UNIVERSIDAD NACIONAL DE ASUNCIÓN

FACULTAD DE CIENCIAS VETERINARIAS

DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

EVALUACION DE ARTICULOS PARA PUBLICACION EN LA REVISTA CIENTIFICA

TITULO: Prevalence of bovine leptospirosis among dairy cows in the Caaguazú department,
Paraguay

TIPO: Trabajo de investigación◉ Artículo de revisión ⃝ Comunicación Breve ⃝

REDACCION EDITORIAL ARBITRO REDACCION EDITORIAL

RECEPCIO Fecha: Fecha: Fecha:
N 27/4/2023

Partes del Evaluación Sugerencias

Artículo (tildar) (Favor emitir sugerencias en caso de evaluación 3, 2 o 1)
Título ⑤④③②① Please remove the word ‘prevalence’ (see below).
The abstract should mention that leptospirosis is a
zoonosis. The word ‘seroprevalence’ should be
removed (see below). It would be better to give the
number of positive samples and number tested as a
fraction as well as the percentage when stating the PCR
Resumen/ and MAT results. It would be better to state (L.12)
⑤④③②①
Palabras clave “Seis de las siete vacas que dieron positivo por PCR
anidada también fue seropositiva a Leptospira spp.”,
rather than the current negative formulation.
Please remove the word “endémico” (L.14) and
replace with “presente”.
Please remove the word ‘prevalence’ (see below).
It would be better to state (L.31) “Six of the seven cows
Title/Abstract/ that tested positive by nested PCR were also
⑤④③②① seropositive to Leptospira spp.”
Key words
Please remove the word “endemic” (L.33) and replace
with “present”.
Introducción ⑤④③②① Please remove the claim that the study aimed to
investigate the prevalence of bovine leptospirosis …in…
Caaguazú department in Paraguay. (See below.)
Just a little more information or a reference about the
dairy production system in the area might be helpful
for readers to interpret the remainder of the
manuscript. For instance, the range and mean or
median herd sizes (if known), whether the cattle are
free-ranging on pasture or housed, the predominant
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

breeds, the extent to which AI or natural service are

used for breeding, how commonly cattle are
vaccinated, especially against leptospirosis, etc.
More details of the Leptospira vaccines available or
used in the area would be helpful.
Materiales y ⑤④③②① This work purports to be a study of
Métodos* prevalence/seroprevalence but ignores many of the
(Texto) fundamental principles of quantitative epidemiology.
The target population is apparently dairy cattle in
Caaguazú Department, but the study population and
sampling frame are not made explicit, and no sample
size calculation is described. Was a sample size
calculation conducted?
The proportions and confidence intervals cited in tables
1 and 2 calculated using Wilson’s score method
represent those of the underlying probabilities of the
samples tested but are not of epidemiological
relevance; neither can inferences be made from these
values about the farms from which the cattle were
sampled nor can they about the wider population of
dairy cattle in the region of interest. These values apply
only to those 50 cattle that were actually tested and
this should be stated in the paper!
It would be preferable to use a standard sample size
calculation (e.g. Thrusfield and Christley 2018) and
decide on the desired absolute precision of the
prevalence/seroprevalence estimates beforehand.
Assuming an expected prevalence of 50% (based on
data from elsewhere and also as the most conservative
estimate when a previous prevalence value is
unavailable) and that all cattle in the population had an
equal chance of being sampled, for a total population
size for the five farms of 500, 750 or 1000 cattle (based
on the stated average herd size of 100-200), sample
sizes of 81, 86 or 88 respectively would be required to
obtain prevalence estimates with even a modest 10%
desired absolute precision. For a large study
population, i.e., a study population corresponding to
the stated target population of dairy cattle in Caaguazú
Department, a sample size of at least 97 would be
required for simple random sampling under which
every cow in the population would have an equal
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

chance of being sampled. However, simple random

sampling is rarely practicable and cluster sampling is
likely to be necessary.
For cluster sampling of 10 animals per herd, (i.e.,
consistent with 50 samples from 5 herds described
here), given an estimated intracluster correlation
coefficient of approximately 0.1 for leptospirosis in
cattle (see for example Otte and Gum, 1997) the design
effect would be 1.9, implying that nearly twice as many
cattle would need to be sampled for the same modest
10% desired absolute precision of the estimated
prevalence. Hence approximately 200 cattle would
need to be sampled, i.e., 10 cattle from each of 20
herds.
The approach used in this work falls short of these
considerations. However, possible sampling strategies
such as these that could be used establish the
prevalence in further studies might be discussed, in
which case the paper might be useful for soliciting
support for such studies.
Please do explain how were the 50 cows selected from
the population on each farm, and how were the farms
selected? Was this a truly random sample, such that
every eligible cow of the 5 farms had an equal
probability of inclusion, or was it a convenience
sample? How were the 50 cows distributed among the
five farms?
Please explain why vaccinated cattle were excluded
from the study. Please discuss this as in practice the
target population presumably includes vaccinated and
unvaccinated cattle. It might have been better to
sample both vaccinated and unvaccinated cattle, to
note whether cattle had been vaccinated possibly to
assess whether vaccination is a risk factor (in either
direction) for testing positive.
Please reconcile the statement that “all the cows were
apparently in good health” (L.84) with the subsequent
statement “that one of the cows had aborted 15 days
before sampling” (L.213).
Please remove the word “serum” (L.85) as blood rather
than serum was collected.
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

How was the serum separated? (L.89) Was a

centrifugation step conducted?
5–500 µL of vaginal mucus seems to be a very wide
range. (L.90) 5µl could easily get ‘lost’ in a process
involving a 4 cm hemisphere and a 15ml tube!
The “Strains that can identify seven serovars” (L.102)
were presumably strains of these serovars; if so, better
to say so.
A little more information about the culture conditions
(L. 106) would be helpful - e.g. within what were the
cultures performed, how was the temperature
maintained?
How was the culture density determined? (L.108)
Each well of what? (L.112) Please name or describe the
microtitration plates used.
Might the endpoint (L.114) possibly be defined better
as the highest serum dilution with at least 50%
agglutination?
Please describe the a control culture (L.115) in more
detail.
Please reconsider use of the Chi-squared test (L.143).
See below under Table 1.
Please remove the word seroprevalence (L.146) and
reconsider the use of this confidence interval as
described above.
Resultados y ⑤④③②① Please refrain from using the words prevalence or
Discusión* seroprevalence in relation to the results of this study –
see above.
The current wording implies incorrectly that the
organisms were detected. Better to say antibodies or
serological responses were detected. (L.153)
For completeness and avoidance of doubt, better to
add that none of the 50 serum samples reacted in the
MAT against any of the other four serovars of
Leptospira (Canicola, Grippotyphosa, Pomona, or
Pyrog). (L.153)
While the proportion of samples with positive Wolffi
MAT is indeed significantly higher, the statistical basis
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

of this statement should be made clear. (L. 154)

Probably better to state (L.156-158): -
1. How many of the 50 bovine serum samples (and %)
reacted in the MAT against each individual Leptospira
antigen.
2. How many of the 50 bovine serum samples (and %)
reacted against more than one antigen, and what the
combinations were.
3. That the overwhelming majority of serological
reactions were against serovar Wolffi antigen.
Better to state the numbers first and give percentages
in parentheses afterwards i.e., 7/49 (14.3%). (L.160)
It could also be stated that overall PCR and MAT results
were in agreement (either both positive or both
negative) for 17 (34.7%) of the 49 samples, but this was
little more than the number expected to agree by
chance alone if the two were unrelated (15.6; 31.8%).
Also, that of the 37 MAT positive samples, just 6
(16.2%) were positive by PCR, whereas of the 7 PCR
positives, 6 (85.7%) were positive by MAT.
The reasons why some samples were positive for one
test but not the other could be discussed – i.e.,
potential causes of false positives and false negatives
for each test.
Please refrain from making comparisons between the
results of this study and prevalence studies conducted
elsewhere – this is not a prevalence (or seroprevalence)
study. (L.171ff)
Please clarify how a denominator of 415 can result
from a study of 31 animals. (L.171-2) Perhaps it should
say “cattle on 15 dairy and 16 beef farms”?
Please clarify whether Sejroe is a serovar or a
serogroup. (L.185).
Care should be taken in making claims about the
identity of serovars based on serology, see ref 15,
section 2.1. (L.187)
Reference #22 does not provide convincing evidence
that serovar Icterohaemorrhagiae directly or indirectly
infects rodents or dogs, causing acquired leptospirosis.
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

(L.189). Please provide a more supportive reference.

For many pathogens, detectible serological responses
persist far longer than the agent may be detected - the
immune system is doing its job! Please discuss why a
higher proportion with MAT responses than detectible
by PCR is unexpected in this case. (L.194-6)
The word “correlated” for two binary variables might
suggest use of the Phi coefficient, which isn’t really
appropriate here; probably better to say “…results
were in poor agreement”. (L.206)
Was there just one owner for all 5 farms? If so, this
raises yet further concerns about the validity of wider
inference of the results for the target population.
(L.212).
By all means discuss that 37/50 (74.0%) of the samples
were seropositive for serovar Wolffi, but please do not
assert that there was a significant association between
the MAT Wolffi result and PCR; Fisher’s exact test (used
because of expected value < 5) p = 0.665. (L.217-9) .
Please make it clear that the proportions and
confidence intervals shown in Tables 1 and 2 are
applicable only to the 50 (or 49) cattle actually tested
but not the entire herds from which they were drawn
and nor indeed from the target population of all dairy
cattle in Caaguazú Department.
Table 1: Usually, the association between the results of
two diagnostic tests for the same disease is not in
doubt, one is simply interested in how well they agree.
However, in this instance the agreement is so poor (the
95% CI for Cohen’s kappa includes zero!) that it
probably is worth testing the association. However,
one should probably use McNemar’s test in this
situation rather than regular Chi-squared since for the
most part (49 samples) the data are paired and not
independent and hence Table 1 is not a true
contingency table. Also note comment about use of
Chi-squared in Cohen’s original paper, p38-39.
Table 2: Again Chi-squared is not appropriate here for
the same reason as for Table 1.
Table 3: Please show Cohen’s kappa and its confidence
interval to at least 2 significant figures e.g., kappa =
 UNIVERSIDAD NACIONAL DE ASUNCIÓN
FACULTAD DE CIENCIAS VETERINARIAS
DIRECCIÓN DE INVESTIGACIÓN CIENTÍFICA
Y TECNOLÓGICA
Casilla de Correo Nº 1061 – Tel. 585 574/7
Fax: 585 577 – email: investigacion@vet.una.py
San Lorenzo - Paraguay

0.0427, 95% confidence interval: -0.153 – 0.238.

Overall, despite the reservations expressed, the
reviewer considers this work is worthy of publication
and that a revised version could perhaps state that five
farms were investigated as a preliminary look at the
presence of leptospirosis in dairy cattle in Caaguazú
Department.
Refs cited in this review: -
Cohen, J. (1960). A Coefficient of Agreement for
Nominal Scales. Educational and Psychological
Measurement, 20(1), 37-46.
https://doi.org/10.1177/001316446002000104
Otte, M. J., & Gumm, I. D. (1997). Intra-cluster
correlation coefficients of 20 infections calculated from
the results of cluster-sample surveys. Preventive
Veterinary Medicine, 31(1), 147-150.
https://doi.org/10.1016/S0167-5877(96)01108-7
Thrusfield, M., Christley, R. In Veterinary Epidemiology
4th Edn. John Wiley & Sons Ltd. 2018. (See Ch.13.)
doi:10.1002/9781118280249.
Please remove reference to high seroprevalence.
Please suggest that further epidemiological studies
Conclusión would be required to establish the prevalence. Please
⑤④③②①
mention the relevance of the findings on serovars to
any possible vaccination programme.
Should a funder be acknowledged, and if not externally
Agradecimientos ⑤④③②① funded possibly the authors’ own institution?
No doubt the editorial office will check consistency of
Bibliografía ⑤④③②① formatting of references.
*En los Artículos de Revisión y Comunicaciones breves pueden ser reemplazados por Texto.
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