An outbreak of Neospora caninum abortion in a dairy herd from the State of Georgia, United States

Abstract An outbreak of 92 abortions out of 1,700 pregnant cows (5.41%) in a period of 3 weeks (19 May to 05 June 2019) occurred in a Georgia Dairy, USA, in cattle that were between 3 and 7 months of gestation. Two sets of samples (aborted fetuses’ organs, placental tissues, aborted cows blood) were submitted for laboratory investigations at the Tifton Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia (TVDIL, Tifton, GA, USA). An abortion panel testing for the major abortion‐causing agents [e.g. Bovine Viral Diarrhoea Virus (BVDV), Infectious Bovine Rhinotracheitis Virus/ Bovine Herpes Virus‐I (IBR/ BHV‐I), Brucella spp., Leptospira spp.] was conducted on several of the samples. On the first set of samples, microbial cultures, serology and PCR tests for the common abortifacient agents revealed the presence of Neospora caninum (N. caninum) DNA, which was positive by PCR on the placenta and fetal tissues. The second set of diagnostic investigations also identified two out of three submitted freshly aborted fetuses to be positive for N. caninum by PCR and immunohistochemistry. Moreover, all three dams were also sero‐positive for N. caninum. The entire herd was being fed on grass silage harvested from a pasture where feral pigs were hunted previously and carcasses were left behind. As a consequence of this action a large population of wild coyotes were attracted to these carcasses, and likely contaminated the pasture with potential N. caninum‐infected feces. After the abortion outbreak was resolved, it was recommended that the farmers should avoid disposal of cadavers of hunted animals in the wild, as it could attract carnivorous and omnivorous animals that may potentially spread the disease to the cattle and other wildlife.


| INTRODUC TI ON
Neospora caninum (N. caninum) is a protozoan parasite from the phylum Apicomplexa affecting several animal species, including canine, bovine, ovine, caprine, deer and rodents (Lindsay & Dubey, 2020). Neosporosis has emerged as an important reproductive disease of cattle worldwide. In cattle, it causes abortion, which was first described in dairy cows in New Mexico, USA, in 1989 (Dubey, Schares, & Ortega-Mora, 2007). In the year 2000, a N. caninum-associated abortion outbreak occurred in a dairy farm in South Carolina wherein greater than 10% of the herd aborted over a 4-month period (Jenkins et.al, 2000). The main economic impact of neosporosis in dairy cattle is attributed to its negative impact on fertility and milk yield. The total annual cost of N. caninum infections/abortions was estimated to range from US $1.1 million in the New Zealand beef industry to US $546.3 million in the US dairy cattle industry (Reichel, Ayanegui-Alcérreca, Gondim, & Ellis, 2013).
Dogs and related canids (coyotes, wolves) are definitive hosts of N. caninum (Klein, Barua, Liccioli, & Massolo, 2019). In addition, Australian dingoes or wild dogs (Canis lupus dingo) have been also identified as definitive host of N. caninum (King et al., 2010). The life cycle consists of three known infectious stages: tachyzoites, tissue cysts and oocysts. Tachyzoites and tissue cysts are the stages found in intermediate hosts, and they occur intracellularly (Dubey et al., 2002). The third stage (oocysts) is an environmentally resistant stage that is excreted with the faeces of definitive hosts in an unsporulated form (Dubey et al., 2007), to become invasive as early as 24 hr under room temperature of 37°C (Lindsay, Dubey, & Duncan, 1999).
The mechanism of survival of N. caninum oocysts in the environment is not well understood. One of the main routes of infection for carnivores and omnivores is the ingestion of tissue cysts being presented in muscles of their prey (Lindsay & Dubey, 2020). Herbivores are likely to become infected by the ingestion of food or drinking water contaminated by N. caninum sporulated oocysts (Dubey et al., 2007).
Ingestion of faecal-contaminated food and water is the major source of infection for cattle that may cause an epidemic abortion storm in naïve population, after transplacental transmission of N. caninum to the fetus (Qian et al., 2017). This paper reports an abortion outbreak associated with N. caninum infection in a large Holstein herd from South Georgia, USA.

| Farm
The outbreak occurred in a dairy farm located in Leesburg, GA, USA (Latitude: 31.68; Longitude: −84.17), which consisted of 3,400 lactating Holstein cows, housed in a free-stall system with sand-bedding, fans and sprinklers. Cows were milked three times a day with an average milk yield of 38.6 kg cow −1 day −1 , 190,000 somatic cell counts and standard plate bacteria count of 1,300 cfu.
Lactating cows were fed three times a day a total mixed ration based on grass silage, corn silage and concentrates. Forages were produced in the same farm.
Right after drying-off, cows were sent to another farm to give birth. Fifteen days after parturition, cows were moved back to the original farm. Reproductive management started at 40 days post-partum (synchronization of oestrus protocols) and first breeding occurred around 70 days in milk. Pregnancy diagnosis was performed by the local veterinarian by ultrasound at 32 days' post artificial insemination. Cows were reconfirmed to be pregnant by rectal palpation between 50 and 70 days' post service. If cows were found non-pregnant, they were resynchronized and bred back. After 70 days of pregnancy cows were moved to a pregnant group until dry-off. Historically pregnancy losses (abortion rate) that occurred from 70 days of gestation until drying-off (7 months of gestation) at this dairy farm were less than 1% per month.

| Abortion outbreak description
Overall, 92 abortions occurred in a span of 18 days (19 May 2019 and 5 June 2019) in cattle that were between 3 and 7 months of gestation. These abortions occurred within a population at risk consisting of 1,700 pregnant lactating cows, accounting for an incidence of 5.45%. Table 1  Records from the dairy computer systems were obtained. A walk through of all lactating and dry-cow barns was carried out and the cows were visually inspected. Cows were observed to have a moderate to excellent body condition score, ranging from 2.75 to 3.25 on a scale 1 to 5 (Ferguson, Galligan, & Thomsen, 1994). They appeared to be very clean and were using almost 95% of the sand bedded freestalls. Faeces showed very good consistency and almost 65% of laying cows was ruminating.
Samples from the corn silage, grass silage and total mixed ration were submitted to the TVDIL to check the presence of N. caninum DNA.

| Materials and Methods
Blood samples from five aborted cows, placental tissues (cotyledons and membranes) from three aborted cows, and sections of liver, spleen, kidneys and lungs from two aborted fetuses (abortions from the week of 19-25 May 2019) were received at the TVDIL for the first round of investigation. Representative tissue samples were fixed in neutral-buffered 10% formalin and processed for routine microscopic examination, stained with haematoxylin and eosin (H&E) and examined under light microcopy. In addition, abortion panel testing that comprised of bacterial culture, virus isolation and fluorescent antibody tests were performed on fetal membranes from aborted cows and organs from aborted fetuses. The fresh tissues were also evaluated by PCR tests (probe-based Taqman real-time qPCR assays; Barry et al., 2019;Brower et al., 2008)

| Results
No significant lesions were observed on histopathological examination in the sections of placenta from the three aborted cows as well as liver, lungs, spleen and kidneys from the two aborted fetuses. caninum. All examined tissues including placenta from the cows and tissues from aborted fetuses were positive for N. caninum by PCR.

| Materials and Methods
Three fetuses and one placental tissue (abortions from 26 to 28 May 2019) were submitted to the TVDIL for necropsy (fetuses 1, 2 and 3) along with samples of blood from the dams. During necropsy, samples were collected for histopathology, bacterial cultures, serology and virology and PCR testing (probe-based Taqman real-time qPCR assays; Barry et al., 2019;Brower et al., 2008) ( Table 2). In addition, ocular fluid from fetuses 1 and 2 were collected for nitrate test. Serology for the following infectious agents was performed on the serum from the dams: BTV, Leptospira spp., Anaplasma spp., N. caninum, BVDV types 1a, 1b and 2, IBRV and BHV-4. In addition, virus isolation was attempted on the buffy coat of dam's blood. Mild multifocal non-suppurative encephalitis with glial nodules was observed throughout the brain (Figure 1). Interestingly, N. caninum ELISA was positive from sera of all three cows. In addition, BTV antibody ELISA was positive in two cows and BHV-4 antibody serum neutralization was positive in one cow. All cows were positive for IBR and BVDV types 1a, 1b and 2 by serum neutralization. Seropositive results indicate prior exposure and/or response to immunization. Virus isolation on samples of blood buffy coat was negative in all three cows.
To further confirm diagnosis, immunohistochemistry (IHC) for N. caninum (PAB-NC, VMRD Inc., Pullman WA) was performed in tissue sections of brain, skeletal muscle and heart from fetus 1 and tissue sections of brain, tongue and heart from fetus 2. Positive immunostaining for N. caninum was observed in sections of skeletal muscle and heart from fetus 1 and sections of heart from fetus 2 ( Figure 2).

| D ISCUSS I ON
This herd's normal abortion rate in cows over 70 days of gestation was less than 1% per month prior to 19 May 2019. The abortion rate escalated to 5.41% in a period of 3 weeks (19 May-5 June 2019).
Unquestionably, this spike in fetal losses within a short period of three weeks classifies this case as an 'abortion storm' or 'outbreak'. Detection of N. caninum specific DNA by PCR is a highly sensitive F I G U R E 1 Fetus 1. Brain. Multifocal glial nodules (arrow). HE stain F I G U R E 2 Fetus 2. Heart. Multifocal positive immunostaining for Neospora caninum (arrow) method for the identification of N. caninum in aborted fetal tissues, particularly for a probe-based Taqman real-time qPCR assay that offers an accurate and rapid detection of the parasite from contaminated tissues. The qPCR results were confirmed by two different assays (Barry et al., 2019;Brower et al., 2008) (Nusinovici, Seegersa, Joly, Beaudeaua, & Fourichon, 2012). In addition, positive serology to one of the aborted cows to BHV-4, also raises a question about the role of this herpesvirus in the present outbreak investigation, because this virus has been isolated from several epidemic events of metritis and abortions. BHV-4 has also been considered as a pathogen associated with uterine disease in dairy cattle (Donofrio et al., 2007).
When the positive results of N. caninum infection were confirmed, the local veterinarian proceeded with an inspection of the pastures where grass was harvested for silage. Looking for source of infection, the local veterinarian searched for canid feces on the farm. Further discussions with the farm manager revealed that as a control measure to an invasion of wild pigs in the pastures where these cows were managed, a company that specialized in the control of pests to eliminate wild hogs was hired. It was determined that several carcasses of the slaughtered pigs remained exposed on the pastures where the presence of a large number of coyotes was subsequently observed consuming the remnants of hog carcasses.
The PCR examination of feed did not confirm the presence of N.
caninum DNA. On the basis of the recent studies cited above, the manager of the farm inspected in detail the field where coyotes were observed in a large number. Unfortunately, faecal material from the canids was not found, recalling the grass from this field was previously harvested for silage production. After the abortion outbreak was resolved it was recommended that the owners of the farm not leave cadavers of hunted animals, which may serve as food for coyotes or other carnivores and omnivores that may potentially spread the disease.
In conclusion, an abortion storm in a large confined dairy herd from Georgia, USA occurred in a period of 3 weeks. A thorough laboratory diagnostic investigation of several samples (aborted fetuses, placentas, blood from aborted dam) led to the following findings: N.
caninum intermediary-stages of the parasite, DNA of the parasite and dam's serum antibodies. These results suggest that the most likely aetiological source for abortion cases in this naïve cattle population was N. caninum.

E TH I C S S TATEM ENT
The authors confirm they followed the ethics policies of the journal and the University of Georgia.

ACK N OWLED G EM ENT
We thank LeeCo Dairy, GA, for allowing us to publish this case report.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.