Protozoa1 encephalomyelitis of dogs involving Neosporum caninum and Toxoplasma gondii in New Zealand

In a retrospective study of 15 cases of encephalomyelitis in dogs, three cases of Neospora caninum and two cases of Toxoplasma gondii infection were identified using immunohistochemical staining of central nervous system sections. All cases of neosporosis showed ataxia and progressive hind limb paralysis due to multifocal non-suppurative meningoencephalomyelitis which was most severe in the spinal cord and base of the brain stem. Neospora tissue cysts could not be distinguished morphologically from those of T. gondii using light microscopy, but electron microscopy confirmed their characteristic features. Although Neospora abortion in cattle has only recently been recognised in New Zealand, this study has shown that neosporosis has been present in dogs since at least 1972.     

Prevalence of antibodies against Neospora caninum and Toxoplasma gondii in dogs and foxes in Austria

Sera from 1770 dogs and 94 red foxes from Austria were examined for antibodies against Neospora caninum using the indirect immunofluorescent antibody test (IFAT). 3.6% of the dogs were seropositive with titres ranging from 1:50 to 1:6400. Dogs from rural areas were significantly more often seropositive for N. caninum than those from the urban area of Vienna (5.3% versus 2.1%). There were no significant differences in sex or breed, but a slight increase in seropositivity with age was apparent, indicating postnatal infection. None of the foxes had antibodies against N. caninum. Additionally, sera from 242 dogs and 94 foxes were examined for antibodies against Toxoplasma gondii using the IFAT. Thirty-five percent foxes and 26% of the dogs were positive; 1.7% of the dogs were positive for both parasites. This is the first report of the prevalence of N. caninum infections in dogs and foxes in Austria.     

Occurrence of antibodies to Neospora caninum and Toxoplasma gondii in dogs from Pernambuco, Northeast Brazil

A serological survey was carried out to assess the occurrence of anti-Neospora caninum antibodies in dogs from the State of Pernambuco. A total of 625 serum samples of dogs (289 from Paulista, 168 from Amaraji and 168 from Garanhuns) were tested by an immunofluorescence antibody assay for the detection of anti-N. caninum antibodies. A total of 177 (28.3%; IC 95%, 24.9-32.1) samples were positive. The seropositivity rates found in Paulista, Amaraji and Garanhuns were 26% (IC 95%, 21-31.4), 26.2% (IC 95%, 19.7-33.5) and 34.5% (IC 95%, 27.4-42.2), respectively. Of the 177 serum samples positive to anti-N. caninum antibodies, 170 were additionally tested for the presence of anti-Toxoplasma gondii antibodies and out of these 57.6% (IC 95%, 49.8-65.2) were positive. The results indicate that dogs from Amaraji, Paulista and Garanhuns are exposed to both N. caninum and T. gondii infections. The presence of dogs infected by N. caninum in Pernambuco represents a potential risk factor for the occurrence of outbreaks of abortion in cattle and small ruminants in this state. This study is the largest serological survey on the presence of anti-N. caninum antibodies in dogs carried out in Brazil and reports for the first time the exposure to N. caninum and T. gondii in dogs from Pernambuco.     

Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum infections in dogs

Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum are related apicomplexans that can cause systemic illness in many species of animals, including dogs. We investigated one breeder's 25 Basset Hounds for these infections. In addition, tissues from dogs and other non-canine hosts previously reported as S. canis infections were studied retrospectively. Schizonts resembling those of S. neurona, and recognized by polyclonal rabbit anti-S. neurona antibodies, were found in six of eight retrospective cases, as well as in two additional dogs (one Basset Hound, one Springer Spaniel) not previously reported. S. neurona schizonts were found in several tissues including the central nervous system, lungs, and kidneys. Fatal toxoplasmosis was diagnosed in an adult dog, and neosporosis was diagnosed in an adult and a pup related to the one diagnosed with S. neurona. No serological reactivity to S. neurona antibodies occurred when S. canis-like liver schizonts were retrospectively assayed from two dogs, a dolphin, a sea lion, a horse, a chinchilla, a black or either of two polar bears. Sequencing conserved (18S) and variable (ITS-1) portions of nuclear ribosomal DNA isolated from the schizont-laden liver of a polar bear distinguished it from all previously characterized species of Sarcocystis. We take this genetic signature as provisionally representative of S. canis, an assumption that should be tested with future sequencing of similar liver infections in other mammalian hosts. These findings further extend the uncharacteristically broad intermediate host range for S. neurona, which also causes a neurologic disease in cats, mink, raccoons, skunks, Pacific harbor seals, ponies, zebras, lynxes, and sea otters. Further work is necessary to delineate the causative agent(s) of other cases of canine sarcocystosis, and in particular to specify the attributes of S. canis, which corresponds morphologically to infections reported from wide range of terrestrial and marine mammals.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in dairy goats from Romania

Little information is available about the seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Romania and even in Europe. During 2007-2010, 735 serum samples were collected from dairy goats located in 4 historical regions (Cri?ana, Maramure?, Transylvania and Muntenia) of Romania. Sera were analyzed for T. gondii and N. caninum antibodies (IgG type) by enzyme-linked immunosorbent assay (ELISA) using two commercial kits (Chekit Toxotest Antibody ELISA and Chekit Neospora caninum Antibody ELISA; Idexx-Bommeli, Switzerland). Three hundred and eighty-eight out of 735 (52.8%) goats presented T. gondii antibodies and 12 out of 512 (2.3%) goats had N. caninum antibodies. The high seroprevalence of T. gondii suggests that infection with this parasite is common in dairy goats in Romania, and less common the infection with N. caninum. This is the first time that infection with N. caninum in goats has been reported in Romania and the first extended study on seroepidemiology of T. gondii.     

Seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Poland

In 2007 a survey on herd-level seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Poland was carried out. Sera were collected from all 49 breeding goat herds, scattered over the entire country, with the vast majority of them located in the western, central and northern provinces. Only adult females (≥12 months of age) were included in the study. A herd was recorded as infected if at least one seropositive female was detected. In each herd, simple random sampling was applied and sample size was determined in a way, which allowed to evaluate serological status of a herd at expected individual-level seroprevalence 10% and level of confidence 95%. In total, 1060 sera were tested using two commercial indirect ELISA kits. Sera positive to N. caninum were subsequently confirmed with IFAT. The true herd-level seroprevalence was 100% for T. gondii and 9.0% for N. caninum infection. Three herds positive to T. gondii infection were randomly selected and all adult goats were tested with an ELISA. Individual-level true seroprevalence in these herds ranged from 30.2% to 100%. This is the first time that antibodies to N. caninum have been detected in goats in Poland.     

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.     

Prevalence of Antibodies to Neospora caninum and Toxoplasma gondii in Swedish Dogs

Neospora caninum is a newly described coccidian parasite which has been found in various species such as the dog, cattle, horse, sheep and goat. Morphologically it resembles Toxoplasma gondii with which it is related (Holmdahl et al. 1994), and with which it has earlier been confused. The life cycle of N caninum is only partially known. Tachyzoites and tissue cysts are the only known stages of the parasite, and transplacental transmission is the only known route of infection. Subclini-cally infected dams can transmit the parasite to their fetuses and successive offspring from the same mother might be born infected (Dubey et al. 1990b). Clinical neosporosis is mostly seen in pups or young dogs, and the majority or all pups in a litter are often affected. The disease is characterized by ascending paralysis of the legs, with the hind legs more severely affected than the front legs, paralysis of the jaw, difficulty in swallowing and muscle flaccidity and atrophy (Dubey 1992, Dubey & Lindsay 1993). Fatal infections with N caninum in dogs have been reported from many countries, e.g. Norway (Bjerkäs & Presthus 1988), USA (Dubey et al. 1988), Sweden (Uggla et al. 1989a,b) and the United Kingdom (Dubey et al. 1990a). Serological surveys for antibodies to N. caninum in dogs from Kansas, USA and England have shown a prevalence of 2 and 13%, respectively (Lindsay et al. 1990, Trees et al. 1993).     

Seroprevalences of antibodies to Neospora caninum and Toxoplasma gondii in zoo animals

Neospora caninum is an apicomplexan parasite that causes neuromuscular disease in dogs and abortions in cattle. Little is known about the prevalence of antibodies to this parasite in zoo animals. Sera from 556 animals, from 13 Czech and Slovak zoos were tested for antibodies to N. caninum and Toxoplasma gondii by indirect fluorescent antibody test. Antibodies to N. caninum were found in 31 of 556 zoo animals (5.6%), representing 18 of 114 species tested: Eurasian wolf (Canis lupus lupus), Maned wolf (Chrysocyon brachyurus), fennec (Vulpes zerda), cheetah (Acinonyx jubatus), jaguarundi (Herpailurus yaguarondi), Eurasian lynx (Lynx lynx), Indian lion (Panthera leo goojratensis), fisher (Martes pennanti), blackbuck (Antilope cervicapra), European bison (Bison bonasus), lechwe (Kobus leche), African buffalo (Syncerus caffer caffer), eland (Taurotragus oryx), sitatunga (Tragelaphus spekei gratus), Thorold's deer (Cervus albirostris), Eastern elk (C. elaphus canadensis), Vietnam sika deer (C. nippon pseudaxis) and Père David's deer (Elaphurus davidianus). Titres ranged from 1:40 to 1:2560. The highest prevalence 50% was found in family mustelidae of the order carnivora. Antibodies to T. gondii were detected in 193 of 556 zoo animals (34.7%) representing 72 of 114 species tested, with titres ranging from 1:40 to 1:40960. The highest prevalence 100% was found in families: hyaenidae, mustelidae, ursidae and viveridae of the order carnivora. The results of this study indicate that zoo animals have more exposure to T. gondii than to N. caninum. It is the first report of seroprevalence of antibodies to N. caninum in European zoo animals.     

Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum

The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added to wells containing either T. gondii or N. caninum tachyzoites in equine dermal (ED) cells. Parasite growth inhibition was measured using 3H-uracil incorporation as compared to untreated controls. T. japonica inhibited T. gondii proliferation by 99.3, 95.5, 73.0 and 54.0% in the range from 156 to 19.5 ng/ml, and S. flavescens inhibited T. gondii proliferation by 98.7, 83.0 and 27.2% in the range from 156 to 39 ng/ml. T. japonica inhibited N. caninum proliferation by 97.8, 97.9, 85.3 and 46.4% in the range from 156 to 19.5 ng/ml. S. flavescens inhibited N. caninum proliferation by 98.6, 97.0, 69.5 and 14.0% in the range from 156 to 19.5 ng/ml. Toxicity to host cells was noted when concentrations of T. japonica and S. flavescens exceeded 625 ng/ml. The herb extracts from S. acutum, Pulsatilla koreana, and U. macrocarpa also showed toxicity at higher levels but did not achieve the same inhibition effects at the lower concentrations against T. gondii and N. caninum as T. japonica and S. flavescens.     

Occurrence of Neospora caninum and Toxoplasma gondii infections in ovine and caprine abortions

Neospora caninum and Toxoplasma gondii are closely related cyst-forming apicomplexan parasites identified as important causes of reproductive failure in cattle and small ruminants, respectively. Protozoan abortion in small ruminants is traditionally associated with T. gondii, but the importance of N. caninum remains uncertain. The aim of this study was to investigate the presence of N. caninum and T. gondii infections in abortion cases in small ruminants submitted for diagnosis. For this purpose, 74 ovine and 26 caprine aborted foetuses were recovered from different areas in Spain. Foetal histopathology was used to detect the presence of protozoal-associated lesions in brain. The presence of N. caninum and T. gondii was confirmed by PCR. Protozoal infection was detected in 17 out of 100 (17%) foetuses examined by at least one of the diagnostic techniques used. Lesions suggestive of protozoal infection were observed in 10.8% (8/74) and 15.4% (4/26) of the ovine and caprine abortions respectively. N. caninum and T. gondii infection was detected by PCR in 6.8% (5/74) and 5.4% (4/74) of sheep foetuses, respectively, of which five showed protozoal-associated lesions. N. caninum DNA was detected in 11.5% (3/26) of goat foetuses, of which two showed protozoal-associated lesions, whereas T. gondii DNA was detected in one goat foetus with no lesions. The simultaneous presence of N. caninum and T. gondii DNA was detected in one sheep foetus with severe lesions. This study demonstrates that N. caninum plays a significant role in abortion in small ruminants in the studied population. In addition, our results highlight the importance of differentiating between protozoa whenever characteristic lesions are observed.     

Neospora caninum infections in Australia and New Zealand

OBJECTIVE: To review the current state of knowledge of Neospora caninum infections with particular reference to Australia and New Zealand. PROCEDURE: Several databases were searched electronically including Medline, Current Contents, Vet CD using several key words (Neospora caninum, neosporosis, abortion, cattle, dogs) and authors names. References in original articles were also traced and use made of the author's own original research in the field. CONCLUSION: N caninum is recognised worldwide and is also widespread, in particular in dairy cattle, in Australia and New Zealand. It has been reported in both countries retrospectively (in dogs) from the early 1970s. Abortion storms in dairy herds appear to be the most common feature. Recent reports indicate that the dog is the definitive host, in which the sexual cycle is completed. Further studies are however required to establish important aspects of the epidemiology, such as mode of transmission. In the absence of an effective vaccine, the most effective control strategy is selective culling of infected animals and the prevention of access of dogs to expelled placentas and foetuses and, possibly, to raw beef.     

The occurrence of Toxoplasma gondii and Neospora caninum as regards meat hygiene

Toxoplasma gondii and Neospora caninum are phenotypically and phylogenetically closely related cyst-forming coccidia, both of which may cause abortion in livestock animals. T. gondii exhibits also zoonotic potential by causing diaplacental infections in the human fetus and harmful infections in immunosuppressed individuals. Humans get infected either by consuming inappropriately prepared cyst-containing meat or by ingesting oocysts originating from cat feces. Therefore, in order to assess infection risk we need to have knowledge on the prevalence of the parasite in consumable meat and thus slaughtered animals. So far, no data indicate any zoonotic potential for N. caninum. Due to its high economic impact in the bovine production in Switzerland, we included this parasite in the present study as well. The prevalence of both parasite species were investigated by PCR in muscle and brain samples of slaughtered bovines, sheep, pigs and horses. Comparatively, a serum sample from each animal was simultaneously tested serologically by a Toxoplasma-P30-ELISA and a Neospora-SA-ELISA. The prevalences determined by the T. gondii-PCR were the followings: adult cows 3%, young bulls 2%, young cows prior to gravidity 6%, calves 1%, sheep 6%, horses and pigs each 0%. For N. caninum, the PCR-prevalence was 2% for adult cows and 0% for all other animal groups. Conversely, the seroprevalences were much higher for both parasite species and all animal groups, with the exception of the fattening pigs. However, as T. gondii was principally detectable in bovine (cows and calves) as well as in sheep meat, the consumption of this meat harbours a potential infection risk for humans. In contrast, the lack of any parasite detectability in fattening pig and horse meat allows to consider this infection source as neglectable when compared to bovine and ovine meat.     

Seroepidemiology of Toxoplasma gondii and Neospora caninum in seals around Hokkaido, Japan

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.     

Presence of Toxoplasma gondii and Neospora caninum DNA in the brain of wild birds

Toxoplasma gondii infections are prevalent in many avian species and can cause mortality in some bird hosts. Although T. gondii has been isolated from various species of birds, the role of many different species of wild birds in the epidemiology of T. gondii remains unknown. Neospora caninum, a closely related parasite to T. gondii, has been recently confirmed to infect domestic chickens and wild birds such as house sparrows (Passer domesticus). The present study reports the presence of T. gondii and N. caninum DNA by PCR in brain tissues of 14 species of wild birds from Spain. From a total of 200 samples analyzed, 12 samples (6%) were positive for T. gondii [5 Eurasian jays (Garrulus glandarius), 5 magpies (Pica pica), 1 black kite (Milvus migrans) and 1 Griffon vulture (Gyps fulvus)], while 3 samples (1.5%) were positive for N. caninum [2 magpies and 1 common buzzard (Buteo buteo)]. This is the first report of detection of T. gondii in magpies, griffon vulture and black kite and of N. caninum in common buzzard and magpies, extending the list of natural intermediate hosts for T. gondii and N. caninum infections to these species.     

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.     

The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina

The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous bovine abortions in Argentina. Based on histopathological results, 70 presumptive cases of apicomplexan protozoal abortion from a total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999 to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available. Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in any aborted bovine fetus.     

Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil.

A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlandia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T. gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N. caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T. gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1%) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T. gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By IP-Nc, two highly immunodominant antigens (29 and 35kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N. caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders.     

Seroprevalences of Neospora caninum and Toxoplasma gondii in nondomestic felids from southern Africa.

Sera from 68 nondomestic captive and free-ranging felids from southern Africa were tested for antibodies to Neospora caninum and Toxoplasma gondii by the indirect fluorescent antibody test. Four of the 68 (5.9%) serum samples were positive for antibodies to N. caninum, with titers ranging from 1:50 to 1:200. All other animals were negative for antibodies to N. caninum at a dilution of 1:50. Fifty of the 68 (74%) serum samples tested positive for antibodies to T. gondii, with titers ranging from 1:50 to 1:26,500. Four animals tested positive for antibodies to both N. caninum and T. gondii. None of these animals displayed clinical signs of disease. Results of this study indicate that nondomestic felids in southern Africa have been exposed to, and are likely infected with, N. caninum and T. gondii.     

Molecular assessment of the transplacental transmission of Toxoplasma gondii,Neospora caninum,Brucella canis and Ehrlichia canis in dogs

Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents.