RESUMO
Doenças infecciosas são as maiores responsáveis por falhas reprodutivas (FR) em cadelas, causando aborto, morte fetal e natimortalidade. Este estudo teve como objetivo investigar a associação entre agentes infecciosos, FR inexplicáveis e anemia em cadelas. Todas as amostras maternas e fetais foram negativas para a presença dos principais agentes infecciosos causadores de FR: herpes vírus canino 1, Neospora caninum, Brucella spp. e B. canis, enquanto agentes como o de Leishmania spp., parvovírus canino, Ehrlichia canis e Anaplasma platys foram encontrados em sangue materno. Coinfecções de A. platys/E. canis e A. platys/Leishmania spp. foram diagnosticadas. Os resultados indicam que os animais com anemia causadas por doenças transmitidas por vetores podem ser mais suscetíveis a sofrerem FR do que animais com valores hematológicos normais.
Palavras-chave:
Anaplasma; Ehrlichia; Leishmania; falhas reprodutivas
Keywords:
Anaplasma; Ehrlichia; Leishmania; reproductive failures
Palavras-chave:
Anaplasma; Ehrlichia; Leishmania; falhas reprodutivas
Many authors consider male infertility and mistimed breeding as the major causes of reproductive failure (RF) in bitches. However, once these causes are excluded, the role of infectious diseases in pregnancy losses should be addressed. The impact of infectious diseases on the normal progression of pregnancy can be associated with the direct action of the infectious agents or their toxins, inducing placentitis that leads to fetal death (Givens and Marley, 2008GIVENS, D.M.; MARLEY, M.S.D. Infectious causes of embryonic and fetal mortality. Theriogenology, v.70, p.270-285, 2008. ). In dogs, the viral action of the canid herpesvirus 1 (CaHV-1) and the bacterium Brucella canis have been found to be the main causes of reproductive problems. In Brazil, leishmaniasis, parvoviruses, and tick-borne diseases (Anaplasma platys and Ehrlichia canis) are endemic, particularly in the North-East region, and all these pathogens can cause anemia. Apart from these agents, previous studies on reproductive disorders in dogs have neglected other pathogens such as Neospora caninum (Greene, 2012GREENE, C.E. (Ed.). Infectious diseases of the dog and cat. St. Louis: Elsevier, 2012. 1376p.). Due to the lack of research concerning potential causes of RF, this study was designed to investigate the association between infectious agents, unexplained RF (abortion, stillbirth, or fetal death), and anemia in bitches.
Samples from 36 pregnant dogs (20 with RF and 16 without RF) treated at the Veterinary Hospital Jerônimo Dix-Huit Rosado Maia-HOVET in the Federal Rural University of the Semi-Arid (UFERSA) in Mossoró/RN, North-East Brazil, were obtained according to the Ethics Committee on Animal Use (CEUA) under license number 23091.006326/2014-88 (date of approval March 3rd, 2015). Depending on the clinical condition: RF (unhealthy) or without RF (healthy), tissue samples were collected through ovariosalpingohisterectomy, Caesarean, parturition, and through the post-mortem examination of the uterus, placenta, and fetus. Blood samples were collected after the RF episodes or after parturition in healthy bitches, and a complete blood count (CBC) was performed in both groups. In both groups, using a STRATEC Molecular kit (Invitek), DNA was extracted from the female dogs (blood/serum for all bitches, and uterus/placenta in unhealthy group) and from fetus (amniotic fluid, fetal abdominal fluid, and pool of liver/spleen and kidney/lung) of the bitches with RF. The samples were subjected to molecular/serological immunodiagnostic tests, both for detection of pathogens related to RF and for anemia (hemoglobin, erythrocytes, and hematocrit below the reference values, Table 1). A Chi-square test was used to determine if there was a relation between the presence/absence of the infectious pathogens and RF. In addition, an analysis of variance (ANOVA) was conducted to determine if RF was related to the blood biochemistry parameters measured (anemia).
None of the animals tested positive for the main infectious causes of RF in bitches in either group (Table 2). Canine parvovirus (CPV) was detected in the maternal blood without hematological alterations, and fetal tissues had signs of autolysis, which probably occurred after the infection (Table 3). For leishmaniasis, five females (C2, C4, C6, C9, C16; RF group) were serologically positive (Table 2). Excepting C16, the other four animals also had co-infection with A. platys; C6, C9 and C16 also had anemia with CBC with characteristics of infection (leukocytosis with neutrophilia), and C9 was positive for leishmaniasis in PCR (Table 2). Some bitches (C1, C3, C7, C13, Tab.2) presented co-infection of E. canis/A. platys, demonstrating anemia and thrombocytopenia (except female C3 which showed only anemia and CBC with characteristics of infection).
Results of molecular/serological tests, CBC and microscopy of bitches with reproductive problems
The other females with ehrlichiosis/anaplasmosis had at least one alteration in the CBC and alterations in the fetuses that suggest an infectious pathogen (Table 3). The presence or absence of the pathogen did not significantly explain the occurrence of RF (χ2 = 0.02, P>0.05, n = 20). Regarding the hematological data, a relation between anemia and RF in the animals was observed when considering the erythrocytes (F 32.2, DF= 1.33, P<0.05, Figure 1a), hemoglobin levels (F = 32.8, DF = 1.33, P<0.05, Figure 1b), and the haematocrit (F = 35, DF = 1.33, P<0.05, Figure 1c). This indicates a significant difference between animals who suffered RF (unhealthy group), and those who did not experience RF (healthy group). About the data in Tab. 2 and 3, CaHV-1, brucellosis, and neosporosis may cause abortion of fetuses in late pregnancy and foetal autolysis (Greene, 2012GREENE, C.E. (Ed.). Infectious diseases of the dog and cat. St. Louis: Elsevier, 2012. 1376p.). A previous work also did not find B. canis or CaHV-1 in maternal tissues of bitches with RF (Mir et al., 2013MIR, F.; FONTAINE, E.; ALBARIC, O. et al. Findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study. Theriogenology, v.79, p.312-322, 2013. ). Since dogs are vaccinated for CPV, RF associated with this disease is unlikely to occur due to mass population immunity (Givens and Marley, 2008GIVENS, D.M.; MARLEY, M.S.D. Infectious causes of embryonic and fetal mortality. Theriogenology, v.70, p.270-285, 2008. ). Leishmaniasis is zoonotic and endemic in North-Eastern Brazil (Costa et al., 2014COSTA, K.F.L.; AMÓRA, S.S.A.; COUTO, C.F. et al. Awareness of visceral leishmaniasis and its relationship to canine infection in riverside endemic areas in Northeastern Brazil. Rev. Soc. Bras. Med. Trop., v.47, p.607-612, 2014.); it is a systemic disease, which can lead to anemia and placentitis (Oliveira et al., 2015OLIVEIRA, V.V.G.; ALVES, L.C.; SILVA JUNIOR, V.A. Transmission routes of visceral leishmaniasis in mammals. Ciênc. Rural.; v.45, p.1622-1628, 2015.).
Post mortem exam of maternal/fetal tissues, fetuses, and gestational age of each female with reproductive disorders
Reproductive failures (RF) in bitches due to anemia. Blood analysis comparing females that suffered of RF (unhealthy) and females that did not suffer of RF (healthy). A, Quantification of erythrocytes (mean ± standard error; in 1x106/mm3); B, Number for hemoglobin (mean ± standard error; in g/dL); C, Quantification of haematocrit (mean ± standard error; in %).
The other four animals also had co-infection with A. platys, so we cannot infer which disease caused the anemia. Their features in fetuses (Table 3) suggest a pathogenesis of infectious origin. Ehrlichiosis and anaplasmosis are related as emerging zoonotic diseases (Arraga-Alvarado et al., 2014), which are not associated with RF in animals. These pathogens may have caused weakness and consequently RF. Thrombocytopenia in dogs with leishmaniasis (C6), ehrlichiosis (C1, C7, C13), and anaplasmosis (C1, C7, C13, C19) is expected; however, RF was not previously described as a clinical sign (Borin et al., 2009). The presence of these pathogens induces anemia, which can increase the likelihood of RF. Anemia has been associated with animals chronically infected with multisystemic diseases (anaplasmosis, ehrlichiosis, or leishmaniasis), and clinical signs vary with the severity of the disease and the presence of co-infections (Borin et al., 2009; Ferreira et al., 2014FERREIRA, R.F.; CERQUEIRA, A.M.F.; CASTRO, T.X. et al. Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. Braz. J. Vet. Parasitol., v.23, p.301-308, 2014.). Genetic diversity of E. canis strains can express different forms of hematological and clinical manifestations of the disease. However, previous studies on clinical signs of anaplasmosis, ehrlichiosis, or leishmaniasis were conducted in nonpregnant animals. The co-infection of E. canis and A. platys in nonpregnant dogs resulted in more pronounced anemia and thrombocytopenia (Gaunt et al., 2010GAUNT, S.; BEALL, M.; STILLMAN, B. et al. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasite. Vector., v.3, p.33, 2010. ). Maternal and perinatal mortality is significantly higher in anemic women (Bencaiova et al., 2012BENCAIOVA, G.; BURKHARDT, T.; BREYMANN, C. Anemia-prevalence and risk factors in pregnancy. Eur. J. Intern. Med., v.23, p.529-533, 2012. ). Maternal systemic diseases can result in fever, anemia, or endotoxemia, which in turn can result in RF (Givens and Marley, 2008GIVENS, D.M.; MARLEY, M.S.D. Infectious causes of embryonic and fetal mortality. Theriogenology, v.70, p.270-285, 2008. ). The endotheliochorial placenta of dogs allows high blood supply to the fetus. Maternal infections by intracellular parasites can lower oxygen levels in the blood transported to the fetus, leading to RF. The principal reproductive pathogens were not found in this study, which suggests that the systemic disease diagnosed in the maternal blood is related to RF. Hence, bitches with anemia caused by vector-borne diseases are significantly more likely to experience RF than those with normal hematological values (Figure 1).
REFERENCES
- ARRAGA-ALVARADO, C.M.; QUROLLO, B.A.; PARRA, O.C. et al. Case report: molecular evidence of Anaplasma platys infection in two women from Venezuela. Am. J. Trop. Med. Hyg., v.91, p.1161-1165, 2014.
- BENCAIOVA, G.; BURKHARDT, T.; BREYMANN, C. Anemia-prevalence and risk factors in pregnancy. Eur. J. Intern. Med., v.23, p.529-533, 2012.
- BORIN. S.; CRIVELENTI, L.Z.; FERREIRA, F.A. Aspectos epidemiológicos, clínicos e hematológicos de 251 cães portadores de mórula de Ehrlichia spp. naturalmente infectados. Arq. Bras. Med. Vet. Zootec., v.61, p.566-571, 2009.
- COSTA, K.F.L.; AMÓRA, S.S.A.; COUTO, C.F. et al. Awareness of visceral leishmaniasis and its relationship to canine infection in riverside endemic areas in Northeastern Brazil. Rev. Soc. Bras. Med. Trop., v.47, p.607-612, 2014.
- FERREIRA, R.F.; CERQUEIRA, A.M.F.; CASTRO, T.X. et al. Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. Braz. J. Vet. Parasitol., v.23, p.301-308, 2014.
- GAUNT, S.; BEALL, M.; STILLMAN, B. et al. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasite. Vector., v.3, p.33, 2010.
- GIVENS, D.M.; MARLEY, M.S.D. Infectious causes of embryonic and fetal mortality. Theriogenology, v.70, p.270-285, 2008.
- GREENE, C.E. (Ed.). Infectious diseases of the dog and cat. St. Louis: Elsevier, 2012. 1376p.
- MIR, F.; FONTAINE, E.; ALBARIC, O. et al. Findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study. Theriogenology, v.79, p.312-322, 2013.
- OLIVEIRA, V.V.G.; ALVES, L.C.; SILVA JUNIOR, V.A. Transmission routes of visceral leishmaniasis in mammals. Ciênc. Rural.; v.45, p.1622-1628, 2015.
Publication Dates
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Publication in this collection
Sep-Oct 2017
History
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Received
09 Dec 2016 -
Accepted
19 Jan 2017
