Neospora caninum infection in cattle in the state of Amazonas, Brazil: seroprevalence, spatial distribution and risk factors

Abstract

Livestock in the Amazon has grown significantly and, although neosporosis in cattle has been reported worldwide, there is no information about N. caninum in production systems in the state of Amazonas. The objective of this study was to determine the prevalence of anti-Neospora caninum antibodies in cattle, their spatial distribution and the risk factors associated with N. caninum infection in the state of Amazonas. Questionnaires were applied to farmers to assess risk factors associated with N. caninum infection. Blood samples were collected from 1,073 animals on 47 farms in 33 municipalities in the four Amazonian subpopulations. IgG anti-N.caninum antibodies were detected by the indirect fluorescence test, with a general prevalence of 30.2%, being seropositive in 43 farms (91.5%), with prevalence ranging from 2.2% to 69.2%. The highest number of high density points was found in subpopulation 3 (municipality of Apuí and other municipalities on the Madeira River and affluent). It was concluded that N. caninum is present with high seroprevalence values, when compared to other cattle producing states in the Amazon region of Brazil. The identified factors can be used as risk indicators so that control measures can be implemented to prevent infection by N. caninum in these herds.

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ISSN 1984-2961 (Electronic)
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Original Article
Braz J Vet Parasitol 2021; 30(1): e020820 | https://doi.org/10.1590/S1984-296120201083
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Neospora caninum infection in cattle in the state of
Amazonas, Brazil: seroprevalence, spatial distribution and
risk factors
Infecção por Neospora caninum em bovinos no estado do Amazonas, Brasil:
soroprevalência, distribuição espacial e fatores de risco
Paulo Cesar Gonçalves de Azevedo Filho1; Müller Ribeiro-Andrade2; Jomel Francisco dos Santos1;
Arthêmio Coelho dos Reis3; José Wilton Pinheiro Júnior4; Sandra Regina Fonseca de Araújo Valença4;
Erika Fernanda Torres Samico-Fernandes4; Rinaldo Aparecido Mota4* 
1 Instituto Federal de Educação, Ciência e Tecnologia do Amazonas – IFAM, Campus Manaus Zona Leste, Manaus, AM, Brasil
2 Instituto de Ciências Biológicas e Saúde, Universidade Federal de Alagoas – UFAL, Maceió, AL, Brasil
3 Agência de Agricultura e Proteção Florestal do Amazonas – ADAF, Manaus, AM, Brasil
4 Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco – UFRPE, Recife, PE, Brasil
How to cite: Azevedo Filho PCG, Ribeiro-Andrade M, Santos JF, Reis AC, Pinheiro Júnior JW, Valença SRFA,etal. Neospora caninum
infection in cattle in the state of Amazonas, Brazil: seroprevalence, spatial distribution and risk factors. Braz J Vet Parasitol 2021;
30(1): e020820. https://doi.org/10.1590/S1984-296120201083
Received August 31, 2020. Accepted November 17, 2020.
*Corresponding author: Rinaldo Aparecido Mota. E-mail: rinaldo.mota@hotmail.com
Abstract
Livestock in the Amazon has grown significantly and, although neosporosis in cattle has been reported worldwide,
there is no information about N. caninum in production systems in the state of Amazonas. The objective of this study
was to determine the prevalence of anti-Neospora caninum antibodies in cattle, their spatial distribution and the
risk factors associated with N. caninum infection in the state of Amazonas. Questionnaires were applied to farmers
to assess risk factors associated with N. caninum infection. Blood samples were collected from 1,073animals on
47 farms in 33 municipalities in the four Amazonian subpopulations. IgG anti-N.caninum antibodies were detected
by the indirect fluorescence test, with a general prevalence of 30.2%, being seropositive in 43 farms (91.5%), with
prevalence ranging from 2.2% to 69.2%. The highest number of high density points was found in subpopulation
3(municipality of Apuí and other municipalities on the Madeira River and affluent). It was concluded that N. caninum
is present with high seroprevalence values, when compared to other cattle producing states in the Amazon region
of Brazil. The identified factors can be used as risk indicators so that control measures can be implemented to
prevent infection by N. caninum in these herds.
Keywords: Amazon rainforest, Neospora caninum, prevalence, IFAT, cattle, Brazil.
Resumo
A pecuária na Amazônia tem crescido significativamente e, embora a neosporose em bovinos tenha sido relatada
em todo o mundo, não há informações sobre N. caninum nos sistemas de produção no estado do Amazonas.
Objetivou-se determinar a prevalência de anticorpos anti-Neospora caninum em bovinos, sua distribuição espacial
e os fatores de risco associados à infecção por N. caninum no estado do Amazonas. Questionários foram aplicados
aos fazendeiros, para avaliar fatores de risco associados à infecção por N. caninum. Amostras de sangue foram
coletadas de 1.073 animais em 47 fazendas, em 33 municípios das quatro subpopulações amazonenses. Anticorpos
IgG anti-N.caninum foram detectados pelo teste de imunofluorescência indireta, com prevalência geral de 30,2%,
com soropositividade em 43 fazendas (91,5%), com prevalência variando de 2,2% a 69,2%. O maior número de
pontos de alta densidade foi encontrado na subpopulação 3 (município de Apuí e demais municípios do rio
Madeira e afluentes). Concluiu-se que N. caninum está presente com altos valores de soroprevalência, quando
comparado a outros estados produtores de gado na região amazônica do Brasil. Os fatores identificados podem
ser usados como indicadores de risco, para que medidas de controle possam ser implementadas para prevenir
a infecção por N. caninum nesses rebanhos.
Palavras-chave: Floresta Amazônica, Neospora caninum, prevalência, IFI, gado, Brasil.

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
Introduction
In Brazil’s Amazon region, composed of municipalities in the states of Rondônia, Acre, Amazonas, Roraima, Pará,
Amapá, Tocantins, Mato Grosso, Maranhão, the herds of the states of the Amapá, Roraima, Amazonas and Acre,
together, represented only 5.9% of the total for the region (Valentim & Andrade, 2009). Amazonas is estimated
to have 1.48 million head of cattle, which corresponds to only 0.69% of the national herd (ADAF, 2019). Several
obstacles hinder the development of beef cattle in this state. Factors such as deforestation, pasture degradation,
high rainfall indices, and lack of information about diseases that affect zootechnical indices all contribute to meager
production and economic rates (Arimaetal., 2005). Infectious diseases are considered an important etiological cause
of losses in cattle herds (Pinheiroetal., 2000; Reicheletal., 2013), above all neosporosis, which is considered the
main disease associated with reproductive impairment in cows in different regions around the world (Dubeyetal.,
2007; Dubey & Schares, 2011; McAllister, 2016). In Brazil, this infection is considered prevalent throughout the
territory (Cerqueira-Cézaretal., 2017).
Neosporosis is caused by the coccidium Neospora caninum. The main pathways of transmission of this disease
are transplacental and the ingestion of sporulated oocysts excreted in the environment by the definitive hosts,
especially domestic dogs (McAllister et al., 1998; Donahoeetal., 2015). The pathogenicity of N. caninum varies
according to its host species. Cattle are the most susceptible hosts, and the disease causes abortions, stillbirths
and the birth of weak calves. N. caninum infection in cattle is reported in every continent where cattle are raised
(Dubey & Schares, 2011). However, prevalence rates of the infection in cattle herds vary significantly, depending on
factors such as production systems, the presence of definitive hosts, herd health management (Ghalmietal., 2012).
Several studies have been conducted in different regions of Brazil to detect anti-N. caninum IgG antibodies in
cattle, whose prevalence ranges from 9.1% to 97.2% (Hasegawaetal., 2004; Viannaetal., 2008; Andreottietal.,
2010; Amaraletal., 2012; Silvaetal., 2017). In the Amazon region, N. caninum infection has been described in the
states of Pará (Minervinoetal., 2008; Silvaetal., 2017), Rondônia (Aguiaretal., 2006; Boasetal., 2015) and Tocantins
(Martinsetal., 2011), with prevalence rates varying from 10.4% to 52%. However, there are no seroepidemiological
studies of N. caninum infection in the state of Amazonas, so there is a gap in information about herd health and
factors associated with infection in this region of the country. Thus, our aim was to determine the seroprevalence,
spatial distribution and risk factors associated with Neospora caninum infection in cattle herds in the state of
Amazonas.
Material and Methods
Farms and sampling
This study was approved by the Ethics Committee on Animal Use (CEUA) of the Federal Rural University of
Pernambuco (UFRPE), under protocol no. 80/2018.
Farms were selected in the four subpopulations in the state of Amazonas, defined by the Ministry of Agriculture,
Livestock and Food Supply – MAPA (Brasil, 2019) (see Figure1). These subpopulations are as follows: Subpopulation
1 – comprising the municipalities of Rio Negro and its tributaries; Subpopulation 2 – composed of the municipalities
of Rio Solimões and its tributaries; Subpopulation 3 – comprising the municipality of Apuí and the other municipalities
of the Madeira River and its tributaries, and Subpopulation 4 – comprising the other municipalities of the state of
Amazonas on its border with the state of Pará.
Blood samples were collected from 1073 reproductively mature cows (older than 24 months) on farms with
more than 10 cattle (primary sampling units) in 33 municipalities in the state of Amazonas. The animals were
identified individually by an ear tag usually attached to the left ear. This tag number was recorded on the label of
the vacutainer tube and on a sample collection form. For the study sample calculation, an expected prevalence of
50%, 95% confidence and 5% statistical error were considered (Thrusfield, 2004).
Sample collection for serology
Blood samples were collected by venipuncture of the external jugular vein into sterile and tagged vacutainer
tubes. Ten mL of blood without anticoagulant were drawn into individual test tubes after prior skin antisepsis with
3.0% iodinated alcohol. The samples were centrifuged at 1000 x g for five minutes to obtain serum. The blood sera
were aliquoted and stored at -20ºC until serological tests were performed.

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
Preparation of antigen for the IFAT
Neospora caninum antigen stored in a culture of MARC-145 cell monolayers was used, according to the conditions
described by Regidor-Cerrilloetal. (2010). The number of viable tachyzoites was determined by counting in a
Neubauer chamber using 0.2% trypan blue. The purified tachyzoites were suspended in 0.6% formalin solution in
PBS, adjusted to a concentration of 1200-1500 parasites/μL, and each well of the slides for the indirect fluorescent
antibody test (IFAT) was sensitized with 10 µL of the tachyzoite suspension. All the microscope slides were fixed
in acetone at -20ºC and stored under refrigeration until use. All slides were fixed in acetone at -20°C and stored
under refrigeration until use.
Survey of anti-N. caninum IgG antibodies
To begin with, serum samples were diluted 1:200 (cutoff) (Gondimetal., 1999) in 1X PBS, distributed in the
wells of the sensitized slides and incubated in a humidifying chamber at 37ºC for 30 min. The slides were then
washed and reincubated with fluorescein isothiocyanate-conjugated anti-bovine IgG serum (Sigma Chemical, USA)
containing 0.02% Evans blue (Sigma Chemical, USA). Lastly, the slides were washed again, covered with buffered
glycerine and coverslips and examined under an epifluorescence microscope (Nikon Eclipse 40x objective lens).
Samples were considered positive when 50% of the tachyzoites in the wells presented total peripheral fluorescence
(Kimetal., 2019). Serum samples known to be positive and negative were included as control samples. Positive
control sera were obtained from cattle with consistently positive serological results by IFI and negative control
sera from healthy cattle with consistently negative serological results by IFI, as mentioned above. The slides were
examined by an epifluorescent microscope (Olympus, Mod. BH2, Tokyo, Japan).
Figure 1. Map of the subpopulations (1, 2, 3, and 4) of the state of Amazonas, according to the Ministry of Agriculture (Brasil,
2019), Livestock and Supply of Brazil.

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
The analysis of the results per farm indicated that 91.5% (43/47) of farms had at least one positive animal. The
KDE of the N. caninum infection per farms in the state of Amazonas revealed the presence of seropositive animals
distributed throughout the all subpopulation of state, indicated rates varied from 2.2 to 69.2%, with the more
points of high density found in subpopulation 3 (Figure2).
A univariate analysis revealed a significant association between N. caninum infection in cattle and the following
variables: type of herd (p=0.009); type of livestock farming system (p=0.012); source of water supply (p=0.000);
presence of dogs (p=0.000); occurrence of abortion (p=0.000); newborn mortality rate (p=0.000); and birth of
weak calves (p=0.000). The following risk factors for cattle in this region were identified by logistic regression:
dairy farming (OR=2.71; p=0.021), beef farming (OR=1.61; p=0.008), herds raised on a combination of dry and
flooded lands (OR=1.54; p=0.007), source of running water in association with standing water (OR=2.10; p=0.000),
presence of dogs (OR=1.80; p=0.000), occurrence of abortion on the farm (OR=2.35; p=0.000) and birth of weak
calves (OR=1.88) (see Table2).
Spatial distribution and of N. caninum infection
The coordinates of each farm were determined by georeferencing, using a TrackMaker® PRO GPS to characterize
the map of the state of Amazonas, Brazil. The georeferenced data were entered into the ArcMap version 12.2.2
program, using the Kernel Density Estimator (KDE), which is a non-parametric technique that allows the variability
of a data set to be filtered while preserving the essential local characteristics of the data (Bailey & Gatrell, 1995).
The parameter used for elaboration of the maps using the KDE was the frequency of N. caninum-positive cattle per
farm studied. This frequency was classified in ranges and the density expressed by color: very high (red color), high
(dark orange color), medium (yellow color), low (green), very low (yellow color) and negative (blue). The number of
farms located in the different areas was counted on the maps resulting from this operation.
Risk factors associated with N. caninum infection
Pre-structured questionnaires were applied the farmers to assess the risk factors associated with N. caninum
infection, variables included in the analysis final model were: type of farming system (type of herd; livestock farming
system; farm size; herd size) and management-related (water source; presence of other domestic and wild animals;
occurrence of abortion; birth of weak calves).
The absolute and relative frequencies were subjected to dispersion. The risk factors associated with Neospora
caninum infection were studied based on an analysis of the variables of interest using Pearson’s chi-square test
or Fisher’s exact test, when necessary. A logistic regression analysis was then performed, considering the IFAT
results (positive or negative) as the dependent variable. The independent or explanatory variables considered in
the final model were those with a statistical significance of <0.05. The statistical calculations were made using Epi
Info version 3.5.1 software.
Results
The prevalence of anti-Neospora caninum antibodies in cattle in the state of Amazonas was 30.2% (324/1073;
CI95%: 27.5% – 33.0). The frequencies of each the subpopulations are shown in Table1. No significant differences
(p>0.05) were observed between the prevalence of subpopulations.
Table 1. Prevalence of Neospora caninum infection in cattle per subpopulations (1,2,3 and 4) in the state of Amazonas, Brazil.
Subpopulation Total No. of animals No. positive animals Neospora caninum infection
(%)
1 64 17 26.6%
2 174 51 29.3%
3 467 136 29.1%
4 368 133 36.1%
Subtotal 1073 324 30.2%

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
Discussion
To the best of our knowledge, this is the first study on the epidemiology of N. caninum infection in cattle herds
in the state of Amazonas, where a prevalence rate of 30.2% (324/1073) was found. The territory of Amazonas state
is covered entirety by the largest tropical forest in the world, the Amazon Rainforest, is a continental size state
with sociocultural, economic and political differences, as well as geographical and ecological characteristics that
may partly explain the widely varying prevalence rates among the farms (Vasconcelos, 2015). In fact, the reported
prevalence rates of N. caninum infection in cattle in Brazil vary widely and in the northern region of Brazil, where
the state of Amazonas is located, search data is limited.
This is an important and sensitive region of world for the preservation of fauna and flora. However, there has
been a growth in deforestation in the region, especially in the last 30 years (IBGE, 2017), one of the main direct
causes of livestock and large-scale agriculture, and among these, the expansion of cattle livestock is the most
important (Riveroetal., 2009). In this sense, is necessary to encourage the development of a livestock based on
productivity supported by scientific data and animal health knowledge.
In Brazil, the presence of N. caninum antibodies in cattle has been studied in several areas (Gennari, 2004),
but only two of these reports were related to the Amazon region, under management conditions similar to that
carried out in this study. Aguiaretal. (2006) in the state of Rondônia and Minervinoetal. (2008) in the state of
Pará, reported prevalence rates ranging from 10.4% to 19%. These studies show different seroprevalence values
for anti-N. caninum antibodies to those found in our study.
This difference may occur due to the cutoff point used for each study, as well as the breeding conditions for
each herd studied. In the Rondônia State, there was not the floodplain system (a kind of wet meadow) and the
animals are raised only in cultivated pastures in more intensive conditions; also there are some different climatic
conditions in special related to the rainy season period. In the states of Pará and Amazonas, the climatic conditions
Figure 2. Kernel density estimation of the prevalence of Neospora caninum infection in cattle in the state of Amazonas, Brazil.

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
are similar, alternating in wetter periods and with greater concentration of rainfall. Condition that can affect the
survival of N. caninum oocysts (Minervinoetal., 2008).
The analysis of the Kernel density estimation map for N. caninum infection in cattle in the state of Amazonas
revealed the presence more points density very high in the subpopulation 3, with at least one animal testing positive
for the infection at 91.5% of the farms involved in this study (Figure2). The subpopulation 3 represents almost
Table 2. Univariate analysis and logistic regression of risk factors associated with Neospora caninum infection in cattle in the
state of Amazonas, Brazil.
Variable N* Positive (%)
Univariate Regression
p value OR p value
Type of herd
Mixed 214 48 (22.4%) 0.009 -
Beef 834 265 (31.8%) 1.61 (1.13 – 2.29) 0.008
Dairy 25 11 (44.0%) 2.71 (1.15- 6.37) 0.021
Livestock farming system
Dry land 810 227 (28.0%) 0.012 -
Floodplain 10 2 (20.0%) 0.89 (0.18 – 4.35) 0.886
Dry land + Floodplain 253 95 (37.6%) 1.54 (1.12 – 2.11) 0.007
Size of farm (hectare)
< 50 170 56 (32.9%) 0.213 -
51 to 500 626 176 (28.1%)
> 501 277 92 (33.2%)
No. of cattle
1 – 10 animals 36 10 (27.8%) 0.595 -
11 – 50 animals 72 25 (34.7%)
51 – 100 animals 236 77 (32.6%)
More than 100 animals 729 221 (29.1%)
Source of water supply
Running water 306 64 (20.9%) 0.000
Still water 230 68 (29.6%) 1.58 (1.06 – 2.35) 0.021
Running + Still water 537 192 (35.8%) 2.10 (1.51 – 2.91) 0.000
Presence of dogs
Yes 737 254 (34.5%) 0.000 1.80 (1.28 – 2.52) 0.000
No 239 54 (22.6%)
Occurrence of abortion
Yes 501 199 (39.7% 0.000 2.35 (1.80 – 3.07) 0.000
No 572 125 (21.9%)
Birth of weak calves
Yes 431 165 (38.3%) 0.000 1.88 (1.44 – 2.45) 0.000
No 642 159 (4.8%)
*Number of animals tested. P – significance probability. Odds Ratio – is a measure of association between an exposure and an outcome.

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Neospora caninum infection in cattle in the state of Amazonas, Brazil
80% of the bovine population in the state, this concentration of hot areas may be due to the animals staying for a
longer time together on the same property, different from subpopulations 1, 2 and 4 that have high turnover of
herds and lower population density.
Dairy farms were identified as a risk factor for N. caninum infection and were 2.71-fold more likely to have
infected cattle. The animal farming system employed by these farms may increase the risk of infection because it
is more intensive, thus facilitating the transmission of the parasite. Almeríaetal. (2009) reported a similar finding
that dairy herds are more susceptible to N. caninum infection than beef cattle, because this livestock farming system
is more intensive, and the animals remain in this system for longer periods.
Raising herds on a combined system of dry and flooded lands, which is exclusively the case of Subpopulation
4, was also identified as a risk factor (OR=1.54). This mesoregion has two characteristic seasons: the dry season,
from July to December, characterized by low water levels of the Amazon River, exposing the naturally fertilized
floodplain lands, and the wet season, from January to June, characterized by the rising level of the river, whose
waters flood the surrounding lowlands, forcing the riverine population and their livestock, including cattle, to move
to higher ground (Filizolaetal., 2006). This seasonal flooding causes cattle herds to concentrate on small areas of
dry land, where they are in direct contact with dogs and other domestic animals in the surroundings, in livestock
sheds and at feed troughs. Conversely, in the dry season, cattle are raised on the floodplains, in collective systems
where several species of livestock occupy the same area. Similar findings were reported by Dubeyet al. (2007),
who demonstrated that husbandry management is a risk factor for environmental contamination by N. caninum.
The presence of dogs on the farms was also considered a risk factor in our study. Cattle raised on farms
where dogs lived were 1.80-fold more likely to be infected with N. caninum. According to Dubeyetal. (2007) and
McAllisteret al. (1998), the presence of dogs is considered a risk factor, since they are the definitive hosts of
N.caninum. Oocysts, the environmentally resistant stage of the parasite, are excreted in dog feces, contaminating the
environment and transmitting the parasite to cattle via horizontal transmission through the ingestion of sporulated
N. caninum. Ghalmietal. (2012) found that water sources on a farm are important reservoirs of N. caninum oocysts,
because they can become contaminated by these infecting forms excreted in the feces of the definitive hosts. The
findings of the aforementioned authors corroborate those of our study, which confirmed that animals that drink
running water in association with standing water were 2.10-fold more likely to become infected.
A strong correlation was found between abortion and the birth of weak calves on farms where cows tested
positive for anti-N. caninum antibodies. This finding is similar to that reported by Boasetal. (2015), who stated that
the birth of weak calves, abortions and the presence of N. caninum are correlated, and that the occurrence of the
disease is closely associated with production losses, as we found in our study. Other studies have demonstrated
that the probability of abortion occurring among seropositive cows is two to seven times higher than among
seronegative animals (Dubey & Schares, 2011), and that this risk is 7.4-fold higher among congenitally infected
heifers (Thurmond & Hietala, 1997).
Conclusions
In this first seroepidemiological study of N. caninum infection in cattle in the state of Amazonas, it was concluded
that N. caninum is present with high values of seroprevalence, when compared to other cattle producing states
in the Amazon region of Brazil. The factors identified in our study can be used as risk indicators, so that control
measures can be implemented to prevent infection by N. caninum in herds in this region.
Acknowledgements
The authors gratefully acknowledge the assistance of the staff at the Laboratory of Infectious Diseases of the
Federal Rural University of Pernambuco – UFRPE and the staff of the Amazonas Agriculture and Forest Protection
Agency (ADAF).
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