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 Contents

Preface................................................................. ix 15. Non-spore-forming anaerobes...............205

Acknowledgements............................................ xi 16. Clostridium species.................................. 215
17. Enterobacteriaceae.....................................239
18. Pseudomonas, Burkholderia and
SECTION 1: General procedures Stenotrophomonas species........................275
in microbiology 19. Aeromonas, Plesiomonas and
Vibrio species...........................................289
1. Collection and submission of
20. Actinobacillus species...............................297
diagnostic specimens.................................. 3
21. Pasteurella, Mannheimia, Bibersteinia
2. Bacterial pathogens: Microscopy,
and Avibacterium species.........................307
culture and identification........................... 9
22. Francisella tularensis................................. 317
3. Serological diagnosis................................49
23. Brucella species........................................325
4. Molecular techniques in diagnostic
24. Campylobacter, Arcobacter and
microbiology . ..........................................59
Helicobacter species.................................335
5. The isolation of viruses and
25. Lawsonia intracellularis............................345
the detection of virus and .
26. Haemophilus and Histophilus
viral antigens.............................................67
species......................................................349
6. Antimicrobial agents................................79
27. Taylorella species......................................355
28. Bordetella species.....................................359
SECTION 2: Bacteriology 29. Moraxella species.....................................369
30. Glucose non-fermenting,
7. Staphylococcus species.............................. 105 Gram-negative bacteria...........................375
8. The streptococci and related cocci......... 121 31. The spirochaetes..................................... 381
9. Corynebacterium species and 32. Miscellaneous Gram-negative
Rhodococcus equi .....................................135 bacteria....................................................399
10. The Actinobacteria..................................147 33. Chlamydiales.............................................407
11. Mycobacterium species............................. 161 34. Rickettsiales and Coxiella burnetii............ 417
12. Listeria species.........................................177 35. The mycoplasmas
13. Erysipelothrix species................................187 (class: Mollicutes)...................................423
14. Bacillus species........................................195 36. Mastitis ...................................................433

vii
 Contents

56. Birnaviridae.............................................. 613

SECTION 3: Mycology 57. Flaviviridae............................................... 617
37. Introduction to the pathogenic 58. Arteriviridae..............................................629
fungi.........................................................457 59. Togaviridae................................................635
38. The dermatophytes................................. 471 60. Orthomyxoviridae......................................639
39. Aspergillus species and 61. Paramyxoviridae........................................645
Pneumocystis carinii.................................. 481 62. Coronaviridae............................................655
40. The pathogenic yeasts.............................487 63. Rhabdoviridae...........................................665
41. The dimorphic fungi..............................497 64. Bunyaviridae.............................................673
42. The pathogenic Zygomycetes...................505 65. Retroviridae...............................................679
43. Fungi causing subcutaneous 66. Bornaviridae............................................. 691
mycoses................................................... 513 67. Prions (proteinaceous infectious
44. Mycotoxins and mycotoxicoses............. 521 agents).....................................................693

SECTION 5: Zoonoses
SECTION 4: Virology
(including prions) 68. Zoonoses.................................................703

45. Parvoviridae.............................................. 541

46. Circoviridae...............................................547 SECTION 6: A systems approach
47. Papillomaviridae....................................... 551 to infectious diseases on
48. Adenoviridae.............................................555 a species basis
49. Herpesviridae............................................559
50. Asfarviridae...............................................575 69. Infectious diseases..................................735
51. Poxviridae.................................................579
52. Picornaviridae...........................................587 Appendix 1 Reagents and stains...................845
53. Caliciviridae..............................................597 Appendix 2 Culture and transport media.... 851
54. Astroviridae...............................................603
55. Reoviridae.................................................605 Index................................................................857

viii
 Preface

A lot of water has passed under the bridge since the popular first edition of this book. Needless to
say the writing of textbooks is not a full-time occupation for any of the authors but rather a com-
plementary activity to teaching, research and diagnostic service. This project was driven by a love
of books. On more than one occasion the writing faltered due to other more pressing commitments
and the vicissitudes of life in general. We have tried to retain the essential structure and character
of the first edition while updating and adding new material as necessary. In particular, the virology
section has been greatly expanded with the addition of a chapter for each of the families containing
viruses of veterinary importance. For taxonomy we have relied heavily on resources such as the List
of Prokaryotic Names with Standing in Nomenclature (www.bacterio.cict.fr/), the virus list of the
International Committee on Taxonomy of Viruses (ictvonline.org/index.asp) and Index Fungorum
(www.indexfungorum.org/). For information on the diagnosis of many of the more important
infectious diseases of farm animals we have frequently consulted the Manual of Diagnostic Tests
and Vaccines for Terrestrial Animals on the website (www.oie.int/en/international-standard-setting/
terrestrial-manual/access-online/) of the OIE (Office International des Epizooties – World Organisa-
tion for Animal Health). We hope we have succeeded in producing a book that is useful, informative
and appealing.

Dublin, 2013

ix
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 Acknowledgements

Assembling material for a book requires forward planning, attention to detail and the means to put
in place the knowledge and ideas of the authors. We were fortunate to have the resources of the
first edition to draw upon in bringing this project to fruition. We are indebted to Professor Emeritus
Joe Quinn for being our mentor and greatest supporter in this endeavour.
Veterinary microbiology lends itself to illustration and we have tried to include suitable illustrative
material in the form of colour images and line drawings. A number of colleagues supplied images
which we would like to acknowledge: Professor KP Baker (38.7), Centres for Disease Control (31.13
and 34.2), Dr SJ Cook and Professor CJ Issel (3.3, 3.16 and 3.22), Dr DO Cordes (11.14, 42.7, 42.8,
42.9 and 44.7), Professor FWG Hill (29.1), Dr L Hoffmann (31.12), Dr G Joseph (18.7, 18.8 and
22.1), Professor JF Kazda (11.5, 11.6 and 11.7), Mr Aonghus Lane (36.21 and 36.22) and Dr N
Seiranganathan (35.8).
We also wish to acknowledge our predecessors in the Dublin Vet School, particularly Mr BT
Whitty, Mr MA Gallaher, Professor PJ Quinn and Dr Margery Carter who were prominent in our
formative training and who left for posterity a wealth of illustrative material and stained smears,
many of which we have used to illustrate individual chapters. We would also like to record our
appreciation to colleagues who furnished material for photography: Dr HF Bassett (10.17), Mr MJ
Casey (68.4), Mr RP Cooney (11.12), Mr P Costigan (5.4, 5.5, 51.1 and 51.2), Dr WJC Donnelly
(12.2, 12.3 and 67.2), Mr E Fitzpatrick (41.2), Dr HA Larkin (35.2), Dr GHK Lawson (33.4), Mr JB
Power (31.8 and 31.10), Dr GR Scott (34.1 and 35.1) and Professor Emeritus BJ Sheahan (39.2).
Finally, the authors would like to record their appreciation to the publishers for their patience
and encouragement while the book was in preparation, in particular Joyce Rodenhuis, Rita Deme-
triou-Swanwick, Louisa Welch, Robert Edwards, Veronika Watkins and Clive Hewat.

xi
This page intentionally left blank
 Section 1
General procedures in microbiology

1
This page intentionally left blank
 Chapter
Collection and submission of
diagnostic specimens
Choosing and Working with
a Laboratory
In choosing a laboratory clinicians must consider the cost
of the service and the ease of access. However, it is equally
important to determine if a laboratory is accredited, has a
specialization in the relevant species, offers a service at
weekends and holidays and has staff with the necessary
expertise and training to assist with disease investigations.
The quality of the dialogue between the veterinary practice
and the laboratory has an enormous influence on the
effectiveness of the service. Good communication is essen-
tial. The clinician and the microbiologist must establish
and maintain contact with each other. An accurate diag-
nosis is based on the interpretation of both clinical and
laboratory data.
If the clinician is not absolutely certain of the correct
sample to collect they should seek advice from the labora-
tory prior to collecting the samples. The samples must be
appropriate for the purpose required which may be diag-
nosis, certification, surveillance or the monitoring of
vaccine efficacy or the response to antimicrobial therapy.
A clinical diagnosis cannot be confirmed by the examina-
tion of inappropriate samples. In disease control situa-
tions samples must be collected systematically for a
definite purpose, for example, the lifting of movement
restrictions or the commencement of breeding. Laboratory
tests can be both expensive and time-consuming. It is
important for all concerned that resources are not wasted
in the generation of irrelevant information.
Both the veterinary practitioner and the laboratory have
an essential role in the isolation of new agents and the
detection of emerging disease patterns. In influenza out-
breaks, for example, the submission of samples to the
laboratory for virus isolation is necessary to identify new
© 2013 Elsevier Ltd
1
strains of viruses. This facilitates the updating of vaccines
with epidemiologically relevant viruses.
Some laboratories are involved in contract research and
perform safety and efficacy studies for the regulatory
authorities. All diagnostic laboratories have a role to play
in the monitoring of the efficacy of existing products. Cli-
nicians have a responsibility to inform the microbiologist
of the vaccination and/or therapeutic history of the animal
to facilitate independent assessment of different products
and treatment regimens. Microbiologists in turn have a
responsibility to advise clinicians with regard to their
findings.
General Principles for
Sample Collection
• Samples should be taken from the affected site(s) as
early as possible following the onset of clinical signs.
This is particularly important in viral diseases as
shedding of virus is usually maximal early in the
infection. This is also true of enteric bacterial
pathogens.
• It is useful to collect samples from clinical cases and
in-contact animals, particularly if there has been
an outbreak of disease. In-contact animals may
be at an earlier stage in the infection with a greater
chance of them shedding substantial numbers of
microorganisms.
• Samples should be obtained from the edge of lesions
and some macroscopically normal tissue included.
Microbial replication will be most active at the
lesion’s edge.
• It is important to collect specimens as aseptically as
possible, otherwise the relevant pathogen may be
overgrown by numerous contaminating bacteria
3
Section | 1 | General Procedures in Microbiology
Figure 1.1 Grossly contaminated blood agar plate
demonstrating the need to collect specimens as carefully as
possible.
(Fig. 1.1). In certain circumstances a guarded swab
should be used to bypass an area with a large
population of normal flora.
• The laboratory should be informed if treatment has
commenced in order that counteractive measures
may be taken to increase the possibility of isolating
bacteria or that an alternative method of detection
such as polymerase chain reaction (PCR) may be
employed.
• When possible a generous amount of sample should
be taken and submitted, such as blocks of tissue
(approximately 2 cm3), biopsy material, or several
millilitres of pus, exudate or faeces. For serology, at
least 5 mL of blood should be obtained to allow a
number of tests to be carried out if necessary and to
allow the sample to be stored and tested with
subsequent samples.
• Cross-contamination between samples must be
avoided. This is essential where a highly sensitive
amplification technique such as PCR is to be used
for the detection of the aetiological agent.
• Precautions must be taken to avoid human infection
where a zoonotic condition is suspected.
Tissue
Postmortem material should be collected as soon as pos-
sible after death. However it is sometimes worthwhile
submitting old and even partially decomposed samples if
that is all that is available. In the outbreak of African horse
sickness in the Iberian Peninsula in the late 1980s virus
was isolated from the bone marrow of a horse that had
been buried for eight days. An organism does not have to
be viable or even entire for its genome to be detected by
polymerase chain reaction. Thus it may be possible to
obtain a diagnosis by PCR from a sample that is totally
unsuitable for histopathological examination or agent
isolation.
4
Figure 1.2 Sterile disposable containers and swabs for
specimen collection.
Tissues from outside the body cavities should be col-
lected first followed by tissues from the thorax and then
the abdomen. Sterile instruments should be used to collect
tissue samples of at least 1 cm3 which should then be
placed in separate sterile screw-capped jars (Fig. 1.2). If
the laboratory is some distance away tissue may be for-
warded in virus or bacterial transport medium. It is impor-
tant to remember that virus transport medium usually
contains antibiotics thus rendering the sample unsuitable
for bacterial examination. Tissue for histopathological
examination should be placed in at least 10 times its
volume of neutral buffered 10% formalin.
In cases of abortion the whole foetus and placenta
should be submitted. If this is not possible then samples
of tissue, a piece of affected placenta, foetal abomasal
contents (ruminants) and uterine discharge (if applicable)
should be forwarded to the laboratory. A clotted blood
sample from the dam for serological examination may
yield additional information.
Swabs and discharges
Fluids are preferable to swabs as the greater sample volume
increases the likelihood of detecting the causal organism.
Samples for agent isolation should be placed in sterile
containers. Viruses and many bacteria are susceptible to
desiccation especially if collected on a dry swab. Formulae
for suitable transport media for viruses, chlamydia and
other organisms are given in Appendix 2. Whenever pos-
sible the sample should be collected from the specific site
of infection. The usual short cotton wool swabs are gener-
ally unsatisfactory for obtaining nasopharyngeal speci-
mens of epithelial cells and mucus for the investigation of
respiratory disease of large animals. Guarded swabs are
necessary for certain bacteriological examinations where
misleading results could be generated due to contami-
nants from adjacent sites that are colonized with bacterial
flora. Similarly, fungal organisms from the environment
 Collection and submission of diagnostic specimens
readily contaminate the nasal passages and upper trachea.
The diagnostic laboratory should be consulted before
collecting samples for the isolation of specific pathogens
that require specialized media or culture conditions, for
example, Taylorella equigenitalis, Chlamydophila psittaci or
Mycoplasma species. The laboratory will either supply spe-
cialist swabs and transport media or recommend a repu-
table source, as appropriate.
Samples from skin lesions
If intact pustules or vesicles are present, the surface
should be disinfected with 70% ethyl alcohol, allowed to
dry, and material aspirated from the lesion with a sterile
syringe and fine needle. A swab may be taken from the
raw surface of ulcers. A biopsy of wound tissue should be
collected after the superficial area has been cleaned and
debrided.
In cases where ringworm is suspected, hair should be
plucked from the lesion and the edge of the lesion scraped
with a blunt scalpel blade until blood begins to ooze.
Plucked hair, skin scrapings (including the scalpel blade
itself) and any scab material that is present should be
submitted. These specimens will also allow detection of
mange or a bacterial infection, if present. In cases of orf
the crust and scrapings from the edge of the lesion should
be collected. In birds feather follicle skin is useful in the
diagnosis of Marek’s disease.
Blood
Blood should be withdrawn using an aseptic technique
into a dry syringe or vacutainer. If collected in a syringe,
care must be taken not to cause haemolysis. The needle
should be removed prior to expelling the sample carefully
into a sterile dry tube. It is not acceptable to submit blood
to the laboratory in a syringe. Glass tubes are fragile.
However, blood clots often retract poorly in plastic bottles
making it difficult to separate the serum. Whole blood
should never be frozen prior to submission to a laboratory
as the ensuing lysis of red blood cells makes it impossible
to perform many serological assays.
Serological tests are usually performed on the serum
harvested from clotted samples. Paired samples are fre-
quently required to make a definitive diagnosis on the
basis of serology. It is advisable to confirm the most appro-
priate sampling interval with the microbiologist who
should also advise as to usefulness of a single blood
sample.
Blood for the isolation of viruses or bacteria should be
prevented from clotting. The laboratory should advise on
the most appropriate anticoagulant. As a bacteraemia can
be intermittent, it is advisable to take more than one
sample within a 24-hour period. The blood should be
added aseptically and without delay to one of the special
commercial blood-culture bottles. Blood samples for
Chapter |1|
culture should be kept cool and submitted to the labora-
tory as quickly as possible.
Faeces
A faeces sample freshly voided or collected from the
rectum is preferable to a rectal swab which often does not
have enough faecal matter for agent detection. A faeces
sample (about the size of the end of a thumb) may be
forwarded to the laboratory without transport medium.
Faecal swabs should be placed in medium such as buffered
glycerol saline to avoid desiccation. Some organisms are
shed intermittently and samples may need to be collected
over several days.
Urine samples
Urine samples may be submitted for urinalysis, bacterial
microscopy and culture or for a bacterial viable count to
establish whether clinical bacteriuria is present. For bac­
teriological procedures the preferred methods of collec-
tion are by cystocentesis, by catheter or mid-stream urine
sample.
Abscesses
If possible about 3 mL of pus should be collected together
with scrapings from the wall of the abscess. Pus at the
centre of an abscess is often sterile. Pus from recently
formed abscesses will yield the best cultural results. Anaer-
obic bacteria can often be cultured from abscesses.
Eye
A conjunctival swab may be taken gently holding the
palpebrae apart. Scrapings may also be taken with a fine
sterile spatula. The cells should be washed carefully into
transport medium.
Bovine mastitic milk samples
Milk samples should be collected from cows as soon as
possible after the mastitis is first noticed and not from
animals treated with either intramammary or systemic
antibiotics. The udder should not be rinsed with water
unless very dirty. If the udder and teats are washed, they
should be dried thoroughly with a paper towel. Usually
it is sufficient to wipe the teats vigorously, using 70%
ethyl alcohol on cotton wool, paying special attention
to the teat sphincters. Antiseptics should be avoided. The
two teats furthest from the operator are wiped first and
then the two nearest teats. The sterile narrow-necked col-
lection bottle must be held almost horizontally. The first
squirt of milk from each teat is discarded and then, for a
composite sample, a little milk from each quarter is
5
Section | 1 | General Procedures in Microbiology
directed into the bottle. The milk should be collected from
the two near teats first and then from the two far teats, so
that one’s arm is less likely to accidentally brush against a
cleaned teat.
Specimens for anaerobic culture
A good collection method is essential because many anaer-
obes do not survive frank exposure to the oxygen in the
air for more than 20 minutes. It is important not to con-
taminate the samples by contact with adjacent mucosal
surfaces as these have a resident anaerobic flora. Speci-
mens from animals that have been dead for more than
four hours are usually unsuitable because of the rapid
postmortem invasion of the animal body by anaerobes
from the intestinal tract. Bone marrow is a good specimen
to collect for the diagnosis of blackleg or malignant
oedema as bone marrow appears to be one of the last
tissues to be invaded by contaminating bacteria. A piece
of rib stripped of the periosteum could be submitted to
the laboratory for the extraction of bone marrow. Any
specimens for the attempted isolation of anaerobes must
arrive at the laboratory as soon as possible after collection.
Collection of samples for anaerobe culture on ordinary
swabs is usually of no value. Acceptable samples include
blocks of tissue (4 cm3) or several millilitres of fluid
placed in a sterile closed container. Anaerobic transport
medium is essential for swabs.
In suspected enterotoxaemia cases, where the demon-
stration of a specific toxin is required, at least 20 mL of
ileal contents should be submitted. A loop of ileum with
contents, tied off at each end, is acceptable or the ileal
contents drained into a secure sterile container.
Sample Submission
Laboratories usually supply a variety of sample containers
and transport media. The laboratory should supply sample
submission forms. These forms must be completed by the
veterinary practitioner and must give specific details of the
tests required as well as clinical history, differential diag-
noses, vaccination history, therapy, age of animal, etc. The
latter will enable the microbiologist to choose the most
appropriate tests and to avoid unnecessary expenditure. A
complete history will also allow the laboratory to identify
a particularly urgent situation and expedite the processing
of the sample. In certain instances owners may be con-
cerned that information in relation to their animals is kept
confidential. In such circumstances the clinician can assign
a code name or number to the animal and the owner but
should never omit clinical detail that will facilitate the
selection of the appropriate tests and the interpretation of
the results. The laboratory must be notified if the differen-
tial diagnosis includes a fungal infection or any agent that
is potentially infectious for people.
6
Many laboratories only set up certain tests on particular
days or at a designated time each day. Clinicians need to
familiarize themselves with the laboratory timetable in
order for them to provide an efficient service to their own
clients. An awareness of the time it takes to perform certain
assays is essential. Some assays may take weeks to com-
plete. Where certification is required, samples should be
submitted in good time and allowance should be made
for repeat testing and/or the collection of a second sample
if necessary.
The clinician should contact the laboratory in advance
if they are not a regular client or if the tests required are
not routine. It is essential to give the laboratory adequate
time to prepare for the receipt of a sample which requires
specialist testing. It is inadvisable, for example, to submit
a sample that needs to be passaged on a cell line that the
laboratory does not use on a regular basis without prior
discussion. In such circumstances the sample may have to
be stored for days if not weeks, while cells are resuscitated
from the freezer. In certain cases it may be necessary to
forward the sample to a specialist laboratory. If samples
are being submitted to a laboratory in another country an
import licence may be required.
Samples should be collected and delivered to the labo-
ratory as early in the day as possible so that processing can
commence on the same day. If a result is required urgently
this must be indicated on the request form and the head
of the laboratory should be notified in advance of the
arrival of the sample. Prompt delivery to the laboratory
will maximize the possibility of obtaining a diagnosis.
Viruses only replicate in living cells and a delay in sample
submission may result in loss of viability. The transit time
needs to be minimized. Samples should be transported in
contact with cold packs or wet ice. If transportation to the
laboratory is delayed, most samples should be held in the
refrigerator at +4°C rather than frozen. Serum harvested
from clotted blood samples can be stored frozen for
extended periods.
The labelling of samples must be clear and unambigu-
ous. Samples must be submitted individually in separate
leak-proof containers that are clearly marked, indicating
the identity of the sample (tissue, exudate, etc.), animal
identification and the date of collection (Fig. 1.3). Con-
tainer caps should be screwed on tightly and taped, if
necessary, to avoid leakage. All samples sent in the post
must be labelled and packed in accordance with the regu-
lations of the postal authorities. Glass tubes and other
fragile containers must be adequately packaged to ensure
they are not broken in the mail. Samples must always be
surrounded by sufficient absorbent material to soak up the
entire sample in the case of breakage or leakage. In general,
triple packaging of diagnostic samples is required. The
secondary or outer packaging must be a rigid container.
The package should be clearly labelled with the words
‘biological substances’.
 Collection and submission of diagnostic specimens
Figure 1.3 Sturdy, leak-proof containers for transporting
microbiological specimens.
Interpretation of Diagnostic Results
There must be full co-operation between the laboratory
and the veterinary practice for the benefit of the patient
and owner. It is the responsibility of the clinician to collect
and submit the appropriate samples accompanied by spe-
cific requests or adequate history. It is the responsibility of
the microbiologist to interpret the results in relation to the
information supplied. The quality of the diagnostic activ-
ity undertaken by the laboratory depends to a large degree
on the clinical and epidemiological information supplied
by the veterinary clinician.
The following points are pertinent when interpreting
reports from a diagnostic microbiology laboratory:
• A negative diagnostic report does not necessarily
mean that the suspected microorganism is not the
aetiological agent of the condition. There may be
many reasons for the failure of the laboratory to
isolate and identify the pathogen, such as a
bacterium being overgrown by contaminants, a
virus or other fragile microorganism having died on
the way to the laboratory or the animal may have
stopped excreting the microorganisms before the
sample was taken. Unexpected negative results
should be discussed with the head of the laboratory
who may decide to perform additional tests for the
detection of the suspect agent. They may also be able
to advise on the collection of alternative samples or
suggest other possible causal agents.
• It must not always be assumed that the detection
of a virus or bacteria establishes a diagnosis. The
laboratory result must be interpreted in light of the
clinical signs, vaccinal history, etc. If the sample is
collected from a site normally colonized with
bacteria, including species that have the potential to
Chapter |1|
cause disease in certain circumstances, the culture
results need to be interpreted cautiously. Some latent
viruses may reactivate and bacteria proliferate in
response to a condition which they have not caused.
Apparently healthy animals can be subclinical
shedders of microorganisms such as salmonellae,
rotaviruses, enteroviruses or coronaviruses in faeces
and leptospires in urine. Even if these potential
pathogens are isolated and identified, the illness
or death might have been due to another cause.
• The clinician should always discuss unexpected
results with the laboratory. This may assist the head
of the laboratory in the early detection of a problem
with a particular technique. The microbiologist must
be prepared at all times to act on information
received and to critically appraise the procedures in
the laboratory.
• It is important that the difficulties associated with
some types of samples and the limitations of certain
assays are flagged to the clinician. This will help to
promote intelligent utilization of the laboratory and
to avoid unrealistic expectations.
• Bacteria such as members of the Enterobacteriaceae
are ubiquitous. Isolation of microorganisms may
represent contamination of the sample by faeces or
soil or their presence could be due to postmortem
invasion.
• Some bacteria such as salmonellae, leptospires or
Mycobacterium avium subsp. paratuberculosis may be
shed intermittently. A repeat sample following a
negative examination report might be worthwhile.
• Escherichia coli in diarrhoeal faecal samples from
farm animals is usually only significant if the animal
is under 10 days of age and if the E. coli isolate
possesses the K88, K99, F41 or 987P fimbrial
antigens, associated with enteropathogenicity. Pigs
are the exception as they are also susceptible to
colibacillosis soon after weaning and to oedema
disease in the growing period.
• Diagnosis of a mycotic disease thought to be due
to a ubiquitous fungus such as Aspergillus fumigatus
should always be confirmed histopathologically. It is
necessary to demonstrate the fungal hyphae actually
invading the tissue and causing a tissue reaction.
• Herd or flock vaccination programmes will modify
the interpretation of serological tests. Serological
diagnosis of recent viral exposure usually requires
the comparison of two samples collected
approximately two weeks apart.
• Conflicting results may reflect the sensitivity of
different assays. Thus a sample that is negative by
enzyme-linked immunosorbent assay (ELISA) and by
isolation may be positive by PCR.
7
This page intentionally left blank
 Chapter
Bacterial pathogens: Microscopy, culture
and identification
MICROSCOPY
A good-quality microscope with a built-in light source
(bright-field microscopy) as well as low-power, high-dry
and oil-immersion objectives is required. A darkfield con-
denser is a useful addition necessary for the visualization
of unstained preparations such as those of spirochaetes.
Microscopes for microbiology require a higher degree of
resolution than those used for haematology or histopa-
thology. Figure 2.1 indicates the size of bacteria relative to
that of an erythrocyte and to viruses. Bacteria are measured
in micrometres (µm) which are 10−6 m while viruses are
measured in nanometres (nm) which are 10−9 m. Using an
oil-immersion objective lens on a light microsope, a mag-
nification of 1000 × can be achieved to visualize bacteria.
On account of their small size viruses are visualized by
using an electron microscope, which utilizes a beam
of electrons to achieve magnifications of the order of
100,000 ×.
Stained Smears from
Pathological Specimens
Stained smears made from lesions can yield a considerable
amount of information inexpensively and quickly. Table
2.1 summarizes the information that can be gained from
the various diagnostic staining techniques.
Preparing Bacterial Smears
Microscope slides are not always clean enough to use
directly from the supplier. Rubbing with a clean, soft cloth
© 2013 Elsevier Ltd
2
and passing quickly through the Bunsen flame may be
sufficient to remove a greasy film. If not, a mildly abrasive
liquid cleaner can be used followed by rinsing the slide
thoroughly and wiping it dry with a clean cloth.
A blunt scalpel and forceps should be kept in a con-
tainer of 70% ethyl alcohol. The instruments are flamed
and cooled before use. Afterwards they should be placed
into a container of disinfectant. When making a smear
from tissue lesions, the specimen is held firmly with
the forceps and the scalpel is used to scrape deep into the
material. A small amount of the scrapings is placed on
the cleaned microscope slide. Another clean slide is used
with a scissor action to prepare a thin smear. With liquid
or semiliquid specimens, a little of the sample is placed
on the slide with a sterile swab. The contents of the swab
are smeared over the surface of the slide, with the aim of
having thick and thin areas of specimen present. The
smears are allowed to dry thoroughly before proceeding
further.
Fixing the Smears
The reasons for fixing the smears include killing the veg-
etative bacteria, rendering them permeable to the stain
and ensuring that the material is firmly fixed to the slide.
Fixed and stained smears should be handled carefully as
not all bacteria, especially endospores, may have been
killed. After use, the stained smears should be autoclaved
or soaked in a reliable disinfectant (24–48 hours) before
discarding.
For routine staining the smears are fixed by passing the
slide, smear side up, quickly through the Bunsen flame
two or three times, taking care not to overheat the smear.
This can be tested on the back of the hand; the slide
should feel warm but not hot enough to burn.
9
Section | 1 | General Procedures in Microbiology

Light microscope Electron microscope

Poxvirus
10 µm 1 µm 100 nm
Staphylococcus
Adenovirus

Parvovirus

Brucella

Chlamydia
Red blood cell

Figure 2.1 Comparison of the relative sizes of a red blood cell, bacteria and viruses.

Table 2.1 Diagnostic uses of stained smears

Disease and species Specimen Pathogens Appearance in stained

affected smears

Gram stain
Abscesses or suppurative Pus or exudate Staphylococcus species Gram + cocci, often in clumps
conditions
Many animal species Streptococcus species Gram + cocci, usually in
chains
Trueperella pyogenes Gram + rods, pleomorphic
Corynebacterium Gram + rods
pseudotuberculosis
Pseudomonas aeruginosa Gram − rods
Pasteurella multocida Gram − rods
Fusobacterium Gram −, long, slender
necrophorum filaments, often staining
irregularly
Strangles Pus Streptococcus equi Gram + cocci, often in chains
Horses subspecies equi
Suppurative broncho- Pus Rhodococcus equi Gram + rods with tendency to
pneumonia or superficial coccal forms
abscesses
Foals and young horses

10
 Bacterial pathogens: Microscopy, culture and identification
Table 2.1 Diagnostic uses of stained smears—cont’d
Disease and species
affected
Dermatophilosis
(Streptothricosis)
Many animals, mainly in sheep,
cattle and horses
Canine nocardiosis and
canine actinomycosis
Bovine actinobacillosis
(wooden tongue)
Bovine actinomycosis
(lumpy jaw)
Clostridial enterotoxaemia
Sheep and calves mainly
Urinary tract infections
Dogs and cats
Dilute carbol fuchsin (simple) stain
Campylobacter infections
Infertility in cattle. Abortion in
sheep and cattle
Swine dysentery
Foot rot in sheep
Modified Ziehl–Neelsen (MZN) stain
Brucellosis in cattle, sheep,
pigs and dogs
Nocardiosis
Canine nocardiosis
Bovine mastitis
Chlamydial infections
Sheep and cattle: abortion
Lambs, calves and pigs:
polyarthritis
Cats: feline pneumonitis
Chapter |2|
Specimen Pathogens Appearance in stained
smears
Scabs Dermatophilus Gram +, filamentous and
congolensis branching with coccal
zoospores arranged two or
more across
Pus or thoracic aspirates Nocardia asteroides Both Gram +, filamentous
Actinomyces viscosus and branching, Nocardia spp.
MZN +
Granules from pus Actinobacillus lignieresii Gram − rods
Actinomyces bovis Gram +, filamentous and
branching
Scrapings from small Clostridium perfringens Gram +, fat rods. Large
intestine of recently numbers suggestive of
dead animal enterotoxaemia
Fresh, carefully collected Escherichia coli, Proteus Gram − rods
urine species, Staphylococcus Gram + cocci
species and Enterococcus Gram + cocci
faecalis
Vaginal mucus or foetal Campylobacter fetus Curved rods that can be in
stomach contents chains giving ‘seagull’ forms
Faeces or scrapings from Brachyspira Numerous long (7.0 µm) but
colon hyodysenteriae finely spiralled bacteria
Exudate from hoof Dichelobacter nodosus Rods with a knob on one or
both ends
Fusobacterium Long, slender filaments,
necrophorum staining irregularly
Foetal stomach contents, Brucella spp. Small, red coccobacilli in
vaginal discharge, clumps
placenta
Pus and aspirates Nocardia asteroides Long-branching filaments,
Sediment from many staining bright red
centrifuged milk
Small, red coccobacilli in
Cotyledons Chlamydophila abortus clumps. Similar to brucellae
Joint fluid Chlamydophila pecorum Small, red coccobacilli
Conjunctival scrapings Chlamydophila felis
Continued
11
Section | 1 | General Procedures in Microbiology
Table 2.1 Diagnostic uses of stained smears—cont’d
Disease and species
affected
Ziehl–Neelson (acid-fast-) stain
Tuberculosis
Cattle and other species
Poultry, other avian species and
cervical lymph nodes of pigs
Feline leprosy
Paratuberculosis (Johne’s
disease) in cattle and sheep
Giemsa stain
Anthrax in cattle, sheep
and pigs
Feline infectious anaemia
Avian spirochaetosis
Dermatophilosis
(Streptothricosis) Many
animals, mainly sheep, cattle
and horses
Polychrome methylene blue
Anthrax in cattle, sheep
and pigs
Wet preparations
Leptospirosis in many
animal species
Ringworm in many animal
species
Other suspected fungal
infections in many animal
species
Bovine trichomoniasis
12
Specimen
Suspect lesions
Suspect lesions
Scrapings from lesions
Faeces, smear from
ileocaecal valve area and
mesenteric lymph nodes
Blood smear from ear
vein or fluid from
peritoneal cavity (pigs)
Thin blood smear
Blood smear taken during
febrile period
Scabs
Thin blood smear from
ear or tail vein. Fluid from
peritoneal cavity (pigs)
Centrifuged deposit of
urine or kidney tissue
Hair and skin scrapings in
20% KOH
Tissue, exudates, biopsies
in 20% KOH
Purulent uterine discharge
in saline. Keep warm and
examine within one hour
of collection
Pathogens Appearance in stained
smears
Mycobacterium bovis Long, thin, bright red rods,
can appear beaded. Usually
not numerous in smears
Mycobacterium avium As above but larger numbers of
acid-fast rods usually present
Mycobacterium Large numbers of red-staining,
lepraemurium acid-fast rods present
Mycobacterium avium Fairly short, red, acid-fast rods
subspecies in clumps
paratuberculosis
Bacillus anthracis Purplish, square-ended rods in
short chains surrounded by a
reddish-mauve capsule
Mycoplasma haemofelis Small, dark-blue coccal forms
on red blood cells
Borrelia anserina Helical bacteria, 8–20 µm long
and 0.2–0.5 µm wide with
five to eight spirals
Dermatophilus Blue, filamentous and
congolensis branching with coccal
zoospores arranged two or
more across
Bacillus anthracis Blue, square-ended rods in
short chains surrounded by a
pinkish-red capsule
Leptospira serovars Helical, approx.15 µm long
and 0.15 µm wide. Appears
beaded with hooked ends.
Dark field microscopy
Microsporum and Chains of refractile, round
Trichophyton spp. arthrospores on hairs.
High-dry objective
Aspergillus fumigatus, Fungal mycelial elements or
Candida albicans and budding yeast cells. High-dry
others objective
Trichomonas foetus Protozoan agent with three
free flagella and an
undulating membrane. Low/
high-dry objectives
Another random document with
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unintentionally sunk as the result of a cannon shot, and spot and red sailed
into harbour. With Thomas's miss I scored eleven. Unfortunately, off my
next stroke, Thomas again went down.

"Billiards," he said.

"You don't think I want to put the rotten thing down, do you? It's such a
blessed rabbit. Directly it sees a hole anywhere it makes for it. Hallo, six
more. I shall now give what they call a miss in baulk."

"Oh, good miss," cried Myra, as spot rested over the middle pocket.

"That was a googly. You both thought it would break the other way."

The game went on slowly. When Thomas was ninety and I was ninety-
nine, there was a confused noise without, and Archie and Miss Blair burst
into the room. At least only Archie actually burst; Miss Blair entered
sedately.

"Who's winning?" cried Archie.

"What an absurd question," I said. "As if we should tell you."

"All right. Dahl—Miss Blair, have you ever seen billiards played really
well?"

"Never."

"Then now's your chance. Ninety, ninety-nine—they've only just begun.

This is Thomas's first break, I expect. There—he's got a clear board. You
get five extra for that, and the other man is rubiconed. Ninety-nine all. Now,
it is only a question of who misses first."

I put down my cue.

"Thomas," I began, "we have said some hard things about each other to-
night, but when I listen to Archie I feel very friendly towards you."

"Archibald," said Thomas, "is a beastly name."

 "So I told Miss Blair. For a man who was, so to speak, born with a
silver billiard-table in his mouth to come here and make fun of two
persevering and, in my case, promising players, is——"

"You'll never finish that sentence," said Myra. "Try some more
billiards."

"It was almost impossible to say what I wanted to say grammatically," I

answered, and I hit my ball very hard up the table at the white.

"It's working across," said Archie, after the second bounce; "it must hit
the red soon. I give it three more laps."

"It's going much more slowly now," said Miss Blair.

"Probably it's keeping a bit of a sprint for the finish. Wait till it gets its
second wind. No, I'm afraid it's no good; it ought to have started sooner.
Hallo, yes, it's—— Got him!"

"It hasn't finished yet," I said calmly. "Look—there!"

"Jove!" said Archie, shaking my hand, "that's the longest loser I've ever
seen. My dear old man, what a performer. The practice you must have had.
The years you must have devoted to the game. I wonder—could you
possibly spare an hour or two to-morrow to play cricket for us?"

CHAPTER IV

A FEW WIRES

A hundred and eighty for none. The umpire waved his lily hand, and the
scorer entered one more "four" in his book. Seeing that the ball had gone
right through a bicycle which was leaning up against the pavilion, many
people (the owner of the bicycle, anyhow) must have felt that the actual
signalling of a boundary was unnecessary; but our umpire is a stickler for
the etiquette of the game. Once when—— But no, on second thoughts, I
sha'n't tell you that story. You would say it was a lie—as indeed it is.

"Rotten," said Archie to me, as we crossed over. (A good captain always

confides in his wicket-keeper.)

"Don't take Simpson off," I said. "I like watching him."

"I shall go on again myself soon."

"Oh, it's not so bad as that. Don't lose heart."

The score was two hundred when we met again.

"I once read a book by a lady," I said, "in which the hero started the
over with his right hand and finished it with his left. I suppose Simpson
couldn't do that?"

"He's a darned rotten bowler, anyway."

"His direction is all right, but his metre is so irregular."

At the end of the next over, "What shall I do?" asked Archie in despair.

"Put the wicket-keeper on," I said at once.

The idea was quite a new one to him. He considered it for a moment.

"Can you bowl?" he said at last.

"No."

"Then what on earth——"

"Look here; you've tried 'em with people who can bowl, and they've
made two hundred and twenty in an hour and a half; somebody who can't
bowl will be a little change for them. That's one reason. The second is that
we shall all have a bit of a rest while I'm taking my things off. The third is
that I bet Myra a shilling——"

Archie knelt down, and began to unbuckle my pads. "I'll 'keep' myself,"
he said. "Are you fast or slow?"

"I haven't the faintest idea. Just as it occurs to me at the moment, I

expect."

"Well, you're quite right; you can't be worse than some of us. Will you
have a few balls down first?"

"No, thanks; I should like to come as a surprise to them."

"Well, pitch 'em up anyhow."

"I shall probably vary my length—if possible without any alteration of

action."

I am now approaching the incredible. The gentle reader, however, must

not be nasty about it; he should at least pretend to believe, and his best way
of doing this is to listen very silently to what follows. When he has heard
my explanation I shall assume that he understands.

Bowling is entirely a question of when you let go of the ball. If you let
go too soon the result is a wide over the batsman's head; if too late, a nasty
crack on your own foot. Obviously there are spaces in between. By the law
of averages one must let go at the right moment at least once. Why not then
at the first ball? And in the case of a person like myself, who has a very
high action and a good mouth—I mean who has a very high delivery, such a
ball (after a week of Simpsons and Archies) would be almost unplayable.

Very well then; I did let go at the right moment, but, unfortunately, I
took off from the wrong crease. The umpire's cry of "No-ball" and the
shattering of the Quidnunc's wicket occurred simultaneously.

"Good ball," said Archie. "Oh, bad luck!"

 I tried to look as though, on the whole, I preferred it that way—as being
ultimately more likely to inspire terror in the batsman at my end. Certainly,
it gave me confidence; made me over-confident in fact, so that I held on to
the next ball much too long, and it started bouncing almost at once.

The Quidnunc, who was convinced by this that he had been merely
having a go at the previous ball, shouldered his bat and sneered at it. He
was still sneering when it came in very quickly, and took the bottom of the
leg stump. (Finger spin, chiefly.)

Archie walked up slowly, and gazed at me.

"Well?" I said jauntily.

"No, don't speak. I just want to look, and look, and look. It's wonderful.
No elastic up the sleeve, or anything."

"This is where it first pitched," said the Major, as he examined the

ground.

"Did you think of letting in a brass tablet?" I inquired shortly.

"He is quite a young man," went on Archie dreamily, "and does not care
to speak about his plans for the future. But he is of opinion that——"

"Break, break, break," said Simpson. "Three altogether."

"Look here, is there anybody else who wants to say anything? No? Then
I'll go on with my over."

Archie, who had begun to walk back to his place, returned thoughtfully
to me.

"I just wanted to say, old chap, that if you're writing home to-night
about it, you might remember me to your people."

Blair was about the only person who didn't insult me. This was because
he had been fielding long-on; and as soon as the wicket fell he moved round
about fifty yards to talk to Miss Fortescue. What people can see in her——
Well, directly my next ball was bowled he started running as hard as he
could to square leg, and brought off one of the finest catches I've ever seen.

"The old square-leg trap," said Archie. "But you cut it rather fine, didn't
you? I suppose you knew he was a sprinter?"

"I didn't cut it at all—I was bowling. Go away."

Yes, I confess it. I did the hat trick. It was a good length half-volley, and
the batsman, who had watched my first three balls, was palpably nervous.
Archie walked round and round me in silence for some time, and then went
over to Thomas.

"He's playing tennis with me this evening," he began.

"I was beaten at billiards by him last night," said Thomas proudly.

"He's going to let me call him by his Christian name."

"They say he's an awfully good chap when you know him," replied
Thomas.

I got another wicket with the last ball of the over, and then we had
lunch. Myra was smiling all over her face when we came in, but beyond a
"Well bowled, Walter" (which I believe to be Brearley's name), would have
nothing to do with me. Instead she seized Archie, and talked long and
eagerly to him. And they both laughed a good deal.

"Arkwright," I heard Archie say at the end. "He's sure to be there, and
would do it like a shot."

Like a wise captain Archie did not put me on after lunch, and Simpson
soon began to have the tail in difficulties. Just after the eighth wicket fell a
telegram came out. Archie took it and handed it to me. "From Maclaren, I
expect," he said with a grin.

"You funny ass; I happen to know it's from Dick. I asked him for a wire
about the Kent match."
 "Oh, did Kent win?" said Archie, looking over my shoulder. As I
opened it, the others came up, and I read—

"Please be in attendance for next Test Match."

"HAWKE"

I got three more that afternoon. One from Fry, one from Leveson-
Gower, and one from Maclaren. They all came from Lord's, and I've half a
mind to take my telegrams with me, and go. Then Myra would probably get
six months in the second division.

"But I shouldn't mind that," said Myra. "You could easily bowl—I mean
bail—me out."

A silly joke, I call it.

CHAPTER V

AT PLAY

I selected a handkerchief, gave a last look at the weather, which was

beastly, and went down (very late) to breakfast. As I opened the door there
was a sudden hush. Everybody looked eagerly at me. Then Miss Fortescue
tittered.

Well, you know how one feels when that happens. I put my hand
quickly to my tie—it was still there. I squinted down my nose, but there
was no smut. To make quite sure I went over to the glass. Then Simpson
exploded.
 Yet nobody spoke. They all sat there watching me, and at last I began to
get nervous. I opened my mouth to say "Good-morning," but before I got it
out Miss Blair gave a little shriek of excitement. That upset me altogether. I
walked up to the tea-pot, and pouring myself out a cup said, with
exaggerated carelessness, "Rotten day, isn't it?"

And then came the laughter—shout after shout.

I held out my hand to Myra. "Good-bye," I said, "I'm going home.

Thank you for a very jolly time, but I'm not going to be bullied."

"Oh, you dear," she gurgled.

"I am rather sweet before breakfast," I admitted, "but how——"

"It was too heavenly of you. I never thought you would."

"I think I shall go back to bed."

"It was rather rough luck," said Archie, "but of course the later you are
the worse it is for you."

"And the higher the fewer. Quite so. If this is from Breakfast Table
Topics in The Daily Mirror, I haven't seen them to-day; but I'll do my best."

"Archie, explain."

Archie took up a piece of paper from the table, and explained. "It's like
this," he said. "I came down first and looked at the weather, and said——"

"Anyone would," I put in quickly.

"Well, then, Blair came in and said, 'Beastly day,' and then Simpson
—— Well, I thought I'd write down everybody's first remark, to see if
anybody let the weather alone. Here they are."

"It's awful," put in Myra, "to have one's remarks taken down straight
off. I've quite forgotten what I said."
 This was the list:

Archie: "Bother." (So he says.)

Blair: "What a beastly day!"

Simpson: "What a jolly day!"

The Major: "Well, not much cricket to-day, hey?"

Myra: "Oh dear, what a day!"

Miss Blair: "What a terrible day!"

Miss Fortescue: "Oh, you poor men—what a day!"

Thomas: "Rotten day, isn't it?"

Me: "Rotten day, isn't it?"

"I don't think much of Thomas's remark," I said.

Later on in the morning we met (all except the Major, that is) in the
room which Myra calls hers and Archie calls the nursery, and tried to think
of something to do.

"I'm not going to play bridge all day for anyone," said Archie.

"The host should lay himself out to amuse his guests," said Myra.

"Otherwise, his guests will lay him out," I warned him, "to amuse
themselves."

"Well, what do you all want to do?"

"I should like to look at a photograph album," said Thomas.

"Stump cricket."
 "What about hide-and-seek?"

"No, I've got it," cried Archie; "we'll be boy scouts."

"Hooray!" cried everybody else.

Archie was already on his hands and knees. "Ha!" he said, "is that the
spoor of the white ant that I see before me? Spoorly not. I have but been
winded by the water-beetle.

"Sound, sound the trumpet, beat the drum,

To all the scouting world proclaim
One crowded stalk upon the turn
Is worth an age without a name."

"Archie!" shrieked Myra in horror. "It is too late," she added, "all the
ladies have swooned."

We arranged sides. Myra and I and Simpson and Thomas against the
others. They were to start first.

"This isn't simply hide-and-seek," said Archie, as they went off. "You've
got to track us fairly. We shall probably 'blaze' door-posts. When you hear
the bleat of a tinned sardine that means we're ready. Keep your eyes
skinned, my hearties, and heaven defend the right."

"We ought to have bare knees really," said Myra, when they'd gone.
"Boy scouts always do. So that when they go through a bed of nettles they
know they've been."

"I shall stalk the stairs to begin with," I said. "Simpson, you go down
the back way and look as much like a vacuum-cleaner as possible. Then
they won't notice you. Thomas and Myra—— Hush! Listen! Was that the
bleat of a fresh sardine or the tinned variety?"

"Tinned," said Myra. "Let's go."

 We went. I took the Queen Anne staircase on my—in the proper
stalking position. I moved very slowly, searching for spoor. Half-way down
the stairs my back fin slipped and I shot over the old oak at a tremendous
pace, landing in the hall like a Channel swimmer. Looking up, I saw
Thomas in front of me. He was examining the door for "blazes." Myra was
next to him, her ear to the ground, listening for the gallop of horses' hoofs. I
got up and went over to them.

"Hast seen aught of a comely wench in parlous case, hight Mistress

Dahlia?" I asked Thomas.

"Boy scouts don't talk like that," he said gruffly.

"I beg your pardon. I was thinking that I was a Cavalier and you were a
Roundhead. Now I perceive that you are just an ordinary fathead."

"Why," said Myra at the foot of the stairs, "what does this button mean?
Have I found a clue?"

I examined it, and then I looked at my own coat.

"You have," I said. "Somebody has been down those stairs quite
recently, for the button is still warm."

"Where is Scout Simpson?"

At that moment he appeared breathless with excitement.

"I have had an adventure," he said hurriedly, without saluting. "I was on
the back stairs looking like a vacuum-cleaner when suddenly Archie and
Miss Blair appeared. They looked right at me, but didn't seem to penetrate
my disguise. Archie, in fact, leant against me, and said to Miss Blair: 'I will
now tell you of my secret mission. I carry caviare—I mean despatches—to
the general. Breathe but a word of this to the enemy, and I miss the half-
holiday on Saturday. Come, let us be going, but first to burn the secret
code.' And—and then he struck a match on me, and burned it."
 Myra gurgled and hastily looked solemn again. "Proceed, Scout
Simpson," she said, "for the night approaches apace."

"Well, then they started down the stairs, and I went after them on my—
scouting, you know. I made rather a noise at one corner, and Archie looked
round at me, and said to Miss Blair: 'The tadpoles are out full early. See
yonder where one lies basking.' And he came back, and put his foot on me
and said, 'Nay, 'tis but a shadow. Let us return right hastily. Yet tarry a
moment, what time I lay a false trail.' So they tarried and he wrote a note
and dropped it on me. And, afterwards, I got up and here it is."

"The secret despatch," cried Myra.

"It's addressed to the Scoutmistress, and it says outside: 'Private, not to

be opened till Christmas Day.'"

Myra opened it and read: "Your blessed scouts are everywhere. Let me
just have five minutes with her in the nursery, there's a dear. I'd do as much
for you."

But she didn't read it aloud, and I didn't see it till some time afterwards.
She simply put it away, and smiled, and announced that the scouts would
now adjourn to the billiard-room for pemmican and other refreshments;
which they did. The engagement was announced that evening.

CHAPTER VI

IN AND OUT

"Well," said Thomas, "how are we going to celebrate the joyful event?"

We were sitting on the lawn, watching Blair and Miss Fortescue play
croquet. Archie and Dahlia were not with us; they had (I suppose) private
matters to discuss. Our match did not begin for another hour, happily for the
lovers; happily also for the croquet-players, who had about fifty-six more
hoops, posts, flags and what not to negotiate.

"It's awfully difficult to realise it," said Myra. "My own brother! Just
fancy—I can hardly believe it."

"I don't think there can be any doubt," I said. "Something's happened to
him, anyhow—he's promised to put me in first to-day."

"Let's have a dance to-morrow night," continued Thomas, relentlessly

pursuing his original idea. "And we'll all dance with Miss Blair."

"Yes. Archie would like that."

"I remember, some years ago, when I was in Spain," said Simpson——

"This," I murmured appreciatively, "is how all the best stories begin."
And I settled myself more comfortably in my chair.

"No," said Simpson, "I'm wrong there. It was in Hampstead." And he

returned to his meditations.

"Tell you what," said Thomas, "you ought to write 'em an ode,
Simpson."

"There's nothing that rhymes with the lady."

"There's hair," I said quite unintentionally.

"I meant with Dahlia."

"My dear man, there are heaps. Why, there's azalea."

"That's only one."

"Well, there are lots of different kinds of azalea."

"Any rhymes for Archie and Mannering?" said Simpson scornfully.

 "Certainly. And Simpson. You might end with him—

"'Forgive the way the metre limps on,

It's always like that with Samuel Simpson.'

You get the idea?"

"Hush," said Myra, "Miss Fortescue has passed under a hoop."

But it is time that we got on to my innings. Archie managed to win the

toss, and, as he had promised, took me in with him. It was the proudest and
most nervous moment of my life.

"I've never been in first before," I said, as we walked to the wickets. "Is
there any little etiquette to observe?"

"Oh, rather. Especially, if you're going to take first ball."

"Oh, there's no doubt about my taking the first ball."

"In that case the thing to remember is, that when the umpire calls 'play'
the side refusing to play loses the match."

"Then it all rests on me? Your confidence in me must be immense. I

think I shall probably consent to play."

I obtained guard and took my stand at the wicket. Most cricketers

nowadays, I am told, adopt the "two-eyed stance," but for myself I still stick
to the good old two-legged one. It seems to me to be less wearing. My style,
I should observe, blends happily the dash of a Joseph Vine with the patience
of a Kenneth Hutchings; and after a long innings I find a glass of—— I've
forgotten the name of it now, but I know I find it very refreshing.

Being the hero (you will admit that—after my hat trick) of this true
story, I feel I must describe my innings carefully. Though it only totalled
seventeen, there was this to be said for it: it is the only innings of less than a
hundred ever made by a hero.
 It began with a cut to square leg, for which we ran a forced single, and
followed on with a brace of ones in the direction of fine slip. After that, I
stopped the bowler in the middle of his run-up, and signalled to a spectator
to move away from the screen. This was a put-up job with Myra, and I
rather hoped they would give me something for it, but apparently they
didn't. At the end of the over, I went up and talked to Archie. In first-class
cricket, the batsmen often do this, and it impresses the spectators
immensely.

I said, "I bet you a shilling I'm out next over."

He said, "I won't take you."

I said, "Then I huff you," and went back to my crease.

My next scoring stroke was a two-eyed hook over point's head, and then
Archie hit three fours running. I had another short conversation with him, in
the course of which I recited two lines from Shakespeare and asked him a
small but pointed conundrum, and afterwards I placed the ball cleverly to
mid-off, the agility of the fieldsman, however, preventing any increment,
unearned or otherwise. Finally, I gave my cap to the umpire, made some
more ones, changed my bat, and was caught at the wicket.

"I hit it," I said, as I walked away. I said it to nobody in particular, but
the umpire refused to alter his decision.

"I congratulate you," said Miss Blair, when I was sitting down again.

"I was just going to do that to you," I said.

"Oh, but you were kind enough to do that last night."

"Ah, this is extra. I've just been batting out there with your young man.
Perhaps you noticed?"

"Well, I think I must have."

"Yes. Well, I wanted to tell you that I think he has quite an idea of the
game, and that with more experience he would probably be good enough to
play for—for Surrey. Second eleven. Yes. At hockey."

"Thank you so much. You've known him a long time, haven't you?"

"We were babes together, madam. At least, simultaneously. We actually

met at school. He had blue eyes and curly hair, and fought the captain on
the very first day. On the second day his hair was still curly, but he had
black eyes. On the third day he got into the cricket eleven, and on the fourth
he was given his footer cap. Afterwards he sang in the choir, and won the
competition for graceful diving. It was not until his second term that the
headmaster really began to confide in him. By the way, is this the sort of
thing you want?"

"Yes," smiled Dahlia. "Something like that."

"Well, then we went to Cambridge together. He never did much work,

but his algebra paper in the Little Go was so brilliant that they offered him
the Senior Wranglership. He refused on the ground that it might interfere
with his training for the tug of war, for which he had just obtained his blue
—and—— It's a great strain making all this up. Do you mind if I stop
now?"

"Of course I know that isn't all true, but he is like that, isn't he?"

"He is. He put me in first to-day."

"I know you really are fond of him."

"Lorblessyou—yes."

"That makes you my friend, too."

"Of course." I patted her hand. "That reminds me—as a friend I feel
bound to warn you that there is a person about in the neighbourhood called
Samuel Simpson who meditates an evil design upon you and yours. In
short, a poem. In this he will liken you to the azalea, which I take to be a
kind of shrubby plant."

"Yes?"
 "Yes, well, all I want to say is, if he comes round with the hat
afterwards, don't put anything in."

"Poor man," smiled Dahlia. "That's his living, isn't it?"

"Yes. That's why I say don't put anything in."

"I see. Oh, there—he's out. Poor Archie."

"Are you very sorry?" I said, smiling at her. "I'm just going, you know."

"Between ourselves," I said later to Myra, "that isn't at all a bad girl."

"Oh, fancy!"

"But I didn't come to talk about her. I came to talk about my seventeen."

"Yes, do let's."

"Yes. Er—you begin."

CHAPTER VII

ALL OVER

"May I have a dance?" I asked Miss Blair.

She put her head on one side and considered.

"One, two, three—the next but five," she said.

"Thank you. That sounds a lot; is it only one?"

"You may have two running then, if you like."

 "What about two running, and one hopping, and one really gliding?
Four altogether."

"We'll see," said Miss Blair gravely.

Myra, who was being very busy, came up and dragged me away.

"I want to introduce you to somebody. I say, have you seen Thomas?"

"It's no earthly good introducing me to Thomas again."

"He's so important because he thinks the dance was his idea; of course
I'd meant to have it all along. There she is—her name's Dora Dalton. I think
it's Dora."

"I shall call her Dora, anyhow."

I was introduced, and we had a very jolly waltz together. She danced
delightfully; and when we had found a comfortable corner she began to
talk.

She said, "Do you play cricket?"

I was rather surprised, but I kept quite cool, and said, "Yes."

"My brother's very fond of it. He is very good too. He was playing here
yesterday against Mr Mannering's team, and made six, and then the umpire
gave him out; but he wasn't out really, and he was very angry. I don't
wonder, do you?"

I had a sudden horrible suspicion.

"Did you say your name was Dora—I mean his name was Dalton?"

"Yes. And just because he was angry, which anybody would be, the
wicket-keeper was very rude, and told him to go home and—and bake his
head."
 "Not bake," I said gently, my suspicion having now become almost a
certainty. "Boil."

"Go home, and boil his head," she repeated indignantly.

"And did he?"

"Did he what?"

"Er—did he understand—I mean, don't you think your brother may

have misunderstood? I can't believe that a wicket-keeper would ever
demean himself by using the word 'boil.' Not as you might say boil. 'Cool
his head' was probably the expression—it was a very hot day, I remember.
And ... ah, there's the music beginning again. Shall we go back?"

I am afraid Miss Dalton's version of the incident was not quite accurate.

What had happened was this: I had stumped the fellow, when he was
nearly a mile and a half outside his crease; and when he got back to it some
minutes later, and found the umpire's hand up, he was extremely indignant
and dramatic about it. Quite to myself, sotto voce as it were, I murmured,
"Oh, go home!" and I may have called attention in some way to the "bails."
But as to passing any remarks about boiling heads—well, it simply never
occurred to me.

I had a dance with Myra shortly after this. She had been so busy and
important that I felt quite a stranger. I adapted my conversation accordingly.

"It's a very jolly floor, isn't it?" I said, as I brought her an ice.

"Oh yes!" said Myra in the same spirit.

"Have you been to many floors—I mean dances, lately?"

"Oh yes!"

"So have I. I think dances have been very late lately. I think when the
floor's nice it doesn't matter about the ices. Don't you think the band is
rather too elastic—I mean keeps very good time? I think so long as the time
is good it doesn't matter about the floor."

"Oh, isn't it?" said Myra enthusiastically.

There was a pleasant pause while we both thought of something else to

say.

"Have you," we began.

"I beg your pardon," we said at once.

"I was going to say," Myra went on, "have you read any nice books
lately, or are you fonder of tennis?"

"I like reading nice books about tennis," I said. "If they are nice books,
and are really about tennis. Er—do you live in London?"

"Yes. It is so handy for the theatres, isn't it? There is no place exactly
like London, is there? I mean it's so different."

"Well, of course, up in Liverpool we do get the trams, you know, now....

I say, I'm tired of pretending I've only just met you. Let's talk properly."

At this moment we heard a voice say, "Let's try in here," and Archie and
Dahlia appeared.

"Hallo! here's the happy pair," said Myra.

They came in and looked at us diffidently. I leant back and gazed at the
ceiling.

"Were you just going?" said Archie.

"We were not," I said.

"Then we'll stay and talk to you."

"We were in the middle of an important conversation."