Neospora caninum in aborted bovine fetuses in Trakya Region, Turkey - histopathological, immunohistochemical and molecular detection

Background: Being the major cause of bovine abortion in the world, Neosporosis is considered to be a very important protozoal infection in dairy cattle. Vertical transplacental transmission is the major route of the infection causing either abortion or birth of calves with persistent infection. As the seropositivity in individual cows and in fetal serology only indicate exposure to the protozoa, the diagnosis of the infection has to be based on histopathology of aborted fetuses. Additional techniques such as immunohistochemistry (IHC) and PCR are required for the detection of the etiological agent. The purpose of the current study was to diagnose Neospora caninum infection in aborted bovine fetuses in Trakya Region of Turkey. For this purpose, serological, histopathological, IHC, and PCR methods were used. Materials, Methods & Results: The blood samples and the fetuses of 55 aborted dairy cattle from various farms located in 3 provinces of Trakya, Turkey constituted the material of the present study. The sera obtained from the blood samples were tested using a Neospora caninum Antibody Test Kit cELISA and anti-N. caninum antibodies were detected in the sera of the dams of the 8 aborted fetuses (8/55; 14.54%). Following the necropsy, samples from the brain, heart, liver, lung, kidney, spleen, and placenta of 55 fetuses were routinely processed for histopathological examination and evaluated under a light microscope. Nonsuppurative encephalitis (15/55; 27.27%), necrosis (5/55; 9%) and gliosis (1/55; 1.8%) in the brain, mild to severe nonsuppurative myocarditis and epicarditis (14/55; 25.45%), and portal to mid-zonal nonsuppurative hepatitis (13/55; 23.63%) were the relevant findings. PCR analysis was performed on fresh frozen fetal tissues. Nested PCR detected N. caninum DNA in the brain, heart, liver, lung, and kidney tissues of 6 fetuses (6/55; 10.9%). IHC was performed on the brain, heart, and liver tissues of all the fetuses using avidin-biotin-complex peroxidase method. Immunoreactivity was observed in the brain of 1 fetus (1/55; 1.8%). Discussion: In the present study, histopathological, immunohistochemical and PCR analyses were performed to detect N. caninum in 55 spontenously aborted bovine fetuses in Trakya Region, Turkey. Histopathologic hallmark of the study was nonsuppurative inflammation found mostly in the brain, heart and liver followed by kidneys and lungs. No protozoa was observed in the microscopic examination supporting the fact that definitive diagnosis of N. caninum infection requires ancillary techniques such as IHC and PCR. Nested PCR detected N. caninum DNA in the tissues of 6 fetuses (6/55; 10.9%). Brain was the most reliable organ for detection by PCR (6/6; 100%), compatible with the previous reports. IHC diagnosis revealed only 1.8% positivity in the present study which was remarkably lower than found in the previous studies. Even though histopathology in conjunction with IHC are accepted as the "gold standard" methods to detect N. caninum infection in aborted bovine fetuses, there are studies claiming that IHC is relatively insensitive in the diagnosis of neosporosis as parasite numbers can be low and thus, false negative results can be obtained. Other factors affecting the sensitivity of the technique are thoroughly discussed by many authors. Supportively, the findings of the current study showed that using both IHC and PCR as complementary techniques, increases the success of detection of N. caninum as recommended in previous studies. In conclusion, the present study demonstrated the first molecular diagnosis of Neospora caninum infection in bovine aborted fetuses in Trakya Region of Turkey which has a critical geographical location bordering Europe.

Improvement of Leptospira spp. diagnosis in aborted bovine fetuses by qPCR.

Leptospirosis is a disease with major economic impact on livestock industry. The objective of this work was to determine the presence of Leptospira spp. DNA by qPCR in bovine fetuses with presumptive diagnosis of leptospirosis as the cause of abortion. Leptospira spp. DNA was detected by qPCR in 11 out of 34 fetuses. These specimens (10/11) had histopathological findings in hepatic and/or renal tissues compatible with leptospirosis. qPCR detection rate (32.4 %) was higher compared with direct immuno-fluorescence antibody test (DFAT) (11.8 %). The concordance coefficient between both techniques was 0.44. qPCR is a rapid and sensitive technique for the diagnosis of leptospirosis and improved the detection rate in fetal tissues compared with DFAT. Implementation of molecular techniques may increase the accurate detection of leptospirosis as a cause of bovine abortion allowing the application of rapid therapeutic and prophylactics measures in order to reduce the impact of this zoonotic disease.

Leptospira sp. vertical transmission in ewes maintained in semiarid conditions.

Leptospirosis is a globally distributed disease associated with reproductive failures in livestock; however, its pathogenesis has not been fully elucidated. Results from the present study indicate there is a presence of Leptospira sp. in organs and fluids of fetuses from ewes slaughtered in the semiarid region of Brazil. Twenty-nine fetuses from 23 ewes determined to be Leptospira sp.-positive using PCR were sampled (14 and 15 in dry and rainy seasons, respectively). Fetal samples of blood, central nervous system (CNS), lung, liver, spleen, stomach contents, peritoneal fluid, kidney, bladder, urine and reproductive system were collected. Diagnostic methods included the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Of the 29 fetuses, 24 (82.8 %) had at least one Leptospira sp.-positive organ or fluid, as determined using PCR, and of a total of 209 samples, 62 (29.7 %) contained leptospiral DNA. Of the 99 samples collected during the dry season, 42 (42.4 %) were positive, and of 110 samples collected during the rainy season, 20 (18.2 %) were positive (P = 0.0001). There was deoxyribonucleic acid (DNA) sequencing of three samples of kidney, CNS and liver, and in all of these, there was 99.3 % similarity with Leptospira interrogans. Leptospires were present in cultures of pooled samples from fetuses with deformities. Results indicate there is vertical (maternal-to-fetus) transmission which would represent an alternative transmission route for the spread of Leptospira sp. in ewes, suggesting molecular detection is essential in the investigation of leptospirosis in fetuses to identify animals that have been infected with this bacterium.

Aborto infeccioso por Neospora spp. em equino ˗ relato de caso / [Neospora spp infectious abortion in the horse ˗ case report]

O abortamento na espécie equina é responsável por grandes perdas econômicas e, entre as possíveis causas, está a neosporose, uma enfermidade que nem sempre é investigada como provável diagnóstico. Assim, o objetivo deste trabalho foi relatar um caso de aborto equino aos 129 dias de gestação, resultante da infecção por Neospora spp.. Amostras sanguíneas da égua e do feto abortado foram enviadas para um laboratório especializado. À necrópsia, constatou-se edema gelatinoso e hemorrágico em subcutâneo, fígado ictérico e friável com autólise de alguns órgãos, como baço, rins e glândulas adrenais. Fragmentos dos órgãos coletados na necrópsia foram submetidos à histopatologia e à pesquisa de agentes infecciosos, em que se identificou hepatite e epicardite não purulentas e onfalite purulenta, bem como exame positivo para Neospora spp. pela técnica de reação em cadeia da polimerase (PCR) convencional e Nested. O soro sanguíneo obtido da égua foi submetido à pesquisa de Neospora spp. pela técnica de imunofluorescência indireta, de herpesvírus por soroneutralização em cultura de células e de Leptospira sp. por PCR, todas com resultados negativos. Dessa forma, é importante considerar a neosporose como diagnóstico diferencial em casos de abortamento e natimortalidade, uma vez que a presença de cães nas propriedades é extremamente comum, representando uma importante fonte de infecção.(AU)

Abortion in the equine species is responsible for major economic losses, and among the possible causes is neosporosis, a disease that is not always investigated as a probable diagnosis. Thus, the objective of this study was to report an abortion at 129 days of gestation resulting from Neospora spp. Blood samples from the mare and the aborted fetus were sent to a specialized laboratory. Under necropsy, gelatinous and hemorrhagic edema was detected in subcutaneous tissue, friable and jaundiced liver with autolysis in spleen, kidneys and adrenal glands. Fragments of the organs collected at necropsy were submitted to histopathology and infectious agent tests, which identified non-purulent hepatitis and epicarditis and purulent omphalitis. Also, a positive result for Neospora spp. by the conventional and nested Polymerase Chain Reaction (PCR) technique. Blood serum obtained from the mare was subjected to analyses to Neospora spp. by indirect immunofluorescence technique, herpesvirus by serum neutralization in cell culture and Leptospira sp. by PCR, all with negative results. Thus, it is important to consider neosporosis as a differential diagnosis in cases of abortion and stillbirth, since the presence of dogs in the properties is extremely common and represent an important source of infection.(AU)

Aborto infeccioso por Neospora spp. em equino - relato de caso / [Neospora spp infectious abortion in the horse - case report]

O abortamento na espécie equina é responsável por grandes perdas econômicas e, entre as possíveis causas, está a neosporose, uma enfermidade que nem sempre é investigada como provável diagnóstico. Assim, o objetivo deste trabalho foi relatar um caso de aborto equino aos 129 dias de gestação, resultante da infecção por Neospora spp.. Amostras sanguíneas da égua e do feto abortado foram enviadas para um laboratório especializado. À necrópsia, constatou-se edema gelatinoso e hemorrágico em subcutâneo, fígado ictérico e friável com autólise de alguns órgãos, como baço, rins e glândulas adrenais. Fragmentos dos órgãos coletados na necrópsia foram submetidos à histopatologia e à pesquisa de agentes infecciosos, em que se identificou hepatite e epicardite não purulentas e onfalite purulenta, bem como exame positivo para Neospora spp. pela técnica de reação em cadeia da polimerase (PCR) convencional e Nested. O soro sanguíneo obtido da égua foi submetido à pesquisa de Neospora spp. pela técnica de imunofluorescência indireta, de herpesvírus por soroneutralização em cultura de células e de Leptospira sp. por PCR, todas com resultados negativos. Dessa forma, é importante considerar a neosporose como diagnóstico diferencial em casos de abortamento e natimortalidade, uma vez que a presença de cães nas propriedades é extremamente comum, representando uma importante fonte de infecção.(AU)

Abortion in the equine species is responsible for major economic losses, and among the possible causes is neosporosis, a disease that is not always investigated as a probable diagnosis. Thus, the objective of this study was to report an abortion at 129 days of gestation resulting from Neospora spp. Blood samples from the mare and the aborted fetus were sent to a specialized laboratory. Under necropsy, gelatinous and hemorrhagic edema was detected in subcutaneous tissue, friable and jaundiced liver with autolysis in spleen, kidneys and adrenal glands. Fragments of the organs collected at necropsy were submitted to histopathology and infectious agent tests, which identified non-purulent hepatitis and epicarditis and purulent omphalitis. Also, a positive result for Neospora spp. by the conventional and nested Polymerase Chain Reaction (PCR) technique. Blood serum obtained from the mare was subjected to analyses to Neospora spp. by indirect immunofluorescence technique, herpesvirus by serum neutralization in cell culture and Leptospira sp. by PCR, all with negative results. Thus, it is important to consider neosporosis as a differential diagnosis in cases of abortion and stillbirth, since the presence of dogs in the properties is extremely common and represent an important source of infection.(AU)

Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico.

The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzyme-linked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87% (n = 246). Seropositive animals were found in six out of nine (66.6%) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51%. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico.

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of São Paulo, Brazil.

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (São Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

Identification of new IS711 insertion sites in Brucella abortus field isolates.

BACKGROUND: Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated). Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. RESULTS: We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. CONCLUSIONS: Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

Isolamento de Campylobacter jejuni em feto ovino abortado: relato de caso / Isolation of Campylobacter jejuni from ovine aborted fetus: case report

Relata-se o isolamento de Campylobacter jejuni de um feto ovino abortado no terço final da gestação. Em uma semana, observaram-se quatro casos de aborto em ovelhas com quatro meses de gestação. Imediatamente após o terceiro aborto, um dos fetos foi submetido a exame patológico, quando se realizou a colheita do conteúdo do abomaso para análise bacteriológica. A necropsia, somente foram evidenciadas alterações autolíticas. Apenas na microaerofilia encontrou-se crescimento bacteriano puro com características morfo-tintoriais de Campylobacter sp., identificado fenotípica e molecularmente como C. jejuni. Alerta-se para a possibilidade de quadros de abortos em ovinos serem causados por esse patógeno.

Isolamento de Campylobacter jejuni em feto ovino abortado: relato de caso / Isolation of Campylobacter jejuni from ovine aborted fetus: case report

Relata-se o isolamento de Campylobacter jejuni de um feto ovino abortado no terço final da gestação. Em uma semana, observaram-se quatro casos de aborto em ovelhas com quatro meses de gestação. Imediatamente após o terceiro aborto, um dos fetos foi submetido a exame patológico, quando se realizou a colheita do conteúdo do abomaso para análise bacteriológica. À necropsia, somente foram evidenciadas alterações autolíticas. Apenas na microaerofilia encontrou-se crescimento bacteriano puro com características morfo-tintoriais de Campylobacter sp., identificado fenotípica e molecularmente como C. jejuni. Alerta-se para a possibilidade de quadros de abortos em ovinos serem causados por esse patógeno.(AU)

Campylobacter jejuni was isolated from aborted ovine fetus at the end of gestation. Three abortion cases within a week and another fourth case one month later were observed. Immediately after the third case, one fetus was submitted to necropsy and samples from abomasal content were collected for bacteriological analysis. At necropsy, only post-mortem and autolytic lesions were observed. Under microaerophilic conditions, a pure bacterial growth was obtained and identified by morphological and tintorial characteristics as Campylobacter sp., being the isolate confirmed as C. jejuni, by phenotypic and molecular assays. This is an alert for the possibility of this pathogen cause abortion in ovine.(AU)

Buenos Aires, a new Leptospira serovar of serogroup Djasiman, isolated from an aborted dog fetus in Argentina.

This study describes the isolation of a new leptospiral serovar from the Djasiman group from an Argentinean aborted fetus of a dog. The strain was isolated from a culture of mixed liver and kidney tissue from one aborted dog fetus. Bitch's serum showed a titre of 1:800 against the new serovar and 1:400 or less against other serovars tested. Microscopic Agglutination test (MAT) with a panel of 38 rabbit anti-Leptospira sera representative for all pathogenic serogroups was performed to establish the putative serogroup of the isolated strain. Serovar identification was done by Cross-Agglutination Absorption Test (CAAT). Species determination was carried out on basis of sequence identity of primer pair G1/G2 generated PCR products from the isolate with those from reference strains belonging to the various pathogenic Leptospira species. Results showed that the new strain named, Baires, belongs to species L. interrogans sensu stricto and the serogroup Djasiman. However, the isolate could not be identified as any of the known serovars within this serogroup. These results allow us to conclude that the strain Baires represents a new serovar (Buenos Aires) of Djasiman serogroup.

Aetiology of bovine abortion in Argentina.

Necropsies were performed on 354 fetuses from dairy and beef herds submitted from 1994 to 2000 to the diagnostic laboratories at Instituto Nacional de Tecnología Agropecuaria, Balcarce, Argentina. Samples from the fetuses were examined for pathogenic organisms and processed for microscopic examination. An aetiological diagnosis was made for 161 (45.5%) of the fetuses. No diagnosis was made for 193 (54.5%) fetuses. Infectious agents were isolated from 122 (34.4%) of the fetuses, bacterial agents being involved in 80 (22.6%) of these. The most common bacterial agents isolated from the fetuses were Brucella abortus in 28 fetuses, Campylobacter fetus in 26 cases, and Escherichia coli in 9 cases. Bovine herpesvirus and bovine viral diarrhoea virus were found in 9 and 6 cases, respectively. Neospora caninum was detected by an immunohistochemical technique in 26 cases (7.3%). Congenital abnormalities, dystocia and mummifications were found in 8, 19 and 11 cases, respectively.