Chapter 5:Small Gram-Negative Rods and Coccobacilli

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 5: Small Gram-negative rods and coccobacilli

Authors: Drs. M.M. Henton & J.A. Picard

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

1 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli INTRODUCTION

The genera Haemophilus, Actinobacillus, Mannheimia and Pasteurella are currently classified in the family Pasteurellaceae. These species are major causes of respiratory and systemic diseases in humans, other mammals and birds. They exist as mucosal parasites, but are capable of being primary or opportunistic pathogens depending on the carrier status and health of the host.

Recently there have been many changes in the taxonomy of this group that now contains seven genera of veterinary importance: Actinobacillus, Avibacterium, Haemophilus, Histophilus, Mannheimia, Pasteurella and Bibersteinia. Important differential characteristics of genera in this family are included in Table 5.1.

Table 5.1: Phenotypic characteristics separating selected genera of veterinary importance in the family Pasteurellaceae

Histophi Haemop Actinob Pasteur lus hilus acillus ella

Haemolysis D D D - Capnophilia + - - - Yellowish pigmentation + - - - V-factor dependence - + D D X-factor dependence - + - - Catalase - - + + Oxidase + + D D Urease - D + D Voges–Proskauer (37 °C) - - - - Indole +d D - +b Ornithine decarboxylase - D - D L-Arabinose - D - D-Fructose - D + + D-Galactose - D D + Meso-Inositol - - - Maltose - + + D Mannitol - D D D-Mannose D D + D-Melibiose - D - D-Sorbitol - -C D Sucrose - - + + Trehalose - - D D ONPF (-fucosidase) - - - ONPG - PNPG - - - +, only positive reactions, -, only negative reactions; D, positive or negative; NK, not known. a Deviating strains occur. b P. avium is indole negative

c Aesculin, amygdalin, arbutin, cellobiose, salicin and NPG (-glucosidase) d Bisgaard taxon 9 late positive

2 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Haemophilus/ Histophilus

There have been a number of taxonomic changes to organisms within the genus Haemophilus in recent years. The species Histophilus somni now includes the bovine commensal and opportunistic pathogen Haemophilus somnus and the ovine organisms Histophilus ovis and Haemophilus agni. A list of Haemophilus and Histophilus species causing disease in animals are included in Table 5.2.

Table 5.2: Histophilus and Haemophilus species of veterinary importance in production animals, dogs and poultry

Species Hosts Disease

Cattle (previously Septicaemia Haemophilus Infarcts in the brain (Thrombotic somnus) meningoencephalitis) Respiratory infections H. somni Genital tract infections

Epididymitis Pneumonia Sheep (previously Mastitis Haemophilus agni Polyarthritis and Histophilus Meningitis ovis) Septicaemia

Polyserositis, meningitis (Glasser’s H. parasuis Pigs disease) Infectious coryza in chickens and A. paragallinarum Poultry respiratory infection in turkeys H. haemoglobinophilus Dogs Balanoposthitis, vaginitis H. paracuniculus Rabbits Mucoid enteritis

Speciation of Haemophilus in the clinical laboratory has been primarily based on the requirement for haemin (a porphyrin also known as X factor) or NAD (also known as V factor or coenzyme I). The methods currently used for the determination of these growth factor requirements rely on the supplements being added to defined agar media or impregnated in disks or strips placed on agar plates.

Unlike human pathogens in this genus, no animal isolate is dependent on both factors. They are all dependent on only one factor with the exception of Histophilus somni , which is X and V factor independent. The X factor is present in adequate amounts in blood agar. In chocolate agar both factors are present by virtue of being released into the medium following haemolysis during heating of the blood. The V factor is also produced by an organism such as Staphylococcus aureus. When the latter is grown together with Haemophilus spp. on blood agar, the resultant enhanced growth of Haemophilus is referred to as satellitism. In veterinary laboratories, routine isolation on chocolate agar in an atmosphere of 5-10% CO2 (or candle jar) at 35-37°C for 3-4 days is satisfactory. Selective media can be used when one specifically suspects the presence of Haemophilus species.

3 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Histophilus somni (formerly Haemophilus somnus, H. agni and Histophilus ovis)

H. somni is an opportunistic pathogen of cattle. It is a common component of the normal urogenital and respiratory flora and sporadically causes thromboembolic meningoencephalitis, abortion, infertility and pneumonia. It is often the only bacterial isolate from the many reported cases of necrotic laryngitis, necrotic tracheitis, fibrinous bronchopneumonia and fibrinous pleuritis in cattle. There are probably many subtypes of H. somni.

This organism is a nutritionally demanding, moderately slow growing, capnophilic, Gram-negative bacillus. Colonies on blood agar are unremarkable. H. somni does not require X or V factors for growth. A selective growth medium for H. somni has been described. It incorporates vancomycin (5 g/ml), neomycin (5 g/ml), sodium azide (50 g/ml), nystatin (100 g/ml) and cycloheximide (100 g/ml) into 5% horse blood agar. Addition of thiamine monophosphate (1 g/ml) also enhances growth of the organism.

When attempting to isolate H. somni from cases of suspected thromboembolic meningoencephalitis, brain tissue that includes visible lesions should be homogenised in sterile saline at the ratio of 5 mg tissue in 20 ml saline. Spread aliquots of 0.1 ml over the surface of blood agar. H. somni is considered to be homologous to Haemophilus agni and Histophilus ovis both found in sheep and goats. There are slight biochemical differences between them e.g. H. agni is usually urease positive and indole negative. Histophilus somni may be weakly indole positive and when scraped together the colonies are usually yellow. Actinobacillus seminis is also very similar to H. somni. Both are sucrose negative, which distinguishes them from the rest of the community found in the Haemophilus/Pasteurella/Actinobacillus (HPA) group, but A. seminis is grey when the colonies are scraped together. Differential characteristics are summarized in Table 5.3.

Table 5.3: Identification of Haemophilus and Taylorella species of veterinary importance

A. Avibact H. H. H. T. H. pleurop erium. haemog Test H. agni parasui paracu equigen somnus neumo paragall lobinop s niculus italis niae inarum hilus

Requirement for X factor - - - - - + - - Requirement for V factor - - + + + - - Growth on MacConkey ------Catalase - - + d - + + + Oxidase + + - d - + + + Capnophilic + + + + - + Haemolysis - - - + - - - Indole +w - - - - + + - Urease - (+) - + - - + - Nitrate reduction + + + + + + + - Glucose (acid) + + + + + + + - Aesculin hydrolysis - - -- d - - - ONPG d d + + d + Phosphatase - + + + - + + Ornithine decarboxylase + + - - - - + - Gas from glucose ------Acid production from: L-arabinose d +/w - - - - Cellobiose ------Dulcitol d ------Galactose d +/w + +/w - - - m-Inositol d - d - - - -

4 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Lactose - - d d - - - Maltose + +/w + + (+) + + Mannitol + +/w - + + + - Mannose + - + + + + Melibiose ------Raffinose - - - d - - - Salicin ------Sorbitol + - - - + - - Sucrose - - + + + + + Trehalose + ------D-Xylose + +/w - + d + -

Haemophilus parasuis

H. parasuis is considered to be part of the normal flora of the respiratory passages of conventionally raised pigs. It causes Glässer’s disease, characterized by polyserositis and meningitis. The disease only occurs sporadically, mainly in pigs two to four weeks of age, subjected to stressful environmental conditions. A selective medium for H. parasuis consists of chocolate agar containing 1.5 g/ml lincomycin, 5 g/ml bacitracin and 0.1 g/ml crystal violet. The specimen material should be spread with a sterile swab to completely cover the agar surface. Haemophilus parasuis grows weakly and may take 2 to 3 days to become apparent. Actinobacillus pleuropneumoniae in comparison grows rapidly (1 day), is CAMP and urease positive.

Tayorella Equigenitalis

This organism was formerly known as Haemophilus equigenitalis. It is an important cause of genital tract infections in horses and is either venereally transmitted or via assisted reproduction (artificial insemination). Swabs of the cervix, clitoral fossa and urethra can be examined in mares, and the urethral fossa and penile sheath (lamina interna) in stallions

For primary isolation agar bases such as Oxoid blood agar base No. 2, or Columbia agar (BBL) can be used with the addition of sodium sulphate 200 mg/L and L-cysteine HCl 100 mg/L. Chocolated blood (preferably sheep blood) are added at 10% concentration. Some plates should be prepared with streptomycin at 200 g/ml media and some without. One of each should be used to examine swab specimens. Plates are incubated in air with 5-10% CO2 for up to 6 days before discarding them. Colonies are small pinpoint to pin head in size.

The minimum profile of tests to be carried out on suspicious colonies is:

Motility non-motile in Robertson's cooked meat medium

Oxidase positive (rapid and strong reaction)

Catalase positive

Urease negative

Phosphatase positive

5 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli

Actinobacillus

Actinobacillus spp. are commensals on the mucous membranes of their hosts. They do not penetrate intact skin or healthy mucosa, but are responsible for endogenous infections following trauma. These organisms are aerobic, Gram-negative coccobacilli. Members of this genus that are of veterinary importance are listed in Table 5.4. Note that A. muris has only been found in rodents and A. actinomycetum-comitans and A. hominis in humans.

Table 5.4: Actinobacillus species of veterinary importance in production animals and dogs

Species Hosts Disease Cattle Wooden tongue Pyogranulomas A. lignieresii Sheep Pyogranulomas

pigs Granulomatous mastitis foals Neonatal foal septicaemia Arthritis

mares Abortion A. equuli Septicaemia

Pigs Arthritis

dogs Opportunistic infections pigs Pneumonia A. suis Septicaemia A. pleuropneumoniae pigs Fibrinous pleuropneumonia A. seminis rams Epididymitis

They can all be isolated on blood agar and incubated aerobically for 24 hours at 37°C. Growth on MacConkey agar may take longer than 24 hours. Members of the genus Actinobacillus typically produce sticky colonies, grow well on MacConkey agar, and hydrolyze urea. The differential characteristics of the species belonging to the genus Actinobacillus are included in Table 5.5.

6 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Table 5.5: Differential characteristics of the species belonging to the genus Actinobacillus

A. A. A. arth A. equ A. acet ritti equ uli ple A. omy dis uli sub A. A. A. uro A. A. A. cap Test cetu sub sp. homi lignier muri pne rossi semi sui sula m- sp. hae nis esii s um i nis s tus comi equ mol oni tans uli ytic ae us Catalase +a (+) d (+) - d + d (+) + + Oxidase + + (+) + + (+) + d (+) d (+) Nitrate reduction + + + + + + + + + + + ONPG reaction - + + d + + d - + (+) -b (+) Phosphatase + + + + + - + - - + Ornithine ------d - decarboxylase Indole production ------Urease - + + + + + + + - + Aesculin hydrolysis - +, L - (-) d - +, L d - d + NAD requirement ------+ - - - Growth on d + - (+) - - (+) - - (+)w - + MacConkey agarc Beta-haemolysis - - - + - - - + d - + (sheep cells) Fermentation f + NF + + + + + + + W, L + Gas production from (+) ------d - - glucosed Acid production from: - L-arabinose - + d (-) (-) - (-) - - + d, L (+) Arbutin - + - NT - + NT - - + Cellobiose - + - + - - +,L - - - + Dulcitol ------Galactose + + + + + + d,w +,w + d,L (+) m-Inositol - - - - - d,w - (+) d - Lactose - + + + + d,L - d + - + Maltose + + (+) + + + + + (-) d,L + Mannitol (+) + + (-) + + + + (+) d,L - Mannose + + + - + + + d - + Melibiose - + + + + +,L - +,L - (-) - + Raffinose - + + + + + d + d (-) - + Salicin - + - (+) d - + - - - + Sorbitol (-) + + - (-) - (-) - - (+) - - Sucrose - + + + + + + (-) - + Trehalose - + + + + - + - - - + D-Xylose d + + + + - + (+) - + G+C content (mol% 42.7- 40- 41.8- 42.4 40.9 46.9 42 41.9 43.7 40.5 range) 47.1 42.9 43.2 a +, ≥90% of the strains are positive; (+), 80 – 89% of the strains are positive; d, 21 to 79% of the strains are positive; (-), 11- 20% of the strains are positive; -, ≤10% of the strains are positive; w, weak reaction; L, late reaction; NT, not tested. b Positive according to Piechulla et al, and negative according to Sneath and Stevens. c Results for A. pleuropneumonia is on media supplemented with NAD d In Hugh-Leifson medium containing glucose, with incubation for 48 h at 37°C.

Actinobacillus lignieresii

7 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli This organism is a normal Gram-negative inhabitant of the oral cavity, and is the recognized cause of “wooden tongue” and local infections of the pharyngeal region, nostrils and neck of cattle. In sheep and goats, skin or lung lesions may be found. Drainage from these lesions tends to be non-putrid and watery. Tissues and pus from lesions may contain firm granules 1-3mm in diameter. They represent the so-called “sulphur” granules. Pus diluted with water and swirled around in a Petri dish may show up the small granules. Staining of crushed granules usually reveals masses of Gram-negative bacteria. In histological sections of granulomatous tissues, the masses of organisms are surrounded by Gram-negative club-shaped bodies.

Actinobacillus lignieresii is often difficult to isolate and only a few colonies may be present. It dies rapidly in the laboratory.

Actinobacillus rossii

This organism has been isolated from pigs and is a small Gram-negative rod. It produces smooth, grey colonies on blood agar incubated aerobically and anaerobically at 37 °C, but not at 42°C or at 25°C. It does not grow on MacConkey or Simmon’s citrate agar.

Actinobacillus suis

It is always strongly beta-haemolytic and can be distinguished from A. rossii on biochemical tests.

Actinobacillus pleuropneumonia

Pneumonic lung specimens are usually cultured on blood agar with a S. aureus streak or on chocolate horse blood agar as rare strains are V factor dependent. The selectivity of the medium is enhanced with the addition of bacitracin (500 units/ml), and cloxacillin (5 l/ml). Plates should be incubated for 24 hours aerobically or in an atmosphere of 5-10% CO2 (or a candle jar) at 37 °C. Colonies on chocolate agar are opaque and reach a diameter of 1-2mm within 48 hours. Two types of colonies often are visible: a round, hard, waxy type and a flatter, soft, glistening type. The haemolytic activity of this organism is characteristic of this species. The intensity of the haemolysis varies not only according to the type of erythrocyte used but also with the different serotypes.

Mannitol fermentation and positive urease reaction distinguish A. pleuropneumoniae from other respiratory pathogens of swine such as Haemophilus parasuis

Actinobacillus seminis

Actinobacillus actinomycetum-comitans mentioned in older texts should be considered to be A. seminis.

This organism is a highly pleomorphic, Gram-negative rod that does not grow on MacConkey agar, is not haemolytic, and does not require CO2 for growth. Colonies on blood agar are small (less than 1 mm), clear and glistening.

Actinobacillus seminis should be distinguished from other Gram-negative pleomorphic rods that can be isolated from the semen of rams such as Histophilus ovis, Histophilus somni and Haemophilus agni. These three organisms cannot be distinguished on the basis of DNA studies, and should therefore be regarded as the same. They differ from A. seminis by the production of a yellow pigment, enhancement of growth in air with 10% CO2, are catalase negative and indole positive. 8 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli Actinobacillus equuli

Bacterial septicaemia in foals usually occurs during the first week of life and incurs a high mortality rate. Various pathogens can cause bacterial septicaemia in foals, also referred to as sleepy foal disease. They include A. equuli, E. coli, Streptococcus equi subsp. zooepidemicus, Salmonella Typhimurium, Klebsiella pneumoniae and S. aureus.

Mannheimia/ Pasteurella/Gallibacterium

The genus Pasteurella is widely parasitic on mammals and birds. Under conditions of stress, these organisms can become invasive and play a significant role in the pathogenesis of a variety of infections in animals, including pneumonia, sinusitis, abortion, mastitis and septicaemia. Table 5.6 lists the most important species that can be isolated from ruminants, horses, pigs, companion animals, rabbits and birds. Colonies from mammals are small, grey and glistening, or larger, shiny colonies with a brown centre. Poultry strains are tiny beta-haemolytic colonies similar to Streptococcus. Pasteurella species are small, Gram- negative rods or cocco-bacilli. Clinical material should routinely be inoculated on blood agar and MacConkey agar. Colonies of all species are usually visible after 24 hours.

Table 5.6: Important Pasteurella and Mannheimia species in animals

Species Hosts Disease Cattle Pneumonia

Sheep Pneumonia Gangrenous mastitis “Bluebag” Mannheimia haemolytica Septicaemia in lambs under 3 months of age

Pigs Pneumonia Sheep Septicaemia in lamds 5-12 months old B. trehalosi Cattle Pneumonia (primary or secondary) Occasional but severe mastitis

Sheep/goats Pneumonia Mastitis

P. multocida type A Pigs Pneumonia (usually secondary)

Rabbits “Snuffles”, pleuropneumonia, abscesses, otitis media, conjunctivitis and genital infections

Poultry Fowl cholera (primary infection) Cattle and Water Buffalo Haemorrhagic septicaemia (type B in SE Asia and type E in Africa) P. multocida types B and E Nasopharynx of carrier animals P. multocida type D Pigs Atrophic rhinitis P. multocida type F Turkeys mainly Role as a pathogen is unclear Dogs and cats Normal nasopharyngeal micro flora P. pneumotropica Rodents Pneumonia and abscesses Commensal in the nasopharynx Dogs (humans) Commensal in oral cavity P. canis From dog bite injuries in humans P. dagmatis Dogs and cats Commensal in oral cavity

9 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli From dog bite injuries in humans P. stomatis Dogs and cats Commensal in respiratory tract Horses Respiratory infections including P. caballi pneumonia Pigs Commensa; in digestive tract P. aerogenes P. anatis Ducks Intestinal commensal Poultry Commensal in respiratory mucosa. P. gallinarum Occasional mild respiratory infections. P. avium, P. langaa & P. volantium Chickens Commensal in the respiratory tract P. testudinis Turtles, tortoises Abscessation P. granulomatis Cattle Fibrogranulomatous disease

The differential characteristics of the species belonging to the genus Pasteurella only are given in Table 5.7 and that of Pasteurella and related species in Table 5.8.

Mannheimia haemolytica and Pasteurella trehalosi

Pasteurella haemolytica biotype A has been divided into the pathogenic Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia variegata and the non-pathogenic Mannheimia glucosida. The former Pasteurella haemolytica biotype T has been allocated to a new species known as Pasteurella trehalosi. Of these, M. haemolytica and P. trehalosi have the greatest veterinary significance. Poultry strains of P. haemolytica retained their name. They are small Gram-positive cocco-bacilli that grow both aerobically and as facultative anaerobes. Mannheimia haemolytica produces acid in arabinose, and P. trehalosi acid in trehalose. The fermentation reaction should be allowed to continue for up to 10 days. On blood agar, M. haemolytica strains produce small, grey colonies with distinct zones of haemolysis. The larger slightly yellow/brown colonies of P. trehalosi are less uniformly haemolytic.

On the basis of specific antisera, 17 serotypes of M. haemolytica have been identified. Mannheimia haemolytica includes serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, 14; 16 and 17 and P. trehalosi includes serotypes 3, 4, 10 & 15. Selected characteristics of M. haemolytica are compared with other respiratory pathogens of cattle in Table 5.9.

Pasteurella multocida

P. multocida colonies are non-haemolytic and may be mucoid or non-mucoid in appearance. They do not grow on MacConkey agar. On the basis of their capsule polysaccharides, they have been divided in types A, B, D, E & F. In Africa, only types A, D & E have been described. Haemorrhagic septicaemia is caused by types B and E.

Pasteurella caballi

Strains isolated from horses, but not fulfilling the characteristics of any established species within the genus were described in 1989. Subsequent biochemical and genetic studies led to the proposed name of P. caballi. These strains differed from other Pasteurella species in that all were aerogenic and catalase-and oxidase negative, and some strains produced acid from myo-inositol and L-rhamnose. They seem to have a limited host range and have only been isolated from horses. They are sporadically isolated from horses where they are opportunistic pathogens. They also produce gas from glucose, are urease-negative and ferment raffinose (slow), mannitol, maltose, dextrin, glucose and xylose. They utilize ODC and don’t hydrolyze aesculin. Colonies are yellow and non-haemolytic.

10 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli P. canis and P. dagmatis

These organisms are part of the normal oral flora of dogs but are potentially pathogenic for humans following bite wounds from dogs. When trying to isolate these bacteria from the buccal cavities of dogs, contaminating bacteria such as Staphylococcus, Streptococcus and Corynebacterium can be inhibited by the addition of 20 mg/litre thiostrepton to the culture media.

Other Pasteurella species

Pasteurella lymphangidis has only been found in cattle in India and Mannheimia granulomatis in cattle in Brazil. Pasteurella mairii causes abortion and septicaemia in pigs and rarely in other animals. Table 5.8: Phenotypic separation of species and taxa within the genus Mannheimia (From Angen et al, International Journal of Systematic Bacteriology, p. 47). Pasteurella testudinis is associated with respiratory infections in tortoises. Pasteurella bettae and P. ureae are only found in humans.

Infections of poultry by members of the Pasteurellaceae

These are important disease causing agents in poultry, and common species infecting poultry are listed in Table 5.7.

These bacteria can easily be confused with Riemerella anatipestifer (formerly Pasteurella anatipestifer) and Cytophagia.

Table 5.7: Identification of bacteria belonging to the genus Pasteurella. (All are non-motile, oxidase positive, nitrate positive and ferment glucose)

P. m. P. P. m. P. m P. P. Past Past P. P. P. mult dag P. Species septi gallic galin anati eurel eurel lang aviu volan ocid mati canis ca ida arum s la A la B aa m tium a s

Catalase + + + + + + + + + - + + Indole + + + + - d - - + - - - Urease - - - + ------Ornithine + + + - - + - - + - d - Glucose gas - - + - - - - d - - - - Acid from Dulcitol - - + - - - - - + - - d Maltose - - - + + - - - + - - + Mannitol + + + - - - + d - - - + Sorbitol + - + ------d Trehalose d + - + + d + + + - + +

Table 5.8: Differential characteristics of the species belonging to the genus Pasteurella and some related species (Lonepinella koalarum and Phocoenobacter uteri are catalase and xylose negative

P. G O Ha Haemoly A. T R. C. C C. C. C. Ta Ta ca all r e tic s ax a .g . oc ca cy xo xo ba ib n m Actinob al o n in hr rni no n n 3 lli ac i op acillus- pi n at gi ac m de 2 te t hil like n 14 ip v ea or g ri h us gi e al e su mi u o pa n st is s m b ra gi if a ga ti er c lli di t na s. e ru s P. r m al p i pi P. n P. P. u n P. M. P. P. P. P. m e te tr P. m gi ae P. P. ha st be da gal ul u st e ur ti Te ro an av em o tti P. canis gm lin P. mairi to m u h e r di st ge ati iu oly m ga ati aru ci ot di al a h a ne s m tic at e s m d ro ni o e i s s a is a pi s si n al c o pi a t n r gi a ti c di h s e s a al l pi e n gi ti di s Ca - tal +, +/ + + d - + + + + + + + + - d / - + + + + - - - + + - + as w d + e Ox + +, (+ ida (+) + - - + + + + + (-) + + + + + / (-) + - + - - - + + w ) se -

Ma cC +, on + - d - - - - + d - d d - d + d - - d + ------d d w ke y - ha em oly sis (+ ------(+) d -w - - - + - - - + + - d ------(sh ) ee p cel ls) Po rp hyr + + + + + + + + + in tes t Nit rat + + + + + + + + + + + + + + + + - + + - d d - - e O (+ NP d + - - + - - - d + d - + - - - + + - - - - + + + + + + ) G Ph os ph + + + + + + + + + + + + + - + + ata se Or nit hin e de d - - - + + - - - + d + - - - - car bo xyl as e Ind (+ + - - - - - d,w + + - - - + + ------ole ) w Ur ------+ - - - + - + - - - + + - + - - d ------/d - ea

se Ae sc (-) ------d d +) - - - + d - - - + - + + + + + (+) (+) - - uli n NA D re qui - - d ------re me nt Fe rm + (+ ent + + + + + + + + + + + + + + + + + + + + + + + + + + L ) ati on Ga s d, glu (+) - - + - +,w - - - - - d ------w co se Aci d fro m L- Ar abi + - - - + - - - + - + d (-) - d - - d - - (-) - - (+) - no se Ar + but ------(-) - - - - d - L in Ce llo ------(-) - - - - - d - bio se Du lcit ------(-) - - (-) ------d - ol Ga lac (+ + + +,L - d + + + + + + + + + - - + - - + + - - - d + d + + tos ) e

m- Ino + (+ + - - - (+) - - - d (+) + - d - d - - - - (-) d + + sit L ) ol La cto + +,L - - (+) - - - d d - - d - (-) - - + - + - - - d + + + + d se Ma (+ (+ (+ (+ lto + - - d + - + + + d - (-) d - + + (+) + - w + + + + - - ) ) ) ) se Ma (+ nni (-) + - - + - - - + d + - - d + d - + d + ------+ + ) tol Ma ( nn + + +,L d + + + (-) + + + + + - + d + + d d w- + + + + os ) e Me libi ------(-) d - + - - os e Ra ffin +, (-) - - (+) - +,w (+) d + - - d - d - - os w e Rh am ------no se Sa lici ------(-) d - - (-) - (-) d - - - + - d - - - - n So rbit ------(-) + d + d - - d + (-) - + d - - d d ol Su cro + + - - + + + + + + + + + + + + - + - + - + + + + + se Tr eh (+ - + + - - d + + - (+) (-) d + d + - - - + + - - - - alo ) se D- Xyl (+ (+) + d - + (-) - d + + + d d - + - - - d - + ------+ + os ) e

Table 5.9: Differential characteristic of Mannheimia species

M. gra M. M. Species M. glucosida nul ruminali variegan Unnamed taxa within Mannheimia M. om s s haemo atis lytica Test Taxon A B C D E F G H I 1 2 1 2 8A 8B 8C 7 9* 10§

Beta- haemolysis (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) - - - d d (+) (+) (+) d (+) d Ornithine decarboxylase - + + + + + - - - + - - - + - - - - - + - L-arabinose - + - + + - - + - + - -¥ - + + - - - + + + D-sorbitol + + + + + + + + + + + d ------+ + D-xylose +w + + + + + + + + + d d - +† + + + - +† + + Maltose + + + + + + + + + + d - + d d + + + + + + Dextrin + + + + + + + + + + d - d d d + +w +w +w + d Glucosides∏ - + + + + + + + + - d - - d‡ ------Gentiobiose - + + - + + + + + - d - - - - + - - - - - NPG (β-glucosidase) - + + + + + + + + + + - - d d ------Meso-inositol d + +w + +w +w +w +w +w + d - - d - w/- + + d - d ONPF (α-fucosidase) + +w +w + + +w - +w +w + - - - d - + - - - - d ONPX (β-xylosidase) d + + - - - + - - d w/- - - - + - d - d - d ONPG (β-galactosidase) d + + + + + + + + + D + + d + - + + + + + Indole ------d - - +w +w d - w/- D-Melibiose ------d ------Origin of isolates BO O O O O O O O O O BLD O BO BP P O B B BO B BO No. of strains 30 34 28 12 51 5 2 3 7 3 8 *meso-inositol- and NPG-negative strains of [P.] haemolytica biogroup 9, the meso-inositol- and NPG-positive strains reclassified as M. glucosidal biovar I.

§ Consisting of two genetically distinct groups that could not be phenotypically separated.

¥Arabinose-positive strains of Bisgaard taxon 18 exist but their taxonomic allocation is still undecided.

† One strain negative.

∏ Amygdalin, arbutin, aesculin (acid), cellobiose and salicin.

‡ Most strains negative for all glucosides; the strains that ferment glucosides also ferment D-Melibiose.

B: Bovine; O: Ovine: P; Porcine; L; Leprine; D: Deer.

17 | P a g e Gallibacterium species

Gallibacterium is a newly described genus incorporating the former Pasteurella haemolytica – A. salpingitis bacteria isolated from poultry. They are members of the family Pasteurellaceae. Colonies on bovine blood agar are mostly strongly β-haemolytic, greyish, non-transparent, but eventually translucent at the periphery, with a butyrous consistency, smooth and shiny, circular, raised with an entire margin and 1·0–2·0 mm in diameter after 24–48 h at 37 °C. They are catalase-, oxidase- and phosphatase-positive. Nitrate is reduced. Porphyrin and alanine aminopeptidase tests are positive. Acid is formed without gas from glycerol, (-) D-ribose, (+) D-xylose, (-) D-mannitol, (-) D-fructose, (+) D-galactose, (+) D-glucose, (+) D-mannose, sucrose and raffinose. ONPG and PNPG tests are positive. Negative in symbiotic growth, Simmons citrate, mucate-acid, malonate-base, H2S/tri-sugar iron (TSI), growth in the presence of KCN, Voges–Proskauer at 37 °C and urease.

Negative results are also observed with arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, indole, gelatinase and Tween 20 and 80. Pigment is not formed. Acid is not produced from m-erythritol, adonitol, (+)D-arabitol, xylitol, (-)L-xylose, dulcitol, (+)D- fructose, (+)L-rhamnose, (-)L-sorbose, cellobiose, (+)D-melibiose, (+)D-melezitose, (+)D-glycogen, inulin, aesculin, amygdalin, arbutin, gentiobiose, salicin, (+)D-turanose or -N-CH3–glucosamid. Reactions for p- nitrophenyl -D-glucopyranoside (NPG), o-nitrophenyl -L-fucopyranoside (ONPF), p-galactosidase, p- nitrophenyl -D-glucopyranosiduronic acid (PGUA), -mannosidase and o-nitrophenyl -D-xylanopyranoside (ONPX) are also negative. Variations are observed in the methyl red reaction at 37 °C, growth on MacConkey agar and acid production from (+)L-arabinose, (-)D-arabinose, m-inositol, (-)D-sorbitol, (-)L- fucose, lactose, maltose, trehalose and dextrin. The type species is Gallibacterium anatis and the identification characteristics are included in Table 5.10.

Table 5.10: Identification characteristics of Gallibacterium species

G. anatis bv. Gallibacterium Gallibacterium Character G. anatis bv. anatis haemolytica genomospecies 1 genomospecies 2 Haemolysis + - + + (-)D-Arabinose (+) - (+) d (+)L-Arabinose - - d d m-Inositol d d - d (-)D-Sorbitol d d - - (-)L-Fucose (+) - + d Maltose d - + + Trehalose d + + d Dextrin d - + +

Ornithobacterium rhinotracheale This organism is a Gram-negative, pleomorphic rod that grows slowly and was isolated as recently as 1991 from cases of respiratory disease in broiler chickens in South Africa. In 1994 it was classified as a new species. This organism is not only linked to air sacculitis and purulent pneumonia in broiler chickens, but also in turkeys. It affects broiler chickens of 28 days and older and is associated with high mortalities during this period. In some cases it is characterized by a subcutaneous and oedematous swelling over the cranium with a severe bacterial osteitis. Other clinical signs include weakness, gasping, severe dyspnoea, mucus discharge and poor growth. It is normally resistant to all antibiotics except tetracyclines and amoxicillin. O. rhinotracheale grows well on 5 % sheep blood agar incubated for 48 hours at 5 to 10 % CO 2 at 37 º C. They appear as grey to grey-white colonies. After primary isolation the size of the colonies may vary from 1 to 3 mm.

Seven serotypes, namely A to G have been demonstrated with agar gel precipitation tests (AGP). There appears to be some cross reactions between serotypes A, B and E and some strains do not fit into the present system. An ELISA has been developed that can detect the presence of antibodies in chickens.

Pasteurella serotyping using the haemagglutination method

Preparation of the Pasteurella multocida extracts

1. Inoculate a lawn of P. multocida on blood agar a day before the extraction is to be done and incubate overnight at 37°C.

2. Set the water bath to 60°C about 1½ hours before required.

3. Just before the water bath has reached 60°C, scrape the bacterial growth off the two blood agar plates into a thick-walled centrifuge tube containing 5 ml of PBS pH 6.0. (NB put the left over PBS in the refrigerator)

4. Seal tube with a cloth plug and place the inoculated tube in the water bath at 60°C for 30 minutes.

5. Cool slightly.

6. Add 0.2 ml hyaluronidase (in freezer) to the tube.

7. Place the extracts in the 37°C incubator for 4 hours. Shake every hour or place on a shaker.

8. Centrifuge for 1 hour at 2000 rpm.

9. Collect the supernatant with a Pasteur pipette, taking care not to collect bacteria and place it in a clean MacCartney bottle.

10. Adjust the pH to 7.0 by adding drops of 4 % sodium hydroxide (NaOH). Check with a pH meter.

11. Label and place the extract in a thick-walled centrifuge tube and store in a beaker in the freezer until required.

Preparation of the Mannheimia haemolytica extracts

1. Inoculate a lawn of M. haemolytica on blood agar a day before the extraction is to be done and incubate overnight at 37°C.

2. Switch on and set the water bath to 56°C about 1½ hours before required.

19 | P a g e 3. Just before the water bath has reached 56°C, scrape the bacterial growth off the two blood agar plates into a thick-walled centrifuge tube containing 5 ml of normal saline.

4. Seal tube with a cloth plug and place the inoculated tube in the water bath at 56°C for 30 minutes.

5. Cool the tubes and centrifuge for 1 hour at 2000 rpm.

6. Collect the supernatant with a Pasteur pipette, taking care not to collect bacteria and place it in a clean MacCartney bottle.

7. Label and place the extract in a thick-walled centrifuge tube and store in a beaker in the freezer until required.

Preparation of guinea pig red blood cells

Day before typing

1. Bleed a guinea pig from the heart using a heparin containing (green cap) vacutainer tube and 21G venoject needle.

2. Mix blood well by inverting it 10 times.

3. Divide the blood equally into two different thick-walled centrifuge tubes.

4. Centrifuge at 3000 rpm for 10 minutes.

5. Remove the plasma (clear supernatant) using a Pasteur pipette.

6. Add Normal saline and mix well by inverting the tube gently 10 times. Make sure that the red blood cells do not haemolyse.

7. Centrifuge at 3000 rpm for 10 minutes.

8. Repeat steps 5 to 7 twice more.

9. Remove the saline supernatant and place the blood cells in the refrigerator.

Day of typing

1. Label and add the following to the appropriate size measuring cylinder dependent on the number of strains to be tested.

1 strain = 0.125 ml healthy rabbit serum (HRS) + 25 ml normal saline

2 strains = 0.25 ml HRS + 50 ml normal saline

4 strains = 0.5 ml HRS + 100 ml normal saline

8 strains = 1 ml HRS + 200 ml normal saline 2. Remove the P. multocida and M. haemolytica extracts from the freezer and place in a water bath to defrost.

3. Once defrosted add 0.2 ml of the washed guinea pig red blood cells to each extract using a 1 ml syringe and pink needle (19G).

4. Mix well and place in an incubator set to 37°C for 90 minutes.

5. Using a multi-channel pipette, place 100μl of saline in each well of 96-well “U” plates (one per P. multocida and two per M. haemolytica extract)

6. Add 100 μl of the respective antiserum to the first wells labelled on the first plate 1 – 12 and on the second plate 13 – 17 (for M. haemolytica) and wells on one plate labelled A, B, C, D and E for P. multocida).

7. Using the multi-channel pipette and starting at row “A” and continuing until row “H” make serial two-fold dilutions of the antiserum. Discarding the last volume in the pipette and rinsing the tips three times in saline.

8. Stack the plates on top of one another to reduce evaporation.

9. Remove the extract together with red blood cells from the incubator.

10. Centrifuge the extract and red blood cells at 2000 rpm for 5 minutes.

11. Remove the supernatant (clear fluid).

12. Fill the tube will Normal saline and mix well.

13. Repeat steps 10 to 12, 2 more times.

14. Remove the supernatant (clear fluid).

15. Add 25 ml saline and HRS mixture to each 50 ml labelled beaker (one per strain to be tested)

16. Mix the washed red blood cells from step 14 with the saline and HRS mixture and mix well.

17. Place the mixture made in step 16 into a white container and using a multi-channel pipette add 100 μl of it into each well allocated for that strain.

18. Rinse out the tips three times in saline before adding a new strain.

19. Place the plates next to each other on white paper and leave for 1 to 2 hours then read.

21 | P a g e Agglutination is noted by the presence of an even suspension of red cells in a well and non-agglutination by a pellet at the bottom of a well.

REFERENCES

1. ANGEN, O., MUTTERS, R., CAUGANT, D.A., OLSEN, J.E., & BISGAARD, M., 1999. Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., mannheimia granulomatis comb. nov., Mannheimia glucosidal sp. nov., mannheimia ruminalis sp. nov., and mannheimia varigena sp. nov. International Journal of Systematic Bacteriology, 49:67-86.

2. PIECHULLA, K., MUTTERS, R., BURBACH, S., KLUSSMEIER, R., POHL, S. & MANNHEIM, W. 1986. Deoxyribonucleic acid relationships of “Histophilus ovis/Haemophilus somnus,” Haemophilus haemoglobinophilus, and “Actinobacillus seminis”. International Journal of Systematic Bacteriology, 36: 1-7.

3. SNEATH, P, H.A., & STEVENS, M., 1985. A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia. Journal of General Microbiology, 131:2711-2738.

4. SNEATH, P, H.A., & STEVENS, M., 1990. Actinobacillus rossi sp. nov., Actinobacillus seminis sp. nov., nom. Rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov, Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov. International Journal of Systematic Bacteriology, 40:148- 153.