Detection of malaria in goats and sheep

Plasmodium species, as the causative agent of malaria, are a significant concern in livestock production. This study aims to investigate the presence of malaria parasites in small ruminants comprised of goats and sheep. Plasmodium infections in ungulates do not cause malaria in humans. However, they might have anopheline mosquitoes as the same vector of this parasite. This study uses molecular techniques, nested PCR, to detect Plasmodium infections in small ruminants, as traditional microscopic examination may lead to misdiagnosis. The results show that Plasmodium infections were found in goat samples from Sumba Barat Daya, Nusa Tenggara Timur, with a prevalence of 7.41% based on nested PCR assay. Notably, Plasmodium was not detected through microscopic examination, underscoring the sensitivity of molecular techniques. It should be noted that none of the goats tested positive for malaria based on microscopic examination, suggesting extremely low parasitemia levels. This study emphasizes the importance of understanding Plasmodium infections in small ruminants, shedding light on their impact on animal health and their potential role in local transmission patterns. The sample size in this study is limited. Further research with larger samples and expanded geographical scope is recommended to comprehensively understand Plasmodium prevalence in small ruminants. 


INTRODUCTION
Blood parasites are considered one of the primary causes of disruptions in livestock production worldwide (Armour et al., 1996).These parasites pose a serious economic threat and remain a major concern for animals in tropical and subtropical regions (Soulsby, 1986) (Soulsby, 1986).Diseases caused by protozoa in the blood also result in significant losses for wild and domestic animals worldwide (Ozubek et al., 2020).Plasmodium is one such blood parasite that affects domestic animals, and it is also found in humans, causing a disease known as malaria.
The economic impact of P. caprae as a cause of malaria in goats is generally unknown in goat farming.Research on P. caprae is limited, and malaria in goats typically does not exhibit clinical symptoms, with most cases presenting as low parasitemia (Kaewthamasorn et al., 2018).Recent studies have reported that goats infected with malaria may exhibit symptoms such as fever, tachycardia, tachypnea, and mucosal jaundice.Still, these observations have yet to be confirmed due to the presence of other concurrent infections (Al-Badrani & Alabadi, 2021).
The prevalence of malaria in goats is reported to be relatively low.Molecular detection of malaria in goats in Thailand showed positive results only in Phetchaburi province and not in Chonburi, Rayong, or Nan provinces.The gradual examination of P. caprae positivity in samples in Phetchaburi was reported as five positives out of 126 samples (4%), 0 positives out of 88 samples (0%), and 5 positives out of 100 samples (5%).The prevalence of P. caprae in goats in Thailand is sporadic, ranging from 0% to 5%, and is much lower than P. bubalis in water buffaloes, which ranges from 16% to 45% (Asada et al., 2018;Templeton et al., 2016).
Specific research on malaria in goats in Indonesia has not been conducted to date.Parasites resembling human Plasmodium in goats in malaria-endemic areas for humans have been reported in West Sumba, Fakfak (Munirah et al., 2020) and Kaligesing (Sumanto et al., 2021).These reports do not specify the possible blood parasite species found based on an approach to P. caprae since they focus on malaria affecting humans.
It is important to conduct investigations into how malaria parasites affect the transmission dynamics in ungulates, including their impact on the health of both animals and humans.By studying ecosystems, we can develop strategies to control and prevent malaria.Hence, this study aims to contribute to understanding Plasmodium infections in small ruminants.In this study, malaria parasites were found and screened in goats and sheep using nested PCR, which targets the cytochrome b oxidase (cytb) gene.The results of this investigation will improve our knowledge of Plasmodium infections in ruminants.Provide valuable insights into the broader epidemiological and evolutionary context of the malaria parasites in small ruminants.The findings from this study could have implications for controlling and preventing malaria among small ruminants.

Ethical statements
The protocol for this study was reviewed and approved by the Universitas Gadjah Mada, Faculty of Veterinary Medicine's Institutional Animal Care and Use Committee (IACUC), with the reference number 006/EC-FKH/Int./2023.This study obtained official veterinary government consent to collect blood samples from goats at defined places.

Sample collection and sampling site selection
A cross-sectional study was conducted to detect the Plasmodium parasite in goats from Nusa Tenggara Timur and Jember.The sampling location was chosen because this area has been documented to have probable human-endemic malaria.Each goat's blood was collected in three milliliters and immediately transferred to an ethylenediaminetetraacetic acid (EDTA) tube for preservation.Following sample collection, the samples were transferred to the Faculty of Veterinary Medicine laboratory at Universitas Gadjah Mada, utilizing an ice box to keep the temperature stable.Morphological and molecular approaches were used to investigate all samples.

Blood smear examination
Blood smears were taken for morphological analysis.Each blood smear was fixed in methanol for 10 minutes before being stained for 45 minutes with 10% Giemsa.The blood smears were then cleaned with clean water and allowed to dry.A drop of immersion oil was applied to each blood smear before being examined under an Olympus DP12 microscope at 1000x magnification.If at least one parasite was found in the blood smear, the sample was termed positive.Following World Health Organization criteria, at least 2,000 red blood cells (RBCs) were counted (WHO, 2015).
A total amount of 12.5 µL was used for the PCR experiment.A master mix containing 0.25 µL of KOD FX Neo Polymerase (Toyobo, Japan), 6.25 µL of 2X PCR Buffer, 2.5 µL of 2 mM dNTPs, 0.3 µM of each primer, and 1.75 µL of sterile distilled water (SDW) were included in each reaction.Each reaction also contained 1 µL of genomic DNA as a template.The initial PCR result was diluted ten times with SDW before being subjected as a DNA template in nested PCR (1 µL).
The PCR method required 40 cycles of denaturation at 98 °C for 10 s and annealing and extension at 62 °C for 3 min, with the first step being a 2-minute denaturation step at 94 °C.A single cycle at 68 °C for 5 minutes was then used for the final extension phase.Nested PCR was done under the same conditions.A mouse culture of Plasmodium berghei was used in this investigation as a positive control, while sterile distilled water (SDW) was used as a negative control.A total of 5 µL of PCR products were electrophoresed for 45 minutes on a 1.5% gel of agarose using 1x TAE at 120 volts and 400 mA.

Data analysis
The binomial Clopper-Pearson confidence interval was used to determine the percentage of samples with positive results from the nested PCR assay.An online statistical analysis tool is available at https://epitools.ausvet.com.au was used to conduct statistical analysis.
with 7.41% of examined samples giving positive results.In concordance with the study by Opara and Nwokedi (2011), the finding of this study implies that Plasmodium parasites are likely present in small ruminants in this area.However, no Plasmodium sp. was detected through microscopic examination, highlighting the superior sensitivity of molecular techniques like PCR in finding these parasites even when they are not readily visible using blood smear examination methods.It is also crucial to note that none of the goats tested positive for malaria, showing that the parasitemia level in these goats is exceedingly low.This finding is consistent with (Nguyen et al., 2023) recent study.
Plasmodium discovery in goats and sheep is crucial not only for understanding its impact on animal health but also for potential human health consequences.While Plasmodium species that infect ungulates do not directly cause malaria in humans, they do contribute to the general epidemiology of these parasites by acting as potential reservoirs and offering insight into local transmission patterns.As reported previously by (Opara & Nwokedi, 2011), Plasmodium sp.infections have been documented in sheep and goats, with reported prevalence rates reaching as high as 56.3%.Remarkably, these infections have been associated with a decline in packed cell volume (PCV), indicative of anemia, as noted previously by Aseme et al. (2020).The study also noted that prevalence rates of 12.0% and 36.0%for Plasmodium sp.infections have been detected in goats at the Trans Amadi and Rumuokoro abattoirs, respectively.Additionally, none of the sheep were detected for malaria infection.This result in contrast to the previous finding by Al-Badrani and Alabadi (2021) which noted that they found malaria parasite in sheep.The prevalence of Plasmodium in goats in areas with a history of human-endemic malaria necessitates additional research into potential zoonotic transmission routes and the involvement of these animals in the malaria ecosystem.
This study has taken a step for malaria detection in small ruminants by PCR assay in domestic goats and sheep, similar to the study by Opara and Nwokedi (2011).Nevertheless, one of the limitations of this study is the small sample size for malaria detection in small ruminants.Therefore, increasing the sample size and expanding the geographical scope might provide a more comprehensive knowledge of Plasmodium prevalence in small ruminants in Indonesia.Advanced research is necessary to identify the Plasmodium species found in goats and to investigate the genetic diversity of these parasites.Such further advanced research would shed light on evolutionary links and the mechanics of potential cross-species transmission.

CONCLUSIONS
Overall, the findings of this study reveal that natural malaria infections were found in Sumba Barat Daya area based on nested PCR assay.The study further revealed that malaria parasite infection in goats was asymptomatic.This suggests that natural malaria infection in small ruminants presenting with no clinical signs may require a sensitive tool for malaria diagnosis.PCR technology could increase the sensitivity of malaria detection in small ruminants since all the animals in this study were asymptomatic, and parasite load could be extremely low parasitemia level for microscopy.Additionally, since human malaria was reported to have a high prevalence in this area, malaria infection in domestic goats may associated with the presence of anopheline mosquitoes as the vector as well as breeding sites in the sample collection area.Therefore, advanced research on malaria in small ruminants is needed in order to develop malaria prevention and control strategies in animals.Further research on malaria in small ruminants can provide insights into potential cross-species transmission dynamics as well as potential implications for human health.

Table 1 .
The estimated proportion of Plasmodium infection based on PCR assay was calculated by the binomial Clopper-Pearson confidence interval method