE is not found in any of the other five sequenced genomes; however, a similar ICE was found in Histophilus somni 2336 and Mannheimia haemolytica PHL213, both of which are bovine respiratory pathogens and thus share the same host niche as P. multocida strain 36950. Strain 36950 also has a DNA segment of 9.5 kbp that contains several genes involved in xylose metabolism (xylA, xylF, xylG, xylH, and xylR). Homologs of this region are present in P. multocida strains P1059, P52VAC, HN06, and 3840 but are absent in strains Pm70 and X73.P. multocida strain Pm70 contains a unique 13.9-kbp region, carrying genes PM1935 to PM1949, that is homologous to a gene cluster found in members of other Pasteurellaceae genera, including H. influenzae R2846 (13 kbp), H. somni 2336 (5 kbp), and Gallibacterium anatis UMN179 (5 kbp). However, strain 36950 is the only other strain of P. multocida that contains a partial sequence (2.1 kbp) homologous to this gene cluster. The genome of the toxinogenic P. multocida strain HN06 has a unique 18-kbp region carrying 14 genes (PMCN06_2106 to PMCN06_2119), including the toxA gene for P. multocida toxin (PMT) (the toxin responsible for atrophic rhinitis) and several phagerelated genes. A 6.7-kbp segment of this sequence lacking the toxA gene is present in the genome of the nontoxinogenic strain 3480. Additionally, there are two regions, a 4.8-kbp region carrying 53 genes (NT08PM_0048 to NT08PM_0100) and a 16-kbp region carrying 22 genes (NT08PM_0622 to NT08PM_0643), in strain 3480 that are also found in strain HN06, albeit fragmented and displaced in multiple loci around the chromosome, further supporting the close relatedness of these two strains. However, there is a 37-kbp fragment (NT08PM_1283 to NT08PM_1334) that is so far unique to strain 3480 and another 33-kbp fragment (PMCN06_1378 to PMCN06_1438) that is so far unique to strain HN06, for which no homologous sequences are found in any of the other strains. It is noteworthy that multiple phage-related genes are present in all of these strain-specific unique sequences, including the segment harboring the toxA gene. Detection, Identification, and Typing of P. multocida Selective culturing and phenotyping of P. multocida. Until very recently, conventional methods for detection and diagnosis of infection with Pasteurella (pasteurellosis) relied on observation of the bacterium by microscopy using staining and/or isolation by in vitro culturing on selective media, followed by phenotypic and/or serological characterization (54). P. multocida is a small, pleomorphic, Gram-negative, nonflagellated coccobacillus. Microscopic analysis of fresh cultures or clinical specimens using Leishman's stain, methylene blue, or Giemsa stain shows bipolarstaining rods. P. multocida isolates are aerobic or facultative anaerobic and grow well at 37°C on 5% sheep's blood (the preferred culture medium) in dextrose-starch, casein-sucrose-yeast (CSY), chocolate, Mueller-Hinton, or brain heart infusion (BHI) agar (81, 82); however, there is no growth on MacConkey agar. Most clinical isolates are catalase, oxidase, indole, and ornithine decarboxylase positive. Most isolates also ferment sucrose, glucose, and maltose. Media containing vancomycin, clindamycin, gentamicin, neomycin, kanamycin, and/or amikacin, either singly or in combination, have been used to select for Pasteurella (83–86), but the results are not always consistent. Although P. multocida grows well on blood agar and chocolate agar, it is easily overgrown by other microbiota in sputum and might be easily misidentified, as it resembles other Gramnegative bacteria such as Francisella tularensis, Yersinia species, and other Pasteurellaceae species (87–89), such as Haemophilus influenzae (90) and on first examination even Neisseria species (91). Phenotypic characterization of P. multocida, based on morphology, carbohydrate fermentation patterns, and serology, is also challenging (92, 93). Identification of P. multocida using biochemical strips (such as API 20E/20NE, Minitek, or Oxi/Ferm strips) remains a rapid method commonly used in diagnostic laboratories, but it has limited accuracy (94, 95) and can lead to confusion of P. multocida with Mannheimia (Pasteurella) haemolytica (95), H. influenzae, or other Pasteurellaceae species (90, 96, 97). For example, two reports of identification of P. gallinarum as a possible cause of disease in humans were later suspected as possible misidentification as Haemophilus aphrophilus due to similarities in phenotype and/ or biochemical properties (98). The authors concluded that the API 20NE system does not differentiate among P. gallinarum, H. aphrophilus, and A. actinomycetemcomitans. Most Haemophilus species, particularly H. influenzae, require chocolate agar or some other source for X and V factors, which Pasteurella species do not. However, a number of Haemophilus species will grow sufficiently on most blood agar media for growth to be discernible, thus necessitating further differentiation from Pasteurella by testing for X and V factor dependency (97). In all, no conclusive diagnostic identification is possible through selective culturing, phenotyping, or direct microscopic examination alone. Serotyping and ribotyping of P. multocida. P. multocida isolates are classified based on a combination of capsular polysaccharide serotyping, which distinguishes isolates into one of the five capsular serogroups A (hyaluronic acid) (99), B (arabinose, mannose, and galactose) (100), D (heparin) (101, 102), E (uncharacterized), or F (chondroitin) (101, 102). Isolates are also subtyped based on their lipopolysaccharide (LPS), which separates isolates further into 16 serovars (103, 104). Isolate designations usually consist of a capsular serogroup letter followed by a somatic serovar number (e.g., A:1, A:2, A:3, B:2, etc.). The polysaccharide structure and biosynthetic genes have been determined for three of the capsular serotypes (99, 101, 102, 105, 106), as well as for the LPSs from a number of isolates (103, 107–117). Leading Article (Pasteurella multocida)

February 2024 13 PCR- plus sequence-based ribotyping analysis using universal primers for 16S rRNA genes, genomics, and other DNA sequencebased molecular techniques have now superseded phenotypic methods for identification, characterization, and differentiation of P. multocida and other Pasteurellaceae (54, 55, 92, 106, 118–120). Conventional ribotyping based on PCR amplification alone is still generally considered a reliable and discriminative method for characterizing clinical isolates of P. multocida (54, 121). However, PCR amplification of 16S rRNA genes, followed by sequencing and sequence comparison against known ribosomal databases, such as the NCBI or the RDP ( database, is now the predominant and most reliable method of taxonomically identifying isolates at the genus and species levels. The availability of additional sequence data for comparing various non-16S rRNA gene clusters has enabled the design of more sophisticated PCR methods for taxonomic identification (16S rRNA gene sequence-based ribotyping) and subtyping (virotyping using non-16S rRNA genes such as those associated with virulence traits) of P. multocida isolates (120–122). For example, the cap locus has been sequenced for capsule serotypes B:2 (strain M1404), A:1 (strain X73), A:3 (strain P1590), and D (strain HN06) (Fig. 4) and has been used as a basis for serotyping (106, 118). As a consequence, the cap loci of previously untyped P. multocida strains can now be subtyped based on clustering with the corresponding loci of known serotypes (118, 123–125). PASTEURELLA DISEASE IN ANIMALS Pasteurellosis Prevalence Pasteurella species cause numerous endemic and epizootic diseases of economic importance in a wide range of domestic and wild animals and birds. P. multocida is a common commensal or opportunistic pathogen found in the upper respiratory tracts of most livestock, domestic, and wild animals (34), including chickens (126–131), turkeys (132, 133), and other wild birds (123, 134–144), cattle and bison (121, 145–147), swine (34, 148–151), rabbits (152– 154), dogs (41, 155–157), cats (domestic house cats as well as large wild cats, such as tigers, leopards, cougars, and lions) (39, 42–46, 49, 157–166), goats (125, 139, 167, 168), chimpanzees (169), marine mammals (seals, sea lions, and walruses) (170), and even komodo dragons (171, 172). The manifestation and pathological symptoms associated with Pasteurella infection, or “pasteurellosis,” range from asymptomatic or mild chronic upper respiratory inflammation to acute, often fatal, pneumonic and/or disseminated disease. Transmission is through direct contact with nasal secretions, where a chronic infection ensues in the nasal cavity, paranasal sinuses, middle ears, lacrimal and thoracic ducts of the lymph system, and lungs (173, 174). Preexisting or coinfection with other respiratory pathogens, particularly Bordetella bronchiseptica (149, 150, 175– 180) or Mannheimia haemolytica (147), significantly enhances colonization by P. multocida, leading to more severe disease. Interestingly, a recent report showed that P. multocida inhibits the growth of M. haemolytica in vitro (181). Primary infection with respiratory viruses or with Mycoplasma species also predisposes animals to secondary infection with P. multocida and/or M. haemolytica (176, 182–186). Environmental conditions, stress, and the overall health of the animal also appear to play important roles in disease severity and likelihood of transmission (147, 187, 188). Pasteurellosis Pneumonia and Atrophic Rhinitis The predominant syndrome of pasteurellosis in endemic and epizootic infections of wild and domestic animal populations is upper respiratory disease in the form of rhinitis (irritation and inflammation of nasal mucosa and nasal secretions) and lower respiratory disease in the form of pneumonia (in cattle also referred to as bovine respiratory distress syndrome). Symptoms of pasteurellosis in most animals range from mild to severe (44, 178, 189–198). Mild symptoms include sneezing, copious mucous secretions, mild rhinitis, mild pneumonia with labored breathing, and fever but can progress to disseminated disease (hemorrhagic septicemia [further discussed in “Pasteurellosis and Hemorrhagic Septicemia” below]) and/or atrophic rhinitis (atrophy of nasal mucosa, seromucinous glands, and turbinate bones) associated with toxinogenic strains. Pasteurellosis pneumonia without symptoms of atrophic rhinitis is most often caused by nontoxinogenic capsular type A strains of P. multocida (147, 151, 199, 200). P. multocida is often endemic in rabbit colonies and swine herds, where the pneumonia and rhinitis disease is commonly called “snuffles” (148, 154). In more severe cases, symptoms progress toward atrophic rhinitis and, in rare cases, renal impairment, testicular and splenic atrophy, and hepatic necrosis. Atrophic rhinitis in rabbits can also result in overall weight loss, growth retardation, and frequently death. Toxinogenic capsular serotype D and some serotype A strains of P. multocida are associated with more severe symptoms of atrophic rhinitis in rabbits and swine (34, 148, 178, 194, 195, 201), with serotype D more prevalent in swine and serotype A more prevalent in rabbits. The primary overt symptom of atrophic rhinitis in swine and rabbits is twisting or distortion of Figure 4 Phylogenetic comparison of the capsule biosynthesis (cap) gene locus among selected Pasteurella multocida strains. The maximum-likelihood tree was calculated by using MEGA5 (575) with distances derived from genes within the cap locus of P. multocida ser otypes B:2 (strain M1404), A:1 (strain X73), A:3 (strain P1590), and D (strain HN06), as well as the cap loci from serotype A strains 3480 and 36950. Leading Article (Pasteurella multocida)

Vetnuus | Februarie 2024 14 « BACK TO CONTENTS the snout due to atrophic rhinitis (148, 202–204), manifested as bone resorption characterized by atrophy of the nasal turbinate bones (Fig. 5). Pathological changes may be mild and restricted to the snout with no overt clinical signs other than shrinkage of the ventral turbinates. However, infection can develop into more severe and progressive disease with complete loss of all turbinate bone structures and septum deviation, which often results in twisting, wrinkling, distortion or shortening of the snout, sneezing, snuffling, nasal discharge, and teary eyes (205–207). Pasteurellosis and Hemorrhagic Septicemia In cattle and other hoofed animals (ungulates), P. multocida causes predominantly respiratory disease. Along with Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, and Arcanobacterium pyogenes, P. multocida is implicated as a common pathogen associated with bovine respiratory disease (BRD), or “shipping fever” (nonsepticemic pneumonia) (208, 209). M. haemolytica is the most predominant isolate from cases of BRD and is associated with acute fulminating, fibrinopurulent pleuropneumonia with hemorrhage or coagulation necrosis due to intense LPS-induced inflammation and production of a ruminantspecific, pore-forming leukotoxin (208). P. multocida serotype A:3 is isolated from about 35% of shipping fever cases in the United States (209), manifesting usually as chronic bovine fibrinopurulent bronchopneumonia and occasionally with fibrinonecrosis (Fig. 6) (147, 208). Hemorrhagic septicemia caused by serotype A strains is only occasionally seen in North America (147, 210, 211); however, the proportion of severe hemorrhagic BRD incidences attributed to P. multocida, particularly as coinfection with M. haemolytica or other pathogens, appears to be rising (121, 146, 176, 183, 208, 209, 212). External stressors such as poor food supply, close confinement, and wet climate conditions are also thought to enhance transmission through contact with nasal secretions of infected animals or fomites (147). Hemorrhagic septicemia (HS) is a serious acute, highly fatal, and highly prevalent disease in livestock, especially cattle and buffalo, in tropical regions of the world, including Asia, India, Africa, southern Europe, and the Middle East (93, 147, 210, 211, 213–220). HS is caused primarily by P. multocida serotypes B:2 and E:2 and is thought to occur at the later stages of pasteurellosis disease (reviewed in reference 221). HS is observed less commonly in swine, sheep, goats, deer, and elk and then is mostly associated with serotype B:2 strains (214, 221). HS may be asymptomatic or unnoticed until onset of the acute stage, which is characterized by rapid onset (within a few hours) and progression. Symptoms usually begin with fever, lethargy, and edema with copious salivation, lacrimation (teary eyes), and nasal discharge, rapidly followed by respiratory distress, septic shock with widespread hemorrhaging, and death within 1 to 3 days. Antibiotic treatment can be effective at early stages, but since acute clinical signs of sepsis manifest so quickly, mortality is nearly 100% after onset (221). Despite extensive research, very little is known to date about the virulence factors and mechanisms involved in the transition from mild chronic pasteurellosis to acute severe disseminated disease. Recently, a mouse model of HS caused by P. multocida serotype B:2 has been developed (222), which might enable further exploration of the roles of different host immune cells and host factors as well as bacterial factors in dissemination of the bacteria in the host. Pasteurellosis and Fowl Cholera P. multocida subsp. multocida is the most predominant cause of fowl cholera worldwide in a variety of avian species (34, 134, 223), although P. multocida subsp. septica and P. multocida subsp. gallicida are also sometimes isolated (223). Capsular serotype A (mainly A:1, A:3, and A:4) is highly correlated to disease predilection for strains associated with fowl cholera (105, 224–227), although capsular serotypes F and D have also been reported (123, 124, 227, 228). Serotype B:3 is often isolated from avian disease cases that manifest as sinusitis (229, 230) with symptoms of nasal discharge, increased lacrimation, swelling, and inflammation. The respiratory tract appears to be the primary site of infection for fowl cholera (126, 129, 223, 231), but isolation of P. multocida subsp. multocida from avian salpingitis has also been reported (232). Fowl cholera tends to be an asymptomatic or mild chronic sinusitis and conjunctivitis (229, 230) or pneumonia-like pasteurellosis, but it can suddenly and rapidly develop into a fatal disseminated disease (223). To date, no single bacterial virulence trait or mechanism has been identified as correlating with observed disease incidence or severity (223, 233), but environmental and host factors appear to contribute to onset and outcome severity. Figure 5 Atrophic rhinitis in swine. Shown are transverse sections of the nasal cavities of pigs exhibiting pathological symptoms of atrophic rhinitis ranging from mild (left panel) to moderate (middle panel) to severe (right panel) caused by infection with toxinogenic P. multocida. (Photos courtesy of the University of Illinois Veterinary Diagnostics Laboratory, Urbana, IL; reproduced with permission.) Figure 6 Bovine pasteurellosis bronchopneumonia lung. Shown is an image of a lung lobe from a calf exhibiting pathological symptoms of bronchopneumonia, including extensive hemorrhagic lesions (dark areas), caused by infection with P. multocida. (Courtesy of Peter G. Moisan, Veterinary Diagnostic Laboratory, Kansas State University; reproduced with permission.) Leading Article (Pasteurella multocida)

February 2024 15 PASTEURELLA AND OTHER PASTEURELLACEAE DISEASES IN HUMANS Pasteurellosis as a Zoonotic Infection in Humans Humans acquire Pasteurella infection primarily through contact with animals, most usually through animal bites, scratches, licks on skin abrasions, or contact with mucous secretions derived from pets (19, 20, 36, 40–42, 46, 48, 90, 158, 160, 163, 165, 185, 234– 244). The prevalence of antisera to P. multocida was 2-fold higher in healthy individuals with occupational or pet exposure than in a control group with no reported exposure (245), indicating that animal exposure increases the likelihood of subclinical carriage or infection. A survey of the literature over the past 30 years suggests that 20 to 30 human deaths due to pasteurellosis occur annually worldwide, but as mentioned above, this rate appears to be rising (Fig. 1), and in nearly all cases death appears to result as a complication from infection acquired through animal exposure. Among the Pasteurella species, P. multocida is the predominant human pathogen encountered, especially in severe disease cases (235, 239), although P. canis may be more prevalent with dog bites (48, 246–248). Common symptoms of pasteurellosis in humans from animal bite wounds are swelling (edema), cellulitis (diffuse, localized inflammation with redness and pain), and bloody or suppurative/ purulent exudate (drainage) at the wound site (39, 41, 48, 49, 160, 165, 241, 249–255). Leukocyte and neutrophil counts are typically high at the infection site, and inflammation develops very rapidly. In more severe cases, pasteurellosis can rapidly progress to bacteremia (fulminant sepsis) (41, 161, 235, 241, 251, 256–266) and other complications such as osteomyelitis (inflammation of the bone) (155, 165, 267–269), endocarditis (inflammation of the heart) (256, 263, 270–285), and meningitis (inflammation of the meninges) (53, 90, 159, 163, 165, 264, 286–293). Respiratory infection in humans is relatively uncommon but can occur in patients with chronic pulmonary disease (44, 48, 152, 239, 247, 294–296). In these instances, pasteurellosis can present as severe bilateral consolidating pneumonia and also can cause lymphadenopathy (swelling of the lymph nodes), epiglottitis, and abscess formation (295, 297). Transmission and Prevalence through Contact with Pets Infections with Pasteurella requiring medical intervention commonly arise as a result of bite or scratch wounds from pets, predominantly cats and dogs (39, 41, 45, 48, 49, 155–157, 161, 240, 242, 243, 249–251, 254, 255, 257, 258, 267, 298–304), but also from other domestic animals (305–308). Bite wound infections with Pasteurella tend to be highly aggressive with skin or soft tissue inflammation, erythema, local lymphadenopathy, fever, pain, and swelling often manifesting within 24 h, but they can present as early as 8 to 12 h (41, 48, 159, 166, 309). Pasteurellosis has an overall mortality rate of 25 to 30% among reported human cases of animal bite wounds (38, 90, 163, 288, 289, 310), with bacteremia found in 40 to 63% of all pasteurellosis patients and meningitis plus neurological complications found in 17 to 29% of patients. Pasteurella infections that do not result from bite wounds are likewise most often associated with P. multocida strains (44, 90, 163, 310) and usually involve contact of skin lesions or naso-oropharynx or other upper respiratory mucosa with animals or animal secretions, particularly in young children, the elderly, or pregnant or immunocompromised individuals (40, 44, 53, 87, 90, 165, 235, 240, 241, 244, 255, 257, 258, 261, 277, 288, 289, 291, 307, 311–339). Neonatal meningitis (usually with septicemia) has been reported (90, 91, 288, 313, 340–342), but in nearly all cases the most likely route of transmission was attributed to direct exposure to pets or other domestic animals. Vertical transmission from mother to child was reported rarely (291, 341). Only three instances of humanto-human horizontal transmission have been reported. In two cases transmission was likely from the father, who had exposure to chickens (286) or sheep (343), and in the third case the mother tested negative for P. multocida colonization but the grandmother tested positive, as did her pet dog (344). Patients with underlying diseases that contribute to an immunocompromised condition, such as cirrhosis (liver dysfunction) (39, 43, 241, 244, 249, 255–257, 260, 262, 263, 265, 266, 273, 275– 280, 292, 312, 314, 316, 317, 321, 322, 328, 329, 335, 337, 345–359), renal failure (kidney dysfunction requiring dialysis or indwelling catheters) (253, 267, 300, 302, 303, 318, 324, 326, 327, 360–366), or HIV-positive status (especially if taking immunosuppressive drugs or experiencing other disease conditions) (290, 310, 315, 367–369), have an increased risk of peritonitis, endocarditis, and/or septicemia caused by P. multocida. This is particularly the case if there is a history of exposure to pets. Indeed, in almost all of the above-mentioned reports, the authors caution patients with these conditions about the risks associated with exposure to pets and/or alert clinicians to consider possible complications with P. multocida infection for cases with pet ownership or a history of animal exposure. It is noteworthy that in human infection cases the subspecies or serotype of the P. multocida clinical isolate is rarely reported (370). However, there are a few studies where this has been examined retrospectively. In one study, 143 isolates collected from human patients over a 12-year period (1983 to 1994) were biochemically characterized for distribution at the species and subspecies levels as well as capsular groups (239). Most of the isolates were determined to be P. multocida subsp. multocida, with the remaining being P. multocida subsp. septica, P. multocida subsp. gallicida, P. canis, P. dogmatis, and P. stomatis. While P. multocida strains were associated with cat and dog bites, P. canis, P. dogmatis, and P. stomatis strains were recovered only from dog bites, and P. multocida subsp. multocida and P. multocida subsp. septica were most frequently associated with cat bites. Most of the animal bite isolates were non-group A capsular strains (serogroup D) and were associated more with disseminated disease. Capsular serogroup A strains were associated more with respiratory infections. Similar findings were observed for isolates recovered from infected patients in four other studies involving 159 strains (247), 107 strains (48), 54 strains (294), and 20 strains (296). Leading Article (Pasteurella multocida) >>> 16